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Functional analysis of the P34 regulatory gene of the baculovirus OpMNPV Huijskens, Ilse 2006

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F U N C T I O N A L A N A L Y S I S O F T H E P 3 4 R E G U L A T O R Y G E N E O F T H E B A C U L O V I R U S O p M N P V by Use Huijskens B.Sc, Brabant Technical College, the Netherlands, 1996 M.Sc, Wageningen University, the Netherlands, 1998 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Doctor of Philosophy THE F A C U L T Y OF G R A D U A T E STUDIES (Plant Science) We accept this thesis as conforming To the required standard THE UNIVERSITY OF BRITISH C O L U M B I A January 11,2006 ©Use Huijskens 2006 Abstract World crops losses attributable to insect pests present a considerable economic problem. Baculoviridae represent a great alternative to pesticides since they are capable of a high speed to kil l and do not threaten non-target organisms or the environment. Baculoviruses also exhibit a narrow host range which allows them to infect only a single or a small group of insects. However, the mechanisms behind host range determination by baculoviruses have not been identified to date. Furthermore, no host proteins have been reported to be involved in insect susceptibility, while several viral proteins have been identified to have a function in host range, virulence a and virus replication. This study investigates the interaction between the Orgyia pseudotsugata nucleopolyhedrovirus (OpMNPV) regulatory protein P34 and host proteins and determines the role of host proteins in viral replication. The p34 gene is a homolog of A c M N P V pe38 but is unique as it is the only baculovirus gene that is known to produce two gene products by alternative transcriptional initiation at early and late times post-infection. The early 1.1 kb p34 transcript initiates from an early promoter and is translated as a 34 kDa protein called P34. The late transcipt is a 0.7 kb mRNA expressed from a late promoter that is internal to the p34 ORF and is translated as a 20 kDa N-terminally truncated protein named P34A. P34 possesses a number of distinct protein domains including an N -terminal basic region, a RING finger, acidic, glutamine rich and leucine zipper domains. P34A consists of the acidic, glutamine rich and leucine zipper domains. Previous studies classified P34 as a regulatory protein that activates viral transcription and replication; however the role of P34A has yet to be determined. This study presents new data about the role and mode of action of P34 and P34A during OpMNPV infection. More precisely, we show that P34 can function as an E3 ubiquitin ligase. i i In addition, P34 forms a homodimer, which is dependent upon all domains, and also interacts with P34A to form heterodimers. Confocal microscopy indicated that P34 is distributed in punctate nuclear foci in Ld652Y cells and colocalizes with P34A. Studies were also performed to identify the interactions of P34 with cellular factors. A yeast 2-hybrid screen was conducted with P34 (as bait) and a cDNA library of OpMNPV infected Ld652Y cells to identify proteins that interact with the P34 baculovirus regulatory protein. To date this is the first study that identified interactions between cellular proteins and a baculovirus protein and showed that they are required for infection. Two cellular proteins, LdUEV and L d S U G l , which are homologs of proteins known to participate in the ubiquitination pathway, were shown to bind to P34 but not P34A. The role of these two cellular proteins in the OpMNPV infection cycle was further analysed using gene silencing (RNAi) studies. This study extends the basic understanding of how baculoviruses interact with the host cell, which is essential for determining the molecular basis for virulence and viral host range. 111 Table of Contents Abstract ii Table of Contents iv List of Figures viii List of Abbreviations xi Dedication xiv Acknowlegdements xv Chapter 1: Introduction and Literature Review 1 1.1 Introduction 1 1.2 Baculovirus infection cycle 2 1.2.1 Baculovirus entry 2 1.2.2 Early gene expression 3 1.2.3 D N A replication 5 1.2.4 Late and Very late gene expression 6 1.2.5 Budded virus production 7 1.2.6 Occluded virus production 8 1.3 Genes involved in early gene expression 8 1.3.1 IEOandlEl 9 1.3.2 The regulatory protein OpMNPV P34 11 1.3.3 The regulatory protein IE2 16 1.3.4 Cellular localization of PE38 and IE2 18 1.4 Regulation of Late and Very late gene expression 19 1.5 The ubiquitination pathway 19 1.5.1 Ubiquitination 19 1.5.1.1 Ubiquitin E l Activating enzyme 21 1.5.1.2 Ubiquitin E2 Conjugating enzyme : 21 1.5.1.2.1 Ubiquitin E2 conjugation variant enzymes 23 1.5.1.3 Ubiquitin E3 ligase 23 1.5.1.3.1 RING-finger E3 ligase 24 1.5.1.3.2 HECT domain E3 ligase 25 1.5.1.4 E4 and U box-containing proteins 25 1.5.1.5 26S proteasome 26 1.5.1.5.1 Ubiquitin recognition by the 26S proteasome 28 1.5.1.6 Ubiquitination and D N A repair 29 1.5.1.7 Regulation of transcription by ubiquitination and the proteasome 30 i v 1.5.2 Virus infection and the ubiquitination pathway 31 1.5.3 Virus infection and the 26S proteasome 32 1.5.3.1 Baculovirus ubiquitination related genes 33 1.6 Objectives 34 1.7 Appendix 37 1.8 References 38 Chapter 2: Functional analysis of the Orgyia pseudotsugata MNPV regulatory protein P34 59 2.1 Introduction 59 2.2 Results 61 2.2.1 Oligomerization of P34, P34A and P34 domain deletions in 2-hybrid assays 61 2.2.2 Co-immunoprecipitation analysis of P34 and P34A using HA-tagged proteins 64 2.2.3 Localization of P34 and P34A in infected and transfected cells 69 2.2.4 P34 plays an important role in viral D N A replication and budded virus production in infected Ld652Y cells 74 2.2.5 P34 is an E3 ubiquitin ligase and depends on the RING finger domain for activity ....78 2.3 Discussion 80 Cellular localization of P34 and P34A 82 2.4 Materials and Methods 86 2.4.1 Cells and virus 86 2.4.2 Yeast 2-hybrid assay 86 2.4.3 Western blot analysis 88 2.4.4 Preparation of dsRNA 89 2.4.5 R N A i time-course experiment 90 2.4.6 Budded virus titration by quantitative PCR (Q-PCR) 91 2.4.7 Analysis of D N A replication using slot blot procedure 92 2.4.8 Localization of P34 and P34A 92 2.4.9 In vitro ubiquitination assay 94 2.4.10 Immunoprecipitation 95 2.5 References 98 Chapter 3: Orgyia pseudotsugata MNPV P34 interacts with LdUEV, a ubiquitin E2 conjugating enzyme variant 102 3.1 Introduction 102 3.2 Results 104 3.2.1 Yeast 2-hybrid screen of a cDNA library from OpMNPV infected Ld652Y cells with P34 identified an interaction between P34 and the cellular protein LdUEV 104 3.2.3 LdUEV forms dimers with P34 but not with P34A 105 v 3.2.4 Localization of P34 and P34A with LdUEV in infected and transfected cells 107 3.2.5 P34 and LdUEV play significant roles in viral D N A replication and budded virus production in infected Ld652Y