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Functional comparison and analysis of protein-protein interactions of Autographa californica multiple… Nie, Yingchao 2010

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FUNCTIONAL COMPARISON AND ANALYSIS OF PROTEINPROTEIN INTERACTIONS OF AUTOGRAPHA CALIFORNICA MULTIPLE NUCLEOPOLYHEDROVIRUS TRANSREGULATORY PROTEINS IE0 AND IE1  by Yingchao Nie M.Sc., Central China Normal University, Wuhan, China, 2004  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Doctor of Philosophy in The Faculty of Graduate Studies (Plant Science)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) February, 2010  © Yingchao Nie, 2010  Abstract Autographa californica multiple nucleopolyhedrovirus expresses two major transregulatory proteins IE0 and IE1 immediately upon infection. IE0 differs from IE1 only by 54 additional N-terminal amino acids (aa). Either IE0 or IE1 can support viral replication; however both are required for a wild-type infection. It is unknown what the different functions of IE0 and IE1. Both IE0 and IE1 can transactivate viral early genes and support viral DNA replication. It is therefore hypothesized that by the addition of Nterminal 54 aa, IE0 acquires different transactivation activity on viral genes and interacts with different viral or host partners. To test this hypothesis, functional comparisons between IE0 and IE1 and the identification of their interaction partners in infected cells were performed.  Comparisons of subcellular localization and transactivation activities between IE0 and IE1 showed no difference. However analyses of the nucleocapsid content of occlusion derived virions (ODV) revealed that IE0 and IE1 appear to regulate the number of nucleocapsids per ODV. Deletion within the IE0 specific N-terminal 54 aa did not affect IE0 transactivation dramatically but reduced its ability to support viral DNA replication. Analyses of interacting proteins did not identify any proteins that were specific to either with IE0 or IE1. However, the viral protein AC16 (BV/ODV-E26) was shown to bind to both IE0 and IE1 via a binding domain at IE1 72-99 aa. Mutation of the binding domain enhanced budded virus (BV) production by viruses expressing only IE0 but not IE1. Deletion of ac16 however resulted in increased levels of IE0 relative to IE1 as the only observable impact. These results would therefore indicate that AC16 regulates ie0 expression. Deletion of ac16 and the overlapping gene ac17, interestingly resulted in a significant delay of viral gene expression for up to 12 hours. However, the delay of viral gene expression was only observed with BV-infected cells and not in cells infected by transfecting viral DNA. AC16 and AC17 are therefore critical for rapid gene expression during the very early events of infection, and highlight the fact that proteins interacting with IE0 and IE1 play key roles in baculovirus biology.  ii  Table of Contents Abstract............................................................................................................................. ii Table of Contents ............................................................................................................. iii List of Tables .................................................................................................................. vii List of Figures ................................................................................................................ viii List of Abbreviations......................................................................................................... x Acknowledgements ........................................................................................................ xiii Dedication....................................................................................................................... xv Co-authorship Statement ................................................................................................ xvi Chapter I Introduction ....................................................................................................... 1 1.1 Baculoviruses .......................................................................................................... 1 1.2 Baculovirus life cycle .............................................................................................. 2 1.3 Baculoviruses genomes............................................................................................ 3 1.4 Baculovirus entry .................................................................................................... 4 1.4.1 ODV entry ........................................................................................................ 4 1.4.2 BV entry ........................................................................................................... 5 1.5 Baculovirus gene expression.................................................................................... 8 1.5.1 Baculovirus genes ............................................................................................. 8 1.5.2 Expression of IE and DE genes ......................................................................... 8 1.5.3 Expression of late and very late genes ............................................................. 11 1.6 Baculovirus DNA replication................................................................................. 13 1.6.1 Origin of replication........................................................................................ 13 1.6.2 Proteins involved in replication....................................................................... 14 1.7 Baculovirus nucleocapsid assembly and egress ...................................................... 17 1.7.1 Virogenic stroma ............................................................................................ 17 1.7.2 Virion structural proteins and assembly........................................................... 18 1.7.3 Nucleocapsid egress........................................................................................ 20 1.8 Baculovirus transactivators IE0 and IE1 ................................................................ 21 1.8.1 AcMNPV ie0-ie1 gene locus........................................................................... 21 1.8.2 IE1.................................................................................................................. 22  iii  1.8.2 IE0.................................................................................................................. 27 1.8.4 Other viral transactivators resemble IE0 and IE1............................................. 30 1.9 Hypotheses and chapter synopsis ........................................................................... 32 1.10 References ........................................................................................................... 38 Chapter 2 Functional Comparison of AcMNPV IE0 and IE1 ........................................... 66 2.1 Introduction ........................................................................................................... 66 2.2 Results................................................................................................................... 69 2.2.1 IE0 colocalizes with IE1 in Sf9 cells during infection. .................................... 69 2.2.2 Comparison of IE0 and IE1 transactivation of viral early promoters................ 71 2.2.3 Comparison of IE0 and IE1 transactivation of viral early promoters containing the hr5 enhancer ...................................................................................................... 72 2.2.4 IE0 regulates a specific group of viral genes during infection.......................... 72 2.2.5 Deletion analysis of IE0 N-terminal specific 54 amino acids ........................... 74 2.2.6 Expression of IE0 or IE1 changes numbers of nucleocapsids per ODV envelope ................................................................................................................................ 76 2.3 Discussion ............................................................................................................. 77 2.4 Materials and methods........................................................................................... 82 2.4.1 Cells and viruses ............................................................................................. 82 2.4.2 Plasmids construction ..................................................................................... 82 2.4.3 Virus construction........................................................................................... 84 2.4.4 Confocal microscopy ...................................................................................... 84 2.4.5 CAT assay ...................................................................................................... 85 2.4.7 DNA quantification by Slot blot...................................................................... 86 2.4.8 Transmission electron microscopy .................................................................. 86 2.4.9 Western blotting ............................................................................................. 87 2.5 References........................................................................................................... 109 Chapter 3 AcMNPV AC16 (DA26, BV/ODV-E26) Regulates the Levels of IE0 and IE1 and Binds to Both Proteins via a Domain Located within the Acidic Transcriptional Activation Domain ........................................................................................................ 114 3.1 Introduction ......................................................................................................... 114 3.2 Results................................................................................................................. 116  iv  3.2.1 Identification of IE0 and IE1 interacting proteins by tandem affinity purification .............................................................................................................................. 116 3.2.2 Co-immunoprecipitation of AC16 with IE0 and IE1 ..................................... 117 3.2.3 Mapping of the IE0 and IE1-AC16 interaction domain by yeast 2-hybrid...... 117 3.2.4 Point mutation analysis of the IE0 and IE1 AC16 interaction domain in vivo 118 3.2.5 Expression of IE0L133L134L140AAA results in higher BV production than IE0.... 119 3.2.6 Deletion analysis of ac16 .............................................................................. 119 3.3 Discussion ........................................................................................................... 121 3.4 Materials and methods......................................................................................... 124 3.4.1 Viruses and cells ........................................................................................... 124 3.4.2 Plasmids construction ................................................................................... 124 3.4.3 Construction of recombinant viruses ............................................................. 127 3.4.4 3×FLAG-6×His tandem affinity purification (TAP) and protein identification. .............................................................................................................................. 129 3.4.5 Immunoprecipitation..................................................................................... 130 3.4.6 Time course analysis of BV production and viral DNA replication ............... 130 3.4.7 Western blot analysis .................................................................................... 132 3.4.8 Yeast 2-hybrid .............................................................................................. 132 3.5 References........................................................................................................... 148 Chapter 4 Deletion of AcMNPV AC16 and AC17 Results in Delayed Viral Gene Expression in Budded Virus Infected Cells but not Transfected Cells ............................ 152 4.1 Introduction ......................................................................................................... 152 4.2 Results................................................................................................................. 153 4.2.1 Transcriptional analysis of ac17.................................................................... 153 4.2.2 Generation of ac16/17KO and repair viruses................................................. 155 4.2.3 Analysis of viral DNA replication................................................................. 155 4.2.4 Analysis of BV production............................................................................ 156 4.2.5 Viral gene expression analysis ...................................................................... 157 4.2.6 Transcription analysis of gp64 and ie0/ ie1 ................................................... 157 4.2.7 Localization of AC17.................................................................................... 159 4.2.8 Expression of viral proteins in transfected cells ............................................. 160 4.3 Discussion ........................................................................................................... 160 v  4.4 Materials and methods......................................................................................... 163 4.4.1 Viruses and cells ........................................................................................... 163 4.4.2 Plasmid construction..................................................................................... 163 4.4.3 Virus construction......................................................................................... 164 4.4.4 Time course analysis of viral DNA replication and BV production ............... 165 4.4.5 Northern blot ................................................................................................ 165 4.4.6 Quantitative RT-PCR.................................................................................... 166 4.4.7 RACE ........................................................................................................... 166 4.4.8 Western blot analysis .................................................................................... 167 4.4.9 Immunofluorescence..................................................................................... 167 4.4.10 BV purification ........................................................................................... 168 4.5 References........................................................................................................... 182 Chapter 5 General Discussion and Future Perspectives.................................................. 187 5.1 References........................................................................................................... 195  vi  List of Tables Table 2. 1 Microarray analysis a of genes regulated by IE0 and/or IE1........................... 105 Table 2. 2 Primers used in the study .............................................................................. 106 Table 3. 1 Primers used in this study ............................................................................. 145  vii  List of Figures Figure 1. 1 Baculovirus life cycle. ................................................................................... 34 Figure 1. 2 Structures of the two virion types of alphabaculoviruses. ............................... 36 Figure 1. 3 AcMNPV ie0-ie1 gene region and functional domains of IE0 and IE1........... 37 Figure 2. 1 Confocal microscopy analysis of IE1 and IE0 localization. ............................ 66 Figure 2. 2 Immunofluorescence confocal microscopy analysis of (A) IE1 and (B) IE0 localization. ............................................................................................................. 91 Figure 2. 3 CAT assays comparing the transactivation ability of IE0 and IE1. ................. 93 Figure 2. 4 CAT assays comparing the transactivation ability of IE0 and IE1 on viral early promoters cis-linked to hr5 element......................................................................... 96 Figure 2. 5 Transactivation analysis of AcMNPV promoters identified by microarray and regulated differentially by IE0 and IE1. ................................................................... 97 Figure 2. 6 Alignment of IE0 N-terminus specific amino acids from (A) alphabaculoviruses and (B) Group I alphabaculoviruses. ......................................... 99 Figure 2. 7 Location of the IE0 N-terminal deletions. .................................................... 100 Figure 2. 8 Transactivation analyses of IE0 deletion mutants on (A) 39K and (B) me53 promoters with or without the cis-linked hr5 enhancer........................................... 101 Figure 2. 9 Slot blot analysis of viral DNA replication................................................... 103 Figure 2. 10 TEM analysis of ODV produced by WT virus or from viruses expressing only IE0 or IE1.............................................................................................................. 104 Figure 3. 1 AC16 co-purifies with TAP tagged IE0. ...................................................... 133 Figure 3. 2 IE0 and IE1 co-immunoprecipitate with AC16. ........................................... 134 Figure 3. 3 Mapping the IE0 and IE1 AC16 binding domain. ........................................ 135 Figure 3. 4 Co-immunoprecipitation and Western blot confirmation of loss of interaction between AC16 and IE0 or IE1 point mutants. ........................................................ 137 Figure 3. 5 Mutation analysis of the IE0 and IE1 AC16 binding domain........................ 139 Figure 3. 6 Schematic diagram of construction of ac16KO and repair viruses ac16KOAC16, ac16KO-AC16HA...................................................................................... 141  viii  Figure 3. 7 BV production and viral DNA replication analysis of ac16KO, ac16KO-AC16 and AcBac. ............................................................................................................ 143 Figure 3. 8 Western blot analyses of the temporal expression of IE0 and IE1, FP25 and P39. .............................................................................................................................. 144 Figure 4. 1 5’ and 3’ RACE analysis of ac17 transcription. ........................................... 152 Figure 4. 2 Construction of ac16/17KO and repair bacmids........................................... 171 Figure 4. 3 Analysis of viral DNA replication by qPCR................................................. 173 Figure 4. 4 Growth curve analysis of ac16/17KO and repair viruses. ............................. 174 Figure 4. 5 Western blot analyses of the temporal expression of the viral early and late genes IE0/IE1, GP64, LEF-3, P143, P35, VP39 and POLYHEDRIN..................... 175 Figure 4. 6 Transcriptional analysis of gp64 and ie0/1. .................................................. 176 Figure 4. 7 Detection of AC17 in infected cells and budded virus. ................................. 178 Figure 4. 8 Temporal analysis of early and late proteins IE0/IE1 and VP39 in transfected Sf9 cells................................................................................................................. 180 Figure 4. 9 Comparison of the egt-ac17 gene locus in the genomes of alphabaculoviruses. .............................................................................................................................. 181  ix  List of Abbreviations A aa AAD AcMNPV AD AdhoNPV AgseGV AgseNPV AngeNPV ATP BD BmNPV bp BV bZIP CAT CfDEFNPV CfNPV ChchNPV CIP CRE C-terminal CTD D DAPI Δ DBD DE DNA dNTP DO DTT E ECFP EppoNPV EHV FBS Fig. GFP g Gly GP64 GSP  alanine amino acid acidic activating domain Autographa californica multiple nucleopolyhedrovirus activation domain Adoxophyes honmai nucleopolyhedrovirus Agrotis segetum granulovirus Agrotis segetum nucleopolyhedrovirus Anticarsia gemmatalis nucleopolyhedrovirus adenosine tri-phosphate binding domain Bombyx mori nucleopolyhedrovirus base pair budded virus basic leucine zipper chloramphenical acetyl-transferase Choristoneura fumiferana defective nucleopolyhedrovirus Choristoneura fumiferana nucleopolyhedrovirus Chrysodeixis chalcites nucleopolyhedrovirus calf intestinal phosphatase cAMP response element carboxy-terminal c-terminal domain aspartic acid 4′, 6-diamidino-2-phenylindole deletion DNA binding domain delayed early deoxyribonucleic acid deoxynucleotide triphophatases dropout dithiothreitol glutamic acid enhanced cyan fluorescent protein Epiphyas postvittana nucleopolyhedrovirus equine herpesvirus fetal bovine serum figure green fluorescent protein gram in the context of weight, gravity force in the context of speed glycine glycoprotein 64 gene specific primer x  GTF GV HA HearNPV His HIV HLH hpi hpt hr HSV HTLV HycuNPV IE IP IPTG K kb kDa KO L or Leu LC50 LC-MS/MS LdMNPV lef M MacoNPV min ml mM MOI NaCl ND10 nt N-terminal NLS NPV OB ODV OpMNPV ORF PBS PCR pi PlxyNPV POLH  general transcription factor Granulovirus heamoglutinin Helicoverpa amigera nucleopolyhedrovirus histidine human immunodeficiency virus helix-loop-helix hour(s) post infection hour(s) post transfection homologous region herpes simplex virus human T-lymphotropic virus Hyphantria cunea nucleopolyhedrovirus immediate early immunoprecipitation` isopropyl-β-D-thiogalactopyranoside lysine kilobase kilodalton knockout leucine lethal concentration 50 liquid chromatography-mass spectrometry/mass spectrometry Lymantria dispar multicapsid nuclear polyhedrovirus late expression factor molar in the context of concentration; methionine in the context of protein sequence Mamestra configurata nucleopolyhedrovirus minutes millilitre millimolar multiplicity of infectivity sodium chloride nuclear domain 10 nucleotides amino-terminal nuclear localization signal Nucleopolyhedrovirus occlusion body occlusion derived virus Orgyia pseudosugata multiple nucleopolyhedrovirus open reading frame phosphate buffered saline polymerase chain reaction post infection Plutella xylostella multiple nucleopolyhedrovirus POLYHEDRIN  xi  PSB psi qPCR R RACE RNA SDS sec SeMNPV Sf SfMNPV SpltNPV TAP TBP TCID50 TEM TnSNPV TPA TRE Tris Trp µ UTR VLF VSV WT EYFP XecnGV Y-2-H  protein sample buffer pound per square inch quantitative polymerase chain reaction arginine rapid amplification of cDNA ends ribonucleic acid sodium dodecyl sulfate seconds Spodoptera exigua nucleopolyhedrovirus Spodoptera frugiperda Spodoptera frugiperda nucleopolyhedrovirus Spodoptera litura nucleopolyhedrovirus tandem affinity purification TATA-binding protein 50% tissue culture infective dose transmission electron microscope Tiichoplusia ni single nucleopolyhedrovirus 12-O-tetradecanoylphorbal 13-acetate TPA response element Tris-hydroxymethyl amino methane tryptophan micro untranslated region very late factor vesicular stomatitis virus wild type enhanced yellow fluorescent protein Xestia c-nigrums granulovirus yeast two hybrid  xii  Acknowledgements First I would like to thank Dr. David Theilmann, my supervisor for giving me the chance to pursue my doctoral study in his lab, and for his excellent guidance and support along the way. I am also grateful to his family for their kindness and support. I would also like to give my sincere thanks to my committee members: Dr. Jim Kronstad, Dr. Tom Grigliatti and Dr. Bob Devlin for committing their time to the committee meetings, and for their valuable suggestions and help.  My special thanks give to Les Willis, who has impressed everybody in the lab including me with his enduring support to the lab members, excellent organization of the lab and great technical assistance, all of which make the lab such a pleasant place to work in. I will always remember the effort you and Joan made to help Minggang and I live a more enjoyable life at Okanagan, the sushi lunch, the movies, the bird watching and of course the fishing without fish∙ ∙ ∙ ∙ ∙ ∙ . I would also like to thank all the people who are currently or have worked in the Theilmann lab, including Virginia, Minggang, Christina, Xiaojiang, and Ilse for their help and support. Many thanks to Michael Weis for his excellent help with transmission electron microscopy and confocal microscopy, especially his great commitment in the summer of 2007, helping to produce TEM data. I would like to thank Joan Chisholm, Rob Linning, Yu Xiang, Jane Theilmann, Anita Quail, Eunice Randall, Melanie Walker and Ron Reade for their suggestions and help to my research. I would also like to thank Pacific Agri-Food Research Centre for allowing me to use their facilities and all the rest of people I haven’t mentioned that helped me on my research.  I would like to thank all my friends who are or once were at the beautiful Okanagan. They and the nature make the place the best I have ever been. They are Yu Xiang and his family, Changwen Lu and his family, Xiao Song and Shibing Qin, Wei Zhou, Dongbao Fu and Shurong Qi, Xin Huang, Shawkat and Lubna Ali, Eunice and Paul Randall, Liz Hui, Julie Boule, Yasantha Athukorala, Albert Hannig, Guangzhi Zhang and his family, Juan Jovel and his family, John and Ikeda Laurie, Xin Hu and his family, Junhuan Xu and Colin Ho. I  xiii  would also like to give special thanks to Zhila and Peter Schofield, for their help and kindness through the years, and wish them both great healths.  My work would not have been completed without the help from these people: Dr. Henry Krause for the plasmid for tandem tags, Suzanne Perry for her assistance in protein identification, Dr. Eric Carstens for the gifts of LEF-3 and P143 antiserum, Dr. George Rohrmann for the gifts of VP39 and POLH antiserum, Dr. Linda Guarino for the gift of IE1 antiserum, Dr. Gary Blissard for the gift of GP64 antiserum, Dr. Sharon Braunagel for the gift of FP25 antiserum.  I would like to thank Lia Maria, Kirsten Cameron and Alina Yukhymets and people from the Faculty of Graduate Studies for their help with administration.  Finally I would like to thank my family and my husband Minggang for their continuous support over the years. I owed a lot to Minggang, who has been always by my side at difficult times, encouraging me, supporting me and helping me. I would not be where I am today without the support and help from him.  xiv  Dedication  To my beloved ones  xv  Co-authorship Statement Dr. Junya Yamagishi and Dr. Gary Blissard are co-authors of paper (chapter 2) as they performed and analyzed the microarray analyses of viral genes regulated by IE0 and IE1.  Dr. Minggang Fang is a co-author of the paper (chapter 3). He provided the backbone vector used for cloning the pie0-3xFLAG-6xHis and pie1-3xFLAG-6xHis constructs and assisted with the protein sample preparation for LC-MS/MS.  Dr. David A. Theilmann is a co-author of all three papers as he and Yingchao Nie designed the experiment and co-wrote the manuscripts.  xvi  Chapter I Introduction  1.1 Baculoviruses The baculoviridae is a large family of arthropod-specific viruses. Over 800 species of insects have been reported to be infected by baculoviruses, most of which are from the order of Lepidoptera but also from the order of Diptera and Hymenoptera (Adams and Bonami, 1991; Jehle et al., 2006; Martignoni and Iwai, 1986; Theilmann et al., 2005). Previously the baculoviridae was divided into two genera: nucleopolyhedrovirus (NPV) and granulovirus (GV). However this taxonomy has recently been updated after extensive phylogenetic analysis due to the wealth of complete genome sequences now available. The baculoviridae is now divided into four genera: alphabaculoviruses and betabaculoviruses, which infect primarily Lepidoptera, and gammabaculoviruses and deltabaculoviruses which infect primarily hymenoptera and diptera respectively (Jehle et al., 2006). Alphabaculoviruses can also be subdivided into Group I and Group II based on their distinct phylogenetic relationships though not recognized taxonomically (Zanotto et al., 1993). Alphabaculoviruses have large occlusion bodies (OBs) containing many virions with single (S) or multiple (M) nucleocapsids per virion. Betabaculoviruses usually have smaller ovoid OBs normally containing only one virion with a single nucleocapsid. Gammabaculoviruses and deltabaculoviruses have intermediate sized OBs containing many S type virions.  Baculoviruses are highly host-specific, making them appealing biocontrol agents, especially in the case of pests that develop resistance to chemical pesticides (Moscardi, 1999). Baculoviruses are being successfully used to control the velvet bean caterpillar, Anticarsia gemmatalis in Brazil (AngeNPV)(Oliveira et al., 2006), cotton bollworm, Helicoverpa amigera in China (HearNPV) (Sun and Peng, 2007), Douglas Fir Tussock moth (Ogyia psedotsugata) and European spruce sawfly, Gilpinia hercyniae (GiheNPV), in North America (Koul et al., 2004). Limitations to the use of baculoviruses in pest control have been hindered in part due to the longer time baculoviruses can take to kill  1  target insects in comparison to chemical insecticides. Various recombinant viruses expressing insect toxin or hormones or deleting the ecdysteroid UDP-glucosyltransferase (EGT) have been investigated aiming to enhance the effective performance on incapacitating target insects (O'Reilly and Miller, 1991). Baculoviruses can also be expensive to produce, but production costs have been reduced in recent years.  In addition to the use as insecticides, baculovirues are one of the most successful and widely used expression vectors for the production of bioactive proteins for research and the pharmaceutical industry (Ikonomou et al., 2003). Advantages of the baculovirus expression vector include post-translational modifications similar to those of higher eukaryotes, including phosphorylation, acylation and amidation (Luckow, 1991; Miller, 1997). Although the glycosylation pathway of insect cells can differ from mammalian cells, expression of foreign proteins in stably transformed insect cells expressing enzymes from certain glycosylation pathway of higher eukaryotes can improve the process of glycosylation (Altmann et al., 1999; Jarvis, 2003).  Recently researchers have shown that baculoviruses are also promising vectors for gene therapy (Huser and Hofmann, 2003; Pieroni and La Monica, 2001). The virus can enter mammalian cells and expresses some viral early genes but is not able to replicate or produce progeny virions. Baculoviruses have also been investigated as a surface display vectors (Grabherr et al., 2001; Oker-Blom and Vuento, 2003).  Above all, research on baculovirus-insect interactions serves as a vehicle to understand the molecular mechanisms by which large DNA viruses infect and kill eukaryotic cells. Indepth understanding of the molecular mechanisms of the baculovirus replication can lead to better exploitation of these viruses for their multiple applications.  1.2 Baculovirus life cycle The characteristic feature of the baculovirus life cycle is its biphasic infection, producing two types of progeny virions: the occlusion derived virus (ODV) and budded virus (BV) (Fig. 1.1). ODV mediates the transmission of virus horizontally between larvae and BV is 2  responsible for spreading the infection to different tissues of infected larvae and in cultured cells. ODV are embedded in OBs, with single or multiple nucleocapsids per ODV envelope (Fig. 1.2), whereas BV is the extracellular form of the virus with only single nucleocapsid per envelope. ODV and BV have the same viral DNA genome but they have different morphology and structural proteins. ODVs obtain an envelope from the inner nuclear membrane, while BVs bud and acquire an envelope from the plasma membrane. Baculovirus OBs are naturally found on the foliage of plants. Insects are infected by feeding on the contaminated plant material and ingesting the virus. OBs disintegrate in the alkaline environment of midgut and release ODVs into the midgut lumen and then penetrate the peritrophic membrane and enter the midgut columnar epithelium cells by direct membrane fusion. Nucleocapsids then translocate to the nucleus and initiate primary infection. For baculoviruses to cause systemic infection, nucleocapsids must bud from the midgut cells through basal lamina to form BV, which spreads the infection systemically via the hemolymph and tracheal system to start secondary sites of infection (Miller, 1997). Systemic infection leads to death and eventually liquefaction of the dead larvae. The liquefaction of dead larvae is also characteristic of baculovirus infection, which contributes to release of OBs into the environment for transmission to additional hosts. Not all baculoviruses cause infection systemically, currently for at least one betabaculovirus, and all gammabaculoviruses and deltabaculoviruses the infection is limited to midgut cells (Jehle et al., 2006; Moser et al., 2001).  1.3 Baculoviruses genomes Baculoviruses have large double stranded circular DNA genomes. The GC content of the genomes varies from 32.4% (Cryptophlebia leucotreta granulovirus (CrleGV)) to 57.5% (Lymantria dispar MNPV ((LdMNPV)), and the size of the genomes ranges from 80 kb (Neodiprion leconteii NPV (NeleNPV)) (Lauzon et al., 2004) to 180 kb (Xestia c-nigrums GV (XecnGV)) (Hayakawa et al., 1999). The variation of genome sizes can be due to repeated sequences including homologous regions (hrs) or genes, such as the baculovirusrepeated ORF (bro). Bro gene homologs are repeated 16 times in LdMNPV (Kuzio et al., 1999). Seventeen hrs have been identified in Spodoptera litura NPV (SpliNPV) (Pang et al., 2001), while no hrs are found in Trichoplusia ni SNPV (TnSNPV) (Willis et al., 2005). 3  As would be expected due to the large variations in genome size, the coding capacity of baculovirus genomes also differs and ranges from 89 (NeleNPV) to 181 (XecnNPV) potentially expressed genes. Baculovirus genes are encoded from both DNA strands. The proportion of genes distributed on the same strand as polyhedrin lies between 38% (NeseNPV) to 56% (CrleGV). So far, over 50 baculovirus genomes have been completely sequenced and more than 800 different orthologous gene groups have been identified. The most studied baculovirus is the alphabaculovirus type species Autographa californica multiple nucleopolyhedrovirus (AcMNPV) with a genome of 133,894 bp and 154 open reading frames (orf) of 150 bp or greater (Ayres et al., 1994).  1.4 Baculovirus entry 1.4.1 ODV entry To initiate primary infection in midgut epithelia cells after release from the occlusion body in the midgut lumen, ODV needs to penetrate the peritrophic membrane (PM) which is a protective physical barrier. It is believed that enhancins encoded by a few NPVs and GVs function to facilitate the process by disrupting the PM (Hashimoto et al., 1991; Lepore et al., 1996; Wang et al., 1994). Enhancin is one of three metalloproteases identified from baculoviruses. It was found that enhancin associated with ODV in LdMNPV (Slavicek and Popham, 2005), but was a component of occlusion bodies in GV (Hashimoto et al., 1991). Enhancin appears to digest the intestinal mucin and allows access of ODV to the microvilli of midgut columnar cells. Nucleocapsids subsequently bind and enter the cells by direct membrane fusion, a process mediated by ODV attachment and fusion proteins. A specific cellular factor is believed to serve as receptor as the attachment sites are saturable and a major reduction in viral binding after protease treatment is observed (Horton and Burand, 1993).  Five viral genes have been identified from AcMNPV to be essential for per os infectivity (pif), including p74 (Faulkner et al., 1997; Kuzio et al., 1989), pif1 (ac119), pif2 (ac22), pif3 (ac115) (Ohkawa et al., 2005) and pif4 (ac96) (Fang et al., 2009a). Homologs of these proteins exist in all baculoviruses and in addition nudiviruses. All five PIFs are ODV  4  structural components, P74 and PIF1 are ODV envelope components, PIF2 and PIF3 are ODV components but their specific location has not yet been determined and PIF4 is an envelope component of both BV and ODV (Fang et al., 2009a; Fang et al., 2006; Faulkner et al., 1997; Kikhno et al., 2002; Li et al., 2007; Pijlman et al., 2003). These proteins are thought to mediate by some mechanism ODV binding, fusion and entry to the midgut microvilli. To date, P74, PIF1 and PIF2 have been shown to specifically affect binding not fusion (Haas-Stapleton et al., 2004; Ohkawa et al., 2005), indicating they might form heterotrimer for contacting with cellular receptor. Recently, Zhou et al. (2005) identified a binding partner of 35 kDa of AcMNPV P74 from Spodoptera exigua, however the identity of the protein is not known. PIF3 is not required for either ODV binding or fusion (Ohkawa et al., 2005); the function of PIF4 in the early phase of ODV infection remains to be analyzed.  After fusion with the microvilli, ODV nucleocapsids have been shown to use actin-myosin to facilitate the journey from the distal end of microvilli to the nucleus of midgut cells either using the motor protein Myosin VI or viral myosin (Volkman, 2007). AcMNPV ODV component AC66 is found to contain an actin binding motif similar to that of myosin VI (Braunagel et al., 2003; Volkman, 2007), however recent studies show that the deletion of ac66, one of the genes conserved in alpha- and betabaculovirus, affect BV production as well as preoccluded virion and occlusion body formation (Ke et al., 2008).  