cells 113 3.3 Discussion 118 3.4 Materials and Methods 125 3.4.1 Cells and virus 125 3.4.2 Construction of the 12 h p.i. OpMNPV cDNA library 125 3.4.3 Yeast transformation of OpMNPV P34 and 12 h p.i. OpMNPV cDNA library and yeast 2-hybrid assay 126 3.4.4 Sequence analysis of clones with P34 - cDNA interaction 126 3.4.5 Western blot analysis 127 3.4.6 Preparation of dsRNA , 127 3.4.7 R N A i time-course experiment 128 3.4.8 Total R N A isolation and Northern blots 128 3.4.9 Budded virus titration by quantitative PCR 129 3.4.10 Analysis of D N A replication using slot blot procedure 129 3.4.11 Localization of P34 and P34A with LdUEV 129 3.4.12 Immunoprecipitation 130 3.5 References 131 Chapter 4: Orgyia pseudotsugata MNPV P34 interacts with the 26S proteasome ATPase homolog L d S U G l 135 4.1 Introduction 135 4.2 Results 137 4.2.1 Yeast 2-hybrid analysis screen of an cDNA library of OpMNPV infected Ld652Y cells with P34 identified an interaction with the cellular protein LdSUGl 137 4.2.2 Comparative analysis of L d S U G l and homologous proteins 138 4.2.3 A l l P34 domains are required the interaction with L d S U G l 138 4.2.4 Co-immunoprecipitation of L d S U G l with P34 and P34A 141 4.2.5 Localization of P34 and P34A with LdSUGl in infected and transfected cells 144 4.2.6 LdSUGl play significant roles in viral D N A replication and budded virus production in infected Ld652Y cells 150 4.3 Discussion 155 4.4 Materials and Methods 160 4.4.1 Cells and virus 160 4.4.2 Construction of the 12 h p.i. OpMNPV cDNA library 160 3.4.3 Yeast transformation of OpMNPV P34 and 12 h p.i. OpMNPV cDNA library and yeast 2-hybrid assay 160 4.4.4 Sequence analysis of clones with P34 - cDNA interaction 160 4.4.5 Yeast 2-hybrid assays of P34, P34A and P34 deletion domains with LdSUGl 161 4.4.6 Western blot analysis 162 4.4.7 Preparation of dsRNA 162 v i 4.4.8 R N A i time-course experiment 163 4.4.9 Total R N A isolation and Northern blots ,163 4.4.10 Budded virus titration by quantitative PCR 163 4.4.11 Analysis of D N A replication using slot blot procedure 163 4.4.12 Colocalization of P34 and P34A with LdSUGl 164 3.4.13 Immunoprecipitation 164 4.5 References 166 Chapter 5: General discussion and future prospectives 171 5.1 References 176 Appendix: Role of AcMNPV IEO in baculovirus very late gene activation* 180 Abstract 180 Introduction 181 Results 186 IE1 and IEO expression and their relative ratios during the life cycle of A c M N P V 186 IEO activation of A c M N P V very late gene expression 186 Activation of very late gene expression with increasing levels of IE1, IEO and IE0M~>A....190 Interaction between IE1 and IEO and affection on very late gene expression 194 Discussion 196 Materials and Methods 199 Cells and Virus 199 Cosmid and Plasmid constructs 199 Transfections 200 C A T assay 201 Western blots : 202 Acknowledgements 204 References 205 vii List of Figures Figure 1.1. Infection cycle of baculoviruses 4 Figure 1.2. Schematic genomic map of the OpMNPV ieO-exonO-iel region 10 Figure 1.3. The expression profile of OpMNPV P34 during infection 13 Figure 1.3. continued 14 Figure 1.4. The ubiquitination pathway 22 Figure 1.5. The 26S proteasome 28 Figure 2.1. Analysis of protein-protein interactions between P34, P34A and P34 deleted domains in yeast 2-hybrid system 62 Figure 2.1. continued 63 Figure 2.2. Analysis of protein-protein interactions between N-terminal deletions of P34A in yeast 2-hybrid system 65 Figure 2.2. continued 66 Figure 2.2. continued 67 Figure 2.3. Analysis of oligomerization of P34 and P34A using co-immunoprecipitation 68 Figure 2.4. Localization of OpMNPV P34 and P34A in uninfected and infected Ld652Y cells 70 Figure 2.4. continued 71 Figure 2.4. continued 72 Figure 2.4. continued 73 Figure 2.5. Analysis of viral D N A replication and budded virus production in untreated Ld652Y cells and in cells depleted of P34 using R N A i 73 Figure 2.5. continued 76 Figure 2.5. continued 77 Figure 2.6. In vitro ubiquitination assay of P34 and P34ARING 79 Figure 3.1. Comparative analysis of LdUEV and U E V homologous proteins 106 viii Figure 3.1. continued : 107 Figure 3.2. Analysis of protein-protein interactions of P34, P34A and with LdUEV using co-immunoprecipitation 108 Figure 3.3. Localization of OpMNPV P34, P34A and LdUEV in uninfected and infected Ld652Y cells 110 Figure3.3. continued I l l Figure 3.3. continued 112 Figure 3.4. Analysis of viral D N A replication and budded virus production in untreated Ld652Y cells and in cells depleted of LdUEV or P34 using R N A i 112 Figure 3.4. continued 115 Figure 3.4. continued 114 Figure 4.1. Comparative analysis of L d S U G l and SUG1 homologous proteins 137 Figure 4.1. continued 140 Figure 4.1. continued 141 Figure 4.2. Analysis of protein-protein interactions of P34, P34A and P34 deletions with L d S U G l ...142 Figure 4.2. continued 143 Figure 4.3. Analysis of protein-protein interactions of P34, P34A and with L d S U G l using immunoprecipitation 145 Figure 4.4. Localization of OpMNPV P34, P34A and LdSUGl in uninfected and infected Ld652Y cells 147 Figure 4.4. continued 148 Figure 4.4. continued 149 Figure 4.5. Analysis of viral D N A replication and budded virus production in Ld652Y cells depleted of P34 or L d S U G l using R N A i 149 Figure 4.5. continued 152 Figure 4.5. continued 153 ix Appendix Figure 1. The nineteen late expression factors (lef) located on cosmids and plasmids used in the A c M N P V transient very late gene expression system 181 Figure 2. Time course analysis of IEO and IE1 expression in A c M N P V infected Sf9 cells 187 Figure 3. Analysis and optimization of A c M N P V JE1, IEO and I E 0 M _ > A activation of very late gene expression 189 Figure 4. Analysis of the effect of increasing levels of A c M N P V IE1, IEO and I E 0 M _ > A on transient very late gene expression 191 Figure 5 . Analysis of the effect of co-expression of IE1 and I E 0 M _ > A on the activation of very late gene expression 195 x List of Abbreviations aa amino acid A alanine A A A ATPases associated with diverse cellular activities Ab antibody AclVTNPV Autographa californica multiple nucleopolyhedrovirus A D activating domain APC anaphase-promoting complex ATP adenosine tri-phosphate BmNPV Bombyx mori nucleopolyhedrovirus B basic domain B D binding domain B L A S T basic local alignment search tool bp base pair B V budded virus C A T chloramphenicol acetyltransferase CIP calf intestinal phosphatase CFP cyan fluorescent protein C-terminal carboxy-terminal A deletion DBP D N A binding protein D N A deoxyribonucleic acid dNTP deoxynucleotide triphophatases DO dropout DTT dithiothreitol E6-AP E6-associated protein Fig. Figure FP fluorescent protein g gram in the context of weight; gravitiy force in the context of speed GP64 glycoprotein 64 h C M V human cytomegaloviruses HECT homologous to E6-AP carboxy terminus His Histidine HTV-l human immunodeficiency