1.4.2 BV entry BV enters cells primarily by adsorptive endocytosis (Volkman and Goldsmith, 1985), a process that includes virion binding, uptake of the virions in clathrin coated vesicles followed by low-pH mediated membrane fusion and release of nucleocapsids into the cytoplasm (Blissard and Wenz, 1992; Long et al., 2006a; Volkman and Goldsmith, 1985). BV binds to specific receptors on the surface of plasma membrane in saturable fashion (Wickham et al., 1992), however the identity of the cellular receptors has not been determined. The fact that BV is able to enter a broad range of cells, including cells that are nonpermissive for replication such as mammalian cells, suggests that the receptors may be highly conserved or there is a non-specific mode of viral entry (Boyce and Bucher, 1996; 5  Brusca et al., 1986; Carbonell and Miller, 1987; Condreay et al., 1999; Hofmann et al., 1995; Sandig et al., 1996; Shoji et al., 1997). Nevertheless, it has been shown that baculoviruses encode two envelope fusion proteins: GP64 and F protein that are used for entry of BV into cells. It is believed that Group I alphabaculoviruses use the glycoprotein GP64; Group II alphabaculoviruses, betabaculoviruses and deltabaculoviruses which lack GP64 use F protein. Gammabaculoviruses however lack both GP64 and F protein (GarciaMaruniak et al., 2004; Herniou et al., 2003). It has been suggested that BV could use an alternative pathway other than adsorptive endocytosis to enter cells, as excessive antibody against BV didn’t neutralize BV completely (Volkman and Goldsmith, 1985).  A monoclonal antibody AcV1 against AcMNPV GP64 is able to neutralize BV at the step post virion binding (Volkman et al., 1984). Hefferon et al. (1999) later showed that incubation of Sf9 cells with inactivated AcMNPV or Orgyia pseudosugata multiple nucleopolyhedrovirus (OpMNPV) or soluble OpMNPV GP64 before infecting with an AcMNPV virus vAchsZ results in significant decrease in the number of cells infected, demonstrating GP64 is required for binding a host cell receptor. A number of studies have shown that GP64 is required and sufficient for a low-pH mediated membrane fusion (Blissard et al., 1992; Hefferon et al., 1999; Volkman and Goldsmith, 1985). The structure of GP64 has also been recently determined and it was shown be the third representative of class III fusion proteins, similar to vesicular stomatitis virus (VSV) entry glycoprotein (G) and herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) (Garry and Garry, 2008; Kadlec et al., 2008). Studies have also shown that if gp64 is deleted BV production is almost completely eliminated indicating that GP64 is critical to BV budding (Oomens and Blissard, 1999).  F protein is a functional homolog of GP64, first identified from Group II alphabaculovirus Spodoptera exigue MNPV (SeMNPV) and LdMNPV (IJkel et al., 2000; Pearson et al., 2000), F proteins SeMNPV Se8, LdMNPV Ld130, HearNPV Ha133 and Agrotis segetum GV (AgseGV) orf25 are able to rescue a AcMNPV gp64 null mutant (Long et al., 2006b; Lung et al., 2002; Yin et al., 2008), but this rescue is not reciprocal (Westenberg and Vlak, 2008). It is suggested that F protein is class I fusion protein and uses different cellular receptor from GP64 to enter insect cells (Garry and Garry, 2008; Westenberg et al., 2007). 6  Alphabaculoviruses also contain F protein homolog, such as AcMNPV AC23, in addition to GP64. AC23 is also present on the BV surface along with GP64, however it is not able to functionally substitute GP64 as it lacks fusogenic activity. It is suggested that the function is lost under the relaxed selection pressure since the acquisition of functional substitute GP64 (Jiang et al., 2009). Deletion of AcMNPV GP64 is lethal to the BV production as infection is limited to single cell indicating that the AC23 F-like protein cannot rescue the gp64 null virus (Lung et al., 2002; Lung et al., 2003). Ac23 however appears to be a regulatory and pathogenic factor, as loss of AC23 results in a virus that takes longer to kill an infected host than control wild type (WT) virus (Lung et al., 2002). In addition, deletion of ac23 results in the production of ODVs that have fewer nucleocapsids (Yu et al., 2009). Wang et al. (2008) also found that AC23 is able to increase the infectivity of the gp64 null AcMNPV mutant pseudotyped by SeMNPV F protein, indicating F-like protein contributes to the binding process and may serve as accessory protein.  After attachment and internalization, the BV envelope fuses with late endosome membrane and BV nucleocapsids are released into the cytoplasm and traverse towards the nucleus. Both actin and microtubule integrity and dynamics have been suggested to play roles in the transport of BV to the nucleus. Three sequential actin rearrangements were observed after BV infected cells: actin cable formation upon nucleocapsids release from endosomes, ventral actin aggregation before viral DNA replication and formation of nuclear F-actin (Charlton and Volkman, 1991). Fluorescence microscopy analysis also found nucleocapsids usually associated with one end of most actin cables, indicating BV use the actin cables for the transport of nucleocapsids into the nucleus (Charlton and Volkman, 1993). The actin cable formation is insensitive to cyclohexamide, indicating BV associated proteins are responsible for the induction of polymerization of existing G-actin to F-actin. The viral capsid protein P78/83 is an N-WASP protein that has been shown to induce actin nucleation and polymerization (Goley et al., 2006). Another structural protein BV/ODVE26 that has homology to tropomyosin was found to complex with FP25 and actin and may also be involved in nucleocapsid transport (Beniya et al., 1998). Treatment with drugs interfering actin dynamics such as Cytochalasin D, Latrunculin A and Jasplakinolide abolishes the uptake of nucleocapsids to mammalian cells, supporting that nucleocapsids 7  use the actin cytoskeleton for transport into the nucleus (Salminen et al., 2005; Van Loo et al., 2001). Early transmission electron microscopy (TEM) analysis by Granados and Lawler (1981) documented the apparent association of nucleocapsids with microtubules, which lead to the suggestion that nucleocapsids travel along the microtubule bundles toward the nucleus and enter the nucleus through nucleopore complex, where they uncoat and release the viral DNA genome to initiate the replication process (Granados and Lawler, 1981; Summers, 1971). However, treatment of infected cells with microtubule depolymerising drugs Nocodazole, Vinblastine and Taxol results in increased number of nucleocapsids being transported to nucleus in mammalian cells suggesting that microtubules are not involved in viral entry (Salminen et al., 2005; Van Loo et al., 2001).  1.5 Baculovirus gene expression 1.5.1 Baculovirus genes The expression of baculoviral genes occurs in an ordered cascade, which can be categorized into immediate early (IE), delayed early (DE), late and very late. Most IE genes play essential roles in successful viral replication as they encode regulatory proteins that likely interact with both host and viral proteins. IE genes activate the expression of other viral early proteins involved in viral DNA replication or are required for the stimulation of late genes expression. Late and very late genes in general encode proteins essential for virion assembly and viral occlusion body formation. Traditionally, the genes of baculoviruses can be functionally subgrouped into five general categories: RNA transcription, DNA replication, structural proteins, auxiliary proteins and proteins of unknown functions.  1.5.2 Expression of IE and DE genes IE and DE genes are transcribed prior to the onset of DNA replication by host RNA polymerase II. The transcripts usually are 5’capped (Jun-Chuan and Weaver, 1982) and 3’ polyadenylated in response to a 3’ polyA signal. The core promoter of baculovirus early genes resembles RNA Pol II responsive genes, usually consists of TATA element at 25-31  8  nucleotide (nt) upstream of transcriptional start site which may also function as an initiator motif. Elements have been identified down stream of transcriptional start sites that regulate expression and have been called downstream activation regions (DAR) (Friesen, 1997). DAR have been identified in the ie1 and gp64 5’untranslated region (UTR), which contributes to the basal transcription presumably by stabilizing protein interactions such as TFIID at the initiator element (Friesen, 1997). Deletion or replacement of TATA causes dramatic decrease in promoter activity of IE genes showing that it is a principal regulatory element (Blissard et al., 1992; Dickson and Friesen, 1991; Theilmann and Stewart, 1991). Baculovirus TATA elements have been associated with directing transcription initiation at the proper site and transcription initiation rate. The baculovirus early promoter initiator motif ATCA(G/T)T(C/T) contributes to the overall activity of TATA-containing early promoters but is not essential (Blissard et al., 1992; Carson et al., 1991a; Guarino and Smith, 1992; Kogan et al., 1995; Pullen and Friesen, 1995a).  The expression of early genes can also be cis-regulated by enhancer elements. Baculovirus enhancers have been shown to be hr elements that are interspersed around the genome of most baculoviruses. An hr sequence is rich in AT content and usually contains multiple copies of a core sequence that may contain palindromic sequences flanked by direct repeats. Core sequences are separated by various lengths of viral DNA. AcMNPV hr element has been shown to increase 39K expression dramatically when transactivated by IE1. In the absence of IE1, the AcMNPV hr5 can increase the activity of p35 and p143 promoters by 2 to 20 fold when linked in cis to the promoters (Guarino and Summers, 1986; Lu and Carstens, 1993; Rodems and Friesen, 1993). OpMNPV hr has also been shown to enhance the expression of ie2 and AcMNPV 39K (Theilmann and Stewart, 1992b). As hr elements can activate expression of viral genes in the presence or absence of viral transactivators it implies that it contains binding sites for viral transactivators and host factors. It is known that AcMNPV IE1 binds to hr palindrome sites as a dimer (Rodems and Friesen, 1995). A 38 kDa Sf9 host protein has been shown to bind AcMNPV hr1 and contributes to hr1 enhancer function (Habib and Hasnain, 1996). Specific nuclear factors from mammalian cells have also been found to bind AcMNPV hr1 (Viswanathan et al., 2003). In support of the idea that hrs contain binding sites for host factors, Landais et al. (2006) showed AcMNPV hrs contain a cluster of bZIP binding sites including cAMP 9  response elements (CRE) and TPA response elements (TRE), and these sites can bind specific insect factors and stimulate RNA Pol II-dependent transcription.  IE genes are distinguished from DE genes as their expression is independent of prior viral gene expression determined by transient assays and the use of metabolic inhibitors such as cyclohexamide (Blissard and Rohrmann, 1990; Choi and Guarino, 1995b; Crawford and Miller, 1988; Ross and Guarino, 1997). Optimum expression of DE genes however usually depends on the expression and transactivation from viral IE genes such as ie0 and ie1. AcMNPV encodes five known IE regulatory genes including ie1, me53 (present in all lepidopteran viruses), ie0 (present in alphabaculoviruses), pe38 and ie2 (present in Group I alphabaculoviruses). IE1 and IE0 are considered as the major transactivators, whereas both IE2 and PE38 have also been shown to be viral transcriptional transactivators (Kovacs et al., 1991; Krappa and Knebel, 1991; Theilmann and Stewart, 1992a; Yoo and Guarino, 1994). Sequence analysis of IE2 revealed the protein has three motifs common to transcriptional regulators: a serine-threonine rich region, a proline-rich region, and a polyglutamine tract in addition to a RING finger, and a leucine zipper (Carson et al., 1991a). Transient assays have shown that IE2 augments the transactivation of the 39K promoter by IE1 (Carson et al., 1988), and also transactivates its own promoter and the promoters of ie1 and ie0 (Carson et al., 1991b; Theilmann and Stewart, 1992a; Yoo and Guarino, 1994). IE2 is also reported to be a strong transactivator of CMV promoter in mammalian cells, and locates in cellular regions of high G-actin content (Liu et al., 2009). IE2 transient expression in Sf21 cells can cause cell cycle arrest at S phase thus may be responsible for the cell cycle arrest during baculovirus infection. Deletion or mutation of the IE2 RING finger eliminates the cell cycle arrest, whereas does not affect its ability to transactivate ie1, indicating the ability of transactivation is independent of cell cycle arrest (Prikhod'ko et al., 1999; Prikhod'ko and Miller, 1998). PE38 is also a RING finger protein that contains a Leucine zipper domain. PE38 has been shown to transactivate the p143 promoter and the transactivation is augmented by IE2, likely due to IE2 transactivation of pe38 promoter (Lu and Carstens, 1993). Bombyx mori nucleopolyhedrovirus (BmNPV) IE2 and PE38 have been shown to have ubiquitin ligase activity and IE2 is suggested to ubiquitinate host factors involved in cell cycle regulation. However, substrates of IE2 and PE38 remain to be identified (Imai et al., 2003). 10  Another possible baculovirus regulatory protein is global transactivator (gta) which has homology to the chromatin remodelling complex protein SNF2. It has therefore been suggested to bind and unwind viral chromatin and regulate the viral gene transcription (Lapointe et al., 2000), however no direct evidence has been collected in support of this suggestion. Recent study shows that deletion of gta has no impact on viral DNA replication and production of BV or OB, yet prolongs the time to death in infected Bombyx mori larvae (Katsuma et al., 2008).  1.5.3 Expression of late and very late genes Late and very late genes are transcribed concomitant with or subsequent to the initiation of viral DNA replication and actively transcribed at late stage of infection by a virally induced RNA polymerase (Fuchs et al., 1983; Huh and Weaver, 1990; Miller et al., 1981). AcMNPV late genes are transcribed primarily between 6 and 24 hours post infection (hpi), whereas very late genes are transcribed from 18 hpi to 72 hpi. Transcription of all late genes initiate from a (A/T/G)TAAG motif which is essential and the most critical element of late promoters (Morris and Miller, 1994; Ooi et al., 1989). Mutations affecting TAAG motif abolish the promoter activity of vp39, and mutations within the 8 bp upstream or 6 bp downstream of TAAG can also reduce the promoter activity by 90 to 60% respectively, indicating the 18 bp consists most important elements of the late promoter. Nevertheless the 18 bp sequence only acts as a weak promoter in the case of very late promoter polh (Morris et al., 1994). The major difference between late and very late promoters is the very high levels of expression that is observed with the hyperexpressed very late promoters. Current data suggests that the very late promoters can contain what is known as a “burst’ sequence in the UTR. Mutations within the UTR can result in 2 to 50 fold reduction of the polh promoter activity (Ooi et al., 1989; Rankin et al., 1988).  The expression of late genes and very late genes are regulated by late expression factors (LEFs). Twenty-one genes have been identified to be required for late gene expression (Hang et al., 1995; Huijskens et al., 2004; Passarelli and Miller, 1993a; Passarelli and Miller, 1993b; Passarelli and Miller, 1993c; Rapp et al., 1998; Yamagishi et al., 2007). 11  These genes are viral IE or DE genes, and support late gene expression by optimizing viral DNA replication or directly affecting late gene transcription. The genes ie0, ie1, ie2, lef-1, lef-2, lef-3, p143, dnapol, p35, and lef-7, lef-11 (Lin and Blissard, 2002b) are replication lef genes, while lef-4, lef-5, lef-6, lef-8, lef-9, lef-10, lef-12, p47, pp31 and vlf-1 are transcription lef genes (Lin and Blissard, 2002a; Lu and Miller, 1995; Yamagishi et al., 2007). Out of the twenty-one lef genes identified in AcMNPV ten are core genes that are found in all baculovirus species that have been completely sequenced todate (lef-1, lef-2, p143, dnapol, lef-4, lef-5, lef-8, lef-9, p47and vlf-1) (Herniou et al., 2003). It is estimated that 84,000 viral genomes may be produced in a single infected cell (Rosinski et al., 2002). This high copy number of genome may enable the hyperexpression of late genes (Rohrmann, 2008). Four of the lef core genes encode components of the viral RNA polymerase: LEF-4, LEF-8, LEF-9 and P47 (Guarino et al., 1998b). LEF-8 and LEF-9 are believed to form the polymerase catalytic pocket as both bear homology to the DNAdependent RNA polymerase of prokaryotes and eukaryotes (Lu and Miller, 1994; Passarelli et al., 1994). LEF-4 has the guanylyltransferase and triphosphatase enzymatic activity which was suggested to carry out the capping process of viral late mRNAs (Guarino et al., 1998a; Jin et al., 1998). However this concept is challenged by the finding that the viral triphosphatase activity is dispensable for the cap formation of viral late mRNA or for viral replication in tissue culture (Li and Guarino, 2008). The function of P47 in the viral RNA polymerase is unclear though it is predicted to have homology to alpha subunits of bacterial RNA polymerases (Rohrmann, 2008).  Possible functions of other LEF factors include LEF-5 which has homology to the transcription elongation factor TFIIS and is required for maximal level of late transcription (Harwood et al., 1998). LEF-6 is not essential for viral DNA replication but seems to accelerate late gene transcription (Lin and Blissard, 2002a). Vlf-1 was originally identified as an essential gene for hyperexpression of polyhedrin and p10 (McLachlin and Miller, 1994; Ooi et al., 1989). VLF-1 has been shown to bind to the burst element in the promoters of polyhedrin and p10 (Mistretta and Guarino, 2005; Yang and Miller, 1999). The predicted amino acid sequence of VLF-1 revealed that it has a motif conserved in lambda phage integrase, a recombinase family protein that functions to catalyze DNA rearrangements through recombination. None of the three conserved residues within the 12  integrase motif can be changed as mutant viruses were unable to be isolated, indicating the putative integrase activity is related to the essential function of VLF-1 (Yang and Miller, 1998a). However mutation of the critical tyrosine did not affect VLF-1 transactivation of polh promoter in transient assays, indicating the integrase activity, if there is any, is not required for the transactivation activity of VLF-1 (Yang and Miller, 1998a). These results imply that VLF-1 plays essential role for viral replication other than transactivation of very late gene expression, and this essential function requires the putative recombinase activity. Recently it has been shown that deletion of vlf-1 does not appear to affect viral DNA synthesis or the production of replication intermediates, but results in the production of long tubular nucleocapsid like structures. It was suggested this was probably due to the failure of the normal packaging of viral genome into capsid sheath or the lack of VLF-1, presumably as a capsid component. Immunogold labelling analysis of VLF-1 indeed showed that VLF-1 associates with one end of nucleocapsids. Repair of the vlf-1 deletion with the vlf-1 mutant bearing the tyrosine mutation in the integrase domain (Y355F) restores the normal morphogenesis of nucleocapsids, confirming that VLF-1 is required for the production of capsids of normal appearance. Nevertheless infectious BV are not produced, indicating VLF-1 putative enzymatic activity may be required to process the nucleocapsid package at final stage prior to egress (Vanarsdall et al., 2006).  1.6 Baculovirus DNA replication 1.6.1 Origin of replication Baculoviruses replicate their DNA genomes in the nucleus of infected cells. The baculovirus genome has been proposed to have multiple origins of replication as transient replication assays have shown that hr elements, a non-hr element and early gene promoters such as ie1 and p35 can act as origins (Kool et al., 1994b; Lee and Krell, 1994; Leisy and Rohrmann, 1993; Pearson et al., 1992; Pearson et al., 1993; Pearson and Rohrmann, 1995; Wu and Carstens, 1996). In AcMNPV infected cells IE1 has been shown to bind the hr elements and may function as an origin binding protein to initiate viral DNA replication (Choi and Guarino, 1995a; Guarino and Dong, 1991; Guarino and Dong, 1994; Rohrmann, 2008). Even though hr sequences have been shown to function as origin of replication in  13  transient assays no direct evidence that they behave as the origins of replication in vivo has been shown. Recently, deletion analysis of individual hr sequences within the AcMNPV genome has shown that none of the 6 hrs analyzed is essential for viral DNA replication (Carstens and Wu, 2007).  The non-hr origins of replication have been identified in AcMNPV, OpMNPV and SeMNPV (Ahrens et al., 1995; Heldens et al., 1997; Kool et al., 1994b; Lee and Krell, 1994; Pearson et al., 1993). A similar non-hr region was found in BusuNPV, however it is not known if it can function as origin of replication (Hu et al., 1998). These non-hr regions contain unique palindromic and repetitive sequences different from those of hr sequences and more closely resemble eukaryotic cellular replication origins (Lee and Krell, 1994). The OpMNPV non-hr sequence was found less effective in transient replication assay than the hr sequence (Ahrens et al., 1995). In vivo analysis using competitive PCR showed that the AcMNPV non-hr origin is more efficient and active compared with the ie1 promoter (Habib and Hasnain, 2000). Both hr and non-hr origins are likely to play important roles in replication and baculoviruses may use multiple origins to rapidly replicate their genomes. It has also been suggested that any site where the viral genome is unwound could allow the access of replication enzyme complex and serve as an origin of replication (Okano et al., 2006).  1.6.2 Proteins involved in replication Like other large DNA virus, baculoviruses encode their own enzymes for DNA replication. Seven proteins including IE0 or IE1, LEF-1, LEF-2, LEF-3, LEF-11, DNA Pol and Helicase P143 have been shown to be required for viral DNA replication. Four proteins P35, IE2, PE38 and LEF-7 have been shown to be accessory factors (Ahrens and Rohrmann, 1995; Kool et al., 1994a; Lin and Blissard, 2002b; Lu and Miller, 1995; Stewart et al., 2005).  As indicated above four of the replication proteins have homologs in all baculovirus genomes HELICASE, LEF-1, LEF-2 and DNA Pol (Herniou et al., 2003). HELICASE has helicase activity and binds and unwinds the DNA duplex (Laufs et al., 1997; McDougal 14  and Guarino, 2000). Helicases also appear to be host specific as replacement of the AcMNPV P143 with CfMNPV P143 fails to support plasmid replication (Chen et al., 2004). But cotransfection of AcMNPV with a 572 bp fragment from BmNPV helicase generated a virus eh2-AcNPV able to replicate both in BmN cells (usually nonpermissive for AcMNPV) and Sf21 cells (usually nonpermissive for BmNPV), however low MOI infection of Sf9 cells with eh2-AcNPV resulted in abortive infection due to defects in DNA replication (Kamita and Maeda, 1996; Maeda et al., 1993). Substitution of the short genome sequence between amino acid 536 and 584 of AcMNPV HELICASE with the homologous sequence of BmNPV can permit high level replication in normally poorly permissive Bm5 cells and kill Bombyx mori larvae (Croizier et al., 1994). Further analysis within the region showed mutation of two amino acids 564 and 577 of AcMNPV P143 is sufficient to enable the protein to be functional in B. mori larvae (Argaud et al., 1998). These results indicate that HELICASE is a multifunctional protein and could be involved in diverse mechanisms during the baculovirus life cycle. LEF-1 functions as primase (Mikhailov and Rohrmann, 2002) and LEF-2 functions as the primase associated factor. It has been shown that LEF-2 binds LEF-1 forming a heterodimer and is thought to stablize the binding of LEF-1 to DNA or other replication proteins (Evans et al., 1997). Purified AcMNPV DNA Pol is active on a singly-primed M13 DNA template and has 3’→5’ but no 5’→3’ exonuclease activity, indicating the enzyme also performs proofreading function (Hang and Guarino, 1999; McDougal and Guarino, 1999; Mikhailov et al., 1986).  LEF-3 is a single-strand DNA binding protein essential for viral DNA replication (Hang et al., 1995). LEF-3 transports P143 to the nucleus, however the requirement of LEF-3 in transient replication assays is independent of the P143 transporter function (Chen et al., 2004; Chen and Carstens, 2005), suggesting SSB function may be essential. LEF-3 also interacts with alkaline nuclease (AN) and is suggested to be involved in the processing of DNA replication intermediates (Mikhailov et al., 2003; Mikhailov et al., 2004). LEF-3 is therefore a multifunctional protein that contains different domains for DNA binding, nuclear localization, P143 and AN interaction and regions involved in oligomerization (Chen and Carstens, 2005). For alphabaculoviruses IE0/IE1 is essential for viral replication and has been shown to bind hr sequences (Rodems and Friesen, 1995). Both LEF-3 and  15  IE1 are absent from deltabaculoviruses (Moser et al., 2001) and gammabaculoviruses (Lauzon et al., 2004).  The stimulatory factors IE2, PE38 and P35 are non-essential in transient DNA replication assays (Kool et al., 1994a). In support of the transient replication assay results AcMNPV mutants containing ie2 deletions exhibit delays in viral DNA synthesis (Prikhod'ko et al., 1999). Deletion of PE38 results in reduced viral DNA synthesis and reduced BV production (Milks et al., 2003). Previous studies have also shown that IE2 colocalizes with the viral DNA binding protein (DBP) and LEF-3 as well as a cellular protein promyelocytic leukemia (PML) ) in nuclear foci of infected cells, although DBP and LEF3 also locate to distinct structures from IE2 (Mainz et al., 2002; Murges et al., 2001). It is suggested that IE2 structures assemble separately from those of DBP and LEF-3 and recruits proteins for viral DNA replication initiation (Krappa et al., 1995). Similarly PE38 also localizes to nuclear foci (Krappa et al., 1995). BmNPV IE1 has also been shown to locate to small foci within the nucleus at early stage of infection, and the foci distribution requires the binding ability to hr sequences (Kawasaki et al., 2004; Nagamine et al., 2005; Okano et al., 1999). The foci expand and occupy half of the nucleus by 20 hpi and also are the sites of BrdU incorporation. The enlargement of the foci is sensitive to aphidicolin treatment, indicating the foci are where viral DNA replication takes place (Okano et al., 1999). Baculovirus DNA replication factories therefore appear to be formed by accumulation of replication essential and non-essential proteins in certain nuclear compartments.  It has been reported that BmNPV IE1 foci resemble a nuclear structure known as nuclear domains 10 (ND10, also known as PML nuclear bodies) (Okano et al., 1999). ND10 structures are dynamic nuclear compartment where certain cellular proteins such as PML accumulate and have been suggested to be the site of DNA virus transcription and replication. It has been shown that input viral genomes of Herpes simplex viruses (HSV) and Adenovirus colocalize with ND10 structures. Viral DNAs associated with ND10 structures have a greater likelihood for the initiation of transcription and replication (Ascoli and Maul, 1991; Ishov and Maul, 1996; Maul, 1998; Stuurman et al., 1992). However it is not clear whether insect cells have ND10 structures and whether 16  baculoviruses use similar mechanism for their replication. Murges et al. (2001) showed that transiently expressed IE2 and PE38 associated with mammalian ND10 structures and IE2 colocalized with human PML in insect cells at early infection. In addition a small ubiquitin-like modifier (SUMO) homolog from Tn-368 cells was recruited to the IE2/PML common domains, which were adjacent to the viral DNA replication sites (Mainz et al., 2002). SUMO modifications of PML have been implicated to be essential for PML deposition at ND10 (Muller et al., 1998). These results would indicate that insect cells may contain ND10-like structures and baculoviruses may deposit viral proteins in the structures or interact with host proteins deposited within the structures to facilitate viral DNA replication.  The third accessory protein, P35 is an inhibitor of apoptosis that when deleted from AcMNPV replication is significantly decreased in Sf21 cells or S. frugiperda larvae due to apoptosis. Apoptosis or effects on viral replication do not occur in Tn-368 cells and p35 mutants have equivalent infectivity in Trichoplusia ni larvae as the WT virus (Clem et al., 1991; Clem and Miller, 1993; Vaughn et al., 1977). Therefore, P35 impacts on viral DNA synthesis may be indirect by preventing cell death and permitting viral replication to occur. Loss of P35 has also been recently shown to result in decreased viral stability suggesting P35 also protects progeny virions from caspase induced damage (Bryant and Clem, 2009).  1.7 Baculovirus nucleocapsid assembly and egress 1.7.1 Virogenic stroma The virogenic stroma (VS) is the center of viral replication, which develops at the onset of viral DNA replication and late gene expression (Knudson and Harrap, 1976; Volkman et al., 1986). VS is first seen as loose granular structures and matures at late times to fibrillar electron dense structures which are interspersed by electron-lucent intrastromal spaces (Harrap, 1972; Summers, 1971; Young et al., 1993). A distinct peristromal compartment of nucleoplasm called ring zone is also seen associated with mature VS (Benz, 1986). The electron-dense region can be intensely stained by DNA specific fluorescent dye hence is believed to be the site for DNA replication (Volkman and Keddie, 1990). The intrastromal  17  spaces are the places where the progeny nucleocapsids are assembled (Harrap, 1972; MacKinnon et al., 1974; Summers, 1971); assembled nucleocapsids are retained in the ring zone at late stage of infection, where they are enveloped to form ODV and ODV are embedded into occlusion bodies (Xeros, 1956).  The VS is clearly observed by microscopy but the molecular components have not yet been clearly identified. The basic protein P6.9 and also PP31 accumulate in the virogenic stroma and both proteins bind DNA (Broussard et al., 1996; Guarino et al., 1992; Wilson, 1988; Wilson et al., 1987). Deleting pp31 or dbp results in the improper formation or failure to form the VS (Gomi et al., 1997; Vanarsdall et al., 2007a). The major capsid protein VP39 accumulates in the VS intrastromal spaces which is in agreement with the observation that the nucleocapsids assemble at this location (Blissard et al., 1989; Pearson et al., 1988; Thiem and Miller, 1989). A number of viral proteins also associate with the ring zone of VS, including P48 (AC103) (Yuan et al., 2008), BV/ODV-C42 (Braunagel et al., 2001), EXON0 (Fang et al., 2007) and BM68 (AC82 homolog) (Iwanaga et al., 2002) and BM61 (AC75 homolog) (Shen et al., 2009), indicating they could be involved in nucleocapsid assembly and maturation or egress.  1.7.2 Virion structural proteins and assembly Baculovirus nucleocapsids are rod-shaped and are approximately 200-300 nm in length and 30 nm in diameter. The nucleocapsids of AcMNPV ODV may consist of up to 29 polypeptides (Braunagel et al., 2003). As summarized in Fig. 1.2, twelve proteins have been identified as the components of both BV and ODV nucleocapsids in AcMNPV, including VP39, P78/83, VP1054, FP25, VLF-1, BV/ODV-C42, P87, P24, EXON0, 38K, AC109 and AC142 (Braunagel et al., 1999; Braunagel et al., 2001; Russell et al, 1997; Fang et al., 2007; Fang et al., 2009c; Lu and Carstens, 1992; McCarthy et al., 2008; Olszewski and Miller, 1997; Thiem and Miller, 1989; Vialard and Richardson, 1993; Wolgamot et al., 1993; Wu et al., 2008; Yang and Miller, 1998b). Some other viral proteins have been found to be common components of both BV and ODV. P91 is detected in the envelope and nucleocapsid of both BV and ODV (Russell and Rohrmann, 1997). AC96 is detected only in the envelope fraction of both BV and ODV (Fang et al., 2009a). 18  AC23 (F-like protein), BmNPV BM61, a homolog of AC75 and AcMNPV ME53 are also found to associate with both BV and ODV, however specific distribution is not clear except AC23 has been shown to locate on the BV envelope along with GP64 (Braunagel et al., 2003; de Jong et al., 2009; Lung et al., 2003; Pearson et al., 2000; Shen et al., 2009). P6.9 which condenses the genomic DNA is also packaged in both BV and ODV(Tweeten et al., 1980). Additional to these common components mentioned above, some ODV specific proteins have been identified, including ODV-E18, ODV-E35, ODV-E25, ODVE66, ODV-EC27, ODV-E56, GP41, p74, AC22/SE35, AC66, AC115, AC119/SpliNPV ORF7 (Braunagel et al., 1996a; Braunagel et al., 1996b; Braunagel et al., 2003; Hong et al., 1994; Kikhno et al., 2002; Ohkawa et al., 2005; Pijlman et al., 2003; Russell and Rohrmann, 1993). Additional BV associated proteins identified are the envelope proteins GP64 (Group I alphabaculoviruses) and F protein (Group II alphabaculoviruses), BmNPV BM68 (AC82) and BM42 (AC53 homolog) (Acharya and Gopinathan, 2002; Iwanaga et al., 2002; Pearson et al., 2000; Volkman, 1986; Volkman et al., 1984).  The assembly of nucleocapsids must involve extensive protein-protein and protein-DNA interactions that occur in a step-wise manner, but little is known about this process. The supercoiled viral genome is found associated with the basic DNA-binding protein P6.9 prior to encapsidation. It has also been shown that phosphorylation of P6.9 was prerequisite for the nucleocapsids uncoating and viral DNA release (Wilson and Consigli, 1985). Later study suggested that dephosphorylation of P6.9 correlated with the assembly of the viral nucleoprotein complex (Funk and Consigli, 1993). The highly basic polyamines putrescine and spermidine are also found to be packaged in nucleocapsids and may also help to neutralize the phosphate of viral DNA (Elliott and Kelly, 1977). The nucleoprotein complex is subsequently inserted into the preformed empty capsid shell at the edge of stromal matte (Fraser, 1986). Nucleocapsid proteins P78/83, VP1054, VLF-1, BV/ODV-C42, ODV-EC27, AC53 and 38K have been shown to be required for the nucleocapsid assembly and in their absence there is either no nucleocapsid production or nucleocapsids are malformed. But in most cases viral DNA synthesis does not appear to be affected (Liu et al., 2008; Vanarsdall et al., 2007b).  19  As described above baculovirus infection also induces the nuclear localization of actin filament and six viral early genes have been shown to be involved in this process. This includes, ie1, pe38, he65, ac004, ac102, and ac152 (Ohkawa et al., 2002). The assembly of infectious progeny requires the filament-actin as infection in the presence of CytochalasinD, which specifically inhibits actin microfilament elongation, results in production of aberrant empty tubular structures without the nucleoprotein core (Volkman, 1988). Mature nucleocapsids are able to migrate into the ring zone surrounding the virogenic stroma, where they could have one of two possible destinations. At the early stages of an infection cycle, progeny nucleocapsids are destined to form BV and egress from the nucleus to cytoplasm and bud at plasma membrane. At very late time post infection, nucleocapsids are retained in the nucleus and enveloped by intranuclear membranes and become occluded in occlusion bodies. Two viral proteins AC142 and P48 have been shown to be involved in the process of ODV envelopment (McCarthy et al., 2008; Yuan et al., 2008).  1.7.3 Nucleocapsid egress Electron microscopy studies suggest the following routes of nucleocapsid egress from ring zone to plasma membrane (Williams and Faulkner, 1997). The nucleocapsids are transported to the inner nuclear membrane which they pass through to the inter-nuclear membrane space. The nucleocapsids become enveloped as they pass through the outer nuclear membrane and form vesicles in the cytoplasm. The vesicle membrane is lost during the BV egress by an unknown mechanism and naked nucleocapsids are released into the cytoplasm. The unenveloped nucleocapsids migrate to the plasma membrane, bud through plasma membrane and obtain a loose-fitting envelope. The sites of budding are pre-enriched with BV envelope proteins including the major envelope fusion protein GP64 or F-protein (Volkman and Goldsmith, 1984; Volkman et al., 1986). Recently it has been shown that the viral protein AC141 (EXON0) is required for the transport of nucleocapsids from the nucleus to the cytoplasm (Fang et al., 2007). AC141 (EXON0) was also shown to colocalize with microtubules suggesting baculovirus egress of BV may utilize the microtubule cytoskeleton to transport nucleocapsids to the plasma membrane (Fang et al., 2009b). Additional viral proteins that have been shown to be associated with efficient BV production but do not affect nucleocapsid assembly include AC66 (Ke et al., 2008) and 20  ME53 (de Jong et al., 2009) and the well studied BV envelope fusion protein GP64 or Fprotein (Long et al., 2006b; Monsma et al., 1996; Oomens and Blissard, 1999).  1.8 Baculovirus transactivators IE0 and IE1 1.8.1 AcMNPV ie0-ie1 gene locus IE0 and IE1 are the primary transregulatory proteins of AcMNPV produced from ie0-ie1 gene complex. The ie0-ie1 gene locus is unique as it is the only known baculovirus spliced gene complex that produces multiple gene products (Fig. 1.3A). Separate promoters are utilized for each mRNA. The ie0 mRNA transcription initiates from an early gene consensus transcription start motif (CAGT) at 45 bp upstream of ac141 (exon0) late transcription start motif, and consists of two exons. The donor splice site is 187 bp downstream from the transcription start site in ac141 (exon0), and the acceptor splice site is 2 bp downstream of the ie1 mRNA initiation site. Therefore exon1 of ie0 consists of 114 bp of ac141 that are spliced to the 5’ end of exon2, which consists of entire ie1 mRNA, after excision of a 4.2 kb intron (Chisholm and Henner, 1988). The transcription of ie0 peaks at about 4 hpi and and declines thereafter but remains detectable up to late times post-infection (Chisholm and Henner, 1988; Guarino and Summers, 1987). Surprisingly, translation of the spliced ie0 mRNA produces both IE0 and IE1 due to the internal initiation of translation at the ie1start codon (Theilmann et al., 2001). The ie1 mRNA is not spliced and consists of only exon2. The promoter of ie1 consists of the consensus immediate early gene TATA-CAGT motif as well as upstream and down stream regulatory sequences (Pullen and Friesen, 1995b). The ie1 promoter is active in uninfected cells in the absence of other viral factors, but the activity can be regulated by viral or host factors. Expression of IE1 can be stimulated by other transactivators such as IE0, PE38, IE2 and itself IE1 (Carson et al., 1991b; Kovacs et al., 1991; Ribeiro et al., 1994; Theilmann and Stewart, 1993). Insect ecdysone and juvenile hormone have been reported to stimulate the expression of BmNPV ie1 promoter both in vitro or in vivo by 2 to 7 fold (Zhou et al., 2002), and an ecdysone-responsive element was recently identified from the BmNPV ie1 promoter (Kojima et al., 2007).  21  Bioinformatic analysis (Herniou et al., 2003) of all baculoviruses genomes sequenced to date has shown that homologs of ie1 are found in alphabaculoviruses and betabaculoviruses but not gammabaculoviruses or deltabaculoviruses, whereas ie0 homologs have only been identified in the alphabaculoviruses. This suggests that the acquisition of the only known baculovirus spliced gene product is a relatively recent evolutionary event.  1.8.2 IE1 IE1 is the best studied baculovirus transactivator and, as indicated previously plays a key role in viral gene expression and DNA replication (Carson et al., 1991b; Choi and Guarino, 1995a; Kovacs et al., 1992; Rodems and Friesen, 1995; Rodems et al., 1997; Slack and Blissard, 1997; Theilmann and Stewart, 1991). The transcript of AcMNPV ie1 starts as early as 15 to 30 min after virus entry and IE1 can be detected soon after (Chisholm and Henner, 1988; Guarino and Summers, 1987). Translation of the AcMNPV ie1mRNA produces a 582 aa protein of 67 kDa which steadily increases throughout infection from immediate-early to late times post-infection (Chisholm and Henner, 1988; Choi and Guarino, 1995b; Huijskens et al., 2004; Leisy et al., 1997; Mainz et al., 2002; Theilmann and Stewart, 1991; Theilmann and Stewart, 1993).  A significant number of studies, predominately using AcMNPV and OpMNPV, have been conducted to dissect the role and mechanism of IE1 action. A number of IE1 functional motifs have been identified (Fig. 1.3) that are involved in transcriptional activation, DNA replication, DNA binding and nuclear localization.  Two domains have been shown to be required for transcriptional activation, the acidic activation domain (AAD) and the acidic domain (AD). The IE1 AAD is similar to the motif that was first identified in yeast GAL4 and herpes simplex virus (HSV)VP16 (Forsythe et al., 1998; Rodems et al., 1997). The common features of an AAD are that it is rich in acidic residues and contain key hydrophobic/aromatic residues; however there is very little sequence conservation (Ptashne and Gann, 1990; Triezenberg, 1995). Acidic residues within the AAD enhance the transactivation, however the hydrophobic/aromatic 22  residues flanking them are essential for the transactivation (Cress and Triezenberg, 1991; Giniger and Ptashne, 1987; Regier et al., 1993). VP16 complexes with the cellular proteins Oct-1 and HCF-1 at specific cis elements present in the HSV IE promoters to activate expression of IE genes, which in turn leads to the cascade expression of viral delayed early and late genes (Roizman and Sears, 1996). VP16 has been shown to involve multiple strategies to activate gene expression. VP16 can directly contact general transcription factors (GTFs) such as TATA-binding protein (TBP) (Ingles et al., 1991), TFIIB (Roberts and Green, 1994), TFIIH (Xiao et al., 1994), suggesting it may activate transcription by stimulating preinitiation complex formation. VP16 stimulates the rate of TFIID and TFIIA complex formation in vitro (Kobayashi et al., 1998) showing that it can overcome the ratelimiting step in initiation. VP16 also interacts with components of mediator to recruit RNA Pol II holoenzyme (Goodrich et al., 1993; Hengartner et al., 1995; Mittler et al., 2003; Neish et al., 1998; Taatjes et al., 2002). Coactivators or adaptors such as CBP/P300 and SWI/SNF are also targets of VP16 (Memedula and Belmont, 2003; Neely et al., 1999; Vo and Goodman, 2001). VP16 was also shown to activate transcription in a similar way to HIV Tat by recruiting P-TEFb, which results in hyperphosphorylation of the large subunit of the RNA Pol II C-terminal domain (CTD) and activation of transcription elongation (Kurosu and Peterlin, 2004; Taube et al., 1999). VP16 is also found to counteract the function of the transcription inhibitors NC2 and Mot (Auble et al., 1997; Kim et al., 1995), similar to the action of the major transcriptional transactivation domain of simian virus 40 (SV40) large T antigen which activates mainly by preventing the binding of inhibitors (Johnston et al., 1996). It is not clear how IE1 acts to transactivate or repress viral gene expression, but one plausible postulation is that IE1 also uses mechanisms similar to that of VP16 and SV40 large T antigen.  The IE1 AD domain (168 to 222 aa in AcMNPV, Fig. 1.3)) differs from the AAD in that there is sequence conservation. The AD domain was shown to have transcriptional activation activity in a heterologous bacterial system (Slack and Blissard, 1997). Deletion analysis also showed that the AAD domain was inhibited by the highly conserved basic domain I (BDI) (AcMNPV residues 152 to 161, Fig. 1.3B) (Kovacs et al., 1992; Rodems et al., 1997; Slack and Blissard, 1997). OpMNPV IE1 constructs that contain deletions of the IE1 AAD but retain AD in the context of the native protein were found to be completely 23  inactive for transcriptional activation (Forsythe et al., 1998). This later result suggests that in vivo AD may not be involved in transcriptional activation or that the BDI domain has a significantly greater impact than observed in the heterologous bacterial system.  Rodems et al. (1997) showed using electrophoretic mobility shift assay (EMSA) in the 28mer binding assays that IE1del 9-52 had the same DNA binding activity as WT IE1, and deletion mutants within other regions of IE1 had impaired DNA binding. The authors also revealed the C-terminus was required for oligomerization of IE1 by testing heterodimerization of IE1 mutants with WT IE1 in the 28-mer binding assay. A later study showed that IE1 could form dimers in the absence of viral DNA in vitro and in vivo, and the oligomerization is mediated by the C-terminal helix-loop-helix domain (residue 543 to 568). Mutation of residues within the hydrophobic face resulted in reduced IE1 stability and loss of hr-dependent or non-hr dependent transactivation ability in transient assays. These results demonstrate that IE1 functions as an oligomer and oligomerization is required for the IE1 transactivation of viral early promoters (Olson et al., 2001). To function as transactivator and viral replication protein, IE1 has to gain access to the nucleus. Basic residues are known to compose nuclear localization signal (NLS) (Kaffman and O'Shea, 1999). IE1 contains BDI and a second conserved basic domain II (BDII) that is located at the carboxyl-terminal (AcMNPV 521 to 538 aa, Fig. 1.3B). BDI was found to have little impact on the IE1 nuclear entry in the absence of other viral proteins. Mutation of R537 and R538 to alanines within BDII however caused accumulation of IE1 in the cytoplasm (Olson et al., 2002). The cytoplasmic IE1 mutants were able to form oligomers, but lost the hr-dependent transactivation on the promoter of p35. The cytoplasmic IE1 mutants also negatively interfere with the transactivation by WT IE1 on an hr-dependent luciferase reporter. It was found that the cytoplamsic IE1 mutants could form oligomers with WT IE1, sequester WT IE1 in the cytoplasm and prevent their transport to the nucleus. Disruption of the oligomerization released the dominant interference of cytoplasmic IE1 mutants on WT IE1 (Olson et al., 2002). These results reveal that IE1 BDII is required for IE1 nuclear entry and IE1 forms oligomer prior to nuclear entry.  Further functional analysis of the BDI domain has revealed that BDI is essential for hr binding and hr-enhancer dependent transactivation (Olson et al., 2003). In BmNPV 24  infected cells BDI was shown to contribute to the IE1 foci formation in the nucleus which requires binding to hr elements (Nagamine et al., 2005). Recently a study of AcMNPV IE1 has also shown that BDI is essential for viral DNA replication as bacmid bearing IE1 mutant deficient for hr-binding is not able to replicate its DNA content (Taggart and Friesen, 2009) (Fig. 1.3B).  As previously indicated, IE1 is essential for viral DNA replication in transient replication of plasmids containing baculovirus replication origins (Kool et al., 1994a; Lu and Miller, 1995). More recent studies using recombinant ie1 knockout (KO) viruses have confirmed that IE1 is essential for viral replication in vivo however it can be interchanged with IE0 (Stewart et al., 2005). Deletion analysis of OpMNPV IE1 has also identified a domain within the AAD that is essential for DNA replication (Pathakamuri and Theilmann, 2002). OpMNPV IE1 mutants that were competent for replication were unable to transactivate genes. This result showed that the replication function was separable from and not dependent on the transcriptional activation function of IE1. A recent study has reported similar findings for AcMNPV IE1 identifying a replication domain located in the AAD between 2 to 32 aa (Taggart and Friesen, 2009).  The accumulated data therefore suggests that IE1 (or IE0) might be involved in viral replication by directly binding to an origin of replication leading to the assembly of a replication complex. AcMNPV IE1 is required for apoptosis induced in both permissive and nonpermissive cells upon infection potentially due to activation of viral DNA replication (Schultz et al., 2009).  Analysis of BmNPV IE1 localization found that IE1 localized to small foci at 4 hpi; the foci expand at 8 hpi and occupied almost the complete nucleus by 20 hpi (Kawasaki et al., 2004; Okano et al., 1999). This temporal distribution of IE1 agrees with sites of DNA replication as shown by BrdU corporation (Okano et al., 1999), therefore supporting that IE1 is involved in viral DNA replication. Several viral proteins have been shown to colocalize with IE1 in the discrete structures, including P143, LEF-3, DBP and BM8 (homolog of AC16-BV/ODV-E26) (Kang et al., 2005; Okano et al., 1999). P143 and LEF3 are deposited in close proximity on viral DNA with IE1 as shown by chromatin 25  immunoprecipitation (Ito et al., 2004). IE1 may require phosphorylation for replication as dephosphorylation results in the loss of its DNA-binding activity (Choi and Guarino, 1995a). IE1 migrates as multiple bands on a SDS-PAGE gel but as a single band when treated with phosphatase (Choi and Guarino, 1995b).  Analysis of purified virions from OpMNPV using a monoclonal antibody showed that IE1 is clearly associated with BV but not ODV (Theilmann and Stewart, 1993). However, IE1 was not detected by Western blot in either AcMNPV BV and ODV (Choi and Guarino, 1995b). Recent proteomic studies using mass spectrometry of ODV from AcMNPV and HearNPV have detected IE1 in virions but the signals was weak and further studies are necessary to confirm these results (Braunagel et al., 2003; Deng et al., 2007). Association of transcription factors with virions can be of significant advantage for the viruses by rapidally accelerating viral gene expression upon infection without the need for prior gene transcription and translation. This strategy is used by other animal viruses, in particular the major HSV transactivator VP16, which is a virion tegument protein (Campbell et al., 1984).  As a transcriptional transactivator, IE1 activates expression of many early genes in transient assays including 39K, p35, gp64, p143, dnapol, lef-1, lef-2 and lef-3 (Blissard and Rohrmann, 1991; Guarino and Smith, 1992; Kovacs et al., 1991; Lu and Carstens, 1993; Nissen and Friesen, 1989; Pullen and Friesen, 1995; Ribeiro et al., 1994; Theilmann and Stewart, 1991). No specific IE1-responsive element has been identified from these promoters, indicating IE1 may achieve transcriptional activation via indirect proteinprotein interaction. However, IE1 has been shown to bind the hr elements which also function as enhancer elements. When hr elements are linked in cis to specific genes and transactivated by IE1 it can result up to 200 to 300 fold induction of gene expression. Therefore genes can be activated by IE1 in an hr-dependent or independent manner (Nissen and Friesen, 1989; Rodems and Friesen, 1993; Theilmann and Stewart, 1991). IE1 can also negatively regulate transcription from promoters of other immediate early genes such as ie2, ie0 and pe38 (Carson et al., 1991b; Kovacs et al., 1991; Leisy et al., 1997; Theilmann and Stewart, 1993). Leisy et al. (1997) showed that IE1 would bind to a sequence similar to half the hr 28-mer repeat between the TATA box and transcription start site of the ie2 promoter resulting in inhibition of gene expression. 26  Even though there has been extensive analysis of IE1 many of the details of how it functions in both replication and transcription remain to be determined. Animal virus transcription regulators can function or affect virtually every aspect of a gene expression including chromatin remodelling and histone modification, binding of transcription activators and co-activators to regulatory elements and promoters, recruitment of basal transcription machinery, switch of transcription initiation to processive elongation and post-transcription/translation regulation. Good examples are human T-lymphotropic virus (HTLV) Tax, human immunodeficiency virus (HIV) Tat, Adenovirus E1A and HSV VP16 (Boxus et al., 2008; Brady and Kashanchi, 2005; Frisch and Mymryk, 2002; Wysocka and Herr, 2003). Many viral transactivators are also capable of modulating a wide range of cellular transcription factors to achieve selective regulation of different genes. It is very likely that the major baculovirus transactivator IE1 also regulates the expression of viral and host gene expression through a variety of mechanisms.  1.8.2 IE0 AcMNPV IE0 contains an additional 54 aa at the N-terminal compared with IE1 due to a splicing event, of which 38 aa are coded by the N-terminal of EXON0 (AC141) and the remaining 16 aa are coded by the IE1 5’ UTR (Fig. 1.3B). IE0 has peak expression at early times post infection and thereafter declines unlike IE1 which increases in steady state levels throughout infection. As a result, the IE0 to IE1 ratio changes throughout the infection (Chisholm and Henner, 1988; Choi and Guarino, 1995b; Huijskens et al., 2004). A surprising finding with the expression of ie0 is that translation of the ie0 mRNA results in the production of both IE0 and IE1. IE1 was found to be produced due to internal translation initiation from the ie1 start codon (Huijskens et al., 2004; Theilmann et al., 2001). Therefore, in vivo, IE1 would always be produced regardless of which gene, ie0 or ie1 is expressed.  IE0 can also function as a viral transactivator. Initial studies examining the function of IE0 were unaware that both IE0 and IE1 were produced by ie0. These studies suggested that AcMNPV IE0 can transactivate the ie1 promoter but not the ie0 promoter. In addition, IE0 27  could only transactivate the delayed early 39K promoter in the presence of cis-linked hr sequence (Kovacs et al., 1991). Later studies analyzing the OpMNPV ie0 (Theilmann et al., 2001) using a construct that was unable to translate IE1 (mutation of internal start methionine) suggested that IE0 was a more potent transactivator than IE1 on specific early promoters, and was able to transactivate its own promoter and 39K in the absence of hr sequences. IE0 can also stimulate the expression of late genes as shown by Huijskens et al. (2004). AcMNPV IE0 was able to replace IE1 in transient very late gene assays, and was shown to be the 20th late expression factor. Additionally, it was found that IE0 and IE1 were mutually antagonistic when coexpressed in transient late gene assays. This could potentially explain why IE1 is expressed at much higher levels at late times post-infection.  The ability of LdMNPV IE0 to function as a transactivator was analyzed using plasmids with viral genomic DNA inserts that contained regions that expressed ie0 and ie1 or just ie1. With these constructs it was found that only LdMNPV IE0 and not IE1 was able to transactivate the AcMNPV 39K delayed early promoter in an enhancer independent or dependent manner (Pearson and Rohrmann, 1997). In addition only LdMNPV ie0 was able to support viral DNA replication in transient replication assays. These results would suggest that for LdMNPV, a group II alphabaculovirus, IE0 may be the dominant transcriptional transactivator. However studies with LdMNPV did not determine if both IE0 and IE1 were translated from the ie0 mRNA.  The development of bacmid technology permitted the construction of AcMNPV recombinant viruses that contained an ie0/ie1 deletion or knockout and viruses that expressed only IE0 or IE1 (Stewart et al., 2005). The ie0/ie1KO virus was unable to replicate showing for the first time that this gene complex is essential for virus replication. The ie0/ie1KO virus could be rescued by either ie0 or ie1, but neither viruses resulted in a WT infection. Repair of the KO virus with either ie0, ie0M→A (internal start M mutated to A) resulted in IE0 having a different temporal expression compared to WT. In the absence of IE1, instead of decreasing at late times post-infection, the levels of IE0 continued to increase. This would agree with previous transient assays that suggested the ie0 promoter could be negatively regulated by IE1 (Kovacs et al., 1991). Expression of ie0 without the methionine mutation produces both IE0 and IE1 but predominantly IE0 which is the 28  opposite of what is observed when IE0 is expressed transiently (Stewart et al., 2005; Theilmann et al., 2001). This suggests there may be other viral factors involved in regulating the internal initiation of IE1 from ie0 mRNA. When only ie1 is expressed the levels of IE1, like WT virus, continue to increase up to late times post-infection but to lower levels. In addition to the different expression profiles viruses expressing only IE0 or IE1 have very different replication properties and neither of them produced WT levels of OBs. Viruses expressing only IE0 produce reduced levels of BV and had lower levels of viral DNA replication for the first 49 hours post-infection compared with WT. Virus expressing only IE1 produces similar levels of BV as WT and the viral DNA replication profiles appear to be unaffected. However, viruses that express only IE1 have reduced OB production. These results indicate that interplay between IE0 and IE1 combined with their different functions is essential to achieve a WT infection.  It has also been reported that if the IE0 expression levels relative to IE1 are changed it can affect host range. Lu et al. (2003) isolated an AcMNPV mutant vAcSL2 that was randomly generated by co-transfecting Spodoptera littoralis (SL2) cells with AcMNPV DNA and overlapping cosmid library representing the entire SpliNPV genome. The vAcSL2 carried a 519-bp insert that disrupted the ie0 promoter, which resulted in reduced expression of IE0 and increased level of IE1 relative to IE0. This change in IE0 and IE1 expression levels permitted vAcSL2 to replicate efficiently in the normally poorly permissive SL2 cell for AcMNPV. Directed mutation of the AcMNPV ie0 promoter by inserting the chloramphenicol acetyltransferase (cat) gene generated a new recombinant virus, which had identical replication properties to that of vAcSL2, confirming that the change of IE0 expression levels was responsible for the host-range switch.  IE0 and IE1 form homodimers and in addition it is known that IE0 and IE1 can form heterodimers (Kremer and Knebel-Morsdorf, 1998; Lu et al., 2003). Each of these complexes potentially has different abilities to support both viral gene transcription and DNA replication. This would also predict that the relative levels of each species could have significant impact on viral replication. Based on the normal expression profiles it would be predicted for AcMNPV infection that at very early times post-infection IE0 homodimers would be dominant, followed by heterodimers at or just prior to viral DNA 29  replication followed by IE1 homodimers thereafter. This would therefore predict that altering the ratio between these two proteins would affect viral replication, which is what is observed.  What or how viral or cellular factors interact with IE0 to result in the differences in regulation compared to IE1 is not known. Determining the mechanism of action of IE0 relative to IE1 and the role of the additional amino acids at N-terminus of IE0 will be critical to a complete understanding of baculovirus pathology, regulation of gene expression and viral DNA replication.  1.8.4 Other viral transactivators resemble IE0 and IE1 Other viral DNA systems express alternatively spliced mRNAs, like ie0 and ie1 that result in primary regulatory proteins that differ by a few amino acids yet have significantly different functions during infection. One of the most intensely studied is the Adenovirus E1A 12S and 13S gene products. E1A is expressed from two alternatively spliced mRNAs, producing proteins of 289 and 243 residues respectively. The two products are identical except the internal 46 aa in the 13S product. The 46 aa is conserved among different adenovirus serotypes and has been designated as conserved region 3 (CR3) in addition to two conserved regions CR1 and CR2 shared by both 12S and 13S. The 13S protein product is considered the primary transactivator of viral gene expression and is required for productive viral infection. The 12S product encodes all the functions necessary for immortalization of primary cells and for transformation of these cells in cooperation with other viral or cellular oncogenes (Svensson and Akusjarvi, 1984; Zerler et al., 1986). 13S, which contains CR3, but not 12S, is able to interact with different DNA binding domains of various transcription factors like c-Jun, Sp1 and USF (Liu and Green, 1994). Transcriptional activation by 13S E1A is inhibited by co-expression of sub-stoichiometric amounts of the 12S E1A. The 12S domain required for inhibition of 13S has been mapped to CR1 which binds to the cellular transcription complex, p300/CBP. The inhibition is reversed by introducing exogenous p300 or CBP. This suggests that E1A 12S inhibits 13S transcriptional activation by sequestering limited cellular transcription resources (Pelka et al., 2009). 30  The equine herpesvirus 1 (EHV-1) IR1 and IR2 gene products differ in their N-terminus acidic activation domain by a splicing event which is also very similar to the baculovirus ie0-ie1 genes. IR1 is expressed under IE conditions whereas IR2 is detected during early and late stages of a EHV-1 productive infection (Harty and O'Callaghan, 1991). IR2 mRNA initiates 1548 bp downstream of the transcription initiation site of IR1 mRNA, thus the IR2 protein is a truncated form of IR1 that lacks a acidic transcriptional activation domain and serine-rich tract (Harty and O'Callaghan, 1991). However, both transiently expressed IR1 and IR2 protein were shown to be potent transcriptional regulators (Caughman et al., 1995). IR1 protein is capable of transactivating EHV-1 early and late promoters, while IR2 functions as a dominant-negative regulator of EHV-1 gene expression. It has been shown that IR2 by itself downregulates EHV early promoters and inhibits in a dose-dependent manner the transactivation of viral promoters by other IE proteins (Kim et al., 2006). Evidence suggests that IR2 inhibits gene expression by blocking IE protein binding to viral promoters or by squelching the limiting supplies of transcription factors TFIIB and TBP (Caughman et al., 1995; Kim et al., 2006).  31  1.9 Hypotheses and chapter synopsis Current data show that AcMNPV IE0 and IE1 play pivotal roles during the viral replication, including regulation of viral gene expression and viral DNA replication. However, to date, the majority of detailed biochemical analyses have focussed on IE1 without considering the role of IE0. As both IE0 and IE1 are required for a WT infection, it is necessary to understand why alphabaculoviruses express IE0 and what advantage it provides. The hypotheses addressed in chapters 2, 3, and 4 in this thesis are as follows:  Chapter 2) IE0 differs from IE1 by an N-terminal 54 aa extension indicating that this region may provide additional functionality. Therefore, it was hypothesized that IE0 would functionally differ from IE1 in cellular localization, early gene transcriptional activation, viral DNA replication or occlusion body development. The subcellular localization was investigated by confocal microscopy using fluorescent fusion proteins or indirect immunofluorescence. Results indicated that IE0 and IE1 colocalized throughout infection and no difference was observed. Early gene transcriptional transactivation was analyzed by transient assays using early gene reporter assays and microarray analysis of all AcMNPV genes. Generally no difference was observed in the ability of IE0 or IE1 to positively or negatively regulate a gene. However, it is possible that levels of expression of IE0 and IE1 could potentially play a role in determining which early gene set is activated. As observed in the microarray analyses IE0 only transactivates a subset of genes that are activated by IE1. The ability to regulate DNA replication was different when the two proteins were compared. Mutation of the IE0 N-terminal domain inhibited the ability of IE0 to support viral DNA replication. Lastly, it was observed that expression of only IE0 resulted in OBs that predominately contain ODVs that only contain single nucleocapsid. Therefore IE0 and IE1 expression could potentially affect the phenotype of single (S) or multiple (M) nucleocapsids per envelope that is observed in alphabaculoviruses.  Chapter 3) In this chapter potential protein-protein interactions between viral or cellular proteins and IE0 or IE1 were investigated. It was hypothesized that IE0 and IE1 interact with a different set of proteins or have altered interactions due to the IE0 N-terminus. To address this both IE0 and IE1 were fused to a tandem affinity tag and interacting proteins 32  were identified. Using this approach a single viral protein AC16 was identified that interacted with both IE0 and IE1. The AC16 interaction domain was mapped and mutated on IE0 and IE1 and the effect on viral replication was determined. In addition an ac16 deletion virus was generated to assess the impact of losing both AC16 and the interaction with IE0 and IE1. The results indicated that the loss of AC16 altered IE0 levels relative to IE1, that is, an IE0 specific effect. In addition, loss of AC16 binding domain on IE0, but not IE1, increased the level of BV produced.  Chapter 4) The gene encoding AC16, an IE0- and IE1-interacting-protein via direct binding to the domain with a predicted coiled-coil structure, is expressed from a evolutionarily conserved pair of ORFs, ac16-ac17, that overlap transcriptionally. The association is such that the promoter of ac17 is contained within the ac16 ORF. It was therefore hypothesized that the function of these two genes were related in the AcMNPV infection cycle and AC17 may also be required for IE0 and IE1 function. To answer this a double ac16-ac17KO virus was generated and the impact on virus replication and IE0 and IE1 expression was investigated. The results of knocking out AC17 were significant such that BV levels were severely reduced. However, the combined effect of deleting both ac16 and ac17 was dramatic delay of up to 12 hours in the expression of not only the immediate early proteins IE0 and IE1 but also all the early and late proteins analyzed. The apparent global delay in gene expression was only observed when cells were infected by BV and not by transfection. Both AC17 and AC16 were found to be possible structural proteins suggested they be required for nucleocapsid transport or uncoating.  The overall objectives of this thesis were to contribute to the characterization of the molecular mechanisms of action and roles of AcMNPV IE0 and IE1 in viral life cycle. A complete discovery of IE0 and IE1 functions and the proteins they interact with will be essential to the understanding of baculovirus biology given the fact that this gene complex appears to produce highly critical regulatory products during alphabaculovirus infection.  33  Figure 1. 1 Baculovirus life cycle. Baculovirus hosts get infected by ingestion of occlusion body (OB) contaminated food. OBs disintegrate and occlusion derived virus (ODV) is released in the alkaline environment of the midgut lumen. ODV enter the columnar epithelia cells via membrane fusion with the microvilli. This process is called primary infection. Nucleocapsids are transported to the nucleus or nucleopore where it uncoatsor viral DNA is released directly. this is followed by viral gene expression. Viral DNA replication takes place in virogenic stroma (VS), where progeny nucleocapsids are assembled. At early stages of infection, progeny nucleocapsids are transported to the plasma membrane. Plasma membrane has been modified by viral proteins and nucleocapsids bud through to produce budded virus (BV). BV then spreads the infection systemically and enters other tissues by clathrinmediated adsorptive endocytosis. This process is named secondary infection. At late stages of infection, nucleocapsids are enveloped within the nucleus forming ODV and become embedded in OBs. Adapted from Blissard and Rohrmann (1990).  34  Figure 1.1 continued  Budded Virus (BV) Budding  Plasma membrane Nuclear membrane Nuclear pore  VS ODV envelope fuse with the midgut microvilli  Uncoating Envelopment Occlusion  Primary infection Occlusion Derived Virus (ODV)  Solubilization of OB in the midgut lumen  Adsorptive endocytosis Systemic Infection  Ingestion of contaminated material by insect host Occlusion bodies (OB) are dispensed into the environment after the infected host liquefies  35  Figure 1. 2 Structures of the two virion types of alphabaculoviruses. ODV is embedded in the OB, and BV is the extracellular virion. BV and ODV have the same DNA genome. Common proteins between the two virions types are shown in the middle panel between the BV and ODV. Proteins and features specific to each virion are shown on the outside (Adapted from Theilmann and Blissard (2008)).  36  Figure 1. 3 AcMNPV ie0-ie1 gene region and functional domains of IE0 and IE1. (A) AcMNPV ie0-ie1gene region. The ie0 mRNA initiates from an early motif (TATA— CAGT) located 5’ to ac141 (exon0) and ie1 mRNA initiates from a similar motif 5’ to ie1 (ac147). The ie0 intron splice donor site is located within ac141, 114 bp downstream of the ac141 start codon. The splice acceptor site is located at the transcriptional start site of ie1, 48 bp upstream of the ie1 start codon. Therefore ie0 exon2 consists of the complete ie1 transcript. (B) Diagram showing the mapped domains of IE1 and counter parts in IE0. IE0 differs from IE1 by 54 additional residues at the N-terminus. R: Replication domain 2-32 aa (Pathakamuri and Theilmann, 2002; Taggart and Friesen, 2009); AAD: Acidic activation domain 8-118 aa; BDI: Basic domain I 152-161 aa; AD: Acidic domain 168-222 aa; DBD: DNA binding domain; BDII: Basic domain II 521-538 aa; HLH: Helix-loophelix 543-568 aa. 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The expression of baculovirus genes occurs in an ordered cascade fashion: early, late and very late (Blissard and Rohrmann, 1990; Miller, 1997). Early genes are transcribed by host RNA polymerase prior to viral DNA replication and can be further subgrouped to immediate early (IE) genes and delayed early (DE) genes. IE genes are expressed immediately upon infection and do not require other viral factors for activated expression. Some IE genes encode transregulators that activate the expression of DE genes, are involved in viral DNA replication and stimulate the expression of late genes. Transcription of late genes utilizes a viral encoded RNA polymerase that is resistant to alpha-amanitin and takes place after or concomitantly with the onset of viral DNA replication. The successive progression from early gene expression to late gene expression is critical to the viral replication.  AcMNPV is the type species of the baculoviridae alphabaculovirus genus. It is the most extensively studied baculovirus and has a wide host range. The AcMNPV genome is 133,894 bp, encoding 154 potentially expressed open reading frames of 150 bp or greater (Ayres et al., 1994). The immediate early IE0 and IE1 are the primary transregulatory proteins of AcMNPV, which are expressed from the only known spliced gene complex. The ie0 mRNA consists of two exons and initiates from an early promoter upstream of ac141. Exon1 of ie0 consists of 114 bp of ac141 that is spliced to the 5’ end of the ie1 mRNA after excision of a 4.2 kb intron. Exon2 composes of 48 bp of ie1 5’ UTR and the complete ie1mRNA. Therefore splicing results in an extra 162 bp added to the 5’ of ie1 1  A version of this chapter will be submitted for publication. Nie, Y., Yamagishi J., Blissard G. and Theilmann DA. Functional Comparison of AcMNPV IE0 and IE1.  66  mRNA and 54 aa added to N-terminus of IE1 (Chisholm and Henner, 1988). The expression of ie0 mRNA results in both IE0 and IE1 being translated due to the internal translation initiation from the ie1 ATG (Huijskens et al., 2004; Theilmann et al., 2001). The ie1 mRNA is not spliced and initiates from the early promoter of ac147. Both ie0 and ie1 mRNAs are transcribed and expressed immediately upon infection, but peak at different times post infection (pi). The steady-state level of ie0 peaks at about 4 hours pi and then decreases rapidly, whereas ie1 keeps accumulating until very late times pi (Huijskens et al., 2004; Ohresser et al., 1994; Pullen and Friesen, 1995; Theilmann and Stewart, 1993).  IE1 was first identified by its ability to transactivate the 39K promoter in transient assays (Guarino and Summers, 1986a). AcMNPV IE1 or the homolog of OpMNPV has been reported to regulate multiple viral early genes including p35, dnapol, p143, ie2, pe38, gp64 and its own promoter (Leisy et al., 1997; Lu and Carstens, 1993; Ohresser et al., 1994; Blissard and Rohrmann, 1991; Kovacs et al., 1991; Nissen and Friesen, 1989; Theilmann and Stewart, 1991), hence is considered as the major transactivator of AcMNPV. IE1 transactivation of viral early promoters is greatly augmented by homologous region (hr) enhancer elements cis-linked to promoters (Guarino and Dong, 1991; Guarino and Summers, 1986b; Nissen and Friesen, 1989). AcMNPV IE1 binds to the 28-mer palindrome of the hr elements as a dimer and binding to both half-sites of hr is required for the stimulation of transactivation by hr (Choi and Guarino, 1995a; Guarino and Dong, 1991; Leisy et al., 1995; Rodems and Friesen, 1995). However similar sequences to the hr palindrome in the promoter of ie2 and pe38 have been shown to be responsible for repression by IE1 (Leisy et al., 1997). Therefore depending on the context, IE1 can function to transactivate or repress viral promoters. IE1 also stimulates the expression of late genes and is one of the twenty-one LEFs (Lu and Miller, 1995; Passarelli and Miller, 1993).  AcMNPV IE0 also functions as a transactivator and initial experiments suggested that it was hr-dependent (Kovacs et al., 1991). However the initial constructs used by Kovacs et al. (1991) likely produced both IE0 and IE1 as they were unaware at the time that ie0 mRNAs were translated as both proteins. Therefore to date it is not clear whether IE0 can 67  transactivate other viral genes by hr-dependent or independent mechanisms or if IE0 can preferentially regulate certain genes. Similar to IE1, IE0 can also stimulate late gene expression as found in transient assay (Huijskens et al., 2004). More recent studies have shown that IE0 can support complete virus replication in the absence of IE1 (Stewart et al., 2005). Pearson and Rohrmann (1997) showed that LdMNPV IE0 can transactivate the AcMNPV 39K promoter with or without hr enhancer. Similarly, OpMNPV IE0 was also shown to transactivate 39K in both hr-dependent or independent way and can autoregulate its own promoter and potentially be a stronger transactivator than IE1 (Theilmann et al., 2001).  As transactivators, IE0 and IE1 have to localize in the nucleus. Studies have shown that BmNPV IE1 accumulates prior to viral DNA replication in discrete subnuclear structures where viral DNA replication occurs, and focal distribution requires binding to hr elements (Kawasaki et al., 2004; Nagamine et al., 2005). BmNPV IE1 co-localizes with DBP and LEF3 in the nuclear structures that are believed to be viral replication factories (Nagamine et al., 2006; 1999). BmNPV IE1 was also found to co-localize with BmNPV BM8 in infected insect cells and interacts with the protein in yeast (Imai et al., 2004; Kang et al., 2005). Ito et al. (2004) found that AcMNPV IE1, LEF3 and P143 bind to closely linked sites on viral DNA in vivo and suggested these proteins may form a replication complex in infected cells (Ito et al., 2004). The colocalization of IE1 with proteins for viral DNA replication in the viral replication factory is consistent with previous finding, that AcMNPV IE1 is one of the six essential genes along with dna polymerase, lef-1, lef-2, lef3, and helicase required for the viral DNA replication in transient replication assays (Kool et al., 1994). Though IE0 has been shown to form dimers with IE1, there is no report on the localization of the protein during infection. Therefore it is unknown whether IE0 also forms foci in the nucleus or locates to different cellular locations compared to IE1.  