virus type 1 H L H helix-loop-helix h p.i. hour(s) post infection h p.t. hour(s) post transfection H P V human papilloma virus HSV-1 herpes simplex virus 1 hr homologous region HTLV-I human T-cell leukemia virus I G Glycine Gly Glycine GST glutathione-S-transferase x i G V Granulovirus IE immediate early IPTG isopropyl-jS-D-thiogalactopyranoside K Lysine kb kilobase kDa kilodalton L Leucine L d 5 0 lethal dose to kil l 50% Ld Lymantria dispar lef late expression factor Leu Leucine Lys Lysine L Z leucine zipper domain M micro m milli M molar in the context of concentration; methionine in the context of protein sequence M A b monoclonal antibody M H C major histocompatibility complex M g magnesium ml millilitre raM millimolar M M S methyl methanesulfonate min minute(s) mRNA messenger R N A M S C multiple cloning site NaCl sodium chloride N D nuclear domain NER nucleotide excision repair VE nanogram nt nucleotides N-terminal amino-terminal N P V Nucleopolyhedrovirus OB occlusion body ODV occlusion derived virus OpMNPV Orgyia pseudosugata multiple nucleopolyhedrovirus ORF open reading frame P pico PBS phosphate buffered saline P C N A proliferating cell nuclear antigen PCR polymerase chain reaction P M L promyelocyte PSMC1 proteasome (macropain) 26S subunit ATPase 1 PVDF polyvinylidene difluoride Q glutamine Q domain glutamine rich domain Xll Q-PCR quantitative PCR R A R retinoic acid receptor RING Real Interesting New Gene R N A ribonucleic acid(s) R N A i R N A interference S Serine S A M S-adenosylmethionine SCF Skp-l-Cullin-F-box protein SDS sodium dodecyl sulfate sec seconds Sf Spodoptera frugiperda ss single stranded SUG Suppressor for Gal l TBP T A T A binding protein TBS Tris buffered saline TCID50 tissue culture infections dose 50% TEF-b transcription elongation factor b TP target protein TRAF6 tumor necrosis factor receptor associated factor 6 TRIP! thyroid-hormone-receptor interacting protein 1 Tris Tris-hydroxymethyl amino methane Tip Tryptophan TsglOl tumor susceptibility gene 101 U B C ubiquitin-conj ugating U E V Ubiquitin E2 conjugating Variant U V ultraviolet V C B VHL-elongin C/elongin B VP16 virus protein 16 vsp vacuolar sorting pathway YFP yellow fluorescent protein WT wild type Zn zinc ZnCl 2 zinc di-chloride Xlll Dedication orn en Joyce, deze prestatie deel ik samen met jullie bij deze bedank ik jullie voor alle steun zonder jullie had ik dit nooit kunnen bereiken xiv Acknowlegdements First I would like to thank my family, Joyce, Sascha, Bjorn and Claudia for their constant support throughout the years and especially this past year. Also many many thanks to Maarten who was willing to wait for me for 2.5 years across the Atlantic Ocean and supporting me during many challenging times. I would like to thank my supervisor Dave for giving me the opportunity to work in his lab and for his guidance during my thesis. I would like to express graditude to the Pacific Agri-Food Research Centre in Summerland for allowing me to conduct the research for my thesis and using their facility. Thanks to my supervisory committee, Dr. Isman, Dr. Chantler and Dr. Grigliatti for their assistance and ideas during the past six years. Many thanks to Les Willis for his endless effort to always help me and great advice; i f I had the finances I would send a container filled with Belgium chocolate as a thank you. I would like to express my graditude to Shawna who gave me the opportunity to let her teach several molecular techniques and for her tremendous help with screening numerous of yeast colonies. Thanks to everybody that are currently or have worked in the Theilmann lab, including Hui, Lulin, Kevin, Minggang, Taryn, Xiaojiang and Yingchao, for their assistance and advice. I am going to miss the sushi lunches!!!! I would like to express my graditude to Alina Yukhymets and Theresa Jones for helping me out with all the administration during the last few months of my thesis. Many many thanks to all my friends here in Canada and the Netherlands. I would especially like to thank Steve for helping me out always in the many difficult situations that I ran into in the past 6 years. I would like to thank John and Lesley ("my Canadian sister") who have taught me so many great Canadian words, made sure I got all my nutrients in the past few months, the great xv Saltspring Island trip and for helping me out many many times. I cannot forget to thank Miss Lisa for the great laughs, hikes, trips and artistic crafting. Thanks Karen and Cody for inviting me in your home in Kelowna and the great talks. I would like thank all my other Canadian friends for their help and good times, including Adam, Caner, Gary, Jeremy, Joan, John, Michelle, Maynard, Misti, Kishore, Oslem, Rob, Ron, Shelly, Sue, Tammy, the summer students, the Oliver film festival group, the "play on sports" soccer team. Thanks to all my friends back in Europe, including Bart en Astrid, Eer, Erik, Helga, Jorrit, Karen en Marc, Melissa, Mireille en Renze, Monique en Rolf, Peter, Patries en Philipe and Sam. xvi Chapter 1: Introduction and Literature Review 1.1 Introduction Insects are major pests worldwide in forests and on crops, resulting in enormous economic and natural losses. The increased use of chemical insecticides is a major public concern due to their potential effect on public health and the environment. In addition to the environmental risks of insecticides, pest populations have become resistant by overuse of these agents. The application of biological control agents either independently or in combination with chemicals can be used to control resistance, as well as protect the environment (Casida, 1990; Fuxa and Richter, 1998). Baculoviruses have great potential as biocontrol agents, because of their narrow host range and no known human health and ecological risks. Baculoviruses have been used very successfully to control pests. For example in Brazil the baculovirus Anticarsia gemmatalis nucleopolyhedrovirus (NPV) is being used on over 1 million hectares of soybean to control the velvet bean caterpillar (Da Silva, 1992; Moscardi, 1990). In the forests of the Pacific Northwest of North America the Douglas fir tussock moth, which causes severe defoliation, has been successfully controlled for many years by Orgyia pseudosugata multiple nucleopolyhedrovirus (OpMNPV) (Martignoni, 1999; Otvos et al, 1998). Limitations to their use as commercial pesticides have been their lack of speed to kil l in comparison to chemicals, their photosensitivity and relatively high economic costs. However, many of these limitations have been overcome by improved spray formulations and large-scale production methods. Baculoviridae consists of rod-shaped viruses that are occluded within a proteinous matrix called polyhedrin and have circular dsDNA genomes varying in size from 80 to 180 kb. 1 Baculoviruses are known to infect Lepidotera, Diptera, Hymenoptera, Trichoptera, and Crustacea and have been classified into two different genera: Nucleopolyhedrovirus (NPV) and Granulovirus (GV) (Murphy et al, 1995; Possee, 1993; Tanada and Kaya, 1993; Theilmann et al, 2004). In addition, based on molecular phylogenetic features, NPVs have also been subdivided into two distinct sub-groups (I and II) (Zanotto et al, 1993). In the occluded form of baculoviruses, N P V virions usually contain one or more nucleocapsids per envelope and there are many virions per occlusion body. GVs typically contain only a single nucleocapsid per virion and occlusion body. In general, NPVs have a broader host range while GVs are restricted to Lepidoptera (Blissard and Rohrmann, 1990). 1.2 Baculovirus infection cycle 1.2.1 Baculovirus entry The infection of NPV starts with the uptake of polyhedrin occlusion bodies (OB) by larvae that are feeding from contaminated crops or trees. The polyhedrin occlusion bodies enter the midgut where their polyhedrin matrix gets dissolved by high pH and local enzymes (Granados, 1978; Granados and Federici, 1986; Granados et al, 1981; Harrap, 1970; Horton and Burand, 1993; Robertson et al, 191 A). The occlusion-derived virions (ODV) migrate through the peritrophic membrane to the membrane of the midgut epithelial cells. The mechanism for the virus transportation to the midgut cell in not clear. There are some suggestions that viral produced proteins called enhancins might facilitate the transport of the virus to the midgut cell (Hashimoto et al, 1991; Lepore et al, 1996; Wang et al, 1994). When the virions arrive at the membrane of the midgut cells, the envelope of the virion fuses to the cell membrane (Granados et al, 1981). The nucleocapsids enter into the cytoplasm and have been shown to migrate to 2 different locations within the cell. They migrate towards the nuclear pore and into the nucleus where virus is released from the nucleocapsid. Alternatively soon after entering the cytoplasm, nucleocapsids transfer directly to the basal cell membrane and exit by budding (Charlton and Volkman, 1993) (Fig. 1.1). 1.2.2 Early gene expression Expression of viral genes in the early phase begins prior to the initiation of D N A replication. Early genes are transcribed by the host R N A polymerase II and many contain in the promoter region a consensus early transcriptional start site that consists of a C A G T sequence located 25 to 31 base pairs downstream from a T A T A box (Friesen, 1997). Several viral genes have shown to be transcribed in the early phase, which are pi 43 (helicase), dnapol,p34 (pe38), lef-1, lef-2, lef-3 and the immediate early (IE) gene ie2. Viral transcriptional regulators have been identified among these genes that up or down regulate early viral protein expression, which include P34 (PE38), IE2, IE1 and IEO. OpMNPV P34 is able to transactivate both the OpMNPV ie2 andp8.9 promoters (Wu et al, 1993a; Wu et al, 1993b) and the Autographa californica M N P V (AcMNPV) PE38 up-regulates the expression of the A c M N P V helicase gene pi43 (Lu and Carstens, 1993). OpMNPV IE2 and A c M N P V IE2 transactivate their own promoter and the iel and ieO promoters (Carson et al, 1988; Theilmann and Stewart, 1992; Theilmann et al, 2001). OpMNPV IEO is auto-regulatory and in addition can transactive the 39K and gp64 early promoters (Theilmann et al, 2001). A c M N P V IEO is able to augment iel and 39K, however not its own promoter (Kovacs et al, 1991b). IE1 has been shown to enhance transcription of its own promoters and most of the early 3 ViflC*B#rnc tlrom* Nuclear membrane Nuclear pore Fusion wm Primary in taction Budaod virus (BV) Poiyrt«df« aw r« '#as«J upon coll lys* and deai> \ of th«hos l logesJton &y niecthost SoluWteittjft ofpcfyNrCrs * n She r cy j : Occlusion derived virus (ODV) Figure 1.1. Infection cycle of baculoviruses. The polyhedra enter the midgut and due to presence of high pH the polyhedrin matrix dissolves. The occlusion-derived virions (ODV) pass through the peritrophic membrane and bind to the surface of the midgut epithelial cells. Subsequently the envelope of the ODV is fused to the cell membrane and the nucleocapsids are released into the cytoplasm. The nucleocapsids migrate to the nuclear membrane and transfer through the nuclear pore into the nucleus and release virion DNA. In the nucleus the viral D N A is replicated and packaged into nucleocapsids. Some of the new nucleocapsids pass through the nuclear membrane and migrate to the cell membrane. The nucleocapsids bud through the cell membrane and obtain an envelope that includes viral proteins such as the GP64 glycoprotein. After release these budded viruses (BV) continue to infect other tissues. In the late stages of baculovirus infection O D V will be occluded by polyhedrin and form polyhedra that accumulate in the nucleus of infected cells. Polyhedra are released by cell lysis. (Adapted from Blissard and Rohrmann(1990)) 4 viral proteins (Blissard and Rohrmann, 1991; Guarino and Smith, 1992; Kovacs et al, 1991b; Lu and Carstens, 1993; Nissen and Friesen, 1989; Passarelli and Miller, 1993c; Pullen and Friesen, 1995; Ribeiro et al, 1994; Theilmann and Stewart, 1991; Theilmann et al, 2001). Many of these viral early genes are utilized for viral D N A replication. 1.2.3 DNA replication D N A replication of Orgyia pseudotsugata M N P V (OpMNPV) takes place between 12 hours (h) postinfection (p.i.) to 18 h p.i. and maintain steady state levels after 24 h p.i. in tissue culture cells (Bradford et al, 1990). In Spodoptera frugiperda (Sf) cells D N A replication of A c M N P V is initially observed at 6 h p.i., continues to increase up to 18 h p.i. and subsequently declines (Tjia et al, 1979). Studies of origin-containing plasmid assays and defective interfering particles of A c M N P V have suggested that D N A replication takes place using the rolling circle mechanism (Oppenheimer and Volkman, 1997). Baculoyirus early genes are essential or have a stimulatory role for replication. Transient viral D N A replication assays with OpMNPV and the related virus A c M N P V have shown five common lef genes to be essential for replication (Ahrens and Rohrmann, 1995; Kool et al, 1994; Lu and Miller, 1995). These essential viral D N A replication genes are ie-1 (transcription factor that also binds the origin of replication) (Choi and Guarino, 1995a; Guarino and Dong, 199.1; Pathakamuri and Theilmann, 2002; Rodems et al, 1997), lef-1 (primase) (Barrett et al, 1996; Evans et al, 1997), lef-2 (primase associated factor) (Mikhailov and Rohrmann, 2002; Passarelli and Miller, 1993c), lef-3 (single stranded (ss) D N A binding protein) (Hang et al, 1995), and pi43 (helicase), (Lu and Carstens, 1993; Tomalski et al, 1988). Viral dnapol (DNA polymerase) is also essential for virus replication (Ahrens and Rohrmann, 1995; Kool et al, 5 1994; Lu and Miller, 1995; Vanarsdall et al, 2005). Additional replication studies