Although IE1 was shown to be an essential gene for transient viral DNA replication it has only been recently that an ie1 deletion could be generated due to the development of bacmid technology to prove that either IE0 or IE1 was essential for baculovirus replication. Using an ie1KO bacmid, Stewart et al. (2005) showed that virus that lacks both IE0 and IE1 was completely non-infectious and was unable to replicate DNA and produce any 68  progeny virus. Deletion of IE0 and IE1 also abolishes the apoptosis induced by viral infection in both permissive and non-permissive cells (Schultz et al., 2009). Rescue of an ie1 deletion virus by ie0 or ie1 showed that either protein can support viral replication though both are required for a wild-type phenotype. Viruses that produced only IE0 or IE1 exhibited very different viral DNA synthesis profiles compared with WT virus. In addition, viruses expressing only IE0 had about 10 times lower BV production compared with WT or IE1 only virus. Expression of only IE0 or IE1 also resulted in viruses producing fewer occlusion bodies compared with WT virus (Stewart et al., 2005). These results show that IE0 and IE1 have both similar and different functional roles in the virus life cycle. The molecular mechanisms by which IE0 and IE1 orchestrate their different roles during infection remains poorly understood. In addition it is unknown how the extra 54 aa at the N-terminal of IE0 contribute to these differences.  In this chapter, it is determined whether AcMNPV IE0 exhibits functional differences relative to IE1 in the transactivation of viral early genes in the presence or absence of cislinked hr enhancer elements. In addition, mutational analysis of the IE0 specific Nterminal domain was conducted to reveal its contribution to the IE0 properties. Cellular localization of AcMNPV IE0 and IE1 was compared using fluorescent tags. Finally the impact of IE0 and IE1 on ODV content of occlusion bodies was assessed by electron microscopy.  2.2 Results 2.2.1 IE0 colocalizes with IE1 in Sf9 cells during infection. IE0 and IE1 are coexpressed throughout the infection, however it is unknown if they distribute to the same or different cellular locations. To determine if there are different subcellular localizations during infection IE0 and IE1 were fused with ECFP and EYFP fluorescent proteins, respectively. The constructs were introduced into an ie0-ie1 deletion backbone (Stewart et al., 2005) to produce the viruses ie1KO-IE0ECFP, ie1KO-IE1EYFP or ie1KO-IE0ECFP-IE1EYFP (Fig. 2.1A). These viruses were used to infect Sf9 cells which were then fixed at various times post-infection, stained with 200 ng/ml 4′, 6-  69  diamidino-2-phenylindole (DAPI) and analyzed by confocal microscopy. As shown in Fig. 2.1B, IE1EYFP formed foci in the nucleus of infected cells at 4 hpi, which become significantly larger by 12 hpi. By 24 hpi, no foci were seen and IE1EYFP was detected evenly across the virogenic stroma within the nucleus. This is consistent with what has been reported for BmNPV IE1, which is shown to locate in the discrete sites at early times post-infection in the nucleus where viral DNA replication takes place (Kawasaki et al., 2004; Okano et al., 1999). At 4 hpi, IE0ECFP expression was very low, and a reliable result of localization could not be produced, however similar to IE1EYFP distribution but at 6 hpi, IE0ECFP formed foci. The IE0ECFP foci became enlarged by 12 hpi and were distributed across the virogenic stroma in the nucleus by 24 hpi (Fig. 2.1C). These results show that when IE0 and IE1 are expressed individually they follow a similar localization pattern during infection. To determine if both IE0 and IE1 distribute to the same locations and if the distribution is affected when both proteins are expressed in the same cell, Sf9 cells were infected by ie1KO-IE0ECFP-IE1EYFP. When both proteins are present the distribution of IE0 and IE1 resemble what is observed with the virus expressing either IE0 or IE1 individually (Fig. 2.1D). In the same cell, IE0 and IE1 were equally distributed in the same small foci at 6 hpi and in the enlarged foci at 12 hpi. By 24 hpi the foci have merge and both IE0ECFP and IE1EYFP encompassing most of the virogenic stroma.  It is possible that fluorescent protein tags, which are quite large, may affect the distribution of IE0ECFP and IE1EFYP so that they did not reflect the real cellular distribution of IE0 and IE1. Therefore to confirm that the fluorescent fusion proteins represent the true distribution, immunofluorescence confocal microscope was performed using viruses expressing IE0FLAG and IE1HA respectively. Though the immunofluorescence was less sensitive than using fluorescent fusion proteins both IE0FLAG and IE1HA were detected in infected cells (Fig. 2.2). At 6 hpi, IE0 and IE1 formed small foci in the nucleus which become enlarged by 12 hpi, followed by an even distribution across the virogenic stroma by 24 hpi. These results therefore confirm the distribution pattern observed using fluorescent fusion proteins. Therefore, IE0 and IE1 appear to have the same cellular distribution, which is not affected by the presence or absence of either protein.  70  2.2.2 Comparison of IE0 and IE1 transactivation of viral early promoters Previously it was shown that IE0 and IE1 had antagonistic impacts on late gene expression (Huijskens et al., 2004). However it is not well understood if they are functionally different in their ability to transactivate early genes. To analyze the ability of IE0 and IE1 to transactivate early genes, reporter plasmids expressing chloramphenicol acetyl-transferase (CAT) under the control of the early promoters ie1, ie0, ie2, me53, p35, gp64 and 39K were constructed. IE0 or IE1 expressing plasmids or a control plasmid pBluescribe + (pBS+) was co-transfected with each reporter plasmid and at 48 hours post-transfection (hpt) the cells were collected and assayed for CAT expression. The results showed that IE1 was unable to transactivate ie1, gp64 and ie2 promoters, but transactivated 39K, me53 and p35 although only to levels slightly above background, and repressed the expression of ie0 promoter (Fig. 2.3B). IE0 also did not show transactivation of gp64, p35 and ie2, but transactivated ie1 (slightly), 39K and me53 and repressed the expression from its own promoter (Fig. 2.3B). Therefore no viral gene that is specifically regulated by IE0 was observed.  Among the viral early genes analyzed, no general difference in the ability of IE0 or IE1 to positively or negatively regulate a promoter was observed. The 39K promoter showed the biggest difference between IE0 and IE1 in the levels of activation. However, as shown by Western blot (Fig. 2.3C) the levels of IE0 were lower compared to IE1. The observed results could therefore be due to the expression level of the transactivators indicating that IE0 may not necessarily be a less potent transactivator compared with IE1 but was just expressed at lower levels. To test this, various amounts of IE0 or IE1 expressing plasmid was co-transfected with 39K reporter, and CAT assays were performed (Fig. 2.3D). Although only limited activation was seen, the preliminary results showed that when transactivated by similar levels of IE0 or IE1 (compare 1 and 2 µg of pAc-IE1 with 2 and 3 ug pAc-IE0, Fig. 2.3D, E) CAT expression levels were similar. Therefore for the 39K promoter IE0 appears to be as strong a transactivator as IE1. Comparison of 2 and 3 ug of pAc-IE1 showed there was no increase in CAT expression even though there was approximately double the level of IE1 expressed in the later transfection (Fig. 2.3D, E).  71  This indicates that saturation levels had been obtained and no further increase in p39K expression could be obtained under these conditions.  2.2.3 Comparison of IE0 and IE1 transactivation of viral early promoters containing the hr5 enhancer No direct comparisons between IE0 and IE1 on their ability to transactivate early gene promoters containing enhancer elements have been previously performed. Therefore, IE0 and IE1 were compared using transient assays using the same set of early gene reporter plasmids used in Fig. 2.3B but with the AcMNPV hr5 enhancer inserted 5’ to the promoter (Fig. 2.3A). Similar to previous reports that hr5 enhanced the activation of 39K and p35 by IE1 (Kovacs et al., 1991; Nissen and Friesen, 1989), in the presence of hr5 expression from 39K, p35 and gp64 was enhanced when transactivated by either IE0 or IE1, however no difference was observed between IE1 and IE0 in terms of levels of activation (Fig. 2.4). Both gp64 and p35 promoters that were unresponsive to either IE0 or IE1 (Fig. 2.3B) become activated by both proteins when the enhancer is present (Fig. 2.4A). The ie0 and ie2 promoters with hr5 element were unresponsive or had slightly lower expression levels when co-transfected with either IE0 or IE1, similar to what was observed with the enhancerless promoter (Fig. 2.3B and Fig. 2.4A). The ie1 promoter was negatively regulated by the hr5 element in the presence of IE0 or IE1 (Fig. 2.4A). The me53 promoter which is positively regulated by both IE0 and IE1 is negatively regulated when the hr5 element is present (Fig. 2.3B and Fig. 2.4A). This switch from positive to negative regulation has not been previously reported for baculoviruses. Even though there were changes in the regulation of the early genes when the hr5 enhancer is present, there was no overall difference between IE0 and IE1 in the ability to regulate the selected genes as was observed in the enhancerless promoters.  2.2.4 IE0 regulates a specific group of viral genes during infection The initial analysis of viral early genes did not identify any specific difference between IE0 and IE1 in their ability to regulate viral genes. To enable a global analysis of viral genes regulated by either IE0 or IE1, microarray analyses were undertaken (Microarray 72  analyses were a collaboration with Dr. Junya Yamagishi and Dr. Gary Blissard and were performed at Dr. Blissard’s laboratory at the Boyce Thompson Institute, Cornell University). Global gene analysis was initially performed on RNA isolated from cells infected by viruses that express only IE0 or IE1 (Stewart et al., 2005) to detect differences in genes expressed. However, the levels of sensitivity offered by using this approach were not sufficient. A second experimental design was therefore used. Sf9 cells were initially transfected with plasmids expressing either IE0 or IE1. At 24 hpt, cells were infected with WT virus in the presence of cyclohexamide and RNA was isolated 24 hours post-infection. Under these conditions the de novo protein expression from the virus was less than one percent compared to the treatment without cyclohexamide as analyzed by Dr. Junya Yamagishi, and only genes activated by previously translated IE0, IE1 or cellular proteins should be expressed. The results from this experiment are shown in table 2.1. A large group of 58 genes having greater than a 2 fold increase in expression were identified to be upregulated by IE1. No gene was found to be down-regulated by IE1. In IE0 expressing cells only a small number of genes were up regulated by IE0 including ac52, ac76, ac79, ac111, lef-3, lef-4, lef-6 and ie1. In addition, unlike IE1, some genes were also found to be down-regulated by IE0 including ac18, ac33, ac91, ie2 and vp39. All the genes upregulated by IE0 were also up regulated by IE1 but to a greater level. However all the IE0 down regulated genes were not impacted positively or negatively by IE1 suggesting they may be specific targets of IE0.  To validate the microarray results and screen potential promoters that may be specifically regulated by IE0 relative to IE1 additional reporter gene constructs were made. Promoters of the IE0 down-regulated genes ac18, ac33 and ac91 and the up-regulated genes ac52, ac76, ac79, ac111, lef-3, lef-4 and lef-6 were inserted into the CAT expression vectors described in Fig. 2.3A. Each construct was transfected into Sf9 cells in the presence or absence of IE0 or IE1 and assayed for CAT expression (Fig. 2.5A). Of the promoters positively regulated by IE0, ac79, ac111, lef-3, lef-4 and lef-6 were consistent with the microarray analysis. However, no up-regulation by IE0 was observed on the ac52 and ac76 promoters but low levels were observed with IE1. Of the negatively regulated promoters tested neither ac33 nor ac91 were activated by IE0 as would be predicted from the  73  microarray results. In contrast to the microarray results however, the ac18 promoter was clearly activated by both IE0 and IE1.  To determine if the enhancer element hr5 affects the ability of IE0 to regulate the set of genes identified by microarray analyses, promoters of IE0 down-regulated genes ac18, ac33 and ac91 and up-regulated genes ac52, ac76, ac79, ac111, lef-3, lef-4 and, lef-6 were cloned into the enhancer CAT expression vector (Fig. 2.3A). In the presence of hr5 the genes up-regulated by IE0 as shown by microarray, ac76, ac79, ac111, lef-3, lef-4 and, lef6 were obviously activated (Fig. 2.5C). The ac52 promoter which exhibited low but detectable increase in expression in the absence of an enhancer was not up-regulated in the presence of hr5 (Fig. 2.5A and Fig. 2.5C). The microarray IE0 down-regulated genes ac18 and ac33 were also up-regulated in the presence of an enhancer, but ac91 remained unresponsive to either IE1 or IE0 (Fig. 2.5C). The ac33 promoter showed only a very slight increase whereas ac18 showed very high levels of activation. Similar to the analyses of early gene promoters (Fig. 2.4A), no major difference between IE0 and IE1 was observed when hr5 enhancer was present (Fig. 2.5C).  The transient CAT assay analysis of promoters with and without enhancer elements therefore agreed with the microarray data to a large degree but with the obvious exceptions of ac18 and ac52. The differences could result from the different context of promoters. In the microarray the promoters are in the viral genome context, whereas in the transient CAT assays, only fragments from the ATG start codon to upstream 350 bp were chosen. Additionally, cyclohexamide treatment at 100 µg/ml does not completely shut down the expression (Ross and Guarino, 1997) therefore there could be effects from viral proteins newly translated at a very low-level in the microarray experiments. Either way this suggests there are auxiliary viral factors that affect the ability of IE0 to up and down regulate viral genes.  2.2.5 Deletion analysis of IE0 N-terminal specific 54 amino acids IE0 differs from IE1 by the additional 54 amino acids at its N-terminal, yet the two proteins have obviously different impacts on the infection process (Stewart et al., 2005). 74  Viruses expressing either of the two proteins exhibit differences in the viral DNA replication profile, BV production and occlusion body production. IE0 and IE1 only differ by 54 N-terminal amino acids present in IE0, therefore the biological differences observed could be due to this domain. To determine if there are conserved regions in the IE0 specific domain, sequences from other alphabaculoviruses were predicted and aligned with AcMNPV IE0 to reveal any conserved pattern or residues (Fig. 2.6). The alignment showed that IE0-specific amino acid sequences are quite variable in length and do not contain any conserved sequences among all alphabaculoviruses (Fig. 2.6A). However, if only the IE0-specific sequences from Group I alphabaculoviruses are aligned, some conserved residues are revealed (Fig. 2.6B). This includes for AcMNPV IE0, MIK/R (1-3), S/T (5) N (19), C (33), E (35) and LQV (39-41) at the exon1-exon2 splice junction. The conservation of these sequences may indicate that are required for IE0 function.  To further analyze the IE0-specific N-terminus a deletion analysis was performed. Nterminus to C-terminus and C-terminus to N-terminus deletion mutants of the IE0 specific amino acids were made as shown in Fig. 2.7. The deletion constructs were analyzed for their ability to transactivate the DE 39K and IE me53 promoters with and without the hr5 enhancer, compared to full length IE0 and IE1 (Fig. 2.8). Each of the IE0 deletion mutants had reduced transactivation of p39K-CAT compared to full length IE0 or IE1. Deletions in the 32 to 54 amino acid region appeared to have a greater impact. This suggests that the deletions were inhibiting IE0 function or residues left within the 54 aa after deletion could inhibit IE1 function. Transactivation of phr5-39K-CAT however showed no significant difference between any of the constructs. For the promoter of me53, transactivation by each construct also did not exhibit any difference in the ability to activate or repress gene expression in the presence or absence of hr5 (Fig. 2.8B), suggesting different protein complexes are involved in the activation of 39K and me53 promoters. The IE0 and IE1 expression levels from each construct were compared by Western blotting (Fig. 2.8C). Mutants IE0∆2-32 and IE0∆2-43 showed similar protein level as full length IE0, whereas the rest of the deletion mutants appeared to express at a lower level compared with IE0. The reduced transactivation activity with p39K-CAT from deletion mutants could in part result from the reduced protein levels. The presence of hr5 allowed full activation of p39KCAT even with variable levels of transactivator which would agree with the previous 75  results of Choi and Guarino (1995b) which showed that the enhancer allows full expression even when levels of transactivator are limiting. Overall the transactivation analysis with the IE0 deletions did not definitively identify any region or conserved residue that represent the relevant function of the additional 54 aa at the N-terminus of IE0.  The transcription analysis results suggest that the primary role of IE0 may not be in transcription but in replication of viral DNA. To address this possibility two viruses were constructed that contained deletions within the IE0 N-terminus, ie1KO-IE02-43 and ie1KO-IE039-54. Viruses expressing only IE0, IE02-43, IE039-54 or IE1 were used to infect Sf9 cells and total DNA was collected at various times post infection and quantified by Slot blot analysis (Fig. 2.9). Both deletion viruses had reduced viral DNA replication relative to either IE0 or IE1 expressing viruses up to 36 hpi. From 48 hpi to 96 hpi ie1KOIE02-43 viral DNA levels were equivalent to IE0 and IE1 whereas ie1KO-IE039-54 had lower levels at all times. It may be that deletions within the 54 aa affect replication via an unidentified transcriptional effect on a cellular or viral protein.  The analysis of viral DNA replication indicated that mutation of the IE0 N-terminus can impact viral DNA replication. IE0 only virus has previously been shown to have reduced viral DNA replication in the first 48 hours post-infection whereas IE1 only virus has WT levels (Stewart et al., 2005). This analysis showed lower levels of DNA replication from IE0 only virus than IE1 only virus by 48 hpi, therefore agrees with the previous results and also showed that mutation of the IE0 N-terminus results in even lower levels of viral DNA replication. In the case of ie1KO-IE039-54 viral DNA replication never reach levels equivalent to IE1 or even IE0 (Fig. 2.9).  2.2.6 Expression of IE0 or IE1 changes numbers of nucleocapsids per ODV envelope Viruses that express only IE0 or only IE1 produce lower levels of occlusion bodies than WT virus. The occlusion bodies however, appear similar to WT in size when examined by light microscopy (Stewart et al., 2005). It is not clear if the packaging of ODV is affected by the expression of only IE0 or IE1 during the viral life cycle. To examine this question 76  and determine if there are differences, transmission electron microscopy (TEM) was performed on OBs produced by viruses expressing only IE1 or IE0 and compared to WT OBs from AcBac. Analysis of the ODV showed that the virions and nucleocapsid structure were the same among the viruses (Fig. 2.10 A). However, there appeared to be a difference in the number of nucleocapsids per ODV. The numbers of nucleocapsids per ODV envelope therefore were counted for each virus from 21 to 26 cross-sections of OBs and the percentage of each type of ODV was determined (Fig. 2.10B). For WT OBs, about 33% of the ODVs contain a single nucleocapsid. The overall distribution of nucleocapsids per envelope ranges from 1 to 11. For OBs produced by the virus expressing only IE1, 65% of the virions contained a single nucleocapsid and the distribution ranges from 1 to 5 nucleocapsids per envelope. For OBs produced by the virus expressing only IE0, 94% of their ODV containing only a single nucleocapsid and a few virions were seen that contained two nucleocapsids. This result clearly shows that the expression of IE0 and IE1 has a dramatic impact on the number of nucleocapsids per ODV. In addition, it shows that IE1 appears to be essential to obtain multiple nucleocapsids per ODV but IE0 also needs to be present to achieve high levels of ODVs with greater than 5 nucleocapsids per envelope. This suggests that the ratio of IE0 to IE1 may determine the numbers of nucleocapsids per ODV envelope.  2.3 Discussion IE0 is the only baculovirus protein known to be expressed exclusively from a spliced mRNA. Even though the ie0 was first identified in 1988 (Chisholm and Henner, 1988) there have only been a few studies conducted to address the function of IE0 compared to the extensively studied nonspliced IE1. This study aims to provide a further understanding of IE0 in comparison of IE1.  Analysis of the localization of IE0 in the absence of IE1 by confocal microscopy showed the protein distributed to discrete foci within the nucleus of infected cells at early times post infection which expanded so that by late times it encompases the whole virogenic stroma (Fig. 2.1C). Similar results were observed with IE1 (Fig. 2.1B). Co-expression of  77  fluorescently tagged IE0 and IE1 showed that IE0 and IE1 colocalized during infection (Fig. 2.1D). This result indicates that IE0 and IE1 have the same cellular distribution which is independent of the other protein. These foci are similar to BmNPV IE1 which was shown to locate within the viral replication factories of the virogenic stroma (Kawasaki et al., 2004; Okano et al., 1999). Therefore, IE0 also appears to locate in the virogenic stroma and replication factories. This would be expected as viruses expressing IE0 can support viral DNA replication and activate gene expression (Stewart et al., 2005).  IE0 and IE1 play pivotal roles in regulating viral gene expression, but there are very limited reports on the comparison of IE0 and IE1 transactivation ability in AcMNPV. Therefore investigations on the ability of the proteins to transactivate viral genes were investigated. Two approaches were taken to examine whether IE0 and IE1 differentially regulate viral genes: 1) transient transactivation analyses of viral early promoter candidates and 2) genome-wide analyses of viral genome expression by by microarray. However neither transient assays nor microarray analysis revealed any viral genes specifically regulated by IE0 or IE1. Nevertheless, these analyses were the first studies to directly compare IE0 and IE1 activation ability within a broad range of potential targets in the absence or presence of an hr enhancer. The results obtained have provided valuable information on IE0 and IE1 transcriptional transregulation including showing that in the absence of hr5, IE0 can also be as strong an activator as IE1 on the early 39K promoter when equivalent amounts of IE0 and IE1 are expressed (Fig. 2.3D). This result is in contrast to the results of Kovacs et al. (1991) who had reported that IE0 only activated 39K in an hr-dependent way. The reason for these differences is unknown, but it could be due to the fact that the previous study used an ie0 construct that was able and likely to express both IE0 and IE1 due to internal translation initiation, resulting in potentially antagonistic effects (Theilmann et al., 2001). In addition, different methods used in this study and that of Kovacs et al. (1991) may also account for the differences observed. In the previous study thin-layer chromatography CAT assays were used which is less sensitive than the method in this study; therefore, it is possible that low levels of transactivation could not be detected.  78  Unlike for the 39K promoter, this study showed no transactivation of the ie0 promoter by IE0 (Fig. 2.3B) which agrees with Kovacs et al. (1991). It does however contrast with the results obtained with the homologous genes from OpMNPV where it was shown that OpMNPV IE0 strongly up regulates its own promoter. Similarly, AcMNPV IE1 has been shown to transactivate the OpMNPV gp64 promoter (Slack and Blissard, 1997), yet our results show that AcMNPV IE0 or IE1 does not activate the AcMNPV gp64 promoter (Fig. 2.3B). This clearly shows that caution must be taken when functionally comparing homologous genes from different species of virus.  Leisy et al. (1997) previously showed that AcMNPV ie2 is negatively regulated by AcMNPV IE1 due to binding of IE1 near the transcriptional start site. These sequences are present in the constructs used in this study but surprisingly no repression of AcMNPV ie2 by AcMNPV IE1 or IE0 was observed (Fig. 2.3B). The two assays used varying amounts of plasmid expressing IE1, and in addition the reporter constructs used different lengths of promoter which may account for differences observed. In the homologous OpMNPV system no activation or repression of ie2 by IE1 or IE0 has been observed (Theilmann et al., 2001).  The differences in regulation by IE0 or IE1 of the same viral genes observed in this study compared to previously reported results could also be due to the length of promoter used in reporter gene constructs. Pullen and Friesen (1995) showed that using reporter constructs with 546 or 161 bp upstream of the ie1 ATG resulted in 8 and 4 fold activation by IE1 respectively. These results again show ie1 promoter length can affect IE1 transactivation. Similarly the promoter of ie1 reporter plasmid in the study of Kovacs et al. (1991) had 600 bp upstream of the ATG start codon of ie1 and was also activated by IE1. In this study 350 bp of the ie1 promoter was used however no activation was seen (Fig. 2.3B) which is surprising considering Pullen and Friesen (1995) achieved activation with promoter of 161 bp.  In the presence of a cis-linked hr5 element, IE0 activation was significantly augmented for some of the viral genes. However, the hr5 element did not always act as an enhancer when cis-linked to viral promoters. IE0 and IE1 showed transactivation of the me53 promoter 79  lacking hr5 (Fig. 2.3B), yet repression instead of activation was observed with the presence of enhancer (Fig. 2.4A). A similar negative effect of hr5 on the expression of ie1was also observed (Fig. 2.4). The negative regulation of immediate early genes by AcMNPV hr enhancer elements has not been previously reported. However whether or not the negative regulation of hr5 on the expression of ie1 or me53 occurs in the viral context is not known.  As revealed by microarray analyses, IE0 transactivated to detectable levels only a subgroup of the genes shown by the same analysis to be regulated by IE1 and down regulated some viral genes (ac18, ac33, ac91, ie2 and vp39). No genes were identified to be down regulated by IE1 using microarray analysis (table 2.1). The transient assay results in general agreed with the microarray results. However one promoter, ac18, which was shown to be down regulated by IE0 in microarrays, was activated by both IE0 and IE1 when examined by transient assays (Fig. 2.5). This result suggests that a viral factor associated with the BV may negatively affect IE0 and IE1 transactivation of genes upon infection.  Transient assays and microarray analysis did not identify any viral genes specifically regulated by IE0 or IE1. The levels of IE0 and IE1 expression and the presence or absence of enhancer elements however can have a significant impact on level of gene expression. Potentially IE0 and IE1 could induce subtle differences in the overall gene expression when expressed at different ratios throughout the viral life cycle.  IE0 differs from IE1 by the additional 54 aa at the N-terminus and deletion within the region reduces the activation ability of IE0 on the 39K but not me53 promoter. However in the presence of the hr5 enhancer all IE0 mutants activated the promoter of 39K to similar levels as IE1. In general therefore, mutation of the IE0 N-terminus does not appear to affect the transcriptional activation of viral genes to a significant degree compared to IE1. However, when viral DNA replication was examined using viruses expressing IE0 mutants viral DNA levels were reduced relative to both IE0 and IE1. These analyses therefore suggest that the biggest impact of IE0 maybe in reducing or delaying viral DNA replication at the early times post-infection. This may indicate that the IE0 N-terminal 54 80  aa could function as a domain to specifically interact with viral or host genes altering the replication process, causing different interactions within replication complexes. Alternatively, IE0 dimers may be less efficient as origin binding proteins compared to IE1 dimers with the addition of the N-terminal 54 aa, and thus have reduced ability to support viral DNA replication. If this is the case, the reduced DNA replication levels observed in viruses expressing IE0 mutants carrying deletions within the 54 aa may suggest that the IE0 deletion mutants form dimers or oligomers that may even have lower binding capacity to the replication compared to either IE0 or IE1.  OBs produced by viruses expressing only IE0 or IE1 appear to be indistinguishable compared to WT when viewed by light microscopy (Stewart et al., 2005). However, in this study additional analysis by TEM revealed differences in the ODV contained with the OBs. Viruses expressing only IE0 produced ODVs which predominantly contained only a single nucleocapsid. Virus expressing only IE1 also showed reduced numbers of nucleocapsids per envelope. The viral protein ac23 F-protein homolog has also been shown to affect the number of nucleocapsids per envelope. Deletion of ac23 results in increased number of ODVs with fewer nucleocapsids (Yu et al., 2009), although the mechanism behind it is not clear. It will be interesting to determine if deletion of ac23 results in a changed ratio of IE0 relative to IE1 or if the expression of AC23 is reduced in the absence of IE0 and IE1. Nevertheless, given the fact that an IE0 and IE1 ratio change could potentially alter the expression level of viral genes on a large scale the observed result could be direct or indirect result from the overall expression of viral genes.  In summary, this study shows AcMNPV IE0 and IE1 have no difference in subcellular localization during infection. IE0 can transactivate a variety of viral genes in both hrdependent and non-hr dependent manner, but no viral gene was identified to be specifically regulated by IE0 by microarray and transient assay analyses. This study also indicated that IE0 can be as strong a transactivator as IE1 and the contribution of Nterminal 54 aa to IE0 properties may be to a large degree an effect on viral DNA replication instead of transcriptional regulation. IE0 and IE1 expression may also be a determining factor of the number of nucleocapsids embedded per ODV envelope.  81  2.4 Materials and methods 2.4.1 Cells and viruses Spodoptera frugiperda clone 9 (Sf9) cells were maintained at 27°C in TC100 medium supplemented with 10% fetal bovine serum. AcMNPV recombinants were derived from bacmid bMON14272 (Invitrogen Life Technologies) in Escherichia coli DH10B cells as described previously (Datsenko and Wanner, 2000; Luckow et al., 1993).  2.4.2 Plasmids construction 2.4.2.1 Construction of pFAcT-AcIE1EYFP, pFAcT-AcIE0ECFP and pFAcTAcIE0ECFP-AcIE1EYFP. To construct pAcIE1-EYFP a fragment containing the AcMNPV ie1 promoter and orf were amplified from AcMNPV E2 using primers 284 and 573, treated with T4 polynucleotide kinase followed by digestion with NcoI and cloned into plasmid backbone (pBS+, Stratagene) containing the pe38 polyadenylation sequence (provided by Minggang Fang) using standard methods. The eyfp and ecfp ORF was PCR amplified with primers 852/924 using pEYFP and pECFP (Clontech) as the templates, respectively. The amplified fragment of eyfp was cloned into 3’ end of the ie1 ORF at NcoI/NotI sites generating an in frame fusion of IE1 with EYFP generating pAcIE1-EYFP. The pAcIE0-ECFP was constructed by the same method except the ie0 promoter and ORF was amplified from pAcie0 (Huijskens et al, 2004) with primers 432 and 573, digested with PstI and NcoI, then ligated into the PstI/NcoI sites of the pBS+ backbone containing pe38 polyA plasmid. To construct the bacmid transfer vectors the fusion genes ie0ecfp was PCR amplified with primers 921/922 using pAcIE0-ECFP as the template and cloned into XhoI/PstI sites of pFAcT (constructed by Taryn Stewart) producing pFAcT-AcIE0ECFP. Ie1yfp was amplified with primers 683/922 using pAcIE1-EYFP as the template and cloned into pFAcT-AcIE0CFP at XhoI site, producing pFAcT-AcIE0CYFP-AcIE1EYFP. The vector pFAcT-AcIE1EYFP was constructed by linearizing pFAcT-AcIE1EYFPAcIE0ECFP with SstI and partial digestion with NotI to drop out the IE0ECFP cassette followed by religation.  82  2.4.2.2 Construction of pFAcT-AcIE1HA, pFAcT-AcIE0FLAG and pFAcTAcIE1HA-AcIE0FLAG. The vector pFAcT-AcIE1HA expressing IE1 tagged with HA (CYPYDVPDYASL) at its C-terminal was constructed by amplifying AcMNPV ie1 with the primer pairs 740/860 using pFAcT-AcIE1 (Stewart et al., 2005) as the template. The amplified fragment was cloned into pFAcT at the XbaI/NotI sites. pFAcT-GFPAcIE0FLAG expressing IE0 C-terminal tagged with FLAG (DYKDDDDK) was made by amplifying the ie0 promoter and ORF with 432/861 using pAcie0 as the template, and cloned into the PstI/XbaI sites of pFAcT-GFP-Tnie1pA (Nie et al., 2009). The AcIE0FLAG cassette was further subcloned into pFAcT at PstI/SstI sites to generate pFAcT-AcIE0FLAG. To make pFAcT-AcIE1HA-AcIE0FLAG the ie1HA cassette from pFAcT-AcIE1HA was isolated by a PstI digest and cloned into pFAcT-AcIE0FLAG.  2.4.2.3 Construction of CAT reporter plasmids. To enable the comparison of transactivation activity between IE0 and IE1 or IE0 deletion mutants, promoters of candidate early genes or genes regulated by IE0 as determined by microarray analysis, were amplified using the primers shown in Table 2.1. The promoters were cloned into pBS-CAT, containing a multiple cloning site upstream of the CAT ORF and the SV40 polyadenylation signal sequence (constructed by Xiaojiang Dai), generating pprm-CAT. Each promoter was also cloned into into phr5-CAT which has AcMNPV hr5 inserted upstream of the multiple cloning sites of pprm-CAT (Fig. 2.3).  2.4.2.4 Construction of IE0 deletion mutants. To identify residues within the N-terminal 54 aa of IE0 that are critical to IE0 properties, IE0∆2-11, ∆2-22, ∆2-32, ∆2-38, ∆2-43, ∆39-54, ∆28-54, and IE0∆19-54 deletion mutants were made by inverse PCR using pAcie0delta (Huijskens et al., 2004) as a template. Primers used were 1444/1447 for IE0∆2-11, 1444/1446 for IE0∆2-22, 1444/1445 for IE0∆2-32, 1444/1170 for IE0∆2-38, 1170/1171 for IE0∆2-43, 1173/1172 for IE0∆39-54, 1451/1173 for IE0∆28-54, 1448/1173 for IE0∆19-54. Primers 432/338 were used to amplify the IE0 mutants IE0∆2-11, ∆2-22, ∆2-32, ∆2-38, ∆28-54, and IE0∆19-54 for cloning into pFAcT-GFP vector (Dai et al., 2004) at PstI/SstI sites, producing pFAcT-GFP-IE0∆2-11, -IE0∆2-22, -IE0∆2-32, -IE0∆243, -IE0∆2-38, -IE0∆39-54, -IE0∆28-54, and IE0∆19-54. All IE0 constructs contain the 83  amino acid 55 (Met to Ala) mutation to prevent internal translation initiation and expression of IE1 (Huijskens et al., 2004). Primers used in this study are listed in Table 2.2.  2.4.3 Virus construction All recombinant viruses expressing IE0 or IE1 used in this study were generated from ie1KO (Stewart et al., 2005) in Escherichia coli DH10B as described previously (Luckow et al., 1993). Transfer vectors pFAcT-AcIE1YFP, pFAcT-AcIE0CFP and pFAcTAcE1YFP-AcIE0CFP were used to make the viruses ie1KO-IE1EYFP, ie1KO-IE0ECFP, ie1KO-IE0ECFP-IE1EYFP respectively; pFAcT-AcIE1HA, pFAcT-AcIE0FLAG and pFAcT-AcIE1HA-AcIE0FLAG were used to produce ie1KO-IE1HA, ie1KO-IE0FLAG, ie1KO-IE1HA-IE0FLAG. Vectors pFAcT-GFP-IE02-11, -IE02-22, -IE02-32, -IE0243, -IE02-38, -IE039-54, -IE028-54, and -IE019-54 were used to generate ie1KOIE02-11, -IE02-22, -IE02-32, -IE02-43, -IE02-38, -IE039-54, -IE028-54, and IE019-54 respectively. AcBac was made by transposing pFAcT-GFP into bMON14272 (Invitrogen Life Technologies) (Dai et al., 2004).  2.4.4 Confocal microscopy Sf9 cells (5x105/35-mm-diameter confocal dish) were infected by ie1KO-IE1EYFP, ie1KO-IE0ECFP and ie1KO-IE0ECFP-IE1EYFP respectively, or infected by ie1KOIE1HA, ie1KO-IE0FLAG and mock infected respectively at multiplicity of infection of 5. For the viruses expressing IE1EYFP, IE0ECFP or both IE1EYFP and IE0ECFP, infected cells were fixed at various times with 3.5% paraformaldehyde, stained with 200 ng/ml DAPI prior to the observation using a Leica confocal microscope. For the immunofluorescence detection, fixed Sf9 cells were permeabilized with 0.2% Triton X100 for 20 min after three washes in PBS, followed by 1 hr incubation in blocking buffer (2% BSA in PBS), and 1 hr incubation with anti-IE1 (1:1000) (Ross and Guarino, 1997) in blocking buffer. Bound antibody was visualized by Alexa 635 conjugated anti-IgG (Molecular probe) (1:500) and nuclei were visualized by 200 ng/ml DAPI staining prior to the confocal microscopy analysis.  84  2.4.5 CAT assay Sf9 cells (1x106 cells/35-mm-diameter well of 6-well plates) were co-transfected in duplicate with 0.5µg of each CAT reporter plasmid and 0.1µg of IE1, IE0, or IE0M to A expressing plasmids or control plasmid pBS+. Transfected cells were collected at 48 hpt and lysed for CAT assay based on the method described by Neumann et al. (1987). Briefly, transfected Sf9 cells were scraped off with rubber policemen and pelleted by centrifugation at 956 x g for 5 min, and then resuspended in 100 µl 0.25 M Tris∙Cl (pH 7.8). To lyse the cells, each sample was freeze-thawed three times (−80°C for 5 min and 37 °C until just thaw). Cell debris was pelleted for 2 min at 10000 x g and cell extracts were incubated at 65 °C water bath for 15 min to inactivate cellular deacetylases. Lysates were then used for diffusion CAT-assay with 3H-Acetyl-CoA. For each assay, cell lysates brought up to 25 ul with 10µl 0.25 M Tris∙Cl (pH 7.8) was added to 100 µl reaction buffer containing 5 mM chloramphenicol, 210 mM Tris∙Cl (pH 7.8), 125 µM acetyl-coenzyme A (Sigma), 0.025 µCi [3H]acetyl-coenzyme A (New England Nuclear, CAT Assay Grade). Each sample was overlaid with 3 ml of toluene-based scintillation fluor (Econofluor-2; Packard BioScience Co.) and the enzymatic reaction was measured using the scintillation counter (Beckman; LS 6500). Cell extracts were was titered to determine the appropriate amount to use to obtain a linear response in the assay.  