have revealed that lef-11 is also required for D N A replication in virus infected SJ9 cells (Lin and Blissard, 2002). Furthermore, the host cell-specific factor 1 (hcf-1) is required for /jr-dependent D N A replication specifically in TN-368 cells (Lu and Miller, 1995; Lu and Miller, 1996). Three lef genes appear to have a stimulatory function for D N A replication, including ie2 (transcriptional factor and cell cycle arrest gene) (Carson et al, 1988; Carson et al, 1991; Prikhod'ko and Miller, 1998),p35 (apoptosis inhibitor) (Clem et al, 1991) andp34 (pe38) (transcription factor) (Lu and Carstens, 1993; Wu et al, 1993a; Wu et al, 1993b). OpMNPV P34 was shown by transient assays to augment D N A replication (Ahrens and Rohrmann, 1995). Studies with ape38 deletion virus that prevented expression of this gene resulted in a significant decrease of viral D N A replication relative to wild type A c M N P V infection (Milks et al, 2003). Recently BmNPV PE38 has been identified to possess E3 ubiquitin ligase activity, which depend on its RING finger structure for maintaining its activity (Imai et al, 2003). 1.2.4 Late and Very late gene expression The final part of baculovirus infection is separated into the late and very late phases. Late gene expression initiates concomitant with the onset of viral D N A replication. The genes expressed during the late phase are primarily structural genes or genes required for virion formation. Late and very late genes are expressed from a late promoter (A/G/T)TAAG and utilize the same unique viral R N A polymerase for transcription (Lu and Miller, 1997; Morris and Miller, 1994; Ooi et al, 1989; Possee and Howard, 1987; Qin et al, 1989; Rankin et al, 1988; Weyer and Possee, 1989). Four lef genes (p47, lef-4, lef-8, and lef-9) have been shown by co-purification to be associated with the viral R N A polymerase (Guarino et al, 1998). Late gene 6 transcription in OpMNPV infected gypsy moth cells (Ld652Y) initiates at approximately 18 h p.i. (Ahrens and Rohrmarm, 1995; Russell and Rohrmann, 1993). Using transient assays nineteen baculovirus genes have been identified as being either essential or stimulatory for late gene expression and have been called late expression factors (lefs) (Li et al, 1999; Rapp et al., 1998). Recently, IEO was identified as the twentieth lef gene since IEO was able to functionally replace IE1 for very late gene activation by IE1 in transient assays (See Appendix). 1.2.5 Budded virus production Baculoviruses are unique insect viruses as they produce two virion forms during the infection cycle, budded virus (BV) and occlusion-derived virus (ODV). BVs are formed by the migration of nucleocapsids through the nuclear membrane and then transport to the cell plasma membrane (Blissard and Rohrmann, 1990). The nucleocapsids bud through the cell membrane and receive an envelope that contains viral proteins, the major one being the glycoprotein GP64 (Blissard and Rohrmann, 1990) (Fig. 1.1). The gp64 gene is transcribed by both early and late promoters and following translation, the expressed proteins accumulate at the plasma membrane during early and late phases (Blissard and Rohrmann, 1989; Hi l l and Faulkner, 1994; Monsma and Blissard, 1995; Monsma et al, 1996; Volkman, 1986; Whitford et al, 1989). The function of B V is to infect other tissues and spread the infection throughout the insect (Blissard and Rohrmann, 1990; Blissard and Wenz, 1992; Granados et al, 1981; Monsma et al, 1996). GP64 is essential for the binding of B V to other tissues and has been shown to act as a membrane fusion protein (Blissard and Wenz, 1992; Hefferon et al, 1999; Monsma et al, 1996). The tracheal system (including hemocytes, tracheal matrix and fatbody) is considered to play the key 7 role for systematic spread throughout the larval tissues (Engelhard et al, 1994). For BVs to escape the midgut cell, they use tracheoblasts to migrate through the basal lamina. The infected tracheoblasts permit transport of BVs and subsequent infection of the tracheal epidermal cells and the hemocoel. The insect lymph system is also found to contain B V and likely aids the rapid spread of the baculovirus throughout the host (Granados, 1980; Volkman and Summers, 1977). 1.2.6 Occluded virus production During the late phase of baculovirus infection nucleocapsids in the nucleus of infected cells are occluded by the hyper-expressed viral protein polyhedrin and form what are known as polyhedra or occlusion bodies. The function of OBs is to provide protection of the virions and viral D N A from the environment and U V light. The production of OBs continues until the nuclear membrane ruptures and the cell lyses. Nuclear lysis appears to be primarily the result of the cooperative action of viral proteases (van Oers et al, 1993). The insect tissues and cuticle are also disrupted by baculoviruses that produce chinitases in addition to the proteases. The combined action of these two proteins results in the liquefaction of tissues and rupture of the cuticle after the death of the caterpillar releasing up to approximately 670 million OBs from a single larva (Hawtin et al, 1995; Martignoni, 1978; Volkman and Keddie, 1990). Disruption of the cuticle gives OBs access to environment where wind, rain or predators will spread the polyhedra enclosed viruses. 1.3 Genes involved in early gene expression Transcription of the early viral genes initiates almost immediately after entry of the viral D N A into the cell. Early phase genes are transcribed from an early promoter (CAGT) by the 8 host R N A polymerase II (Friesen, 1997). Four early viral proteins have been identified to stimulate or activate early gene expression, including P34 (PE38), IE2, IE1 and IEO. 1.3.1 IEO and IE1 The transcriptional transactivators IEO and IE1 are the primary baculovirus regulatory proteins that are essential for transcription and replication during infection. The ieO-iel gene complex is unique as it is the only known baculovirus spliced gene that produce two different proteins (Kovacs et al, 1991b; Theilmann and Stewart, 1991). The OpMNPV ieO transcriptional start site and promoter region are located 4730 bp upstream of the iel transcriptional start site (Fig. 1.2). The ieO transcript contains 135 nucleotides from exonO spliced to the 5' end of the 1.7 kb iel mRNA resulting in a 1.9 kb ieO transcript (Chisholm and Henner, 1988; Kovacs et al., 1991a; Kovacs et al, 1991b; Theilmann et al, 2001). Transcription of OpMNPV ieO and iel starts within 1 h p.i.; ieO peaks at approximately 4 h p.i. but remains detectable up to very late times p.i. In contrast iel continues to increase in steady state levels right up to late times p.i. (Choi and Guarino, 1995b; Theilmann and Stewart, 1991). The transcription profile of A c M N P V ieO-iel is similar to OpMNPV, but displays a more accelerated profile (Chisholm and Henner, 1988; Kovacs et al, 1991a; Kovacs