2.4.6 Microarray Sf9 cells were transfected by 2 µg plasmid expressing IE0 or IE1 and GFP for 106 cells with transfection buffer (calcium phosphate method). A GFP-expressing plasmid was also transfected and used as the control. Only when the transfection efficiency was greater than 75%, did the microarray analysis proceed. After incubation for 24 hours to allow protein expression from the plasmids, cells were treated with 100 µg/ml of cyclohexamide for 2 hours to stop de novo synthesis of protein, and then infected by WT AcMNPV at MOI of 5 in the presence of cyclohexamide. At 24 hpi under the CHX treated condition, cells were harvested for mRNA isolation and microarray analysis. The AcMNPV microarray contains 12 replicates of 289 individual oligos corresponding to all predicted AcMNPV genes, 85  representative cellular Sf9 and Trichoplusia ni genes, and oligos corresponding to spike RNAs. The oligos were designed with a bias to the 3’ end of each ORF but without utilizing regions which overlapped known 5’ or 3’ UTRs. Except in some cases, each oligo printed on the array was a 50 mer. To make probes, cDNA was synthesized with aminoallyl-dUTP. Synthetic RNA was added for normalization. The cDNAs were reacted with N-hydroxysuccinimide (NHS) activated cy3 or cy5. Two independent experiments with reciprocal dye were performed for ie1 transfection and one experiment with reciprocal dye was performed for ie0 transfection. After hybridization and acquisition of the images, signal intensities were normalized according to those of the spikes.  2.4.7 DNA quantification by Slot blot To compare levels of viral DNA synthesis Sf9 cells (2x106cells/35-mm-diameter well of 6well plates) were infected by ie1KO-IE0, ie1KO-IE0∆2-43, ie1KO-IE0∆39-54 and ie1KOIE1 at MOI of 5, respectively. The infected cells were washed once with PBS and collected at various time post infection. Cells were scraped off with rubber policemen and pelleted by centrifugation at 956 x g for 5 min and then resuspended in with 1 ml freshly made 0.4N NaOH, 0.125mM EDTA followed by incubation at 95°C for 10 min to denature the DNA. 20 µl of each sample was loaded onto a Schleicher & Schuell slot blot apparatus in duplicate and blotted under vacuum onto a Hybond-N nylon membrane (Amersham). The Slot blot was hybridized to an α-32P-dCTP labelled vp39 fragment of AcMNPV DNA by standard methods (Sambrook et al., 1989). The blot was visualized by exposure to Perkin Elmer Multisensitive Phosphorscreens, which was scanned using a Cyclone PhosphorImager (Perkin Elmer) and analyzed with Optiquant Acquisition and Analysis Software V5.0 (Perkin Elmer).  2.4.8 Transmission electron microscopy Sf9 cells (2.0 × 106 cells/ml) in 50 ml spinner flasks were infected with ie1KO-IE1, ie1KO-IE0 and AcBac control at multiplicity of infection of 0.5, respectively. At 6 days post infection cells were collected for occlusion body purification following protocol described previously (Gombart et al., 1989). Purified OBs were then immobilized with 50 86  µl of 3% low-melting-point agarose and placed on ice for 30 min. The agarose fraction was removed and OB pellets were fixed in 2.5% glutaradehyde in PBS for 1 h, followed by staining with 2% uranyl acetate for 1 h. After dehydration through a series of ethanol washes from 30% to 100%, OB pellets were embedded in Spur resin (TED Pella, INC.). Ultra thin sections were obtained and subsequently stained with 1% uranium acetate and Sato's lead (Takagi et al., 1990). Images were obtained using a Hitachi transmission electron microscope.  2.4.9 Western blotting To detect the protein levels of IE0 and IE1 in Sf9 cells transfected for CAT assay, total cells transfected were mixed with 4×SDS protein sample buffer (PSB, 0.25 M Tris-Cl, pH6.8, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.02% bromophenol blue) and boiled for 10 min. Protein samples were separated by 7.5% SDS-PAGE and transferred to Millipore Immobilon-P membrane with Bio-Rad Mini-Protean II and liquid transfer apparatus. Membranes were probed with Mouse monoclonal IE1 antibody 1: 10,000 (Ross et al., 1997). To detect bound primary antibodies membranes were incubated with 1:10000 goat anti-mouse peroxidase-conjugated secondary antibodies. Enhance Chemiluminescence System (ECL, Perkin Elmer) was used to detect the signals.  87  Figure 2. 1 Confocal microscopy analysis of IE1 and IE0 localization. (A) Schematic diagrams showing the recombinant viral constructs that express the fusion proteins IE1-EYFP or IE0-ECFP used for the confocal analysis. The epitope tags HA or FLAG were inserted at the C-terminal of IE1 and IE0 resepctively for indirect immmunofluorescence. Confocal microscopy of Sf9 cells infected by (B) ie1KO-IE1EYFP, (C) ie1KO-IE0ECFP and (D) ie1KO-IE0ECFP-IE1EYFP at an MOI of 10, respectively. Cells were fixed with 3.5% paraformaldehyde at 4 or 6, 12 and 24 hpi, stained with 200 ng/ml DAPI and then observed with a Leica confocal microscope. IE1 distribution was shown by yellow and IE0 distribution was shown by cyan. Colocalization of IE0 and IE1 appeared as white in the merged panel.  88  Figure 2.1 continued EM7 prm  SV40 pA Zeocin  A.  ie1 locus  IE1/0 Knock-Out polh locus  ie1KO-IE1EYFP  polh  IE1  EYFP  ie1KO-IE0ECFP  polh  IE0  ECFP  ie1KO-IE0ECFP-IE1EYFP ie1KO-IE1HA  polh  IE0  polh  ECFP  EYFP  IE1  IE1 HA  ie1KO-IEOFLAG  polh  IE0 Flag  ie1KO-IEOFLAG-IE1HA  polh  IE0  IE1 Flag HA  IE1EYFP  DAPI  merge  4 hpi  12 hpi  24 hpi  89  Figure 2.1 continued C.  IE0ECFP  DAPI  merge  4 hpi  12 hpi  24 hpi  D.  IE1EYFP  IE0ECFP  DAPI  merge  4 hpi  12 hpi  24 hpi  90  Figure 2. 2 Immunofluorescence confocal microscopy analysis of (A) IE1 and (B) IE0 localization. Sf9 cells were infected by ie1KO-IE1HA and ie1KO-IE0Flag at MOI of 10 and fixed with 3.5% paraformaldehyde at 6, 12 and 24 hpi for immunostaining. IE0 and IE1 were visualized by 1 hr incubation with anti-IE1 (1:1000) followed by 1 hr incubation with Alexa 635 conjugated anti-IgG (1:500). Nuclei were visualized by staining with 200 ng/ml DAPI. Microscopy was performed with a Leica confocal microscope.  91  Figure 2.2 continued A.  DAPI  Alexa 635  merge  6 hpi  12 hpi  24 hpi  B.  6 hpi  12 hpi  24 hpi  92  Figure 2. 3 CAT assays comparing the transactivation ability of IE0 and IE1. (A) Schematic diagram of CAT reporter constructs pprm-CAT and the enhancer containing construct phr5-prm-CAT. Sequence shows example of how each promoter was cloned into the CAT expression vectors. Promoters of each candidate genes were amplified from the translational start site to 350 bp upstream. The fragments were then cloned into pBS-CAT or phr5-CAT to drive the expression of cat. Prm stands for all the viral promoters analyzed using CAT assays, including ie1, ie0, ie2, 39K, p35, gp64, me53, ac18, ac33, ac52, ac76, ac79, ac91, ac111, lef-3, lef-4 and lef-6. (B) Transactivation analysis of selected AcMNPV early promoters by IE0 and IE1. For each transfection, 0.5 µg of each reporter constructs was co-transfected into Sf9 cells with 0.1 µg pAcIE1, pAcIE0 or pBS+ in duplicate. Cells were collected and lysed for diffusion CAT-assay with 3H-Acetyl-CoA at 48 hpt. (C) Western blot analysis of the relative levels of IE0 and IE1 in the transfected cells. Transfected cells were collected and subjected to 7.5% SDS-PAGE followed by Western blot analysis using a mouse monoclonal antibody to detect IE1. (D) Impact of varying levels of IE0 or IE1 on the expression of p39K-CAT. Sf9 cells were co-transfected with 0.5 µg of p39K-CAT and 0, 0.1, 0.5, 1, 2 and 3 µg pAcIE1 or pAcIE0 in duplicate respectively. Total DNA transfected was equalized by supplementing with pBS+. Cells were collected and lysed for diffusion CAT-assay (see Materials and Methods) at 48 hpt. (E) The relative levels of IE0 and IE1 expressed were detected by Western blot using a mouse monoclonal antibody against IE1. Experiment was repeated twice in duplicate.. Error bars indicate standard errors. A. XhoI XbaI HindIII  CAT SV40 polyA  pBS-CAT XbaI XhoI pprm-CAT HindIII XbaI XhoI phr5-prm-CAT hr5  promoter  350 bp  XhoI  pie1-CAT GTAT· · · · · · TATA · · · · · · CAGT· · · · · · TCTCGAGATG CAT start codon ie1 locus GTAT· · · · · · TATA · · · · · · CAGT· · · · · · TATG ie1 start codon  93  Figure 2.3 continued  B. 350 IE1 IE0  300 CAT activity (CPM)  reporter 250 200 150 100 50 0 ie1  ie0  39k  gp64  me53  p35  ie2  vector  C. 1  IE  0  IE  m  k oc  94  Figure 2.3 continued  CAT activity (CPM)  D. 20 18 16 14 12 10 8 6 4 2 0  IE1 IE0  0  0.1  0.5  1  2  3  plasmid expressing IE0 or IE1 (µg)  . E. pAc-IE1 0  0.1 0.5  pAc-IE0 1  2  3 mock 0  0.1  0.5  1  2  3 µg  95  Figure 2. 4 CAT assays comparing the transactivation ability of IE0 and IE1 on viral early promoters cis-linked to hr5 element. (A) Sf9 cells were co-transfected with 0.5 µg of each hr5 containing CAT reporter plasmids with 0.1 µg of pAcIE1, pAcIE0 or pBS+ in duplicate, respectively. Cells were collected at 48 hpt for (A) diffusion CAT-assay at 48 hpt and (B) IE0 and IE1 expression as detected by Western blot using a mouse monoclonal antibody against IE1. Experiment was repeated twice in duplicate. Error bars indicate standard errors.  A.  CAT activity (CPM)  250  IE1 IE0 reporter  200 150 100 50 0 ie1  ie0  39K  gp64  me53  p35  ie2  vector  hr -CAT reporters  B. 1 IE  0 IE  m  k oc  96  Figure 2. 5 Transactivation analysis of AcMNPV promoters identified by microarray and regulated differentially by IE0 and IE1. Sf9 cells were co-transfected with 0.5 µg of each CAT reporters (A) without or (C) with the cis-linked hr5 enhancer and 0.1µg pAcIE1 or pAcIE0 in duplicate. Cells were collected and lysed at 48 hpt for diffusion CAT-assay with 3H-Acetyl-CoA at 48 hpt. The relative levels of IE0 and IE1 were detected by Western blot using a mouse monoclonal antibody against IE1 (B and D). “-” stands for down regulation by IE0 in the microarray analysis and “+” stands for upregulation by IE0 in the microarray analysis. One repetition was performed. Error bars indicate standard error.  A.  CAT activity (CPM)  300  IE1 IE0 reporter  250 200 150 100 50 0 18 ac  33 ac  -  91 ac  52 ac  f6 79 111 le ac ac CAT reporters  76 ac  +  f4 le  f3 le  or ct e v  microarry  B. 1  IE  0 IE  m  k oc  97  Figure 2.5 continued  CAT activity (CPM)  C. 180 160 140 120 100 80 60 40 20 0  IE1 IE0 reporter  18 ac  33 ac  -  91 ac  52 ac  76 ac  1 79 11 ac ac  f6 le  hr -CAT reporters  +  f4 le  f3 le  or ct ve  microarry  D. 1 IE  0 IE  m  k oc  98  Figure 2. 6 Alignment of IE0 N-terminus specific amino acids from (A) alphabaculoviruses and (B) Group I alphabaculoviruses. The alignment of the amiono acids specific to IE0 at the N-terminus relative to IE1 was performed using Vector NTI using the ClustalW algorithm (Thompson et al., 1994). The different shadings of the amino acids represent the following: black background show 100% conservation, dark grey represents 80% and light grey 60% respectively. Numbers on the right stands for the amino acid position of each individual species. A. AcMNPV BmNPV RoMNPV OpMNPV CfMNPV CfDefNPV AdhoNPV EcobNPV HzNPV LdMNPV MacoNPV-A MacoNPV-B TnSNPV HearNPV SpltNPV  : : : : : : : : : : : : : : :  ----MIRTSSHVLNVQENIMTSNCASSPYSCEATSACAEAQQLQV ----MIRTSSHVLNVQENIMTSNCASSPYSCEATSACAEAQQLQV ----MIRTSNHVLNVQENIMTSNCASSPYSCEATSACAEAQQLQL -------------------MIKGTHWPNLVSKSYTDACETSKLQV -------------------MIKGTYWPNVVSESYIESCEINKLQV -------------------MIKGSYWPNVLDRS--DSCEANKLQV --------------MNFTVSSEHMGEEEYGFQSNVMNNYINEFQI -------------MTSSSSFILNVDDNNLKWPESSWNAEYFELVN MSGTLKRILYDDISDDSDQAKLFRYNSEMQPPASQQMNTAVDYEI -----------MDQTSSSTQILVSLMENAQPDPTAGQPVAQLIVG ----------------------MQKYNNELSTQSARSHVVN-------------------------MQKYNNELSIQSARSHVVN-------MNTFDYQNDLYKRIAMESAAVASILLSPRISDDGNSNNLNS MSGTLKRILYDDISDDSDQAKLFRYNSEMQPPASQQMNTAVDYEI --MILHQSDNEINKPLSQVFKHLREMGSMYDDTEMAAIYAAATTT  AcMNPV BmNPV RoMNPV OpMNPV CfMNPV CfDefNPV AdhoNPV EcobNPV HzNPV LdMNPV MacoNPV-A MacoNPV-B TnSNPV HearNPV SpltNPV  : : : : : : : : : : : : : : :  DTGGDKIVNNQVT------------------DIGGDTIVNNQTT------------------DTGGDKIVNNQVT------------------GCGARSTFA----------------------DRDARSTS-----------------------GSHTRKQRLPT--------------------TIAGHDSSPLIN-------------------QNVCVNNK-----------------------DVENLLKLNFTAS------------------GNAEWQVDSL-----------------------------------------------------------------------------------FLNMRNDNNNNNNNGEKDSTDFETELSLTSVV DVENLLKLNFTAS------------------ATDEKCNKNDSPNSKCEEVEEI----------  : : : : : : : : : : : : : : :  : : : : : : : : : : : : : : :  41 41 41 26 26 24 31 32 45 34 19 19 41 45 43  54 54 54 35 34 35 43 40 58 44 73 58 65  B. AcMNPV RoMNPV BmNPV OpMNPV CfMNPV CfDEFNPV  : : : : : :  MIRTSSHVLNVQENIMTSNCASSPYSCEATSACAEAQQLQVDTGGDKIVNNQVT MIRTSNHVLNVQENIMTSNCASSPYSCEATSACAEAQQLQLDTGGDKIVNNQVT MIRTSSHVLNVQENIMTSNCASSPYSCEATSACAEAQQLQVDIGGDTIVNNQTT MIKGT----------HWPNLVSKSY----TDAC-ETSKLQVGCGARS--T--FA MIKGT----------YWPNVVSESY----IESC-EINKLQVDRDARS--T---S MIKGS----------YWPNVLDRS------DSC-EANKLQVGSHTRK--QRLPT  : : : : : :  54 54 54 35 34 35  99  Figure 2. 7 Location of the IE0 N-terminal deletions. The IE0 N-terminal 54 amino acids are shown at the top in yellow and the conserved amino acids are indicated as described in Fig. 2.6. Yellow bars indicate the region retained in each deletion.  IE0 constructs  N-  -C  N-terminal 54aa IE0 IE0 ∆2-11 IE0 ∆2-22 IE0 ∆2-32 IE0 ∆2-43 IE0 ∆39-54 IE0 ∆28-54  100  Figure 2. 8 Transactivation analyses of IE0 deletion mutants on (A) 39K and (B) me53 promoters with or without the cis-linked hr5 enhancer. Sf9 cells were co-transfected with 0.5 µg of p39K-CAT, phr5-39K, pme53-CAT or phr5me53 and 0.1 µg of pAcIE0, pAcIE02-11, -2-22, -2-32, -2-43, -28-54, -39-54 or pAcIE1 in duplicate, respectively. (C) Cells were collected at 48 hpt and lysed for the diffusion CAT-assay at 48 hpt. The relative levels of IE0 or IE1 was determined by Western blot analysis using a mouse monoclonal antibody against IE1. Error bars represent standard errors.  A. 400 350 reporter  CAT activity (CPM)  300  IE0 IE0∆2-11  250  IE0∆2-22 200  IE0∆2-32 IE0∆2-43  150  IE0∆28-54 IE0∆39-54  100  IE1  50 0 p39k-CAT  phr5-39k-CAT  101  Figure 2.8 continued  B. 250  200  CAT activity (CPM)  reporter IE0 IE0∆2-11  150  IE0∆2-22 IE0∆2-32 IE0∆2-43 100  IE0∆28-54 IE0∆39-54 IE1  50  0 pme53-CAT  C. IE1  IE0  phr5-me53-CAT  4 4 2 9-5 -32 -43 8-5 -1 1 2-2 3 2 2 2 2 ∆ ∆ ∆ ∆ ∆ 0 0∆ IE0 IE IE0 IE0 IE0 IE  102  Figure 2. 9 Slot blot analysis of viral DNA replication. Sf9 cells were infected by ie1KO-IE0, ie1KO-IE0∆2-43, ie1KO-IE0∆39-54 and ie1KOIE1 at MOI of 5, respectively. At the designated times pi, cells were harvested and cell lysates were prepared and and total cellular DNA was subjected to a slot blot analysis. A 32  P-dCTP labelled vp39 probe was used to detect viral DNA and levels were quantified by  phosphorimager analysis. Each column represents the average from two independent infections. Error bars indicate standard error.  16000000  ie1KO-IE0∆2-43 ie1KO-IE0∆39-54 ie1KO-IE0 ie1KO-IE1  DNA density units  14000000 12000000 10000000  ie1KO-IE0∆2-43 ie1KO-IE0∆39-54  8000000  ie1KO-IE0  6000000  ie1KO-IE1  4000000 2000000 0 6  12  24  36  48  72  96  hpi  103  Figure 2. 10 TEM analysis of ODV produced by WT virus or from viruses expressing only IE0 or IE1. (A) Representative TEM micrographs of OB cross sections produced from WT virus (left), ie1KO-IE0 (middle) and ie1KO-IE1 (right). (B) Distribution of the number of nucleocapsids per ODV for WT virus, ie1KO-IE0 and ie1KO-IE1. Nucleocapsids were only counted from ODV for which there were only clear cross sections. The number of OBs counted for each virus were 26 for ie1KO-IE0, 23 for ie1KO-IE1 and 21 for WT AcBac. The number of cross sectioned ODVs for each virus were 295 for ie1KO-IE0, 83 for ie1KO-IE1 and 101 for AcBac. WT  A.  ie1KO-IE0  ie1KO-IE1  percentage of Nucleocapsids/ODV (%)  B. 100 90  WT AcBac ie1KO-IE1 ie1KO-IE0 ie1KO-IE0  80 70 60 50 40 30 20 10 0 1  2  3  4  5  6  7  8  9  10  11  Nucleocapsids/ODV  104  Table 2. 1 Microarray analysis a of genes regulated by IE0 and/or IE1 IE1 transfection ac111 ac25 ac15 ac22 ac79 ac30 pp31 ac55 lef-3 lef-6 lef-4 ac70 ac101 ie1 ac63 ac52 ac132 P6.9 ac70 da16 ac97 ODV-E56 ac75 35K ac29 ac69 P15 lef-2 PK2 ac81  Ratiob 5.50 5.48 5.41 4.35 4.12 4.02 3.95 3.68 3.63 3.55 3.46 3.25 3.22 3.15 3.09 3.03 3.02 2.90 2.90 2.89 2.83 2.83 2.82 2.79 2.77 2.76 2.75 2.71 2.66 2.62  IE1 transfection TLP gp41 ac76 lef-8 ac38 alk-exo ac122 lef-11 ac54 ac1629 P43 lef-9 DNA pol ac108 cg30 ac74 P40 lef-12 ac53 ac84 ac106 ac117 lef-5 P25 ac110 ac145 ac4 ac43 P26 ac146  Ratiob 2.57 2.56 2.51 2.50 2.45 2.44 2.43 2.42 2.42 2.37 2.36 2.33 2.33 2.31 2.30 2.29 2.29 2.25 2.21 2.21 2.11 2.11 2.10 2.09 2.08 2.08 2.06 2.05 2.02 2.01  IE0 transfection ac111 lef-4 lef-3 lef-6 ac76 ac79 ie1 ac52 ac91 ac18 ac33 ie2 vp39  Ratiob 3.75 2.89 2.61 2.54 2.20 2.15 2.14 2.01 0.48 0.48 0.48 0.47 0.45  a: In this experiment IE1 (or IE0) was expressed transiently from a plasmid, then translation was inhibited by 100 µg/ml cyclohexamide at 24 hpt and wt AcMNPV was used to infect the cells. Cells were maintained in cyclohexamide to permit viral transcription (and activation by the transiently expressed IE1 or IE0), but inhibit any viral translation. At 24 hpi, RNA was isolated and transcription was analyzed by microarray. b: Ratio stands for IE1 or IE0 expressing plasmid versus plasmid vector transfection in log2 format. “0” means no change of expression and “1” refers to 2 fold increase.  105  Table 2. 2 Primers used in the study Number  Sequence (5’→3’)  284  AGCCATATGCCAGTTGCACAACACTATTA  338  TGGAGCTCCGTATTACCGCCTTTGAGTGA  432  CCTCGAGACTGCAGGCGCTGGCAAAGATT CCATGCCATGGAAGTTCGAATTTTTTATATTTAC AATTTA  573 683  CGTAACTCGAGCCAGTTGCACAACACTATTA  740  TGGTACCGGTGAATTCGAGACTGCAGGCTC  852  GCGCCATGGGTGAGCAAGGGCGAG  860 861 921 922 924  GCGGGAATTCTTAGGCGTAGTCGGGCACGTCGT AGGGGTAATTAAATTCGAATTTTTTATATTTACA A GCGGTCTAGATTACTTGTCGTCGTCGTCCTTGTA GTCATTAAATTCGAATTTTTTATATTTACAA GCGGCTGCAGAGATTTGGCACGACCCTTGC GCGGCTCGAGGATGTGCTGCAAGGCGATTA ATAAGAATGCGGCCGCTTACTTGTACAGCTCGTC CAT  1144  GCGTCTAGAGTATGCAAATAAATCTCGAT  1145  GCGCTCGAGAGTCACTTGGTTGTTCAC  1146  GCGTCTAGATTTGGCACGACCCTTG  1147  GCGCTCGAGTTAGCTTCGTTGCCCGTTAT  1148  GCGAAGCTTCATTGCTTGTCATTTATT  1149  GCGTCTAGATAGCTTGCAAATGAAT  1150  GCGTCTAGACTGCACGACTGATAAGAC  1151  GCGCTCGAGGCTTATTGGCAGGCTCTC  1152  GCGTCTAGAGGGGACAGATAACAGAAA  1153  GCGCTCGAGCTGGGCTGGTAGGATA  1154  GCGTCTAGATGAAAAAGATAGACAACGAT  1155  GCGCTCGAGGTTTGCTTCTTGTAAACCTT  1156  GCGTCTAGAAACTGCCGTTGCTAAGAAAA  1157  GCGCTCGAGCTTGCTTGTGTGTTCCTTAT  1158  GCGTCTAGAGCGATGACGCACAGTTTG  Note Ac ie1 promoter with NdeI and EcoRI sites 3' primer of polyA in p2Zop2EIE0 upper primer for Ac ie0 5’ primer of ie1 ie1 promoter primer with XhoI site upper primer for Ac ie1 fusion to eyfp upper primer of ecfp for fusion with Ac ie1/0 Primer for tagging AcMNPV ie1 with HA at C -term with EcoRI site C-term Flag tagged ie0 with XbaI site 5' primer of Ac ie0 promoter 3' primer of pe38 polyA primer for ie1/0 fusion 5' primer of ie1 promoter with XbaI site 3' primer of ie1 promoter with XhoI site 5' primer of ie0 promoter with XbaI site 3' primer of ie0 promoter with XhoI site 5' primer of hr5 with HindIII site 3' primer of hr5 with XbaI site 5' primer of pe38 promoter with XbaI 3' primer of pe38 promoter with XhoI 5' primer of ie2 promoter with XbaI site 3' primer of ie2 promoter with XhoI site 5' primer of 39K promoter with XbaI site 3' primer of 39K promoter with XhoI site 5' primer of gp64 promoter with XbaI site 3' primer of gp64 promoter with XhoI site 5' primer of me53 promoter with XbaI site  106  Number  Sequence (5’→3’)  1159  GCGCTCGAGAGTTAGCACTCAGAAATCAA  1162  GCGTCTAGATTTGTCGCAACTACTGAT  1163  GCGCTCGAGTTTGCAATGGTAAAGCTC  1164  GCGCTCGAGATTGTTGCCGTTATAAATAT  1165  GCGTCTAGATCAAGTTGTGCGAAAGAGTC  1166  GCGTCTAGAAAGTTGGTACGAAAGC  1167  GCGCTCGAGGGCCACCACAAATGCTAC  1168  GCGTCTAGAGGAGTGTGTTGCTTTA  1169  GCGCTCGAGCAAAACTGTTACGAAAA  1170  TTGCAAGTTGACACTGGCGG  1171  CATGTTAGCTTCGTTGCCC  1172  CTGCTGAGCTTCTGCGCAAG  1173  GCGACGCAAATTAATTTTAA  1345  GCGCTCGAGATTGCTGTTGTCGATATGTGG  1346  GCGTCTAGAAACACGCTCAGCAAAACTAT  1347  GCGCTCGAGGTTGCTTAAATATAAAAATTAAA  1348  GCGTCTAGACATGTGCCGCGATCATTATA  1349  GCGGGGATCCATGGCGCTAGTGCCCGT  1350  GCGGGGATCCTTAGACGGCTATTCCTCC  1395 1396 1397 1398 1399 1400 1401 1402 1403 1404 1405 1406 1407 1408  GCGTCTAGACGTACGTGATTGGCATACTA GCGCTCGAGTTTATTACACCACAAATATTTT GCGTCTAGATCGGCTTGCATCATACTGTTT GCGCTCGAGATTTATAACGGCAACAATATGGCG GCGTCTAGAAAACGGGCAGTTGTAGCGAT GCGCTCGAGAGTGCAGCACAGGCAATGTC GCGTCTAGAAAATATCTAAAAACACCTCGACG GCGCTCGAGTTTTATTCCCTTACTCTATTCG GCGTCTAGACGCGAGTCATGAGTTTAAAAGA GCGCTCGAGGATTATGCAGTGCGCCCTTT GCGTCTAGATTAAATTTTTTGGAAAACGATA GCGCTCGAGGGTGAATGCATCTTACTCAA GCGTCTAGATGAACAAGTTTATTTATTTATTTT GCGCTCGAGAATGAATAGCGGCGACGCAA  1413  AGCCGCGCGTTATCTCATGC  Note 3' primer of me53 promoter with XhoI site 5'primer of p35 promoter with XbaI site 3' primer of p35 promoter with XhoI site 3' primer of vp39 promoter with XhoI site 5' primer of vp39 promoter with XbaI site 5' primer of p78 promoter with XbaI site 3' primer of p78 promoter with XhoI site 5'primer of polh promoter with XbaI site 3' primer of polh promoter with XhoI site primer of ie0 for deleting 38 aa from exon0 reverse primer for deleting 38 aa from exon0 primer of ie0 for deleting 16 aa from ie1 UTR reverse primer of ie0 for deleting 16 aa from ie1 UTR sense primer for lef-3 promoter antisense primer for lef-3 promoter sense primer for ac91 promoter antisense primer for ac91 promoter vp39 sense primer with BamHI site vp39 anti-sense primer with BamHI site 5' primer of lef-6 promoter 3' primer of lef-6 promoter 5' primer of lef-4 promoter 3' primer of lef-4 promoter 5' primer of ac111 3' primer of ac111 5' primer of ac76 3' primer of ac76 5' primer of ac79 3' primer of ac79 5' primer of ac33 3' primer of ac33 5' primer of ac18 3' primer of ac18 sequencing primer located within Opie2 promoter  107  Number 1414 1419 1420  Sequence (5’→3’) GGTGTACGACGCGTTAAAAT GCGTCTAGATGATAAACGCGTTTGTAAAC GCGTCTAGATATGTTCTGCACCTTTTGTT  1421  GCGAAGCTTTGATAAACGCGTTTGTAAAC  1444  CATGTTAAGCTTCGTTGCCCGTTATC  1445 1446 1447 1448 1451  GCAGAAGCTCAGCAGTTGCAAGT CCATATTCGTGCGAGGCAACG GAAAATATAATGACGTCAAACTGTG TGACGTCATTATATTTTCCTGGACGT GCACGAATATGGCGATGACGCACAGT  Note sequencing primer within ie0 5' primer of ac52 promoter 3' primer of ac52 with XbaI 5' primer of ac52 promoter with HindIII site upper primer for IE0 N-term deletion lower primer for IE0∆2-32 lower primer for IE0∆2-22 lower primer for IE0∆2-11 upper primer for IE0∆19-54 upper primer for IE0∆28-54  108  2.5 References Ayres, M. 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AcMNPV AC16 (DA26, BV/ODV-E26) regulates the levels of IE0 and IE1 and binds to both proteins via a domain located within the acidic transcriptional activation domain. Virology 385(2), 484-495. Nissen, M. S., and Friesen, P. D. (1989). Molecular analysis of the transcriptional regulatory region of an early baculovirus gene. J. Virol. 63(2), 493-503. Ohresser, M., Morin, N., Cerutti, M., and Delsert, C. (1994). Temporal regulation of a complex and unconventional promoter by viral products. J. Virol. 68(4), 2589-2597. Okano, K., Mikhailov, V. S., and Maeda, S. (1999). Colocalization of baculovirus IE-1 and two DNA-binding proteins, DBP and LEF-3, to viral replication factories. J. Virol. 73(1), 110-119.  111  Passarelli, A. L., and Miller, L. K. (1993). Three baculovirus genes involved in late and very late gene expression: ie-1, ie-n, and lef-2. J. Virol. 67(4), 2149-2158. Pearson, M. N., and Rohrmann, G. F. (1997). Splicing is required for transactivation by the immediate early gene 1 of the Lymantria dispar multinucleocapsid nuclear polyhedrosis virus. Virology 235(1), 153-165. Pullen, S. S., and Friesen, P. D. (1995). Early transcription of the ie-1 transregulator gene of Autographa californica nuclear polyhedrosis virus is regulated by DNA sequences within its 5' noncoding leader region. J. Virol. 69(1), 156-165. Rodems, S. M., and Friesen, P. D. (1995). Transcriptional enhancer activity of hr5 requires dual-palindrome half sites that mediate binding of a dimeric form of the baculovirus transregulator IE1. J. Virol. 69, 5368-5375. Ross, L., and Guarino, L. A. (1997). Cycloheximide inhibition of delayed early gene expression in baculovirus-infected cells. Virology 232(1), 105-113. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). "Molecular Cloning." Second ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Schultz, K. L., Wetter, J. A., Fiore, D. C., and Friesen, P. D. (2009). Transactivator IE1 is required for baculovirus early replication events that trigger apoptosis in permissive and nonpermissive cells. J. Virol. 83(1), 262-272. Slack, J. M., and Blissard, G. W. (1997). Identification of two independent transcriptional activation domains in the Autographa californica Multicapsid Nuclear Polyhedrosis Virus IE1 protein. J. Virol. 71, 9579-9587. Stewart, T. M., Huijskens, I., Willis, L. G., and Theilmann, D. A. (2005). The Autographa californica multiple nucleopolyhedrovirus ie0-ie1 gene complex is essential for wild-type virus replication, but either IE0 or IE1 can support virus growth. J. Virol. 79(8), 4619-4629. Takagi, I., Yamada, K., Sato, T., Hanaichi, T., Iwamoto, T., and Jin, L. (1990). Penetration and stainability of modified Sato's lead staining solution. J. Electron. Microsc. (Tokyo) 39(1), 67-68. Theilmann, D. A., and Stewart, S. (1991). Identification and characterization of the IE-1 gene of Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus. Virology 180(2), 492-508.  112  Theilmann, D. A., and Stewart, S. (1993). Analysis of the Orgyia pseudotsugata multicapsid Nuclear Polyhedrosis Virus trans-activators IE-1 and IE-2 using monoclonal antibodies. J. Gen. Virol. 74(9), 1819-1826. Theilmann, D. A., Willis, L. G., Bosch, B. J., Forsythe, I. J., and Li, Q. (2001). The baculovirus transcriptional transactivator ie0 produces multiple products by internal initiation of translation. Virology 290(2), 211-223. Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22(22), 4673-4680. Yu, I. L., Bray, D., Lin, Y. C., and Lung, O. (2009). Autographa californica multiple nucleopolyhedrovirus ORF 23 null mutant produces occlusion-derived virions with fewer nucleocapsids. J. Gen. Virol. 90(6), 1499-1504.  113  Chapter 3 AcMNPV AC16 (DA26, BV/ODV-E26) Regulates the Levels of IE0 and IE1 and Binds to Both Proteins via a Domain Located within the Acidic Transcriptional Activation Domain2  3.1 Introduction AcMNPV is the type species of the baculoviridae alphabaculovirus genus. The baculoviridae consists of a large group of enveloped double stranded DNA viruses. Baculovirus genes can be divided into immediate early, early, late and very late based on the expression cascade (Kelly and Lescott, 1981; Miller, 1997). The primary AcMNPV transregulatory proteins are the immediate early IE0 and IE1 which are produced from the only known AcMNPV spliced gene complex. The ie0 mRNA consists of two exons that initiates from the early promoter of ac141. Exon 1 of ie0 consists of 114 bp of ac141 that are spliced to the 5’ end of the ie1 mRNA after excision of a 4.2 kb intron (Chisholm 1988). The ie1 mRNA is not spliced and consists of only exon 2 and initiates from the early promoter of ac147. Both ie0 and ie1 are transcribed and translated immediately upon infection but peak at different times post infection. The steady state levels of IE0 peak prior to viral DNA replication at about 4 hours pi, whereas IE1 keeps accumulating until very late times pi (Huijskens et al., 2004). Expression of ie0 mRNA results in both IE0 and IE1 being translated from the ie0 mRNA due to internal translation initiation (Theilmann et al., 2001). The amino acid sequence of IE0 includes 54 extra amino acids at the Nterminus of IE1 that are derived from the ac141 ORF and the ie1 5’ untranslated region.  IE0 and IE1 appear to have primary functions at different stages of the viral life cycle. Both IE1 and IE0 are capable of transactivating viral early genes and stimulating late gene expression (Huijskens et al., 2004; Theilmann et al., 2001). Either IE0 or IE1 can support viral replication independently however both are required for a wild-type phenotype 2  A version of this chapter has been published. Nie Y, Fang M and Theilmann DA. (2009) AcMNPV AC16 (DA26, BV/ODV-E26) regulates the levels of IE0 and IE1 and binds to both proteins via a domain located within the acidic transcriptional activation domain. Virology 385: 484-495.  114  (Stewart et al., 2005). Reduced expression level of IE0 enables AcMNPV to replicate in the normally non-permissive SL2 cells therefore suggesting that IE0 might influence host range determination . However it is not clear how IE0 and IE1 orchestrate their functional similarities or differences and little is known about the identity of the proteins with which IE0 or IE1 interacts. AcMNPV IE1 is well known as one of the six essential genes along with dna polymerase, lef-1, lef-2, lef-3, and helicase required for the viral DNA replication in transient replication assays (Kool et al., 1994). It has also been shown that BmNPV IE1 co-localizes with DBP and LEF3 in the nuclear structures that are believed to be viral replication factories (Kawasaki et al., 2004; Okano et al., 1999). Ito et al. found that AcMNPV IE1, LEF3 and P143 bind to closely linked sites on viral DNA in vivo using chromatin immunoprecipitation and suggested these proteins may form replication complexes in infected cells (Ito et al., 2004). Besides the proteins involved in viral DNA replication, BmNPV IE1 was also found to co-localize in infected insect cells and interact in yeast 2-hybrid assays with BmNPV BM8 (Imai et al., 2004; Kang et al., 2005). BM8 is the homolog of AcMNPV AC16, which is one of the 17 Group I NPV specific genes (Herniou et al., 2001). Further identification of proteins that interact with IE0 and IE1 and elucidation of the biological relevance of the interaction is needed to enable the complete understanding of the essential roles that IE0 and IE1 play during the viral life cycle.  In this study tandem affinity purification (TAP) has been used to identify proteins that interact with TAP-tagged AcMNPV IE0 and IE1. Using this approach AcMNPV AC16 (also known as DA26, BV/ODV-E26) (Beniya et al., 1998) was identified. The interaction was confirmed by immunoprecipitation using anti-HA agarose beads which pulled down both IE0 and IE1 along with HA tagged AC16. Using yeast 2-hybrid assays the AC16 interaction domain of IE0 and IE1 was mapped to the acidic activation domain (AAD). Viruses were constructed that expressed only IE0 or IE1 with mutated AC16 interaction domains and the impact on the virus replication cycle was analyzed. In addition, the impact of ac16 deletion on the virus replication was also determined.  115  3.2 Results 3.2.1 Identification of IE0 and IE1 interacting proteins by tandem affinity purification AcMNPV IE0 and IE1 are the primary viral transregulatory proteins and identifying other proteins that complex with them is essential to understand how AcMNPV replicates. TAP purification has been shown to enable the purification of large transcriptional complexes (Rigaut et al., 1999) and therefore we attempted to use this method with IE0 and IE1. To enable the TAP purification for the identification of interaction partners of AcMNPV IE0 and IE1, bacmid derived viruses that express IE0 or IE1 tagged with 3xFLAG-6xHis at the C-terminus (ie1KO-IE0-3xFLAG6xHis and ie1KO-IE1-3xFLAG6xHis respectively) were made using a previously constructed ie1KO virus (Stewart et al., 2005). The IE0 and IE1 TAP tagged viruses and control virus AcMNPV E2 were used to infect Sf9 cells. At 12 hpi and 24 hpi, cells were collected and TAP purification was performed using anti-FLAG beads followed by Ni-NTA beads. The purified proteins were separated on a gradient SDSPAGE gel (4-12%) and stained by SYPRO ruby. There were significant background bands using this approach and very few clear differences were observed, however a protein band between 32.5 kDa to 47.5 kDa was enhanced in the proteins bound to IE0-3xFLAG6xHis at both 12 hpi (Fig. 3.1A) and 24 hpi (data not shown). The band was excised from the gel of 12 hpi samples and subjected to LC-MS/MS for protein identification. One peptide was identified matching the viral protein AC16 (Fig. 3.1B). The predicted molecular weight of AC16 is 26 kDa, however, previous results (Beniya et al., 1998; Braunagel et al., 2009; Burks et al., 2007) have shown that higher molecular weight forms of AC16 are observed in infected cells. In addition, we also observed higher molecular weight forms of AC16 on Western blots (data not shown). BmNPV BM8, a homolog of AC16, has been shown to interact with BmNPV IE1 (Kang et al., 2005).  116  3.2.2 Co-immunoprecipitation of AC16 with IE0 and IE1 The TAP results suggested that AC16 interacted with IE0 but a corresponding enhanced band was not detectable above background in protein complexes isolated from IE1 TAP tagged virus infected cells (Fig. 3.1A). Therefore a second approach, coimmunoprecipitaton was taken to analyze the interaction between IE0/IE1 and AC16. To enable co-immunoprecipitaton a virus was constructed that expressed AC16 and AC16 tagged with the HA epitope at the C-terminus (AcBac-AC16HA, Fig. 3.2A). AcBacAC16HA and the control virus AcBac which expresses only AC16 without HA tag were used to infect Sf9 cells for co-immunoprecipitaton using anti-HA agarose beads. The purified proteins were analyzed by SDS-PAGE followed by Western blot using anti-HA and anti-IE1 antibodies.  The results showed that both IE0 and IE1 were co-immunoprecipitated with HA-tagged AC16 when immunoprecipitated with anti-HA agarose beads. No IE0 or IE1 was detected from cells infected with the control virus expressing untagged AC16 confirming the specificity of the interaction. This result confirms the TAP results and in addition shows that IE1 also interacts with AC16 (Fig. 3.2B). The interaction with IE1 would agree with the previous studies as stated above that have shown the BmNPV homolog BM8 colocalizes with IE1 in infected cells (Imai et al., 2004).  3.2.3 Mapping of the IE0 and IE1-AC16 interaction domain by yeast 2-hybrid The results described above showed that IE0 and IE1 both complex with AC16 therefore suggesting that a region or domain common to IE0 and IE1 is required for the interaction. To map the IE0 or IE1 interaction domain for AC16 the yeast 2-hybrid system was used. To define the amino acid sequences required for interaction with AC16, a series of IE1 deletions and point mutants were constructed in yeast 2-hybrid vectors. Initial large deletions localized the binding domain to amino acids 2-168 (Fig. 3.3A). This fragment was then subjected to a series of N-terminus to C-terminus and C-terminus to N-terminus deletions to map the N and C borders. The results localized the AC16 binding domain to amino acids 72-99, a region that overlaps with a predicted coiled-coil structure (Fig. 3.3B). 117  To confirm the mapping results 3 point mutants were generated: one within the AC16 domain and two outside the domain (Fig. 3.3A, B). Only the point mutant within the mapped AC16 domain (L79L80L86AAA) abolished IE1-AC16 2-hyrbrid formation. The corresponding amino acids were also mutated in a full length IE0 2-hybrid construct (IE0 mt, Fig. 3.3A) and similar to IE1, the interaction between IE0 and AC16 was abolished. These results showed that IE0 and IE1 have the same AC16 interaction domain. The AC16 interaction domain is a new functional component of the transactivators IE0 and IE1 that maps within the highly variable acidic activation domain (Fig. 3.3C).  The mapping of the AC16 interaction domain also identified a second domain at the Cterminus that surprisingly inhibited the interaction between IE1 and AC16. The inhibitory domain mapped to amino acid 454-489, which is within the generally defined DNA binding domain (Fig. 3.3A, C). However, the inhibition effect of this domain was only observed in the absence the C-terminal sequence amino acids 520-582, which contains the nuclear localization signal basic domain II and the oligomerization domain (Olson et al., 2001; Olson et al., 2003; Rodems et al., 1997). At this time it is not clear if this inhibitory domain plays a role in the context of the complete native protein in vivo and will be the subject of future investigations.  3.2.4 Point mutation analysis of the IE0 and IE1 AC16 interaction domain in vivo To examine whether the IE0 and IE1 AC16-interaction domain identified in yeast is indeed responsible for the interaction in vivo, viruses were constructed that expressed either IE0 or IE1 containing the AC16 interaction domain or domain mutants that lack interaction in the yeast 2-hybrid assay. IE0, IE1, and their mutants were inserted into an ie1KO bacmid (Stewart et al., 2005) along with AC16 tagged with the HA epitope and viruses ie1KOIE1–AC16HA, ie1KO-IE0–AC16HA, ie1KO-IE1L79L80L86AAA –AC16HA, ie1KOIE0L133L134L140AAA –AC16HA were generated. The viruses were used to infect Sf9 cells and co-immunoprecipitaton was performed with cells collected at 24 hpi using anti-HA agarose beads. Anti-HA and anti-IE1 antibodies were used for the Western blot analysis to see if IE0 and IE1 co-immunoprecipitated with AC16-HA. In cells infected with the positive 118  control viruses ie1KO-IE1–AC16HA, ie1KO-IE0–AC16HA, which expresses WT IE0 and IE1 and AC16-HA, both IE0 and IE1 were immunoprecipitated confirming the interaction between these proteins. Whereas AcBac infected cells which only express untagged AC16, no IE0 or IE1 was immunoprecipitated. Similarly in cells infected by ie1KOIE1L79L80L86AAA –AC16HA or ie1KO-IE0L133L134L140AAA –AC16HA, neither IE0 nor IE1 was co-immunoprecipitated with AC16HA (Fig. 3.4). These results confirm the domain mapping by yeast 2-hybrid assays and show that the AC16 interaction domain is functional in virus infected insect cells.  