et al, 1991b; Theilmann et al, 2001). Translation of OpMNPV ieO mRNA produces a 68 kDa protein, consisting of 23 amino acids encoded by the upstream exon (exonO), 12 amino acids from the nontranslated leader sequence of the iel mRNA and the entire IE1 protein (Chisholm and Henner, 1988; Pearson and Rohrmann, 1997; Theilmann et al, 2001). A c M N P V has 54 amino acid added to the N-terminus of the 582 amino acid IE1 (Chisholm and Henner, 1988; Pearson and Rohrmann, 1997). In contrast to A c M N P V , OpMNPV IEO also has an additional 4 amino acid minicistron located 9 130 nt Figure 1.2. Schematic genomic map of the OpMNPV ieO—exonO-iel region. The ieO—exonO-iel region contains the ORFS exonO, iel, ieO, orfl39, odv-el7, odv-e27, orfl42, hrf-1 and orfl44. In this genomic map the ORFs are presented in their relative positions and orientation. Splicing of the ieO transcript is illustrated above the genomic map by presenting a solid line as the ieO intron. The ieO transcript consists of regions derived from exonO, iel untranslated region and iel, which are indicated in a black and grey arrow. The iel transcript is indicated by a black arrow (Adapted from Theilmann et al. (2001)) upstream of the ieO ORF (Theilmann et al., 2001). In vivo analysis of OpMNPV or A c M N P V ieO mRNA translation revealed that in addition to translation of the predicted IEO, the smaller IE1 was produced due to internal translational initiation (Huijskens et al., 2004; Theilmann et al., 2001). IE1 and IEO are transcriptional activators that activate early, late and very late genes in an enhancer dependent or independent fashion (Blissard et al., 1992; Choi and Guarino, 1995b; Guarino and Summers, 1986a; Guarino and Summers, 1986b; Guarino and Summers, 1988; Huijskens et al., 2004; Passarelli and Miller, 1993c; Pathakamuri and Theilmann, 2002; Theilmann and Stewart, 1991; Theilmann et al, 2001). A c M N P V IE1 has also been shown to 10 down-regulate the expression of certain early genes (Kovacs et al, 1991b; Leisy and Rohrmann, 2000). In addition both IE1 and IEO support viral D N A replication and are believed to be part of the replication complex (Kool et al, 1994; Pathakamuri and Theilmann, 2002; Rodems and Friesen, 1995; Stewart et al, 2005). Detailed analysis of OpMNPV and A c M N P V IE1 identified the following functional domains: N-terminal acidic activation domain, required for both transcription and replication; basic domain I, essential for enhancer binding; a second acidic domain, also potentially involved in activation of transcription; a D N A binding domain; basic domain II, required for nuclear entry; and an oligomerization domain (Olson et al, 2002; Olson et al, 2003; Pathakamuri and Theilmann, 2002; Rodems et al, 1997; Slack and Blissard, 1997). Previous studies have revealed that A c M N P V IE1 is able to form homodimers and heterodimers, which are necessary for binding to homologous region (hr) enhancers (Guarino and Dong, 1994; Kremer and Knebel-Morsdorf, 1998; Leisy et al, 1995; Olson et al, 2003; Rodems and Friesen, 1995). The helix-loop-helix (HLH)-like domain located at the C-terminus of A c M N P V IE1 appears to be necessary for establishing a homodimer (Olson et al, 2001). Immunofluorescence studies using a green fluorescent protein tagged IE1 revealed that in Bombyx mori N P V (BmNPV) infected BmN cells IE1 initially was found in small nuclear foci prior to the onset of D N A replication (Nagamine et al, 2005). After viral D N A synthesis the IE1-linked foci expand steadily and is believed to be the site for replication factories and nucleocapsid assembly (Kawasaki et al, 2004; Okano et al, 1999). 1.3.2 The regulatory protein O p M N P V P34 During OpMNPV infection, the early promoter of p34 produces a 1.1 kb mRNA that is 11 expressed from 1 to 48 h p.i. and whose translation product is a 34 kDa protein (Fig. 1.3 A and B) (Wu et al, 1993a). A second 0.7 kb transcript is also expressed from 18 to 120 h p.i. by a late internal promoter, which is translated into a 20 kDa N-terminally truncated P34 protein named P34A (Fig. 1.3A and B). OpMNPV P34 has been shown in various transient assays to enhance viral D N A replication and to be associated with transcriptional activation since P34 was able to transactivate both the OpMNPV iel and p8.9 promoters (Ahrens and Rohrmann, 1995; Kool et al, 1994; Wu et al, 1993a; Wu et al, 1993b). P34 possesses a number of distinct protein domains that are known to function in a number of different regulatory pathways (Carson et al, 1988; Krappa and Knebel-Morsdorf, 1991; Wu et al, 1993a; Yoo and Guarino, 1994b). P34 includes a basic, a RING finger and acidic, glutamine rich and leucine zipper domain (Fig 1.3B). These domains have also been identified in the homologs A c M N P V and BmNPV PE38. The N-terminally truncated 20 kDa P34A consists of only the acidic, glutamine rich and leucine zipper domains (Fig. 1.3B) and the PE38 truncated protein is predicted to contain the acidic and leucine zipper domains. The basic domain is characterized as a region that is rich in both arginines and lysines. This type of domain has been shown to be involved in D N A binding (Garcia-Bustos et al, 1991; Hanover, 1992; Mujeeb et al, 199'4; Newmeyer, 1993; Nigg et al, 1991; Osborne and Silver, 1993; Stochaj and Silver, 1992; Voronova and Baltimore, 1990). Furthermore, the basic domain of A c M N P V PE38 is believed to function as a putative nuclear localization signal (NLS) since a deletion of this domain prevented the transport of PE38 to nucleus but definitive proof remains to be determined (Prikhod'ko and Miller, 1999). RING (Really Interesting New Gene) domains have a typical motif (CI-x2-C2-x(9-39)-C3-x(l-3)-Hl-x(2-3)-C4-x2-C5-x(4-48)-C6-x2-C7) (Freemont, 1993). The cysteine and histidine 12 Figure 1.3. The expression profile of OpMNPV P34 during infection. (A) Schematic presentation ofp34 transcription illustrating a 1.1 kb transcript that is expressed from an early transcriptional initiation site (CACAGT). In addition, a second transcript named is expressed from the late transcriptional initiation site (GTAAG) and has a size of 0.7 kb. The transcription patterns of p34 and 5' truncated p34 during the course of infection are shown in the Northern blot. The transcripts were labelled with a radioactive ssRNA probe from the KpnI-PstI fragment of the p34 ORF. The numbers on the top of the Northern blot indicate the hours post infection (h p.i.) of the harvested samples. The sizes of the R N A ladder is presented on the right and the sizes of both transcripts are shown on the left. (Adapted from Wu et. al, (1993a)) (B) Schematics of P34 and P34A with the domains located in these two proteins. The domains include the N-terminal basic region, the RING finger, acidic, glutamine rich and leucine zipper domains. The number of amino acids encoding P34 (307) and P34A (182) are indicated on the right. The start codon of P34 and P34A are symbolized by • and t> respectively. The Western blot shows a time-course analysis of P34 and P34A expression during OpMNPV infection of Ld652Y cells. The numbers on the top of the Western blot indicate the hours post infection (h p.i.) of the harvested samples and the molecular weight of P34 (34 kDa) and P34A (20 kDa) are presented on the right. The proteins were detected using a P34 monoclonal antibody (Adapted from Wue/ al. (1993a)). 