3.2.5 Expression of IE0L133L134L140AAA results in higher BV production than IE0 To determine the function of AC16 interaction with IE0 and IE1, we analyzed the impact of losing this interaction on the viral life cycle. Time course analyses were performed to compare BV production and viral DNA replication between viruses that express only IE1 or IE1L79L80L86AAA and IE0 or IE0L133L134L140AAA (Fig. 3.5A). Mutation of the IE0 AC16 binding domain (ie1KO-IE0L133L134L140AAA) did not affect viral DNA replication during the first 36 hpi compared to cells infected with ie1KO-IE0 (Fig. 3.5B). At late times pi, ie1KOIE0 appeared to have slightly higher levels of viral DNA. However, when BV production was compared, ie1KO-IE0L133L134L140AAA produced nearly a log higher BV compared to ie1KO-IE0 reaching similar levels as AcBac which expresses both WT IE0 and IE1. Therefore, for viruses that express only IE0, the loss of the AC16 binding domain results in an increased ability to produce BV. For ie1KO-IE1, the levels of viral DNA replication were the same as AcBac and no differences were observed when the AC16 binding domain was mutated (Fig. 3.5C). However unlike viruses expressing IE0 L133L134L140AAA there is no impact on BV production observed when the IE1 AC16 binding domain is mutated. These results suggest that AC16 plays a greater role in the function of IE0 rather than IE1.  3.2.6 Deletion analysis of ac16 Previous attempts to generate an ac16 or bm8 null virus only resulted in C-terminal deletions that lead to the suggestion that the 5’ half of ac16 or N-terminal of the protein was essential (Burks et al., 2007; Imai et al., 2004). To test this hypothesis and to further 119  analyze the function of the IE0 and IE1 interaction with AC16, we generated an ac16KO virus that deleted the 5’ end of ac16 including the promoter and first 70 amino acids of the ORF (Fig. 3.6). Unlike the previously reported AC16 deletion viruses, this construct leaves the potential promoter for ac17 intact (O'Reilly et al., 1990). The deletion of correct sequences and insertion of zeocin cassette were confirmed by PCR.  To determine the effect of ac16 deletion on BV production and viral DNA replication, Sf9 cells were infected with ac16KO (ac16KO-GFP-PH), ac16 repair (ac16KO-AC16) (Fig. 3.6) and the control virus AcBac. BV supernatant and infected cells were collected at 6, 12, 18, 24, 48, 72 and 96 hpi for analyses. End point dilution assays and quantitative real time PCR were used to analyze the impact of deleting ac16 on BV production and viral DNA replication respectively. The results showed that deletion of ac16 has no effect on BV production (Fig. 3.7A) and does not affect viral DNA replication (Fig. 3.7B).  The results above did not detect any effect on viral DNA replication and BV production upon deletion of AC16. However, this study and others have showed that AC16 interacts with IE0, IE1 and FP25 (1998; Kang et al., 2005). We therefore determined if the deletion of ac16 impacts the expression of the viral proteins IE0 and IE1, FP25 and the representative late structural protein VP39. The temporal expression of these proteins expressed from ac16KO, ac16KO-AC16 and AcBac was compared by Western blot analysis. For ac16KO the expression of IE0 and IE1 was significantly changed from that observed with ac16KO-AC16 and AcBac. In ac16KO infected cells, IE0 and IE1 were also detected by 6 hpi. However, the relative levels or ratio of IE0 to IE1 increased significantly from 6 hpi to 24 hpi compared to ac16KO-AC16 or AcBac infected cells (Fig. 3.8). This suggests that deletion of ac16 results in an increase in the expression of IE0. Previous results have shown that altering the expression of IE0 or IE1 affects both early and late events in the infection cycle and the correct IE0/IE1 ratio is critical for a wild-type phenotype (Huijskens et al., 2004; Stewart et al., 2005). AC16 has also been reported to bind to FP25, however no difference in the expression of this protein was observed (Fig. 3.8). Similarly, the late structural protein VP39 showed no difference in expression when AC16 is not present in infected cells. In addition no difference in the very late protein POLYHEDRIN expression was observed (data not shown). Therefore the major 120  observable effect of ac16 deletion was the change of relative level of IE0 to IE1 detected by Western blots.  3.3 Discussion IE0 and IE1 are the primary transregulatory proteins of AcMNPV that are required for both DNA replication and transcriptional activation. Elucidating the functional mechanisms by which these proteins facilitate these activities is a key requirement for the understanding of the baculovirus replication cycle. In this study we have shown that IE0 and IE1 interact with AC16 (DA26, BV/ODV-E26) and the binding domain is mapped to IE0 and IE1 amino acids residues 126-153 and 72-99 respectively. The AC16 binding domain is located within the previously characterized acidic activation domain (Forsythe et al., 1998; Pathakamuri and Theilmann, 2002; Slack and Blissard, 1997; Theilmann and Stewart, 1991). Comparison of the AAD of baculovirus IE1s has shown the AAD has very little conserved amino acid homology however the region maintains an overall acidic profile. This is similar to the classic “acid blob” transcriptional activation domain first identified in HSV VP16 and the yeast protein GAL4 (Sadowski et al., 1988; Triezenberg et al., 1988). However reanalysis of the region containing the AC16 binding revealed some amino acid conservation (Fig. 3.3B). Within this domain the IE1 Leu79 and Leu86 are highly conserved hydrophobic residues of IE1 among group I viruses and mutating these residues along with Leu80 to Ala abolished the interaction between IE0 and IE1 with AC16. Comparison with other IE1 AAD domains shows very low sequence conservation however, bioinformatics analysis predicts that the AC16 binding domain forms a coiledcoil domain. Analysis of all other known IE0 or IE1 AAD domains predicts that they contain a potential coiled-coil domain (data not shown). Interestingly the AC16 BmNPV homolog BM8 was shown to bind to BmNPV IE1 and required the sequences BM8 1-110 which also contains a predicted coiled-coil domain (Kang et al., 2005) that is conserved in AC16. These results therefore strongly suggest that the protein-protein interaction between IE0 or IE1 and AC16 is facilitated by coiled-coil domains which have been extensively characterized in other proteins (Delahay and Frankel, 2002; Lupas, 1996).  To address the function of the IE0-IE1 interaction with AC16 two approaches were taken. 121  The first approach mutated the IE0-IE1 binding domain in viruses that express either IE0 or IE1 and the second approach was to delete AC16. Mutating the AC16 binding domain in IE0 had the effect of increasing BV production but this was not observed for IE1. The reason for the increase in BV is unknown but it could be due to a number of effects which include changes in ie0 transcription or alternatively IE0 translation, post-translational modification and/or cellular localization. AC16 has been shown by immunoelectron microscopy to locate predominantly in intranuclear microvesicles of infected cells (Beniya et al., 1998; Burks et al., 2007), but it has also been shown that BmNPV BM8, the AC16 homolog, colocalizes with IE1 and requires the viral hr sequences and IE1 for localizing at specific nuclear sites (Kang et al., 2005). Sequestering IE0 to discrete cellular or genomic locations could result in regulating a different repertoire of genes compared to IE1. Alternatively the binding of AC16 to IE0 could decrease its activity relative to its transregulation of genes involved in BV production which may be determined by the 54 Nterminal amino acids not found in IE1. This would agree with our results which show that deletion of the AC16 binding site in IE1 had no effect on BV production or replication (Fig. 3.5C). If the major role of AC16 is to interact with and regulate IE0 activity, this may explain why AC16 bound to detectable levels in ie1KO-IE0-3xFLAG6xHis infected cells but not in ie1KO-IE1-3xFLAG6xHis infected cells (Fig. 3.1).  The second approach to examining the role of the IE0 or IE1 AC16 interaction was to generate an AC16 null or knockout virus. Previous studies that attempted to generate ac16 or BmNPV bm8 knockout viruses resulted in C-terminal deletions only and were unable to isolate a full deletion, suggesting that ac16 or bm8 was essential (Burks et al., 2007; Imai et al., 2004). Both of these knockout viruses potentially expressed the AC16 or BM8 Nterminus that contains the domain required for binding to IE1 (Kang et al., 2005). In contrast, in this study we generated a knockout virus by deleting the ac16 promoter and the N-terminus which does not affect the downstream ac17 promoter. Analysis of ac16KO showed that there was no effect on either BV production or viral DNA replication when compared to the control virus AcBac. No impact on tissue culture growth is consistent with the results from C-terminal ac16 and bm8 deletions previously reported (Imai et al., 2004; O'Reilly et al., 1990). However O’Reilly et al. (1990) did observe a decrease in LC50 when bioassayed in Trichoplusia ni and Spodoptera frugiperda larvae. Further studies will have 122  to be performed to determine if in vivo differences are observed with the ac16 knockout virus constructed in this study.  Even though no differences were observed in viral DNA replication or BV production in cells infected with ac16KO, we compared the temporal expression pattern of proteins that interact with AC16, which are IE0, IE1, and FP25, and representative late and very late proteins VP39 and POLYHEDRIN. No difference was observed for FP25, however the relative expression levels of IE0 to IE1 significantly altered. At 6 hpi in ac16KO infected cells IE0 was the dominant protein compared to AcBac or ac16KO-AC16 when normally there are approximately equal levels of IE0 and IE1. The higher relative levels of IE0 continue right up to 24 hpi. The impact of deleting ac16 therefore appears to affect predominately IE0 permitting higher levels of expression. These results are consistent with the results of the TAP purification and the IE0-IE1 AC16 binding domain mutations both of which showed greater impact with IE0. Interestingly our previous study (Stewart et al., 2005) suggested that IE0 up regulates IE1. Therefore it would be expected that the increase in IE0 observed in this study would result in increased IE1 but this was not observed. This could be due to multiple reasons including that IE1 levels are already at maximum and an increase in IE0 therefore has no additional effect. Alternatively AC16 may be required for IE0 up-regulation of IE1.  No difference was observed with the late proteins VP39 (Fig. 3.8) or POLYHEDRIN (data not shown). Other studies reported that AC16 stimulates viral late gene expression together with AC18 in transient assays, but deletion AC16 C-terminal did not causes any observable difference in viral late gene regulation (O'Reilly et al., 1990) similar to the results of this study .  AC16 has been shown to interact with FP25 and probably is involved in the trafficking of the occlusion body protein ODV-E66. It has been proposed that AC16, FP25, ODV-E66 may form a complex with actin potentially facilitating nucleocapsid transport for production of both BV and ODV (Beniya et al., 1998; Burks et al., 2007). AcMNPV EXON0 which is demonstrated to be critical for the transport of nucleocapsids out of the nucleus also interacts with FP25. EXON0 also contains the N-terminal 38 amino acids of 123  IE0 but not the AC16 binding domain. As discussed above loss of the IE0 AC16 binding domain results in increased BV production and therefore AC16 could be involved in the regulation of BV levels by interacting with FP25 and IE0. These interactions may also impact on the number of ODV within occlusions bodies. We did not detect any gross differences in the occlusion bodies produced by the AC16 knockout virus when examined by light microscopy, but differences may be observed using electron microscopy.  In conclusion, in this study we have mapped a new IE0 and IE1 functional domain that is utilized for binding to the viral protein AC16. The domain is predicted to form a coiledcoil that likely interacts with the similar type of domain predicted in AC16. Mutation of the IE0 and IE1 domain combined with an ac16 deletion analysis suggests that a primary role of AC16 involves interaction with IE0 as opposed to IE1, potentially regulating the early events of AcMNPV infection involved in BV production. However, these interactions are not essential for viral growth in tissue culture but may play a key role during virus infection in vivo.  3.4 Materials and methods 3.4.1 Viruses and cells Cells and viruses were maintained as described in Chapter 2.  3.4.2 Plasmids construction 3.4.2.1 Construction of transfer vectors pFAcT-GFP-IE0-3xFLAG6xHis and pFAcTGFP-IE1-3xFLAG6xHis. To enable the tandem affinity purification described by Yang et al. (2006), primers 1369 and 1371 were used to amplify the 3xFLAG-6xHis tag from from pCaSpeR-hs-act-Tetra tag plasmid kindly provided by Dr. H. Krause (University of Toronto). The 3xFLAG-6xHis fragment was cloned into plasmids containing the ie0 promoter and ORF or the ie1 promoter and ORF at the NotI/NcoI sites. The resulting plasmids were called pie0-3xFLAG-6xHis and pie1-3xFLAG-6xHis which encoded the 124  TAP tag in frame with ie0 and ie1 at the C-terminus and were confirmed by sequencing. To make the bacmid transfer vectors, pie0-3xFLAG-6His was digested with PstI and NotI and cloned into the vector pFAcT-GFP-Tnie1pA resulting in pFAcT-GFP-IE03xFLAG6xHis. The plasmid pie1-3xFLAG-6His was digested with HindIII and blunt ended with Klenow DNA polymerase followed by NotI digestion and insertion into pFAcT-GFP-Tnie1pA, resulting in pFAcT-GFP-IE1-3xFLAG6xHis. The vector pFAcTGFP-Tnie1pA was made by inserting TnSNPV ie1 polyA that was amplified using primers 517/810 from TnSNPV genomic DNA into pFAcT-GFP (Dai et al., 2004) at the SacI/NotI sites. The sequences of the primers were listed in table 3.1.  3.4.2.2 Construction of IE1 or IE0 plasmids for yeast 2-hybrid analysis. To make the fusion construct of pAD-IE1 or IE1 mutants, primers 1417 and 1496 were used to amplify the complete IE1 ORF, IE1 2-520, IE1 2-168 and primers 1472/1496 were used to amplify IE1 169-582. The PCR products were inserted into pAD-Gal4 at the XbaI/BamHI sites. The AcMNPV IE0 ORF was amplified with primer pair 1424/1416 using pAcie0delta (Huijskens et al., 2004) as the template. The PCR product was inserted into pAD-Gal4 at the XbaI/BamHI sites to generate pAD-IE0. The remaining IE1 mutants or IE0 mutant used for the yeast 2-hybrid were made by standard methodology of inverse PCR using pAD-IE1, pAD-IE1 2-168, pAD-IE1 169-582 or pAD-IE0 as the templates. Three IE1 point mutants were amplified by inverse PCR using pAD-IE1 as the template. They were 1529/1530 for pAD-IE1L79L80L86AAA, 1531/1532 for pAD-IE1K154R156K160K161AAAA, 1533/1534 for pAD-IE1D137E138D141AAA. Primer pair 1529/1530 was also used to inverse PCR pAD-IE0 to make pAD-IE0L133L134L140AAA. For the construct of pBD-AC16, ac16 was PCR amplified with primer pair 1469/1474 and inserted into pBD-Gal4 in frame with Gal4 BD at the EcoRI/PstI sites. Right constructs were screened by restriction enzyme digestion and confirmed by sequencing. The sequences of the primers were listed in table.1.  3.4.2.3 Construction of pFAcT-GFP-IE0, pFAcT-GFP-IE1, pFAcT-GFPIE0L133L134L140AAA and pFAcT-GFP-IE1L79L80L86AAA. To enable the analysis of viruses that express IE0 or IE1 lack of the binding domain for AC16, pFAcT-GFPIE0L133L134L140AAA and pFAcT-GFP-IE1L79L80L86AAA were made. AcMNPV ie0 and ie1 promoter were amplified from the wild type genomic DNA using primer pairs 870/871 and 125  740/869 respectively. The ie0 promoter was cloned into pFAcT-GFP-Tnie1pA at the XbaI/NotI sites generating pFAcT-GFP-ie0prm and ie1 promoter was cloned into pFAcTGFP-Tnie1pA at the PstI/XbaI sites generating pFAcT-GFP-ie1prm. Primer pair 1529/1530 was used to inverse PCR amplify pAcie0delta to generate pAcie0deltaL133L134L140AAA which was confirmed by sequencing. Primers 1542/1543 was used to amplify IE0L133L134L140AAA ORF using pAcie0delta-L133L134L140AAA as the template, and the PCR product was cloned into pFAcT-GFP-ie0prm at NotI and screened by NotI and EcoRI for right orientation, resulting pFAcT-GFP-IE0L133L134L140AAA. The ORF of IE1L79L80L86AAA was PCR amplified with primers 1544/1545 using pAD-IE1L79L80L86AAA as the template and cloned into pFAcT-GFP-ie1prm at XbaI/NotI sites resulting pFAcT-GFPIE1L79L80L86AAA. To make pFAcT-GFP-IE0 and pFAcT-GFP-IE1, ie0 were PCR amplified with primers 870/1542 from pAcie0delta and ie1 were PCR amplified with primers 868/1545 from pAcie1DT, respectively. The fragments were subsequently cloned into pFAcT-GFP-Tnie1pA at the NotI/XbaI sites, generating pFAcT-GFP-IE0 and pFAcTGFP-IE1.  3.4.2.4 Construction of pFAcT-GFP-AC16, pFAcT-GFP-AC16-HA, pFAcT-GFP-IE0AC16HA, pFAcT-GFP-IE1-AC16HA pFAcT- IE1L79L80L86AAA-AC16HA and pFAcTIE0L133L134L140AAA-AC16HA. pFAcT-GFP was also used as the backbone for making transfer vectors pFAcT-AC16 and pFAcT-AC16HA. Briefly, ac16 including the promoter and open reading frame was amplified with primer 1430 paired up with 1560 or 1431 which contains the influenza HA epitope tagged at the C-terminus of AC16, the amplified PCR fragments were cloned into pFAcT-GFP at the XhoI/PstI sites, generating pFAcTGFP-AC16-pA- and pFAcT-AC16HA-pA- respectively. In order to reduce the chance of intragenomic homologous recombination, an OpMNPV ie2 polyA signal was used for both ac16 and ac16HA. OpMNPV ie2 polyA was amplified using primer 1519 paired with 1561 or 1518, the PCR fragments were digested with PstI/SacI or PstI alone and cloned into the corresponding sites of pFAcT-AC16-pA- and pFAcT-AC16HA-pA- respectively. Sequencing was performed to screen the right orientation of the polyA insertion into pFAcT-AC16HA-pA-. The transfer vectors produced were named as pFAcT-AC16 and pFAcT-AC16HA. The ac16HA cassette which includes the ac16 promoter and ORF and polyA signal was PCR amplified with primers 1430/1520 using pFAcT-AC16HA as the 126  template, and the PCR product was cloned into pFAcT-GFP-IE0, pFAcT-GFP-IE1, pFAcT-GFP-IE0L133L134L140AAA and pFAcT-GFP-IE1L79L80L86AAA at XhoI site, generating pFAcT-GFP-IE0-AC16HA and pFAcT-GFP-IE1-AC16HA respectively, pFAcTIE0L133L134L140AAA-AC16HA and pFAcT- IE1L79L80L86AAA-AC16HA respectively. XbaI was used to screen colonies in same orientations as pFAcT- IE1L79L80L86AAA-AC16HA and pFAcT- IE0L133L134L140AAA-AC16HA.  3.4.3 Construction of recombinant viruses 3.4.3.1 Construction of viruses using ie1KO as the backbone. The pFAcT-GFP vectors were used to transpose the ie1KO bacmid to make different recombinant viruses as previously described (Luckow et al., 1993). The vector pFAcT-GFP contains polyhedrin driven by its own promoter, multiple cloning sites and gfp driven by OpMNPV ie1 promoter between the two Tn7 transposition excision sites. Genes cloned between the two transposition sites are transposed into the mini ATT region located in the AcMNPV bacmid. pFAcT-GFP-IE0-3xFLAG6xHis and pFAcT-GFP-IE1-3xFLAG6xHis were used to transpose the ie1KO bacmid (Stewart et al., 2005) to generate viruses ie1KO- IE03xFLAG6xHis and ie1KO- IE1-3xFLAG6xHis respectively for TAP analysis. pFAcTGFP-IE0L133L134L140AAA and pFAcT-GFP-IE1L79L80L86AAA were used to transpose the ie1KO to generate ie1KO-IE0L133L134L140AAA and ie1KO-IE1L79L80L86AAA respectively for the analysis of impacts of loss of AC16 binding domain on the virus behavior. The viruses used for the comparison ie1KO-IE1 and ie1KO-IE0 were named as AcBacIE1 and AcBacIE0M→A respectively (Stewart et al., 2005). All IE0 constructs contain the IE1 start methionine mutated to alanine to prevent internal translation initiation (Stewart et al., 2005). pFAcT-GFP-IE0-AC16HA, pFAcT-GFP-IE1-AC16HA, pFAcT-IE0L133L134L140AAAAC16HA and pFAcT- IE1L79L80L86AAA-AC16HA were used to transpose the ie1KO bacmid to make ie1KO-IE0-AC16HA, ie1KO-IE1-AC16HA, ie1KO-IE0L133L134L140AAA-AC16HA and ie1KO-IE1L79L80L86AAA-AC16HA respectively for the confirmation of loss of interaction between AC16 with IE0L133L134L140AAA or IE1L79L80L86AAA.  127  3.4.3.2 Construction of ac16 N-terminal knockout AcMNPV bacmid. AcMNPV bacmid (bMON14272, Invitrogen) was used to generate an ac16 N-terminal knockout virus by recombination in E. coli as previously described (Datsenko and Wanner, 2000; Hou et al., 2002). A zeocin resistance cassette with ac16 flanking regions was amplified using primers 1434 and 1437 with p2ZeoKS as the template. These primers contain 50 bp homologous sequence to the 5’ flanking and upstream coding regions of ac16. The PCR fragment of zeocin resistance cassette was gel purified and electroporated into E. coli BW25113-pKD46 cells which contained the AcMNPV bacmid bMON14272. The electroporated cells were incubated at 37oC for 4 h in 1 ml of LB medium and plated onto agar medium containing 25 µg/ml of zeocin and 50 µg/ml of kanamycin. Plates were incubated at 37oC overnight and colonies resistant to both zeocin and kanamycin were selected and further confirmed by PCR. Three different pairs of primers were used to confirm the correct knockout of ac16 had been produced. Primers 1430 and 520 were used to detect the correct insertion of the zeocin cassette in the promoter region of the ac16 locus and primers 1239 and 1439 were used to detect the correct insertion of the zeocin cassette in the upstream coding region of the ac16 locus. A third primer pair 1440 and 1439 was used to confirm the deletion of the desired sequence. One recombinant bacmid confirmed by PCR was selected and named AcBac-ac16KO.  3.4.3.3 Construction of AC16 knockout and repair bacmids containing polyhedrin and gfp. To generate the ac16KO and repair viruses with Polyhedrin and GFP, pFAcT-GFP was used to transpose AcBac-ac16KO as previously described to make ac16KO-GFP-PH. pFAcT-GFP-AC16 and pFAcT-GFP-AC16-HA vectors were used to AcBac-ac16KO to produce ac16KO-AC16 and ac16KO-AC16HA. The pFAcT-GFP-AC16-HA was also used to transpose bMON14272 to generate AcBac-AC16HA for the reciprocal immunoprecipitation to confirm the interaction between AC16 and IE0/IE1.  128  3.4.4 3×FLAG-6×His tandem affinity purification (TAP) and protein identification. The TAP method was adapted from Yang et al. (2006). Briefly, 3 x 108 Sf9 cells were infected with ie1KO-IE0-3×FLAG6×His, ie1KO-IE1-3×FLAG6×His and control virus AcMNPV E2 at a multiplicity of infectivity (MOI) of 2 respectively. Cells were collected at 12 hpi by centrifugation at 500 x g for 5 min and washed twice with 50 ml phosphate buffered saline (PBS, 137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, pH 7.4). To lyse the cells for TAP, 2 ml lysis buffer (15 mM HEPES pH 7.6, 10 mM KCl, 0.1 mM EDTA, 0.5 mM EGTA,1 mM DTT, 1% proteinase inhibitor cocktail (Sigma)) was used to resuspend the cells, which were then passed twice through a pre-chilled French press at 8.27 MPa (1000 psi). Cell debris was removed by centrifugation at 18,000 x g for 10 min and supernatant was transferred to 5 ml tubes with 150 µl equilibrated anti-FLAG M2 affinity beads (Sigma) and incubated overnight at 4 oC on an orbiting platform. The incubation products were then transferred to Bio-Rad mini disposable columns, washed once with 1 ml cold lysis buffer and 6 times with 1 ml TBS buffer (50 mM HEPES, pH 7.6, 150 mM NaCl, 0.1% Triton). Proteins bound on the FLAG beads were eluted with 500 µl of 300 µg/ml 3×FLAG peptide (Sigma) in TBS. The eluate proceeded to further purification with Talon cobalt resin (BD Biosciences Clontech). 400 µl FLAG eluate were incubated with 50 µl equilibrated Talon cobalt resin for 1 h at 4 oC. The resin was washed 4 times with 1 ml TBS and eluted with 160 µl TBS containing 300 mM imidazole. The eluate was vacuum concentrated to 45 µl, mixed with 15 µl 4× SDS-PSB, heated at 100 oC for 10 min, separated by NuPAGE® Novex Bis-Tris gel (4-12%, Invitrogen), stained with SYPRO Ruby (Invitrogen). Protein bands specific to ie1KO-IE0-3×FLAG6×His, ie1KO-IE13×FLAG6×His or enhanced were excised from the gel. The gel slides were subjected to ingel digestion and identified by Liquid Chromatography Mass Spectrometry/Mass Spectrometry (LC-MS/MS) at Proteomics Core Facility of University of British Columbia.  129  3.4.5 Immunoprecipitation For the confirmation of interaction between IE0 and AC16, 6.0×107 Sf9 cells were infected with AcBac-AC16HA and control virus AcBac at an MOI=10. For the confirmation of loss of interaction between AC16 and IE1L79L80L86AAA or IE0L133L134L140AAA, Sf9 cells were infected at MOI=2 with ie1KO-IE0-AC16HA, ie1KO-IE1-AC16HA, ie1KOIE0L133L134L140AAA-AC16HA and ie1KO-IE1L79L80L86AAA-AC16HA and control AcBac respectively. Cells were collected at 18 or 24 hpi by centrifugation at 500 x g for 5 min and washed twice with PBS prior to lysing for immunoprecipitation. Cells were resuspended with 1 ml EBC buffer (50 mM Tris-Cl pH 8.0, 120 mM NaCl, 0.5% Nonidet P-40, 0.2 mM sodium orthovanadate, 1% sodium fluoride, 1% protease inhibitor cocktail (Sigma)) and passed through a pre-chilled French Press at 8.27 MPa (1000 psi) twice. Cell debris was removed by centrifugation at 18000 x g in a bench-top centrifuge at 4°C and supernatant was incubated with 50 µl equilibrated anti-HA agarose beads (Sigma) for 4 h at 4°C on a orbiting platform. The incubation products were transferred to a Bio-Rad column and the beads were washed once with 1 ml EBC buffer, followed by 6 washes with 1 ml NETN buffer (20 mM Tris-Cl pH 8.0, 1 mM EDTA, 0.5% NP-40) containing 400 mM NaCl. Proteins bound to the beads were eluted twice using 60 µl 100 mM glycine-HCl (pH 2.2) after 1 min incubation and the pH was raised to 8.0 with 1.5M Tris·cl. The eluate was vacuum concentrated to 45 µl, mixed with 15 µl 4 × PSB, heated for 10 min at 100°C, and subjected to SDS-PAGE and Western blot.  3.4.6 Time course analysis of BV production and viral DNA replication Sf9 cells (2.0 x 106 cells/35mm diameter well of a six-well plate) were infected by AcBac, ie1KO-IE1, ie1KO-IE1L79L80L86AAA, ie1KO-IE0 and ie1KO-IE0L133L134L140AAA respectively or AcBac, ac16KO-GFP-PH and ac16KO-AC16 respectively at a MOI of 5 in duplicate. At various hours post infection, supernatant containing BV was collected and stored at 4 oC after the cell debris was removed by centrifugation at 8000 x g for 5 min. Infected cells were washed once with PBS, scraped off with rubber policemen, pelleted by centrifugation at 2000 x g, for 5 min supernatant was removed and the pellets were stored at -80 oC until analysis. BV titer of the supernatant was determined by end-point dilution analysis in Sf9 130  cells and real-time quantitative PCR . The analysis of BV production by qPCR was adapted from Lo et al. (2004). Briefly, 100µl of BV supernatant collected during the time course and series dilution of titrated AcMNPV E2 stock were aliquoted and incubated at 50 oC overnight in the lysis buffer (10mM Tris·Cl pH8.0, 100mM EDTA, 0.5% SDS, 80µg/ml Proteinase K). Viral DNA then was extracted with phenol-chloroform-isoamyl alcohol followed by extraction with chloroform. 2 µl viral DNA extracts were used directly for the qPCR with primers 850 and 851 as well as 2×DyNAmo HS Master Mix (DyNAmo HS SYBR Green qPCR Kit, New England Biolabs) in a 20 µl reaction to amplify a 100 bp fragment of ac126 (chitinase). The thermal profile used was based on McCarthy et al. (2008). The results were analyzed by the MX4000 software (Stratagene).  Analysis of viral DNA replication was performed using real-time quantitative PCR as previously described which is based on the amplification of 100 bp fragment of gp41 gene (McCarthy et al., 2008; 2004). Briefly, Sf9 cells collected during the time course were resuspended in 1 ml cell lysis buffer (10mM Tris·Cl pH8.0, 100mM EDTA, 0.5% SDS, 20 µg/ml RNAase A) and incubated at 37 oC for 30min. A 50µl aliquot of the lysed cells was removed and added into 250 µl Tris-Cl pH 8.0 and 80µg/ml Proteinase K followed by overnight incubation at 50oC. The overnight digestion reaction was then extracted with 300 µl phenol-chloroform-isoamyl alcohol followed by extraction with 300 µl chloroform. 90 µl aqueous was carefully transferred to another clean 1.5 ml micro test tube. Prior to the PCR, 5 µl of the DNA extracts from each time point were diluted 10 times by adding 45 µl distilled water. 2 µl diluted DNA extracts was used directly for the qPCR with primers 1483 and 1484 as well as 2×DyNAmo HS Master Mix (DyNAmo HS SYBR Green qPCR Kit, New England Biolabs) in a 20 µl reaction. The thermal profile used was based on McCarthy: 1 cycle of 95oC for 15 minutes; 40 cycles of 95oC for 30 seconds, 52oC for 24 seconds, 72oC for 30 seconds; 1 cycle of 95oC for 1 minute; 41 cycles of 55oC for 30 seconds (McCarthy et al., 2008). The results were analyzed by the MX4000 software (Stratagene).  131  3.4.7 Western blot analysis Proteins eluted from affinity beads or total cells colleted during the time course analysis were mixed with 4×SDS-PSB and boiled for 10 min. Protein samples were separated by 10% or 7.5% SDS-PAGE and transferred to Millipore Immobilon-P membrane with BioRad Mini-Protean II and liquid transfer apparatus. Membranes were probed with one of the following different antibodies: 1) Mouse monoclonal HA antibody 1:1000 (Covance, HA11); 2) Mouse monoclonal IE1 antibody 1: 8000 (Ross and Guarino, 1997); 3) Mouse monoclonal OpMNPV VP39 antibody (1:3000) (Pearson et al., 1988); 3) rabbit polyclonal FP25 antibody 1:5000 (Braunagel et al., 1999). To detect bound primary antibodies membranes were incubated with 1:10000 goat anti-mouse or goat anti-rabbit peroxidaseconjugated secondary antibodies. Enhance Chemiluminescence System (ECL, Amersham) was used to detect the signals.  3.4.8 Yeast 2-hybrid Yeast 2-hybrid was performed using Saccharomyces cerevisiae strain YRG2 (MATa ura352 his3-200 ade2-101lys2-801 trp1-901 leu2-3 112 gal4-542gal80-538 LYS2::UAS GAL1-TATA GAL1-HIS3URA3::UAS GAL4 17mers(x3)-TATACYC1-lacZ) (Stratagene). Co-transformation of the fusion plasmids were performed according to the manufacturer’s instructions using the lithium acetate method (Stratagene). Transformants were screened on medium lacking the appropriate amino acids, and selection of histidine reporter gene expression was performed on histidine and tryptophan, or histidine, tryptophan and leucine, dropout agar plates.  132  Figure 3. 1 AC16 co-purifies with TAP tagged IE0. (A) Sf9 cells were infected with viruses expressing TAP tagged IE0 (ie1KO-IE03xFLAG6xHis), IE1 (ie1KO-IE1-3xFLAG6xHis) or with the control virus AcMNPV-E2 that expresses untagged IE0 and IE1 and collected for TAP at 12 hpi. Complexes were affinity purified and separated on gradient SDS-PAGE gel (4-12%, Invitrogen) and stained with SYPRO Ruby. A protein was found to be enhanced in proteins purified from ie1KOIE0-3xFLAG6xHis infected cells (arrow). (B) Amino acid sequence of AC16 and the LCMS/MS analysis of the enhanced band identified a peptide homologous to the sequences shown in red.  133  Figure 3. 2 IE0 and IE1 co-immunoprecipitate with AC16. Protein extracts of Sf9 cells infected with AcBac-AC16HA expressing HA tagged AC16 or the control virus AcBac expressing non-tagged AC16, respectively, were immunoprecipitated with anti-HA agarose beads at 18 hpi. SDS-PAGE and Western blots were used to analyze the precipitated proteins. AC16 and IE0 and IE1 were detected using anti-HA or anti-IE1 antibodies respectively. Top panel shows AC16HA, bottom panel shows IE0 and IE1. The input and eluate lanes represent 0.5% and 30% of the total input and eluate samples respectively.  134  Figure 3. 3 Mapping the IE0 and IE1 AC16 binding domain. (A) Summary of yeast 2-hybrid analyses between AC16 and the IE1 deletions or the IE1 and IE0 point mutants. “+” indicates that there was interaction and “-”stands for no interaction between the IE0 or IE1 constructs and AC16 construct. The IE1 point mutants are as follows, 1, IE1L79L80L86AAA; 2, IE1D137E138D141AAA; 3, IE1K154R156K160K161AAAA. IE0 mt refers to IE0L133L134L140AAA. The red rectangle shows the interaction domain and green shows a potential inhibitory domain at 454-489 aa. (B) Alignment of part of the IE1 Nterminus from nine group I viruses containing the location of the AC16 binding domain (solid box), the predicted coiled-coil domain (dashed line box) and the location of the point mutations. The alignment was performed with Clustal W (Thompson et al., 1994). The different shadings of the amino acids represent the following: black background show 100% conservation, dark grey represents 80% and light grey 60% respectively. The software used for the coiled-coil structure prediction was COILS (http://www.ch.embnet.org/software/COILS_form.html). The residues mutated to alanine are indicated with arrows. (C) Schematic diagram of IE0 and IE1 functional domains that have been identified from this study and others (Kovacs et al., 1992; Olson et al., 2001; Olson et al., 2003; Pathakamuri and Theilmann, 2002; Rodems et al., 1997; Theilmann and Stewart, 1991).  135  Figure 3.3 continued A.  C. IE1 mutants  2-582 2-520 2-168 169-582 22-168 42-168 62-168 72-168 82-168 102-168 122-168 142-168 2-150 2-122 2-110 2-99 2-89 2-77 2-69 2-489 2-453 del169-229 del169-289 del169-347 del169-419 del420-453 del420-489 point mutations IE0 IE0 mt  1  2  3  IE0  Y-2-H + IE1 + + + + + NLS (BDII, IE0 54 aa DBD (223521-538 aa) Acidic activation 520 aa) domain AD (168-222 aa) (AAD, 8-118 aa) + + Basic Cluster + (BDI,152-161 aa) + Oligomerization (543-568 aa) AC16 binding domain 72-99 aa + AC16 inhibitory domain 454-489 aa + 1, -; 2,+; 3,+ + -  B. AcMNPV BmNPV CfDEFNPV CfMNPV EppoNPV HycuNPV OpMNPV AngeNPV PlxyNPV  : : : : : : : : :  ---------------------MTQINFNASYTSASTPSRASFDNSYSEFCDKQ-PNDYLSYYNHPTPDGADTVISDSETA ---------------------MTQINFNASYTSAPTPSRASFDNGYSEFCDKQQPNDYLNYYNNPTPDGADTVVSDSETA -------------------MSKRIDNFRRSYVTPSTPSRALFN-------------------ATEVPIDVMVNSPAEETN ------------------MPKNMAALQQSLYTGPSTPSHTQFSR------------------STEFPENLNFD--VLNDS -----------------MMPKQMADLHRSLYTTPGTPIRALFNT------------------ATELPDNMDADTMDNNWD ------------------MPKNMAALHRSMFTGPPTPSHTLFNT------------------ATELPDNLN----LINGT ------------------MPKNMETLQR-SYMGPSTPNHNLFNN------------------ATELPDDLNFS--TMDVP MLVFVSVASWQPHAQRSPTIMKRIDNYRRSYVTPSTPSHALFN-------------------ATEVPIDVMVNSPAAETN ---------------------MTQINFNASYTSASTPSRASFDNSYSEFCDKQ-PNDYLSYYNHPTPDGADTVISDSETA  : : : : : : : : :  58 59 42 42 45 40 41 61 58  : : : : : : : : :  130 132 113 106 107 106 105 138 130  : : : : : : : : :  202 204 191 183 183 184 184 214 202  1.L79L80L86AAA AcMNPV BmNPV CfDEFNPV CfMNPV EppoNPV HycuNPV OpMNPV AngeNPV PlxyNPV  : : : : : : : : :  AASNFLASVNSLTD------N-DLVECLLKTTDNLEEAVSSAYYSESLEQPVVEQPSPSSAYHAESFEHSAGVNQPSATAASNFLASVNSLTD------DNDIMECLLKTTDNLGEAVSSAYYSESLELPVAEQPSPSSAYNAESFEQSVGVNQPSAAWESMSLTEFDPPIG------TEDLENMLQTPSDNLTTLENASQVLANDVGSSLLSEY--LSFNPRAMEPEVQLSEPSTSYETFSSVSLTTAEQ------DNQIDKILQESAA-MNRDVNSELA-QFTASEYVTG------FRADTMEPEVIVETIGD-LDITPANFETPSLE------NEELVNLLENESNNLARDVMSQYLIFNNN-----------NTQNAMMEPEVTLSEPSTSYEAVAPDDFAPVFN------ETSLMTLLEEQTNNLTHDVNFELARESTASAFVTG------FRANSLEPEVQLVEQQK-YDGSMPMNMS----------SDSLMNLLEDRSKKLACAVDTELARESTASEFVAG------FSADSPQAQLAETGAETGA WENISLTENMSLTEFDPPIGKEDLETMLQTPCDNLTTLENASQVLANDVGSSLLSEY--LSFNPRAMEPEVQLSEPSTSAASNFLASVNSLTD-------NDLVECLLKTTDNLEEAVSSAYYSESLEQPVVEQPSPSSAYHAQSFEHSAGVNQPSAT-  2. D137E138D141AAA AcMNPV BmNPV CfDEFNPV CfMNPV EppoNPV HycuNPV OpMNPV AngeNPV PlxyNPV  : : : : : : : : :  3. K154R156K160K161AAAA  --GTKRKLDEYLDNSQ----GVVGQ--FNKIKLRPKYKKSTIQSCATLEQTINHNTNICTVASTQEITHYFTNDFAPYLM --GAKRKLDEYLDDSQ----SVVGQ--FNKNKLKPKYKKSTIQSCATLEQTINHNTNICTVASTQEITHYFTNDFAPYLM --GTKRKASEDICVDSDDDFSSRGKKHVNKNKIRPRYKKATVQNHTTLQEEQRYTTEICTVAPAEQIAQYFSQDFSVYLD --SMKRKASELDSDSDSGESS-KGKKRVIKPKMRQRYKKATIQNKTSLTEECNYNTEICTVAPTDQIAEYFKHDFSVYLE --GTKRKGAPDSDNDMEDACK--GKKIVNKSKIRPRYKKATIQDKTTLQEEQRYTTEICTVAPANEIAHYFSQDFSTYLN --GGKRKMSELESDESDSEDSSKGKTLVNKPKMRQRYKKATIQNKTTLTEEQHYSTEICTVAPSEQIAKYFSQDFSVYLD AGGSKRKASEVDSDSDSDDSS-KGKKLVNKPKIRQRYKKATIQNRTSLTEERQYSTEICTVAAPDQIAKYFAQDFSAHLN --GTKRKASEDIDVDSDDDFR--GKKHVNKNKIRPRYKKATVQNHTTLQEEQRYTTEICTVAPAEQIAQYFSQDFSVYLD --GTKRKLDEYLDNSQGVVGQ------FNKIKLRPKYKKSTIQSCATLEQTINHNTNICTVASTQEITHYFTNDFAPYLM  136  Figure 3. 4 Co-immunoprecipitation and Western blot confirmation of loss of interaction between AC16 and IE0 or IE1 point mutants. (A) Schematic diagrams of of ie1KO-IE0-AC16HA, ie1KO-IE1-AC16HA, ie1KOIE0L133L134L140AAA-AC16HA and ie1KO-IE1L79L80L86AAA-AC16HA. The schematic diagram for AcBac is shown in Fig. 3.2A and ac16KO-AC16HA is shown in Fig. 3.6. (B) Co-IP results. Sf9 cells were infected by positive control ie1KO-IE0-AC16HA, ie1KO-IE1AC16HA, ie1KO-IE0L133L134L140AAA-AC16HA, ie1KO-IE1L79L80L86AAA-AC16HA and negative control virus AcBac. Cells were collected at 24 hpi for immunoprecipitation with anti-HA agarose beads. Antibodies against IE1 and against HA were used for Western blots. IE1 mt stands for IE1L79L80L86AAA and IE0 mt stands for IE0L133L134L140AAA.  137  Figure 3.4 continued A.  B. IP: anti-HA  AC16HA IE1 +  +  +  +  +  +  -  -  -  IE1 mt  -  -  -  +  -  IE0  +  -  +  -  -  IE0 mt  -  -  -  -  +  input anti-IE1 eluate input anti-HA eluate  138  Figure 3. 5 Mutation analysis of the IE0 and IE1 AC16 binding domain. (A) Schematic diagrams of the bacmid viruses expressing IE0, IE0L133L134L140AAA, IE1 or IE1L79L80L86AAA (B) Comparison of BV production (right panel) and viral DNA replication (left panel) of IE0 and IE0L133L134L140AAA repaired ie1KO viruses, or (C) IE1 and IE1L79L80L86AAA repaired ie1KO viruses. Also shown in all graphs is the control virus AcBac that expresses wild-type levels of IE0 and IE1, and AC16. 50% tissue culture infective dose per ml (TCID50/ml) refers to BV titer and the number of gp41 copies is used as the reference to viral genome copies. The graph shows the average values of two independent samples. Error bars indicate standard error.  139  Figure 3.5 continued  140  Figure 3. 