13 Figure 1.3. continued. A Early transection initiation site r Late tr ante rip (ion initiation site I r CACAGT GTAAG | p34 open reading frame 1.1 kb pi<4 EARLY raRNA J^. 0.7 kb p34 L A T E mRNA AATAA OpMNPV genomic DNA hrp.1. * 2 4 S 8 12 IB 24 36 46 72 96 120 I T Ladder (kb) 7.45 4.40 257 1.1 kb • • 0.7 kb «* 155 - 0.24 B basic RING finger glutamine leucine acidic rich zipper P34A l j r pi_ M 2 4 6 8 12 18 24 36 48 72 96 120 1 34.0 P34 5 S 2 S FL -«-21.5 20.0 P34A 14 residues of the RING domain are required to bind two zinc atoms and as a result form a unique cross-brace complex. The RING finger domains of transcription factors have also been shown to form either homo- or heterodimers (Jensen et al, 1998; Tanimura et al, 1999; Wu et al, 1996). Increasing numbers of proteins with a RING finger motif have been shown to function in the ubiquitination pathway as E3 ubiquitin ligases. Among the baculovirus proteins homologous to P34 only the BmNPV PE38 has been demonstrated to have E3 ubiquitin ligase activity (Imai et al, 2003). E3 ubiquitin ligases participate in the ubiquitination pathway by transferring ubiquitin from E2 conjugating enzymes to target proteins. Many transcriptional activators and repressors have been identified to contain glutamine rich domains. In many proteins the glutamine rich domains are described to form coiled coil structures, which are known to promote or facilitate protein-protein interactions (Chen and Courey, 2000; Chen et al, 1998; Grbavec et al, 1998; Kim et al, 2000; Majello et al, 1997; Mitchell and Tjian, 1989; Miyasaka et al, 1993; Pinto and Lobe, 1996; Ren et al, 1999; Triezenberg, 1995; Xiao and Jeang, 1998). However, the function of the glutamine rich domain in P34 and its homologous proteins is not yet known. The P34 leucine zipper domain consists of heptad repeats of leucine, methionine, serine and alanine residues that are able to form an a-helix and are predicted to form coiled coil structures (Wu et al, 1993a). Many studies have shown that leucine zippers of regulatory proteins are involved in protein-protein interactions such as the formations of homo- or heterodimers (Ellenberger et al, 1992; Glover and Harrison, 1995; Jensen et al, 1998; Lupas, 1996; Tanimura et al, 1999; Wu et al, 1996). Transient assays indicated that the leucine zipper domain of A c M N P V PE38 is required to enhance IE1 induced apoptosis (Prikhod'ko and Miller, 1999). The A c M N P V genome contains a homolog of OpMNPV P34 known as PE38. In a similar 15 manner to OpMNPV, A c M N P V PE38 is transcribed as a 1.3 kb transcript from 1 until 12 h p.i., but decreases at late times p.i. (Krappa and Knebel-Morsdorf, 1991). The pe38 mRNA is translated into a 38 kDa protein. Although the presence of the N-terminal truncated PE38 protein similar to P34A has been identified by Western blot analysis, an R N A transcript coding for it has not been detected (Krappa et al, 1995). A c M N P V PE38 has also been shown to up-regulate the expression of the A c M N P V helicase genepl43 (Lu and Carstens, 1993). PE38 is also capable of augmenting IE1 induced apoptosis but it is unclear how this is achieved (Prikhod'ko and Miller, 1999). Transient replication assays revealed that A c M N P V PE38 is capable of enhancing viral D N A replication, however is not essential (Ahrens and Rohrmann, 1995; Kool et al, 1994). Furthermore A c M N P V PE38 expression has been shown to be negatively regulated by high levels of IE1 (Leisy et al, 1997). Recently Milks et al (2003) generated a virus that had PE38 deleted from the A c M N P V genome. The pe38 deletion virus was able to replicate, but the levels of viral D N A replication and budded virus production were severely impaired. In addition, bioassays with infected Heliothis virescens larvae have shown that the L D 5 0 of the pe38 deleted A c M N P V was significantly increased relative to wild type infection. The previous studies therefore show that both P34 and PE38 are not essential for viral replication but are stimulatory. Nevertheless P34 and PE38 have a dramatic impact on the viral life cycle both in tissue culture and in vivo. 1.3.3 The regulatory protein IE2 OpMNPV ie2 is a second auxiliary regulatory protein that has functional similarities to P34 (and PE38) in that it functions as a transcriptional transactivator and augments viral D N A replication (Ahrens and Rohrmann, 1995; Theilmann and Stewart, 1992; Theilmann et al, 2001). 16 Ie2 is expressed in OpMNPV infected Ld652Y cells as a 1.3 kb transcript from as early as 0.5 h p.i., reaches maximum steady state levels by 6 h p.i. and declines slightly by 12 h p.i. However ie2 transcription remains detectable up to very late times p.i. (Theilmann and Stewart, 1992). During OpMNPV infection the ie2 transcript is translated into 46 kDa protein that has peak expression from 18-24 h p.i. and declines to barely detectable levels by 48 h p.i. (Theilmann and Stewart, 1993). Structurally IE2 is similar to P34 in that the protein contains similar domains, which include a basic, coiled coil, RING finger, and a leucince zipper domain. Cotransfection experiments revealed that IE2 transactivates ieO, iel,p34 and an OpMNPV opep-2 gene with an unknown function (Shippam-Brett et al, 2001; Theilmann and Stewart, 1992; Theilmann and Stewart, 1993). In addition, ie2 expression was shown to be down-regulated by IE1 (Theilmann and Stewart, 1993). Similar activities have been described for A c M N P V IE2 (Carson et al, 1988; Guarino and Summers, 1986b; Prikhod'ko et al, 1999; Prikhod'ko and Miller, 1998; Yoo and Guarino, 1994a; Yoo and Guarino, 1994b). Transient replication assays have shown that IE2 augments viral D N A replication but is not essential similar to P34 (Ahrens and Rohrmann, 1995; Kool et al, 1994). However when both IE2 and P34 are present in transient assays replication is inhibited suggesting an antagonistic interaction between these two OpMNPV proteins (Ahrens and Rohrmann, 1995). IE2 is capable of arresting cell cycle development and is dependent on the RING finger domain for this function (Prikhod'ko and Miller, 1998). However the transregulatory activation of IE1 was unaffected by the RING finger deletion suggesting that cell cycle and transcription regulation are separable functions of IE2. 