6 Schematic diagram of construction of ac16KO and repair viruses ac16KO-AC16, ac16KO-AC16HA. The ac16 partial knockout was made by replacing the sequence of ac16 promoter to 70 aa by an EM7-zeocin cassette amplified with primers 1434 and 1437 containing the 50 bp flanking sequence F1 and F2 respectively. The right deletion was confirmed by PCR. Primers from the flanking region paired with primers from the zeocin cassette 1430/520 and 1439/1239 amplified fragments of predicted size 452 bp and 1110 bp respectively from the ac16KO but not from the AcBac control, indicating the right insertion of the zeocin cassette. Primer 1440 from the deleted region paired with 1439 only gave amplification from control AcBac not from ac16KO confirming the loss of the desired ac16 region. The ac16KO bacmid was repaired with polyhedrin and gfp (ac16KO-GFPPH), HA-tagged ac16, polyhedrin and gfp (ac16KO-AC16HA) or untagged ac16, polyhedrin and gfp (ac16KO-AC16) at polh locus.  141  Figure 3.6 continued  142  Figure 3. 7 BV production and viral DNA replication analysis of ac16KO, ac16KOAC16 and AcBac. (A) End point dilution analysis of BV production and (B) quantitative real time PCR analysis of viral DNA replication. Sf9 cells were infected by ac16KO (ac16KO-GFP-PH), ac16KO-AC16 and AcBac at an MOI=5. BV supernatant and cells were collected at various times post infection for analysis. The graph shows the average values of two independent samples. Error bars represent standard errors.  143  Figure 3. 8 Western blot analyses of the temporal expression of IE0 and IE1, FP25 and P39. Sf9 cells were infected with ac16KO-GFP-PH, ac16KO-AC16 and control AcBac at an MOI of 5. Infected cells were harvested at the times indicated in the presence of protease inhibitors and analyzed by SDS-PAGE (7.5% gel for IE0/1 and 10% for VP39 and FP25) and Western blot.  144  Table 3. 1 Primers used in this study Number 517  Sequence 5’→3’ ATACGCGAGCTCATGCATATGA  520  CCGGAACGGCACTGGTCAACTT  740  TGGTACCGGTGAATTCGAGACTGCAGGCTC  810  ATAAGAATGCGGCCGCATAATTTCAAATGTT GCGGGAATTCTTAGGCGTAGTCGGGCACGTC GTAGGGGTAATTAAATTCGAATTTTTTATATT TACAA GCGGTCTAGAAGTCACTTGGTTGTTCAC GCGGTCTAGAGGCAGGCGCTGGCAAAGATT ATAAGAATGCGGCCGCCGTTGCCCGTTATCA ATTAC  860 869 870 871 1239  CTGACCGACGCCGACCAA  1369 1371  GCGCCATGGGACTACAAAGACCATGACGG ACAGCGGCCGCTTAGCTGCCGCGCGG  1416  GCGGGATCCATAAGAACCAGCAGTCACGT  1417  GCGGGATCCACGCAAATTAATTTTAACGCGT CG  1424  GCGTCTAGATTAATTAAATTCGAATT  1425 1430 1431 1434  1437  GCGTCTAGAGCCCCTCGATAATAAAAGACAA AAA GCGCTCGAGCTACCTACAAAAAACACATGG GCGCTGCAGTTAGGCGTAGTCGGGCACGTCG TAGGGGTAATAGGCGTTAATATCACTTT TTGTGCGACTGCGCACTTCCAGCCTTTATAA ACGCTCACCAACCAAAGCATTCGGATCTCTG CAGCAC TTGCAAATGCCGCAGTTTCTTTTTATGTACAG ACTGTATCTTATTGAAATCGAGGTCGACCCC CCTG  1439  ATAGTTAATAGCTGTCTACCCGTA  1440  CTCGAGGTGCCAGTAGCAATCAATTT  1464  TACCCCTACGACGTGCC  1465  TTGAATTACTTGACTAACGTCT  1466  TGCACAGCTTTGAATTGTG  1467 1468  ACCCTTGAACAGACAATTAA CATAGTCACTTGGTTGTTCA GCGCTGCAGTTACTTGTCGTCGTCGTCCTTGT AGTC ATAGGCGTTAATATCACTTT  1469 1472  GCGGGATCCACCCTTGAACAGACAATTAATC  1474  GCGGAATTCGAGTCTGTTCAAACGCGCTT  Note 3’ primer of TnSNPV ie1 polyA primer in zeocin facing to ATG for KO PCR detection upper primer for AcMNPV ie1 fusion to eyfp 5' primer of TnSNPV ie1 polyA C-term HA tagged AcMNPV ie1 with EcoRI site Lower primer of Acie1 promoter Upper primer of Acie0 promoter Lower primer of Acie0 promoter primer within zeocin cassette for KO PCR detection C-term fusion of 3xFLAG-6xHis C-term fusion of 3xFLAG-6xHis 5' primer of ie0 with BamHI site for AD fusion 5' primer of ie1 with BamHI site for AD fusion 3' primer of ie0 with XbaI site for AD fusion 3' primer of ie1 with XbaI site for AD fusion 5' primer of ac16 with XhoI 3' primer of ac16 fused with HA tag plus PstI site upper primer flanking ac16  lower primer within ac16 lower primer flanking ac17 for PCR confirmation of deletion upper primer of ac16 with XhoI site upper primer at HA tag to AcIE1 truncation mutants lower primer to make IE1 1-520 lower primer combining with 1464 to make IE1 1-168 truncant upper primer to make IE1 169-582 lower primer to make IE1 169-582 FLAG tag ac16 at the C-terminus upper primer of AcIE1 169-582, pair up with 1473 for AD fusion upper primer of ac16 for BD fusion, pair up with 1431 or1469  145  Number  Sequence 5’→3’  1496  GCGTCTAGATTAGGCGTAGTCGGG  1504  GGATCCTAATTCGATCTC  1505  GACAACAGCTATTCAGAGT  1506 1507 1508 1509 1510 1511  CATCCCACCCCGGATGGA AACTTTTTGGCAAGCGTCAACT ACCACTGATAATCTCGAAG CCTGTTGTGGAGCAACCAT GCTGGTGTGAACCAACCAT AATTCACAAGGTGTGGTGG  1512  AAAAAAAGTGAAGTGAAGCCGTTTG  1513  TCTCAAGATGTGTGCAACGAC  1514  TTTCTTTTGAGCAAGTTGTACGAA  1515  AACAAAAAAAGTACGCTCACGTAC  1518 1519 1520  GCGCTGCAGAATAATTTGAGTGAGCATCGTT CCT GCGCTGCAGGGATCGATATCTGACTAAATCT GCGCTCGAGAATAATTTGAGTGAGCATCGTT CCT  1521  GTTAAACTGGCCCACCAC  1522  AGCAGAATGCTCAAAAGATTC  1523  CGAGTTGACGCTTGCCAAAAA  1524  TTTGAAAGACAATACTATAAAATTGTG  1525  ATTTTCTGCGTTATTATTACTCGC  1526 1527 1528  TTAAAAAAGGTTAAGAAGGAGGAC GTTAACAATTAAATTTAAATTGTCCAC AACGAGGAGCGATTGACTATA  1529  GATAATGCCGAAGAAGCAGTT  1530  AGTGGTCTTGGCCGCACATTCCACTAA  1531  GCGGCAAGCACAATTCAAAGCTGT  1532  GTATTTAGGCGCCAATGCAATTTTGTTAAA  1533  TACTTGGCCAATTCACAAGGTGTG  1534  TGCGGCCAGCTTCCGTTTAGT  1537  GATAATGATTTAGTGGAATGT  1538  ACTGGGCGATGGTTGCTCCAC  1539  AAGGGATTCCGAATAATAAGCAGA  Note lower primer of AcIE1HA for AD fusion upper primer for making pAD-IE1 1-168 further deletion lower primer for making pAD-IE1 22-168 lower primer for pAD-IE1 42-168 lower primer for pAD-IE1 62-168 lower primer for pAD-IE1 82-168 lower primer for pAD-IE1 102-168 lower primer for pAD-IE1 122-168 lower primer for pAD-IE1 142-168 del169-229 within pAD-IE1 1-520, combine with primer 1466 del169-289 within pAD-IE1 1-520, combine with primer 1466 del169-347 within pAD-IE1 1-520, combine with primer 1466 del169-419, combine with primer 1466 Lower primer of Opie2 polyA upper primer of Opie2 polyA Lower primer of Opie2 polyA with XhoI site combine with primer 1464 to delete 151-168aa of IE1 combine with primer 1464 to delete 123-168aa of IE1 combine with primer 1464 to delete 70-168aa of IE1 combine with 1464 to delete 490520 combine with 1464 to delete 454520 upper primer for deletion 420-453 lower primer for deletion 420-453 upper primer for deletion 420-489 upper primer contain mutation L86A lower primer combine with 1529, contain L79L80 to AA upper primer containing K160K161 mutated to AA lower primer combine with 1531, contain R156 to A and K154 to A upper primer contain D141A of ie1 lower primer combine with 1533 contain D137A and E138A lower primer combine with 1504 to make 72-168 (kept)IE1 primer combine with 1464 to delete 111-168 of AcIE1 primer combine with 1464 to delete 100-168 of AcIE1  146  Number  Sequence 5’→3’  1540  TGCTTCTTCGAGATTATCAGTGGT  1541  TTCCACTAAATCATTATCAGTTAA  1542 1543 1544 1545  GCGGCGGCCGCCAGAATTCTTAATTAAATTC GAA GCGGCGGCCGCCGAAGCTTAACATGATAAG AA GCGTCTAGAATGACGCAAATTAATTTTAACG CGTCG GCGGCGGCCGCTTAATTAAATTCGAATTTTT TATATTTACAA  1560  GCGCTGCAGTTAATAGGCGTTAATATCACT  1561  GCGGAGCTCTTTGAGTGAGCATCGTTCCT  Note primer combine with 1464 to delete 90-168 of AcIE1 primer combine with 1464 to delete 78-168 of AcIE1 lower primer of AcIE0 with NotI site upper primer of AcIE0 with NotI site upper primer of AcIE1 with XbaI site lower primer of AcIE1 with NotI site lower primer of ac16 stop codon region with PstI site, combine with primer 1430 lower primer of Opie2 polyA with SacI site, combine with primer 1519  147  3.5 References Beniya, H., Braunagel, S. 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Proteomics 6(3), 927-935.  151  Chapter 4 Deletion of AcMNPV AC16 and AC17 Results in Delayed Viral Gene Expression in Budded Virus Infected Cells but not Transfected Cells3  4.1 Introduction The baculoviridae consists of a large group of insect viruses with circular double stranded DNA genomes that range in size from 80 kb to 180 kb (Miller, 1997). The viruses are divided phylogenically into four genera: alpha-, beta-, gamma- and deltabaculovirus (Jehle et al., 2006). Alpha- and betabaculoviruses infect larvae from lepidoptera; gamma- and deltabaculoviruses infect larvae from hymenoptera and diptera respectively. The infection of baculoviruses is a sequential process that is divided into early and late phases. Baculoviral genes are categorized into early, late and very late genes (Miller, 1997). Early genes are transcribed before viral DNA replication using host RNA polymerase II (Friesen, 1997); whereas late genes are transcribed following or concurrently with the onset of viral DNA replication using the viral RNA polymerase (Fuchs et al., 1983). The appropriate expression and regulation on viral early genes is critical for the success of viral replication.  In this study we investigated the roles of the overlapping AcMNPV gene pair ac16 and ac17 during viral replication. Ac16 is an early gene (O'Reilly et al., 1990) that is one of the 17 genes specific to Group I alphabaculoviruses (Herniou et al., 2001). It encodes the BV and ODV envelope protein and may be involved in ODV-E66 trafficking along with FP25 (Beniya et al., 1998). We have previously shown that AC16 interacts with both AcMNPV IE0 and IE1, suggesting it might play a role in viral transcription or replication, however deletion of AC16 does not affect viral DNA synthesis or BV production but loss of AC16 does result in increased levels of IE0 relative to IE1 (chapter 3) (Nie et al., 2009). BmNPV  3  A version of this chapter will be submitted for publication. Nie Y. and Theilmann DA. Deletion of AcMNPV AC16 and AC17 results in delayed viral gene expression in budded virus infected cells but not transfected cells.  152  BM8 is a homolog of AC16 and has been shown to co-localize in an hr dependent manner with BmNPV IE1 in the nucleus of infected cells (Kang et al., 2005).  HearNPV HA128 is a homolog of ac17 and is suggested to be a gene common to all alphabaculoviruses (An et al., 2005), however further analysis of additional viral genomes revealed that ac17 homologs are not present in the genomes of the closely related TnSNPV and Chrysodeixis chalcites NPV (ChchNPV). Ac17 and its homolog bm9 (from BmNPV) have been reported to be early genes (An et al., 2006; Yang et al., 2009). However the HearNPV homolog, ha128, was reported to be a late gene (An et al., 2005). In addition AC17, HA128 and BM9 are reported to be cytoplasmic proteins (An et al., 2005; An et al., 2006; Yang et al., 2009). The deletion of bm9 had no effect on viral DNA replication, but results in reduced BV production and infectivity (Yang et al., 2009).  To further clarify the functional roles of AcMNPV AC16 and AC17, a double knockout virus was made in which the region containing the overlapping ac16 and ac17 orfs was deleted. The double knockout virus was repaired with either ac16, ac17, or both ac16 and ac17. This approach permitted the analysis of the impact of deleting the genes jointly or deleting them individually. Deletion of AC17 was shown to decrease BV production but did not affect DNA synthesis. Significantly, deletion of both ac16 and ac17 was shown to delay viral gene expression when cells were infected by BV but not by transfection of naked viral DNA. The delay was not observed when either ac16 or ac17 was deleted singly suggesting a synergistic role of ac16 and ac17 immediately upon infection of cells by virions. Transcriptional analysis showed that ac17, in contrast to previous reports is a late gene. In addition, AC17 was also located in both the cytoplasm and nucleus at late times post infection.  4.2 Results 4.2.1 Transcriptional analysis of ac17 To design viruses that contain deletions of ac17 it was necessary to identify the transcriptional start site of ac17. A previous study using RT-PCR concluded that ac17 was 153  an early gene however the design of that experiment could not distinguish between the overlapping ac16 transcript and the ac17 transcript (An et al., 2006), as it has been showed that the early gene ac16 transcription terminates downstream of the ac17 orf (Guarino and Summers, 1988). The HearNPV homolog ha128 has also been transcriptionally analyzed and is reported to be a late gene (An et al., 2005).  Rapid amplification of cDNA ends (RACE) analysis was performed to identify the transcription start and termination sites of ac17. At 4 hpi, the ac17 gene specific primer pair produced a single specific 5’RACE product of approximately 0.77 kb that was mapped to the reported CATT early promoter for ac16 (Burks et al., 2007) (Fig. 4.1). At 24 hpi the ac17 gene specific primer pair produced a major 5’RACE of approximately 0.52 kb that when sequenced mapped to the baculovirus late promoter ATAAG, 430 bp upstream of the start codon of ac17 (Fig. 4.1A). These results indicate that ac17 is a late gene and is transcribed with a leader sequence that incorporates two thirds of the ac16 orf. Analysis of the leader sequence reveals the presence of two potential mini-cistrons of 27 and 30 nt (Fig. 4.1B). Previous studies have shown that mini-cistrons in baculovirus leader sequences can regulate the expression of downstream orfs (Chang and Blissard, 1997). The late expression of ac17 is consistent with the expression of the HearNPV ha128 (An et al., 2005). Interestingly there is a second late promoter motif (GTAAG) closer to the ac17 orf however no RACE product was mapped to this location (Fig. 4.1A).  The 3’ RACE produced a major 0.65 kb band specific to virus infected cells at both 4 and 24 hpi. Sequence of this band showed that the transcription terminated at 11 bp downstream of a predicted polyadenylation signal AATAAA in which the TAA is also the stop codon of ac17 (Fig. 4.1C). As the 3’ RACE product is observed at both 4 and 24 hpi it would indicate that the ac17 and ac16 transcripts utilize the same termination site producing 1073 nt late transcript and an 1324 nt early transcript not including the poly (A) tail. This would agree with the approximate sizes of the early and late transcripts previously described by Northern blot for this gene region (O'Reilly et al., 1990).  154  4.2.2 Generation of ac16/17KO and repair viruses As shown in Fig. 4.1 ac16 and ac17 are intimately associated orfs that overlap. We have recently shown that ac16 could be deleted and viable virus could be recovered. This result is in contrast to previous studies which suggested that this gene was essential (Burks et al., 2007; Imai et al., 2004; Nie et al., 2009). One possible explanation for these different results is that the previous studies unintentionally interrupted ac17 expression when deleting ac16. To address the question of ac17 function, ac16-ac17double knockout virus was constructed (Fig. 4.2) using the bacmid system and repaired with ac16 or ac17 individually or together in the normal context. The ac16-ac17 double KO (bMON14272ac16/17KO) was constructed via homologous recombination in E. coli as previously described (Datsenko and Wanner, 2000). The region from the ac16 transcription initiation site to the sequence up to and including amino acid 165 of ac17 was replaced with a zeocin resistance gene cassette (Fig. 4.2). The resulting bacmid was confirmed by PCR (data not shown) and named bMON14272-ac16/17KO. To enable observation of virus propagation and occlusion body formation, gfp and polyhedrin were transposed into polh locus of bMON14272-ac16/17KO, generating ac16/17KO. To examine the function of ac17, the double KO virus bMON14272-ac16/17KO was repaired with ac16, ac17, ac16-ac17 or ac16-ac17HA via transposition along with gfp and polyhedrin. The viruses generated were named ac17KO, ac16KO, ac16/17repair and ac16/17HArepair (Fig. 4.2). The control virus AcBac was generated by repairing the native bacmid bMON14272 with gfp and polyhedrin (Fig. 4.2) as introduced by Dai et al. (Dai et al., 2004).  4.2.3 Analysis of viral DNA replication Transfection of each bacmid DNA showed that all virus constructs replicated and produced BV (data not shown). BV stocks were prepared and used to compare replication properties for each of the viruses. To determine the impact of deleting ac17 or both ac16 and ac17 on viral replication, viruses ac16/17KO, ac17KO, ac16KO, ac16/17repair and AcBac control were used to infect Sf9 cells. Cells were collected for the analysis of viral DNA replication at various times post infection using real-time quantitative PCR (Fig. 4.3). The results showed that deleting both AC16 and AC17 (ac16/17KO) from the viral genome resulted in 155  significantly lower levels of viral DNA replication between 6-48 hpi but similar levels as AcBac were obtained by 72 hpi. The double repair virus ac16/17repair had equivalent viral DNA replication levels as AcBac showing that the observed decrease in DNA replication was due to the loss of ac16 and ac17. The ac17 knockout virus ac17KO, showed similar levels of viral DNA replication as the wild type control AcBac or the double repair virus ac16/17repair. This indicates that deletion of ac17 does not have a detectable impact on viral DNA replication which is similar to the results reported for the homologous BmNPV bm9 gene (Yang et al., 2009). The ac16 knockout virus ac16KO had reduced levels of DNA replication from 6 to 24 hpi but reached normal levels relative to AcBac or the double repair virus ac16/17repair by 48 hpi. This is not exactly the same as what was observed with the single gene knockout backbone virus ac16KO (Nie et al., 2009) which showed no difference in viral DNA replication compared with wild type. One possible reason is that the expression of ac17 is different in the two viruses. These results show that deletion of AC17 alone has no impact on DNA replication, but when deleted in conjunction with AC16 there is a synergistic effect causing an increased reduction in viral DNA replication during the first 6 h to 72 hpi.  4.2.4 Analysis of BV production To investigate the impact of deleting ac17 and ac16 on budded virus production, viral growth curve analysis of ac17KO, ac16KO, ac16/17repair and AcBac was performed. Both of the single gene knockout viruses (ac16KO and ac17KO) had reduced BV production (Fig. 4.4). The deletion of ac17 had greater impact than deleting ac16 with levels being reduced 1-2 logs. Loss of AC16 also resulted in reduced BV levels up to 72 hpi but levels were equivalent to AcBac by 96 hpi. Deletion of both ac16 and ac17 produced the same synergistic negative effect on BV production that was observed with DNA replication. By 72 hpi ac16/17KO BV levels were equal to ac17KO but remained nearly a log lower than AcBac. The reduced BV levels could be related to the lower viral DNA replication level as it shown in the viral DNA replication analysis (Fig. 4.3).  156  4.2.5 Viral gene expression analysis The reduced levels of viral DNA replication in the ac16/ac17 and ac16 knockout viruses outlined above suggests that early viral gene expression may be affected by these deletions. To determine if viral early or late gene expression was affected in cells infected with the ac16/17KO or repair viruses, the temporal expression of early genes (IE0/1, GP64, P35, LEF3, P143) and late, and very late genes (VP39, POLH) were detected by Western blots from cells infected by ac16/17KO, ac17KO, ac16KO, ac16/17repair and control AcBac viruses respectively.  Analysis of ac17KO, ac16KO, ac16/17repair viruses showed no significant differences in the timing of expression of the early genes IE0/IE1, GP64, P143, or P35 (Fig. 4.5), however there was a significant delay in expression in the cells infected by the double knockout virus ac16/17KO. Cells infected by ac16/17KO showed very low level of IE0 at 12 hpi and under detectable levels of IE1 until 12-18 hpi (Fig. 4.5). Additionally the relative levels of IE0 to IE1 were increased, which is similar to what we observed in the ac16 knockout virus, ac16KO (Fig. 4.5) and as previously reported (Nie et al., 2009). The other early proteins analyzed (P143, LEF3, P35 and GP64) also exhibited the same significant delay of expression relative to the control virus AcBac. Similar results were also observed with the late and very late viral proteins VP39 and POLYHEDRIN. This analysis therefore suggests there was a global delay of viral gene expression in cells infected with the ac16/17KO virus.  4.2.6 Transcription analysis of gp64 and ie0/ ie1 The Western blot analyses showed that there was a delay in viral gene expression at the level of translation in ac16/17KO BV infected cells. However this could be due to the delayed transcription initiation of viral genes. To determine if the viral transcription was delayed, we analyzed the expression of three early genes, gp64, ie0 and ie1 for ac16/17KO, ac17KO, ac16KO, ac16/17repair and AcBac. The temporal expression of gp64 was analyzed by Northern blot and the expression of ie0 and ie1 was determined by real time quantitative reverse transcription PCR (qRT-PCR). The Northern blot result (Fig. 4.6A) 157  showed that gp64 mRNA was detected at 4 hpi and increased up to 24 hpi for the AcBac, single repair viruses (ac17KO, ac16KO) and double repair virus ac16/17repair. However in ac16/17KO infected cells gp64 transcription was not detected until 12 hpi (Fig. 4.6A). In addition, the levels of the gp64 transcript from ac16/17KO infected cells were also reduced at 16-24 hpi compared with AcBac, ac17KO, ac16KO and ac16/17repair. These data shows that the delayed expression of GP64 detected by Western blot (Fig. 4.5) was due at least in part to the delayed and reduced transcription of gp64.  The levels of expression from the primary baculovirus transcription factors ie0 and ie1 were measured using qRT-PCR with two sets of primer pairs. The first pair is specific for ie0 and only detects ie0 transcripts (Fig. 4.6B). The second pair detects both ie1 and ie0, since the ie0 transcripts incorporates the entire ie1transcript (Fig. 4.6C). At 4 hpi, the ac17KO, ac16KO and ac16/17repair had ie0 expression levels equivalent to the control AcBac, whereas ac16/17KO had no detectable expression above background (Fig. 4.6B). At 12 hpi ie0 expression from ac16/17KO was detected above background indicating an increase in transcript levels. This is in contrast to the other viruses which all show a decline in ie0 expression between 4-12 hpi. Analysis of combined levels of both ie0 and ie1transcripts (Fig. 6C) showed that ac17KO, ac16KO, ac16/17repair had significant levels of expression at 4 hpi which increased by 12 hpi similar to AcBac. However, for ac16/17KO no detectable expression above background was observed at 4 hpi. At 12 hpi low levels of expression from ac16/17KO were detected but were significantly lower than any of the other viruses. However, the levels detected with the ie1 primers were approximately 5.5 fold higher than ie0 alone, which indicates both ie1 and ie0 are being expressed by 12 hpi for ac16/17KO. These results are similar to the Northern blot results of gp64 and show that deletion of both ac16 and ac17 results in a significant delay in the transcription of the essential immediate early genes ie0 and ie1.  158  4.2.7 Localization of AC17 To determine the subcellular localization of AC17, Sf9 cells infected by ac16/17HArepair were collected at 6, 12, 24, 48, 72, 120 hpi. Cytoplasmic and nuclear fractions of infected cells were prepared and subjected to SDS-PAGE followed by Western blot detection with HA antibody. AC17HA was found to locate mainly in the cytoplasm up to 48 hpi but from 72 to 120 hpi it was observed at approximately equal levels in both the nucleus and the cytoplasm (Fig. 4.7A). This result differs from previous studies which reported that AC17, a truncated AC17-GFP fusion protein and the homologue HA128 were mainly found in the cytoplasm or at the nuclear envelope (An et al., 2005; An et al., 2006). To confirm our Western blot results that AC17 was observed in both the cytoplasm and the nucleus we used confocal microscopy to analyze AC17 cellular localization. A virus expressing AC17 (full length) fused to YFP was constructed and used to detect AC17 by direct immunofluorescence in infected cells. In addition, indirect immunofluorescence was attempted using a virus expressing AC17-HA. However, the signals for AC17-YFP and AC17-HA were similar to background levels and therefore reliable localization could not be determined. The low level of expression from each virus may have been due to the native ac17 late promoter used to drive expression of AC17-YFP or AC17-HA, which contains two mini-cistrons. In order to increase the expression level of AC17 a third virus AcBac-AC17HA was constructed. This virus expresses AC17-HA under the control of polyhedrin promoter. Under these conditions detectable levels of AC17-HA were obtained (Fig. 4.7B). At 72 hpi AC17 is detected in both the cytoplasm and the nucleus similar to results obtained using the Western blot. The nuclear AC17 was localized on the periphery of the virogenic stroma and potentially had enhanced signal at the nuclear envelope.  To determine if AC17 is a component of BV, virions were purified from the supernatant of ac16/17HArepair infected Sf9 cells and the proteins were subjected to SDS-PAGE and Western blot analyses. BV purified from AcBac was used as an HA negative control and antibodies against VP39 and GP64 were used as markers for BV purification (Fig. 4.7C). The results showed that AC17 is associated with BV and therefore may be a structural protein.  159  4.2.8 Expression of viral proteins in transfected cells Analysis of the transcription of ie0/ie1 and gp64 revealed that the deletion of ac16–ac17 resulted in a delay and reduction of transcription. Western blot analysis (above) and previous studies showed that both AC16 and AC17 are found in the BV particle. Therefore as structural components of BV it is possible that they are able to accelerate gene expression upon infection. If this is the case, no delay in viral gene expression between ac16/17KO and AcBac should be observed if the viral genome is delivered to the nucleus as naked DNA by transfection. To test this hypothesis, we transfected Sf9 cells with bacmid DNA of ac16/17KO, ac16KO, ac16/17repair and control AcBac. Cells were collected at 4, 7, 10, 13, 16 and 24 hpt and total cell proteins were analyzed by Western blot for the expression of the early proteins IE0/IE1 and the late structural capsid protein P39. The results showed that there was no difference in the temporal gene expression of either the early proteins IE0 and IE1 or the late protein P39 between AcBac and the double KO ac16/17KO (Fig. 4.8). The same result was also observed for ac16KO and ac16/17repair (Fig. 4.8). This fundamental difference between viral infection and nucleic acid transfection indicates that AC16 and AC17 are structural component of the BV and are able to accelerate gene expression during the BV infection.  4.3 Discussion In this study, the analysis of an ac17 knockout virus has shown that AC17 is required for the efficient production of BV. In the absence of AC17 BV yields are reduced approximately 10 fold however viral DNA levels are unaffected (Fig. 3 and 4). This would therefore suggest that AC17 affects post-replication events such as the efficiency of assembly of nucleocapsids or nucleocapsid egress and budding. A number of baculovirus genes have been reported to affect BV production but do not appear to impact viral DNA synthesis, including gp64, f- protein, gp41, exon0, pp31, ac66, vlf-1, 38K, BV/ODVC42(ac101), ac142, ac143 and me53 (Dai et al., 2004; de Jong et al., 2009; Fang et al., 2007; Ke et al., 2008; McCarthy et al., 2008; McCarthy and Theilmann, 2008; Monsma and Blissard, 1995; Monsma et al., 1996; Olszewski and Miller, 1997; Oomens and Blissard, 1999; Pearson et al., 2000; Vanarsdall et al., 2004; Vanarsdall et al., 2007; Wu et  160  al., 2006; Yamagishi et al., 2007). The deletion or mutation of ac142, ac143, gp41, vlf-1, 38k, ac101, gp64, f-protein abolished infectious BV production (McCarthy et al., 2008; McCarthy and Theilmann, 2008; Olszewski and Miller, 1997; Oomens and Blissard, 1999; Vanarsdall et al., 2004; Vanarsdall et al., 2007; Westenberg and Vlak, 2008; Westenberg et al., 2002; Wu et al., 2006). Whereas deletion of pp31, exon0 and me53 results in reductions of BV production by 100 to 1000 fold (Dai et al., 2004; de Jong et al., 2009; Yamagishi et al., 2007). Three genes vlf-1, 38K, and ac101 have been shown to impair the assembly of nucleocapsids (Vanarsdall et al., 2004; Vanarsdall et al., 2007; Wu et al., 2006). Loss or reduced BV production in the absence of GP41, EXON0, AC66 or AC142 however is suggested to be due to the compromised nucleocapsid transport from the nucleus to the cytoplasm (Fang et al., 2007; Ke et al., 2008; McCarthy et al., 2008; Olszewski and Miller, 1997). Loss of GP64 or F-protein affects the budding of nucleocapsids directly at the plasma membrane (Monsma et al., 1996; Oomens and Blissard, 1999; Westenberg and Vlak, 2008; Westenberg et al., 2002). AC17 in comparison to the above proteins therefore appears to be more of an auxiliary factor for BV production. The 10 fold higher levels of BV achieved in the presence of AC17 represent a large increase in the number of virions produced per cell and could have a potentially significant impact in vivo enabling rapid systemic infection in lepidopteran larvae. Given the viability of the ac17KO BV, it is unlikely that the assembly of nucleocapsids are severely impaired in the absence of AC17. However, it is still possible that the loss of AC17, a BV associated protein, could result in structurally compromised nucleocapsids, which have reduced stability, inefficient transport or budding. The precise nature of the AC17 in BV assembly and production however, remains to be determined.  In addition to the impact on BV production the deletion of ac17 in combination with ac16 had a dramatic impact on the virus life cycle compared to the deletion of either gene independently. This includes significantly delayed early and late viral gene expression, a further reduction in BV production and decreased levels of viral DNA replication. The most intriguing result however was the approximately 6 to 12 hours delay in early and late gene expression when cells were infected with ac16/17KO BV. However, when cells were infected by transfecting viral DNA no difference in gene expression was observed, which indicates that the delay is specific to the BV infection process and that AC16 and AC17 are 161  required for the rapid start of viral gene expression. Interestingly no delay is observed with the single gene deletions indicating a synergistic impact on the virus life cycle when both genes are absent. AC17 is expressed late and as a result de novo ac17 expression could not affect the early events that occur upon BV infection. However, both AC16 and AC17 are found in the BV particles (Fig. 7; (Beniya et al., 1998)) and therefore could influence the early events of the infection process by being introduced into the cells by the virion. The delay of viral gene expression in cells infected but not transfected with ac16/17KO would suggest a defect in the BV entry, nucleocapsids transport and uncoating or recruitment of the RNA Pol II to early viral promoters. Entry may be affected but previous studies have clearly shown that GP64 is required for BV binding to target cells and low-pH-dependent membrane fusion (Blissard and Wenz, 1992; Monsma and Blissard, 1995; Oomens and Blissard, 1999). After fusion of the BV envelope with membranes of a late endosome, nucleocapsids are released and traverse the cytoplasm. AC16 is involved in the sorting of ODV envelope proteins to the inner nuclear membrane (Braunagel et al., 2009) and is also reported to share homology to tropomyosin (Beniya et al., 1998). Potentially AC16 could facilitate the endosome-virion envelope fusion and release of nucleocapsids, which then induce the formation of F-actin bundles that are thought to enable the transport of nucleocapsids to the nucleus (Lanier and Volkman, 1998). Possible functions of AC17 could be attachment of nucleocapsids at the nuclear pore complex, uncoating and release of the viral genome into the nucleus. Once in the nucleoplasm, AC16 could also be involved in enabling access to the viral early promoters by the host RNA Pol II system as we and others have shown that AC16 binds the major viral transcriptional transactivators IE0 and IE1 and therefore could be directly affecting transcription initiation (Kang et al., 2005; Nie et al., 2009).  Comparison of all baculovirus genomes sequenced to date has shown that AC17 is conserved in all the alphabaculoviruses reported with the exception of TnSNPV and ChchNPV. Although ac16 is reported to be one of 17 genes specific to Group I alphabaculoviruses, there is an ORF present at the same locus as ac16 and overlaps with ac17 homologs in the group II alphabaculoviruses. It is possible that the Group II proteins could be functional homologs of AC16. Analyses show that the predicted protein products of these ORFs from group II alphabaculoviruses also contain a similar predicted structure 162  as AC16. This includes the N-terminal predicted coiled-coil structure which is essential for the binding of AC16 to IE0 and IE1 (Kang et al., 2005; Nie et al., 2009). Therefore the ac16-ac17 gene pair and their homologs could be playing a critical role in accelerating the early events of most alphabaculovirus infections. The acquisition of this gene cluster compared to beta-, gamma- and deltabaculoviruses may have contributed to the diversification of alphabacululoviruses.  4.4 Materials and methods 4.4.1 Viruses and cells Cells and viruses were maintained as described in Chapter 2.  4.4.2 Plasmid construction The ac17 orf was amplified with 1558 (5’GCGTCTAGACGCACTTGAATTTCAATAAG-3’) and 1559 (5’GCGGAGCTCCTAACAATACATTTATTTAAATTT-3’) using AcMNPV genomic DNA as the template and cloned into pFAcT-GFP at XbaI/SacI sites, generating pFAcT-GFPAC17. To tag ac17 with HA epitope, primer pair 1558/1781 (5’GCGGCGGCCGCTTAGGCGTAGTCGGGCACGTCGTAGGGGTATTTAAATTTAAA AATAAATAAAATAGT-3’) were used to amplify ac17 and the fragment was cloned into pFAcT-gfp-Tnie1pA at XbaI/NotI, generating pFAcT-GFP-AC17-HA. The ac17-HA cassette was further subcloned into pFAcT at XbaI/SacI to produce pFAcT-AC17-HA. To drive ac17-HA with polyhedrin promoter, ac17 was also amplified with primers 1818 (5’GCGGGATCCATGAATCTCAAAGTGATATTAACGC-3’) and 1783 (5’GCGGAGCTCTTAGGCGTAGTCGGGCACGTCGTAGGGGTATTTAAATTTAAAAA TAAATAAAATAGT- 3’) and subsequently cloned into pFastbacI at BamHI/SstI sites, generating pFastbac-AC17HA. The fragment containing both ac16 and ac17 was amplified with primer pair 1430 (5’-GCGCTCGAGCTACCTACAAAAAACACATGG-3’) and 1477 (5’-GCGGAGCTCAAGCGGTTTATGTCATGTAT-3’) using AcMNPV genomic DNA as the template, and the PCR product was cloned into pFAcT-GFP at XhoI/SacI,  163  generating pFAcT-GFP-AC16/17. The ac16/17 fragment was also amplified with primer 1430/1781 and cloned into pFAcT-GFP-Tnie1pA generating pFAcT-GFP-AC16-AC17HA.  4.4.3 Virus construction 4.4.3.1 Construction of ac16/17 double KO AcMNPV bacmid. AcMNPV bacmid (bMON14272) was used to generate an ac16/17double knockout virus by recombination in E. coli as previously described (Datsenko and Wanner, 2000; Hou et al., 2002). A zeocin resistance cassette with ac16/17 flanking regions was amplified using primers 1434 (5’-TTGTGCGACTGCGCACTTCCAGCCTTTATAAACGCTCACCAAC CAAAGCATTCGGATCTCTGCAGCAC-3’) and 1438 (5’-ATTTTTTTTATTAATATT ATAATTTTTATCTACCTTTATAAATTTTACTACATCGAGGTCGACCCCCCTG-3’) with p2ZeoKS as the template. These primers contain 50 bp homologous sequences to the 5’ flanking region of ac16 and 50 bp homologous region to the 3’ of ac17. The PCR fragment of zeocin resistance cassette was purified and electroporated into E. coli BW25113-pKD46 cells which contained the AcMNPV bacmid bMON14272. The electroporated cells were incubated at 37oC for 4 h in 1 ml of LB medium and plated onto LB agar medium containing 25 µg/ml of zeocin and 50 µg/ml of kanamycin. Plates were incubated at 37oC overnight and colonies resistant to both zeocin and kanamycin were selected and further confirmed by PCR. Three different pairs of primers were used to confirm the correct knockout of ac16/17 had been produced. Primers 1430/520 (5’CCGGAACGGCACTGGTCAACTT-3’) and primers 1239 (5’-CTGACCGACGCCGA CCAA-3’) and 1439 (5’-ATAGTTAATAGCTGTCTACCCGTA-3’) were used to detect the correct insertion of the zeocin cassette and primer pair 1440 (5’-CTCGAGGTGCCAG TAGCAATCAATTT-3’) and 1439 was used to confirm the deletion of the desired sequence. One recombinant bacmid confirmed by PCR was selected and named bMON14272-ac16/17KO.  4.4.3.2 Construction of ac16/17KO and repair bacmids containing polyhedrin and gfp. To generate the ac16/17KO and repair viruses with Polyhedrin and GFP, pFAcT-GFP was inserted into bMON14272-ac16/17KO by transposition as previously described (Luckow et al., 1993) to make ac16/17KO; pFAcT-GFP-AC16, pFAcT-GFP-AC17 and pFAcT164  GFP-AC16-AC17-HA vectors were used to transpose bMON14272-ac16/17KO to generate ac17KO, ac16KO and ac16/17HArepair respectively. To enable the immunofluorescence confocal analysis of AC17, pFastbac-AC17HA was used to transpose bMON14272, producing AcBac-AC17HA.  4.4.4 Time course analysis of viral DNA replication and BV production Sf9 cells (2.0 x 106 cells/35mm diameter well of a six-well plate) were infected by AcBac, ac16/17KO, ac17KO, ac16KO, ac16/17repair, respectively at a MOI of 5 in duplicate. For BV production analysis, at various hours post infection, supernatant containing BV was collected and stored at 4 oC after the cell debris was removed by centrifugation at 8000 x g for 5 min. For viral DNA replication analysis, infected cells were washed once with PBS, scraped off with rubber policemen, pelleted by centrifugation at 2000 x g for 5 min and cell pellets were stored at -80oC until analyses. BV titers and viral DNA synthesis were analyzed by qPCR as described in Chapter 3.  4.4.5 Northern blot Sf9 cells were infected by ac16/17KO, ac17KO, ac16KO, ac16/17repair and AcBac at MOI=5 and cells were collected at 6, 12, 16, 24 hpi. Total RNA was extracted from Sf9 cells using Trizol (Invitrogen). 10 µg of total RNA from each sample were separated on a 1 % formaldehyde gel and blotted and hybridized with -32P labeled single stranded RNA gp64 probe (Fourney et al., 1988). The blot was visualized by exposure to Perkin Elmer Multisensitive Phosphorscreens, which was scanned using a Cyclone Phosphor Imager (Perkin Elmer) and analyzed with Optiquant Acquisition and Analysis Software V5.0 (Perkin Elmer). For the synthesis of a strand specific probe, a gp64 fragment of 300 bp was amplified using primer 1867 (5’-TCATAATACGACTCACTATAGGGTCAG CTCCTCT TGAATATGCA-3’) containing the T7 promoter sequence (underlined) and gp64 homologous sequence and primer 1868 (5’-GTATGATTCTCAAACAAAAGTCTACG3’). The probe was labelled with -32P-UTP and synthesized using GeneScribeTM T7 RNA probe kit (USB).  165  4.4.6 Quantitative RT-PCR Total RNA was extracted using Trizol (Invitrogen) from Sf9 cells infected at an MOI of 5 by ac16/17KO, ac17KO, ac16KO, ac16/17repair and control AcBac collected at 4 hpi and 12 hpi. To synthesize cDNA, 5 µg of total RNA was used for the reverse transcription using Superscript III (Invitrogen) following the manufacture’s protocol. Background was obtained by omitting the Superscript III reverse transcriptase in the cDNA synthesis reactions with total RNA of 4 hpi, and was substracted from each sample values in the analysis. For qPCR, the synthesized cDNA was diluted 5 times with sterilized distilled water before using as templates for the qPCR. Series of amounts of an ie0 expressing plasmid pAcie0delta (Huijskens et al., 2004) was used as the standard for the qPCR. The qPCR reaction was set up using the SYBER green qPCR kit (DyNAmo HS SYBR Green qPCR Kit, New England Biolabs) with primers 1446 (5’-CCATATTCGTG CGAGG CAACG-3’) and 1414 (5’-GGTGTACGACGCGTTAAAAT-3’) for ie0 and 1505 (5’GACAACAGCTATTCAGAGT-3’) and 1523 (5’-CGAGTTGACGCTTGCCAAAAA-3’) for ie1. The program setting for the qPCR was one cycle of 95oC for 15 min, 40 cycles of 95oC for 30 sec, 52oC for 24 sec, 72oC for 30 sec; 1 cycle of 95 oC for 60 sec, 41 cycles of 55oC for 30 sec. The results were analyzed by MX4000 software (Stratagene).  4.4.7 RACE To map the transcription start site for ac17, total RNA was extracted using RNeasy kit (Qiagen) from mock Sf9 cells or cells infected with AcMNPV-E2 virus and collected at 4 hpi and 24 hpi. 5 µg total RNA was used to generate cDNA using the GeneRacer Kit (Invitrogen) following the manufacture’s protocol. For 5’ RACE PCR, gene specific primer 1 (GSP-1) 1731 (5’-AGCCATCTACAATAATCA-3’) was paired with GeneRacer 5’primer for the initial amplification. GSP-2 1732 (5’-GCGGGATCCTGGCATTATGGT AATGCG-3’) was paired with GeneRacer 5’ nested primer to specifically amplify the ac17 transcript. Another gene specific primer 1432 (5’-GCGCTGCAGTTTGAAAGG  166  TGAGGAAGA-3’) was paired with GeneRacer 3’ primer for the 3’ RACE analysis. The PCR RACE products were cloned and sequenced with M13 forward and reverse primers.  4.4.8 Western blot analysis Total cell protein collected during time course analyses or purified BV were mixed with 4×SDS-PSB and boiled for 10 min. Protein samples were separated by 10% or 7.5% SDSPAGE (Laemmli, 1970) using a Bio-Rad Mini-Protean II and transferred to Immobilon-P membrane (Millipore) using a liquid transfer apparatus (Bio-Rad). Western blot hybridizations were performed following the standard protocol (Harlow and Lane, 1988). Membranes were probed with one of the following antibodies: Mouse monoclonal HA antibody 1:1000 (Covance, HA11); Mouse monoclonal IE1 antibody 1: 8000 (Ross and Guarino, 1997); Mouse monoclonal OpMNPV VP39 antibody (1:3000) (Pearson et al., 1988); Mouse monoclonal GP64 antibody 1:250 (Hohmann and Faulkner, 1983); Mouse monoclonal OpMNPV POLH antibody 1:10000 (Quant et al., 1984); Rabbit polyclonal anti-AcMNPV LEF3 antibody 1:2000 (Chen et al., 2004); Rabbit polyclonal antiAcMNPV P143 antibody 1:2000 (Ito et al., 2004); Rabbit polyclonal AcMNPV P35 antibody 1:1000. To detect bound primary antibodies membranes were incubated with 1:10000 goat anti-mouse or goat anti-rabbit peroxidase-conjugated secondary antibodies. Enhanced Chemiluminescence System (ECL, Perkin Elmer) was used to visualize bound antibodies.  4.4.9 Immunofluorescence Sf9 cells infected by AcBac-AC17HA at 72 hpi were washed once in PBS and fixed with in 3.5% paraformaldehyde in PBS for 15 min. The fixed cells were washed three times in PBS for 5 min, followed by permeabilization in 0.15% Triton X-100 in PBS for 20 min. Cells were then blocked for 60 min in 2% bovine serum albumin in PBS and incubated with anti-HA antibody (1:100, HA11, Covance). After three washes in PBS, cells were incubated with Alexa 635 conjugated goat anti-mouse IgG (Molelular Probes) for 60 min  167  followed by staining with 200 ng/ml DAPI (Sigma) for 2 min and examined with a Leica confocal microscope after three washes in PBS.  4.4.10 BV purification Sf9 cells were infected at a MOI of 0.1 at a cell density of 2×106/ml with ac16/17HArepair or AcBac in two spinner flasks for each viruses. At 4 days post infection, the BV supernatants (80mL) were harvested. The purification of BV was performed as previously described (O'Reilly et al., 1992). The supernatant was cleared of cell debris by centrifugation at 8000 x g (Beckman F50C rotor) for 10 min, followed by centrifugation for 60 min at about 100,000 x g (28,000 rpm) in Beckman SW28 rotor. Pelleted BV was resuspended in 1 ml PBS with 1% protease inhibitor cocktail (Sigma) and loaded onto a 25%-65% sucrose (w/w) gradient and centrifuged for 90 min in a SW41 (Beckman) at 80,000 x g (28000 rpm). The BV band was collected followed by centrifugation for 60 min at 100,000 x g (28,000 rpm) in Beckman SW28 rotor and the pellet was cleared of sucrose by washing in PBS. Purified BV was resuspended in 120 µl of PBS with 1% protease inhibitor cocktail, and 15 µl was used for SDS-PAGE and Western blot analysis.  168  Figure 4. 1 5’ and 3’ RACE analysis of ac17 transcription. Sf9 cells were infected by AcMNPV E2 virus and collected at 4 hpi and 24 hpi for total RNA extraction along with mock infected Sf9 cells. (A) Agarose gel analysis of ac17 5’ RACE products at 4 and 24 hpi. Sizes of products are shown on the right in kb. M, mock infected cells. Numbers on the left show sizes of markers in kb. The schematic below the gel shows the ac16 and ac17 ORFs and the location of potential transcription start sites and the location of the transcribed ac16 and ac17 transcripts (arrows). (B) Location of the ac17 transcription start site as determined by 5’ RACE. The arrowhead shows the initiation site of ac17 transcription and the baculovirus late promoter motif (ATAAG) and ac17 translation start codon (ATG) are shown in bold. Two mini-cistrons within the UTR are underlined with the predicted translation products shown underneath. (C) 3’ RACE analysis of ac17 transcription. Both the sequencing result of the 3’RACE products and the AcMNPV genome sequence are shown. The canonical polyadenylation (AATAAA) is shown in bold and the ac17 stop codon is in bold and underlined. Transcription of ac17 was found to terminate 11 bp downstream of the polyadenylation site.  169  Figure 4.1 continued A. M  4  24  1.65 1.00 0.85 0.65 0.50  0.77 0.52  T AAG AAG TT CA CAG AT GT  ac16  AATAAA AATAAA ATG  ac17 4 and 24 hpi 24 hpi  B. ac17 transcription start site | AATAAGATACAGTCTGTACATAAAAAGAAACTGCGGCATTTGCAAAATTTGCTAAGAAAAAAGAACGAAATTA TTGCCGAGTTGGTTAGAAAACTTGAAAGTGCACAGAAGAAGACAACGCACAGAAATATTAGTAAACCAGCTCA TTGGAAATACTTTGGAGTAGTCAGATGTGACAACACAATTCGCACAATTATTGGCAACGAAAAGTTTGTAAGG AGACGTTTGGCCGAGCTGTGCACATTGTACAACGCCGAGTACGTGTTTTGCCAAGCACGCGCCGATGGAGACA M E T AAGATCGACAGGCACTAGCGAGTCTGCTGACGGCGGCGTTTGGTTCGCGAGTCATAGTTTATGAAAATAGTCG K I D R H M K I V A CCGGTTCGAGTTTATAAATCCGGACGAGATTGCTAGTGGTAAACGTTTAATAATTAAACATTTGCAAGATGAA G S S L | M N ac17 start codon  C.  ac17 stop codon | 5’-TTTAAATTTAAATAAATGTATTGTTAGAAAAGTTGTGTTGT-3’AcMNPV 5’-TTTAAATTTAAATAAATGTATTGTTAG(A)n-3’ 3’RACE  170  Figure 4. 2 Construction of ac16/17KO and repair bacmids. The sequence encoding both ac16 and ac17 was replaced by the zeocin resistance gene cassette amplified with primers 1434 and 1438 via homologous recombination in E. coli, generating bMON14272-ac16/17KO. The right deletion were confirmed by PCR using primer pairs 1430/520, 1239/1439 and 1440/1439. The relative positions of these primers are shown on the diagram. Shown in the lower part of the figure are the genes inserted into the polh locus of bMON14272-ac16/17KO to generate the knockout and repair bacmids ac16/17KO, ac17KO, ac16KO, ac16/17repair, ac16/17HArepair. The wild type bacmid (bMON14272) was repaired at the polyhedrin locus with polyhedrin (polh) and gfp to generate AcBac. For confocal microscopy bMON14272 was repaired with ac17-HA under the control of the polyhedrin promoter, generating the virus AcBac-AC17HA.  171  Figure 4.2 continued HA  polh  AcBac-AC17HA  ac17  AcBac  polh  gfp  polh locus bMON 14272 ac16/17 locus  ac16 E ac17 L pA 1434 egt  pA  ac16 1430  ac17  1440  ac18 1438  pA 1434 egt  1439  pA ac18  zeocin 1430  520  1239  1438  1439  ac16/17 locus bMON14272-ac16/17KO polh locus  ac16/17KO ac17KO ac16KO ac16/17repair ac6/AC17HArepair  gfp  polh ac16 E ac16 ac17 L ac17 ac16 E ac17 L ac16 ac16 E ac17 L  ac17  ac16  ac17  HA  Tnie1pA  172  Figure 4. 3 Analysis of viral DNA replication by qPCR. Sf9 cells were infected by ac16/17KO and ac17KO, ac16KO, ac16/17repair and AcBac at an MOI of 5 and collected at various times post infection for the analysis of viral DNA replication using qPCR. The viral genome copies are reflected by the gp41 copies. Each value represents the average of two samples from two independent infections. Error bars represent standard error. The results have been confirmed by one repetition.  173  Figure 4. 4 Growth curve analysis of ac16/17KO and repair viruses. Sf9 cells were infected by ac16/17KO and repairs ac17KO, ac16KO, ac16/17repair and AcBac at an MOI of 5. BV supernatant was collected at various times post infection and titred using qPCR as shown in TCID50/ml values. Each value represents the average of two samples from two independent infections. Error bars represent standard error. The results have been confirmed by one repetition.  174  Figure 4. 5 Western blot analyses of the temporal expression of the viral early and late genes IE0/IE1, GP64, LEF-3, P143, P35, VP39 and POLYHEDRIN. Sf9 cells were infected with ac16/17KO (2), ac17KO (3), ac16KO (4), ac16/17repair (5) and AcBac (1) at an MOI of 5. Infected cells were harvested at the times indicated in the presence of protease inhibitors and analyzed by SDS-PAGE and Western blot.  6  12  IE0-IE1 18 24 48  72  6  96  12  18  LEF3 24 48  1  1  72  6  96  12  18  VP39 24 48  72  96  1  2 2  3  2  4 3  3  5  P143 4  4  1 5  2  5  GP64  3  1  4  2  5  POLYHEDRIN 1  P35  3 4  2  3 1 4  5 2  5  3  4  5  175  Figure 4. 6 Transcriptional analysis of gp64 (A) and ie0 and ie1 (B and C). (A). Sf9 cells were infected with AcBac, ac16/17KO, ac17KO, ac16KO, ac16/17repairat an MOI of 5 and collected for total RNA isolation at 4, 12, 16, 24 hpi. For Northern blot analysis of gp64 expression (A), 10 µg of total RNA was separated on 1% formaldehyde agarose gel, blotted and probed with a strand specific gp64 RNA probe. Below the Northern blot is a picture of the ethidium bromide stained ribosomal bands of the 1% formaldehyde agarose gel. For the qRT-PCR analyses of (B) ie0 and (C) ie0+ie1 transcripts, 5 µg of total RNA from 4 hpi and 12 hpi were used for reverse transcription using Superscript III (Invitrogen). Background was obtained by omitting the Superscript III reverse transcriptase in the cDNA synthesis reactions and was substracted from each sample values in the analysis. A pair of primers specific to ie0 was used for qPCR measurement of ie0 transcript, and a pair of primers that amplifies both ie0 and ie1 was used for the measurement of ie0 and ie1 total transcripts. Each value represents the average of two samples. Error bars represent standard error.  176  Figure 4.6 continued  B.  250 AcBac ac16/17KO ac17KO ac16KO ac16/17repair  ie0 transcript (pg)  200  150  100  50  0 4 C.  12  900 800 700  ie0/ie1 transcript (pg)  hpi  AcBac ac16/17KO ac17KO ac16KO ac16/17repair  600 500 400 300 200 100 0 4  12 hpi  177  Figure 4. 7 Detection of AC17 in infected cells and budded virus. (A) Sf9 cells were infected with ac16/17HArepair at an MOI of 5 and cytoplasmic and nuclear fractions were prepared at the indicated times post infection. Fractions were analyzed by Western blot with anti-HA antibody to detect AC17-HA. To confirm the correct fractionation, the samples were also probed with anti-IE1 and anti-GP64 antibodies. T stands for total cells. (B) Immunofluorescence confocal microscopy analysis of AC17 distribution in Sf9 cells. Cells were infected with AcBac-AC17HA at an MOI of 10. Cells were fixed at at 72 hpi. AC17HA was visualized by incubation with anti-HA followed by incubation with Alexa 635 conjugated goat anti-mouse IgG, and subjected to observation with a Leica confocal microscopy after DAPI staining. (C) Western blot analysis of purified BV for the presence of AC17. BV was isolated from both the virus ac16/17HArepair expressing HA labelled AC17 and AcBac which does not express the HA epitope. Total BV proteins were separted on 10% SDS-PAGE and subjected to Western blot analyses with anti-HA, anti-VP39 and anti-GP64 antibodies.  178  Figure 4.7 continued  6  A.  12  24  48  72  96 120  M  T  C.  cyto  ir pa e r HA 7 c /1 Ba 16 c c a A  anti-HA Nu  anti-HA cyto anti-IE1  anti-GP64 Nu anti-VP39 cyto  anti-GP64 Nu  B.  DAPI  AC17  merge  72hpi  mock  179  Figure 4. 8 Temporal analysis of early and late proteins IE0/IE1 and VP39 in transfected Sf9 cells. Sf9 cells were transfected with 1µg bacmids DNA of ac16/17KO, ac16KO, ac16/17repair and AcBac and total cell proteins were collected at various times post transfection and analysed by 7.5% (anti-IE1) or 10% (anti-VP39) SDS-PAGE and Western blot using the antibody against IE1 and VP39.  180  Figure 4. 9 Comparison of the egt-ac17 gene locus in the genomes of alphabaculoviruses. The egt-ac17 gene locus shows a conservation of gene order with an overlapping ac16 homolog in the group I alphabaculoviruses or an orf that encodes a structurally similar protein in the group II alphabaculoviruses. Exceptions are the closely related TnSNPV and ChchNPV. Arrows in blue and cyan represent egt and ac16 and its homologs respectively. Arrows with blue diagonal lines and red stand for Mamestra configurata nucleopolyhedrovirus (MacoNPV) orf40 and ac17 homologs respectively.  egt  ac16 ac17  ac18  AcMNPV BmNPV  Group I  CfDEFMNPV CfMNPV OpMNPV HycuNPV  AgseNPV AdhoNPV LdMNPV SeMNPV HearNPV MacoNPV-A  Group II  MacoNPV-B TnSNPV ChchNPV SfMNPV SpltNPV  181  4.5 References An, S. H., Wang, D., Zhang-Nv, Y., Guo, Z. J., Xu, H. J., Sun, J. X., and Zhang, C. X. (2005). Characterization of a late expression gene, Open reading frame 128 of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus Arch. Virol. 150(12), 2453-2466. An, S. H., Xing, L. P., Shi, W. J., and Zhang, C. 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A. (2008). Autographa californica multiple nucleopolyhedrovirus ac142, a core gene that is essential for BV production and ODV envelopment. Virology 372(2), 325-339. 184  McCarthy, C. B., and Theilmann, D. A. (2008). AcMNPV ac143 (odv-e18) is essential for mediating budded virus production and is the 30th baculovirus core gene. Virology 375(1), 277-291. Miller, L. K., Ed. (1997). The Baculoviruses. New York: Plenum Press. Monsma, S. A., and Blissard, G. W. (1995). Identification of a membrane fusion domain and an oligomerization domain in the baculovirus GP64 Envelope Fusion Protein. J. Virol. 69(4), 2583-2595. Monsma, S. A., Oomens, A. G. P., and Blissard, G. W. (1996). The GP64 Envelope Fusion Protein is an essential baculovirus protein required for cell to cell transmission of infection. J. Virol. 70, 4607-4616. Nie, Y., Fang, M., and Theilmann, D. A. (2009). AcMNPV AC16 (DA26, BV/ODV-E26) regulates the levels of IE0 and IE1 and binds to both proteins via a domain located within the acidic transcriptional activation domain. Virology 385(2), 484-495. O'Reilly, D. R., Miller, L. K., and Luckow, V. A. (1992). "Baculovirus expression vectors, a laboratory manual." W. H. Freeman and Co., New York. O'Reilly, D. R., Passarelli, A. L., Goldman, I. F., and Miller, L. K. (1990). Characterization of the da26 gene in a hypervariable region of the Autographa californica nuclear polyhedrosis virus genome. J. Gen. Virol. 71(5), 1029-1038. Olszewski, J., and Miller, L. K. (1997). A role of baculovirus GP41 in budded virus production. Virology 233(2), 292-301. Oomens, A. G. P., and Blissard, G. W. (1999). Requirement for GP64 to drive efficient budding of Autographa californica Multicapsid Nucleopolyhedrovirus. Virology 254, 297-314. Pearson, M. N., Groten, C., and Rohrmann, G. F. (2000). Identification of the Lymantria dispar nucleopolyhedrovirus envelope fusion protein provides evidence for a phylogenetic division of the Baculoviridae. J. Virol. 74(13), 6126-6131. Pearson, M. N., Quant-Russell, R. L., Rohrmann, G. F., and Beaudreau, G. S. (1988). P39, a major baculovirus structural protein: Immunocytochemical characterization and genetic location. Virology 167, 407-413. Quant, R. L., Pearson, M. N., Rohrmann, G. F., and Beaudreau, G. S. (1984). Production of polyhedrin monoclonal antibodies for distinguishing two Orgyia pseudotsugata baculoviruses. Appl. Environ. Microbiol. 48(4), 732-736. 185  Ross, L., and Guarino, L. A. (1997). Cycloheximide inhibition of delayed early gene expression in baculovirus-infected cells. Virology 232(1), 105-113. Theilmann, D. A., Willis, L. G., Bosch, B. J., Forsythe, I. J., and Li, Q. (2001). The baculovirus transcriptional transactivator ie0 produces multiple products by internal initiation of translation. Virology 290(2), 211-223. Vanarsdall, A. L., Okano, K., and Rohrmann, G. F. (2004). Characterization of a baculovirus with a deletion of vlf-1. Virology 326(1), 191-201. Vanarsdall, A. L., Pearson, M. N., and Rohrmann, G. F. (2007). Characterization of baculovirus constructs lacking either the Ac 101, Ac 142, or the Ac 144 open reading frame. Virology 367(1), 187-195. Westenberg, M., and Vlak, J. M. (2008). GP64 of group I nucleopolyhedroviruses cannot readily rescue infectivity of group II f-null nucleopolyhedroviruses. J. Gen. Virol. 89(2), 424-431. Westenberg, M., Wang, H., IJkel, W. F., Goldbach, R. W., Vlak, J. M., and Zuidema, D. (2002). Furin is involved in baculovirus envelope fusion protein activation. J. Virol. 76, 178-184. Wu, W., Lin, T., Yu, M., Li, Z., Pang, Y., and Yang, K. (2006). Autographa californica multiple nucleopolyhedrovirus nucleocapsid assembly is interrupted upon deletion of the 38K gene. J.Virol. 80(23), 11475-11485. Yamagishi, J., Burnett, E. D., Harwood, S. H., and Blissard, G. W. (2007). The AcMNPV pp31 gene is not essential for productive AcMNPV replication or late gene transcription but appears to increase levels of most viral transcripts. Virology 365(1), 34-47. Yang, Z. N., Xu, H. J., Thiem, S. M., Xu, Y. P., Ge, J. Q., Tang, X. D., Tian, C. H., and Zhang, C. X. (2009). Bombyx mori nucleopolyhedrovirus ORF9 is a gene involved in the budded virus production and infectivity. J. Gen. Virol. 90(1), 162-169.  186  Chapter 5 General Discussion and Future Perspectives The major AcMNPV transcriptional transactivators, IE0 and IE1, are produced from the only known baculovirus spliced gene complex. IE0 and IE1 are essential for virus replication and the objective of this study was to investigate and identify functional differences between these two proteins. Understanding the individual roles that IE0 and IE1 play during the virus life cycle will enhance the understanding of baculovirus pathology, and potentially lead to improved exploitation of baculoviruses for their multiple uses in biocontrol, protein expression and gene therapy.  Direct comparisons of IE0 and IE1 subcellular localization and ability to transiently transactivate viral early promoters were performed (Chapter 2). Confocal microscopy using IE0ECFP and IE1EYFP as well as immunofluorescence showed IE0 and IE1 had similar distribution in Sf9 cells during infection. Both IE0 and IE1 formed foci in the nucleus at early stages of infection, which expanded and occupied almost the entire nucleus later in infection. However at early times post-infection when IE0 and IE1 are analyzed individually, it appeared that the foci structures of IE0 are smaller in volume and fewer in number. But to confirm this, a quantitative analysis is needed. It is not clear if the a smaller volume and fewer number of IE0 discrete structures correlates with the lower levels of DNA replication and BV production in viruses expressing only IE0 (Stewart et al., 2005). A further analysis on the localization of other viral replication proteins HELICASE, DNA Pol, LEF-1, LEF-2, LEF-3 and DBP to the IE0 and IE1 structures may provide additional information on the role of these two proteins in viral DNA replication.  This is the first study to examine IE0 localization during infection in the presence or absence of IE1. The results support the reported functions of IE0 in enabling viral DNA replication and production of infectious progeny virions. It also excludes in general the  187  possibility that IE0 might regulate the function of IE1 by sequestering IE1 to different cellular compartments via dimerization, though it still might be possible that IE0 and IE1 locate to different microcompartments which can not be distinguished by confocal microscopy. Additionally, from 4 to 6 hpi (or even earlier), when the level of IE0 are below the sensitivity of confocal microscopy IE0 may play critical roles and there still exists the possibility that there are differences in cellular localization of dimers of IE0-IE0, IE0-IE1 and IE1-IE1.  To identify differences between IE0 and IE1 as transcriptional transactivators, transient assays and a global analysis by microarray (Dr. Junya Yamagishi and Dr. Gary Blissard) were conducted. Both methods however did not identify any viral promoter that is specifically regulated by IE0. Nevertheless, IE0 only activated a subgroup of the genes that were regulated by IE1. Even though the studies presented here suggest that IE0 and IE1 are equally strong transactivators, the fact that IE0 only appears to stimulate a subset of genes suggests that there may be subtle differences in promoter recognition that are not detected by transient assays. It is possible that IE0 preferentially activates a set of genes at early stage of infection to favour the taking place of viral replication. Two of the genes, ie1 and lef3, identified in the microarray analysis are known to be involved in viral DNA replication.  As previously reported (Lu and Carstens, 1993; Rodems and Friesen, 1993), hr elements cis-linked to viral promoters dramatically enhance the transactivation of IE0 on most genes analyzed by transient assays. However in the presence of cis-linked hr5, IE0 and IE1 showed repression of the me53 promoter (Fig. 2.4A). This is the first reported case that IE0 and IE1 transactivation on baculoviral gene is inhibited by the hr5 element. Additionally IE0 was previously reported to be unable to transactivate 39K in the absence of hr enhancer (Kovacs et al., 1991). However, this study clearly shows that both IE0 and IE1 can activate 39K to similar levels when equal amounts of protein are expressed. Even though there does not seem to be differences in the viral genes activated by IE0 and IE1, a possible alternate method to reveal genes specifically regulated at certain stage of infection would be transient analyses, while co-expressing IE0 and IE1 at ratios equivalent to those observed during a wild type virus infection. Using constructs in which ie0 and ie1 are both 188  driven by the same promoter to express equal levels may also provide a better picture of the transcriptional transactivation capacity of IE0 and IE1. Attempts were made to identify the critical residues for IE0 function within the IE0 N-terminal 54 aa. The alignments of known and predicted IE0 N-terminal specific sequences showed no conservation among the alphabaculoviruses, but did reveal some conserved residues among Group I alphabaculoviruses (Fig. 2.6). Deletion of the N-terminal residues reduced the ability of IE0 to transactivate 39K, and reduced viral DNA replication (Fig. 2.8, Fig. 2.9). It is possible that the extra residues at N-terminal of IE0 forces a different tertiary conformation compared with IE1 so that the protein acquires different binding capacity for its interaction partners. Alternatively, other binding domains were formed for specific interaction partners that may be unique to IE0 but which yet remain to be identified.  One of the most interesting findings in this study was the observation that the virus expressing only IE0 has a greater proportion of ODVs that contain only a single nucleocapsid per envelope. The virus that expresses only IE1 has a greater number of multiples but the proportion of single nucleocapsids per envelope was also higher compared to WT. This result suggested that the ratio of IE0 to IE1 is a major determining factor of the number of nucleocapsids that get packaged per ODV envelope. It is not clear how baculoviruses determine nucleocapsid numbers to be packaged into an ODV envelope but a recent study did show that an ac23-null virus produces higher percentage of ODVs with a single nucleocapsid (Yu et al., 2009). It is therefore possible that the increased number of ODVs with a single nucleocapsid for viruses expressing only IE0 or IE1 may be due to an indirect effect from changes of expression of other viral genes such as ac23. This however remains to be determined and initial experiments comparing expression levels of proteins such as AC23 in viruses expressing only IE0 or IE1 with WT virus may enable the identification of viral genes involved in ODV packaging.  To further understand the functional differences between IE0 and IE1, it is essential to identify the proteins with which they interact. Towards this goal a TAP purification approach was performed to identify proteins that interact with IE0 and IE1 (Chapter 3). The TAP method has been reported to be very efficient and sensitive in the isolation of protein complexes (Yang et al., 2006), however in this study significant levels of non189  specific binding were observed. Nevertheless, some specific bands were identified that copurified with both IE0 and IE1, but no band was observed that was specific for either protein. One band with increased intensity was excised from the IE0 protein complex and identified as the viral protein AcMNPV AC16, previously known as BV/ODV-E26 or DA26 (Beniya et al., 1998; O'Reilly et al., 1990). AC16 was shown to interact with both IE0 and IE1, which agrees with the colocalization of BmNPV IE1 and BM8, an AC16 homolog (Imai et al., 2004). The AC16 interaction domain was mapped to a predicted coiled-coil domain which is located within the acidic transcriptional activation domain. The region of BmNPV BM8 required for the interaction with BmNPV IE1 was mapped to N-terminal predicted coiled-coil domain (Kang et al., 2005). Computer-assisted analysis shows AC16 and its homologs all contain a predicted coiled-coil domain at the N-terminus. This strongly suggests that IE0 and IE1 interaction with AC16 is facilitated by coiled-coil domains which are known to mediate the oligomerization of many proteins (Beck and Brodsky, 1998; Lupas, 1996; McAlinden et al., 2003). This study has therefore added another functional domain to the primary baculovirus regulatory proteins IE0 and IE1. To date, the domains identified in IE1 and IE0 include transcriptional activation, DNA replication, oligomerization, nuclear localization, DNA binding and enhancer binding and now AC16 binding. These results highlight the complexity of IE0 and IE1 roles in baculovirus replication.  The significance of IE0 and IE1 interaction with AC16 was analyzed by generating viruses that contained mutated IE0 or IE1 within AC16-binding domains and in addition viruses that deleted AC16. Loss of the AC16-binding domain for IE1 or IE0 does not result in any significant impact on the viral DNA replication. However in viruses expressing only IE0 the IE0 mutant with the mutated AC16 domain produced higher BV titres, while no impact on the BV yield from the IE1 only viruses was observed. This would suggest that binding of AC16 specifically impacts the function of IE0 but not IE1 and inhibits the production of BV. In support of this conclusion, the levels of IE0 increased relative to IE1 when ac16 is deleted (Fig.3.8). Detection of expression levels of IE0 and IE0 domain-mutant, IE1 and IE1 domain-mutant by Western blot showed IE0 mutant seemed to accumulate at higher levels than IE0, while IE1 mutant appeared to express at similar level as its counterpart (data not shown). The change in the IE0 expression levels also indicates a potential role of 190  AC16 on the regulation of IE0 expression. Future experiments using transient assays coexpressing AC16 with either IE0 or IE1 could indicate whether AC16 directly regulates the expression of ie0. In initial analysis of ie0 and ie1 transcription in cells infected by the viruses expressing IE0 or IE0 AC16 domain mutant, the ie0 mutant transcript accumulated to a higher level than ie0. However, in cells infected by virus expressing IE1 or IE1 AC16 domain mutant, no obvious difference was seen between the ie1 and ie1 mutant transcripts (data not shown), although this result needs to be confirmed before a solid conclusion can be drawn. Other preliminary experiments on the stability of IE0 and IE1 using cyclohexamide treatment did not reveal any difference between IE0 or IE1 and their AC16-binding domain mutants (data not shown). A more accurate comparison of the halflife of IE0 and IE1 with their AC16-domain mutants will help to understand if AC16binding impacts the turn over of IE0 and IE1 and may provide further insight into the role of AC16.  Interestingly, the ac16 deletion virus showed a different phenotype compared to the viruses that had the IE0 and IE1 AC16 binding domain mutated. The ac16 deletion virus had no significant impact on either viral DNA replication or BV production, but resulted in increased IE0 levels relative to IE1. This is another indication that AC16 might regulate the expression of the primary transactivators IE0 and IE1. Both IE0 and IE1 have the potential to autoregulate their own expression (Kovacs et al., 1991; Theilmann et al., 2001), and it has been suggested IE0-IE0 dimer, IE1-IE1 dimer and IE0-IE1 dimer may have different regulatory targets. It is possible that AC16 prevents IE0 activation of the ie0 promoter by forming an AC16-IE0 complex so that less IE0 homodimer is formed. When AC16 is deleted or IE0 AC16-binding domain is mutated, more IE0 homodimer can act to enhance its own expression, which therefore results in higher level of IE0 expressed as seen in the Western blots (Fig. 3.8). In the case of IE1 it is probable that the ie1 promoter appears to be more active than that of ie0 and may be less sensitive to the autoregulation by IE1 homodimer. Therefore, even though AC16-IE1 may compete with IE1-IE1, no increase of expression of IE1 was observed when AC16 was deleted or the IE1 AC16binding domain was mutated. However other unknown mechanisms could be responsible for the observed increased levels of IE0 relative to IE1.  191  The BmNPV AC16 homolog BM8 was previously shown to colocalize with BmNPV IE1 in nuclear foci believed to be the viral replication factories (Imai et al., 2004). AC16 has also been reported to stimulate late gene expression along with ac18 (Guarino and Summers, 1988), and as discussed above appears to repress the expression of IE0. The AC16 properties are similar to Herpes simplex virus ICP22, which is a multifunctional IE regulatory protein that localizes to replication and transcription complexes (Leopardi et al., 1997; Stelz et al., 2002) and also activates late gene expression (Orlando et al., 2006; Poffenberger et al., 1993; Sears et al., 1985). However, ICP22 also represses the expression of early genes (Bowman et al., 2009; Cun et al., 2006; Kwun et al., 1999; Prod'hon et al., 1996). The mechanism of repression is not clear, however ICP22 has been shown to induce loss of specific phosphorylated species of RNA Pol II (Fraser and Rice, 2007) and repression of the major transregulatory protein ICP0 (Bowman et al., 2009). Like ICP22 it is possible that AC16 may have other activities that may include modifying RNA Pol II as well as regulation of other viral genes including IE0 or IE1. Further quantitative analysis on the IE0 and IE1 expression or ie0 and ie1 transcripts, along with the analysis of the impact of loss of IE0 and IE1 AC16-binding domain on their transactivation ability will help dissect the significance of these interactions.  The deletion analysis of ac16 clearly shows the protein is not essential for viral viability, even though previously there have been strong suggestions that the N-terminal of the protein is essential (Burks et al., 2007; Imai et al., 2004). The suggestions were made based on the failure to isolate an ac16 or bm8 complete knockout virus. The results of this study and the reanalysis of previous studies suggested that the cause of the previous results may have been due to the interruption of the expression of the conserved ac17 which overlaps with ac16. This leads to the study presented in chapter 4 which included the construction and analysis of an ac16-ac17 full knockout virus which when repaired with individual genes results in complete ac16 or ac17 knockout viruses. AC17 was found to be a late protein associated with BV, and is required for the efficient production of BV which agreed with a recent report on the BmNPV homolog bm9 (Yang et al., 2009). The double knockout virus had even greater reduction of viral DNA replication and BV production compared with either of the single ac16 or ac17 knockout viruses. However, the most interesting result from the analysis of ac16 and ac17 is the striking global delay of viral 192  gene expression due to the delayed and reduced transcription which was only observed when cells were infected with BV as opposed to transfection with bacmid DNA. Therefore deletion of both AC16 and AC17 renders a virus with distinctly different phenotype from either single gene knockout. The synergistic impact and evolutionary conservation of ac16 and ac17 suggests a functional relationship between these two genes. It is unlikely that AC16 and AC17 are redundant functionally overlapping genes as the single KO virus presented different phenotypes. The possibility that AC16 and AC17 might interact with each other or are in the same complex with IE0 and IE1 has been initially investigated using the yeast 2-hybrid assay. However no interaction between AC16 with AC17 or AC17 with IE0 and IE1 was observed, suggesting that at least there is no direct interactions (data not shown). Nevertheless the possibility these proteins are in a same complex can not be ruled out.  The deletion of ac16 and ac17 results in what appears to be a global delay in both early and late gene transcription but only when cells are infected with BV. This includes the delay of ie0 and ie1`which are expressed within the first 60 minutes of infecting a cell. Bypassing the BV entry processes by transfecting viral bacmid DNA showed no further delay of viral gene expression from the double knockout when compared to WT. This suggests that the journey BV needs to make before their genes get expressed, that is, viral attachment, entry, nucleocapsid transport through the cytoplasm, attachment to the nucleopore, uncoating and finally unwinding of the viral genome to allow access of cellular transcription machinery, is somehow compromised by deletion of ac16 and ac17. F-like protein AC23 has been suggested to facilitate the binding of GP64 to its cellular receptor, thus enhances the viral entry event (Zhou and Blissard, 2008). AC16 is an envelope protein of both BV and ODV and one of its functions late in infection is to facilitate the trafficking of ODV envelope proteins from the endoplasmic reticulum to the inner nuclear membrane (Braunagel et al., 2009). It is therefore possible that AC16 might participate in the endosomal membrane fusion event that releases naked nucleocapsid into the cytoplasm. This study showed that AC17 is a nucleocapsid protein and therefore could be involved in the events subsequent to endosomal release. Alternatively it is possible that AC23 or GP64 distribution in BV is affected in the ac16-ac17 double knockout, therefore viral entry at the plasma membrane maybe delayed or the entry efficiency is reduced. 193  Further analysis is needed to answer why viral gene transcription is delayed and reduced in cells infected with the ac16-ac17 double knockout virus. Possible future experiments could be to fractionate the cytoplasm and the nucleus of cells infected by the double knockout virus or WT virus, and followed by DNA isolation and qPCR analysis to detect copies of the viral genome in each fractionation and when they get delivered into the nucleus. This would potentially reveal the difference between ac16-ac17 double knockout and WT viruses in terms of entry into the cell and the nucleus. Alternatively TEM analysis could be conducted to compare the distribution of nucleocapsids within cells infected by the double knockout and WT viruses, so that a conclusion can be drawn on whether the BV entry process is affected or at which step it is affected in the absence of AC16 and AC17. Cellular microRNAs have recently been shown to regulate the insect ascovirus, DNAdependent RNA polymerase expression (Hussain and Asgari, 2010). However, to date there is no evidence that cellular microRNA regulates baculovirus gene expression. Comparisons of cellular microRNA profiles in cells infected by WT BV and the double knockout BV or by transfecting WT and double knockout bacmids, could potentially reveal if cellular microRNAs play a role in the observed phenotype of the double knockout virus.  The outcomes of this study on ie0, ie1, ac16 and ac17 have lead to an excellent opportunity to study the fundamental mechanisms of how alphabaculoviruses initiate the infection after BV binds to a host cell. Additional analyses on the infectivity of the mutant viruses developed in this thesis in vivo could also lead to new insights into the molecular pathology of baculoviruses.  194  5.1 References Beck, K., and Brodsky, B. (1998). Supercoiled protein motifs: the collagen triple-helix and the alpha-helical coiled coil. J. Struct. Biol. 122(1-2), 17-29. Beniya, H., Braunagel, S. C., and Summers, M. D. (1998). Autographa californica nuclear polyhedrosis virus: subcellular localization and protein trafficking of BV/ODV-E26 to intranuclear membranes and viral envelopes. Virology 240(1), 64-75. Bowman, J. J., Orlando, J. S., Davido, D. J., Kushnir, A. S., and Schaffer, P. A. (2009). 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