17 1.3.4 Cellular localization of PE38 and IE2 Several studies have employed immunofluorescence studies to determine the cellular localization of A c M N P V PE38 and IE2 during infection and it has been found that they follow similar patterns. Initial analysis using confocal microscopy revealed that A c M N P V PE38 was found to localize predominantly in the nucleus as punctate foci in transfected cells. However in infected cells the PE38 punctate pattern became more diffuse (Krappa et al, 1995; Murges et al., 2001). Immunofluorescence analysis could not distinguish between PE38 and the late 20 kDa product. However following sub-cellular fractionation and Western blot analysis the 20 kDa form was found to reside almost exclusively within the cytoplasmic fractions (Murges et al., 2001). Immunofluorescence studies with A c M N P V IE2 have shown similar punctate foci in the nucleus of transfected cells and at early times p.i. (Krappa et al, 1995). Recent studies indicated the formation of the BmNPV IE2 punctate foci relies on the leucine zipper coiled coil domain (Imai et al, 2005). Interestingly A c M N P V IE2 was shown to colocalize with PE38 and the mammalian RING finger containing protein called promyelocytic leukemia protein (PML) in insect and mammalian cells (Krappa et al, 1995; Murges et al, 2001). P M L is a structural component of the P M L nuclear body (NB) or NDlO/PODs and has been associated with gene regulation, anti-viral response, tumour suppression, proteasomal degradation, apoptosis and D N A repair (Regad and Chelbi-Alix, 2001; Seeler and Dejean, 1999; Strudwick and Borden, 2002; Zhong et al, 2000; Zhu et al, 2001). NDlO-like structures were observed in BmNPV infected cells and these complexes seem to function as sites for viral D N A replication and transcription (Okano et al, 1999). Additionally A c M N P V IE2 and P M L were observed juxtaposed to the viral D N A replication centers (Mainz et al, 2002). PE38 and IE2 as 18 determined by confocal studies appeared earlier in infected cells at the punctate foci than the replication related protein LEI, DBP (DNA binding protein) and LEF-3 (Imai et al, 2005; Mainz et al, 2002; Murges et al, 2001; Okano et al, 1999). 1.4 Regulation of Late and Very late gene expression Late and very late expression is regulated by the late expression factors or lef genes. Late and very late genes are expressed from promoters with a (A/G/T)TAAG motif (Lu and Miller, 1997; Morris and Miller, 1994; Ooi et al, 1989; Possee and Howard, 1987; Qin et al, 1989; Rankin et al, 1988; Weyer and Possee, 1989). Initially, eighteen A c M N P V genes have been shown to be essential for transactivation of expression from the late vp39 and 6.9 promoters and the very late polh andplO promoters, which include iel, ie2, lefsl-11, dnapol,pl43,p47,p35 and 39K (Li et al, 1993; Lu and Miller, 1994; Morris et al, 1994; Passarelli and Miller, 1993a; Passarelli and Miller, 1993b; Passarelli and Miller, 1993c; Passarelli and Miller, 1994; Todd et al, 1996; Todd et al, 1995). Studies by Rapp et al. (1998) identified another gene named A c M N P V lefl2 to be involved in late gene expression. L i et al. (1999) demonstrated that A c M N P V orffl was essential and orf69 stimulated gene expression from the vp39 capsid promoter. Recent transient assays identified A c M N P V ieO as the 20 t h lef gene to be required for activation of the polyhedrin promoter (See Appendix, (Huijskens et al, 2004)). 1.5 The ubiquitination pathway 1.5.1 Ubiquitination The role of the ubiquitination pathway during viral infection has become more apparent recently. Ubiquitination (or ubiquitylation) is a pathway that degrades cellular proteins to 19 regulate levels in the cell for various purposes. The ubiquitination pathway is also involved in many processes such as cell cycle progression, apoptosis, regulated cell proliferation, protein transport, D N A repair, organelle biogenesis, cellular differentiation, quality control in the endoplasmic reticulum, inflammation, antigen processing and stress responses. The 76 amino acid ubiquitin protein is highly conserved among eukaryotes and plays a key role in the ubiquitination pathway. Ubiquitin forms polyubiquitinated structures via its C-terminal glycine (Gly76) and forms an isopeptide bond with one of the side chain lysines of ubiquitin itself or a side chain lysine on a target protein. Proteins tagged by ubiquitination or polyubiquitin then become targets for further processing or function. The ubiquitination pathway is ordered in a hierarchical manner regulated by E l , E2, and E3 enzymes (Fig. 1.4). For any given organism there appears to be only a single E l enzyme, several E2s and hundreds of E3s. The E3 proteins provide the specificity towards target proteins. The ubiquitination pathway initiates with the ATP dependent thiol-ester formation of the C-terminal glycine (Gly76) of ubiquitin to the conserved cysteine of the E l ubiquitin-activating enzyme (Fig. 1.4). Subsequently ubiquitin is transferred to the E2 ubiquitin-conjugating enzyme by forming a thiol-ester bond of Gly76 with the conserved catalytic cysteine residue of E2. The E3 ubiquitin ligase enzyme transports a target protein to the E2 conjugating enzyme tagged ubiquitin to form a complex and catalyzes the transfer of Gly76 of ubiquitin to a side chain lysine on the target protein (Fig. 1.4). The E3 ubiquitin ligase continues to assemble a multi-ubiquitin chain by attaching Gly76 of ubiquitin proteins to the Lys-48 or Lys-63 side chain of ubiquitin that is bound to the target protein (Fig. 1.4). Polyubiquitination of target proteins ultimately results in activation of that protein for some function or in relocation to the proteasome for degradation. 20 1.5.1.1 Ubiquitin £1 Activating enzyme Ubiquitination pathways in many organisms consist of single E l activating enzyme to initiate the ubiquination process (McGrath et al, 1991; Zacksenhaus and Sheinin, 1990). E l utilizes Adenosine Triphosphate (ATP) to activate ubiquitin and forms an ubiquitin adenylate intermediate. Ubiquitin adenylate donates ubiquitin to a cysteine in the E l catalytic site (Haas and Rose, 1982). The E l enzyme can only bind with two activated ubiquitin proteins by forming an interaction with ubiquitin as a thiol-ester and binding of the other ubiquitin as an adenylate (Pickart, 2001). Subsequently, the thiol-ester coupled ubiquitin is transferred to the E2 conjugating enzyme in the ubiquitination pathway. 1.5.1.2 Ubiquitin E2 Conjugating enzyme E2 conjugating enzymes belong to a family of small proteins (14-32 kDa) that share a common conserved core domain of 14-16 kDa with a similarity of 35%. Subsequent to the thiol-ester binding of ubiquitin to the activated E l the ubiquitin is transferred to the catalytic cysteine in the common core (UBC) of the E2 conjugating enzymes and forms another thiol-ester intermediate. The U B C domain is required for binding of distinct E3s, however there are a small 21 Figure 1.4. The ubiquitination pathway. The C-terminus of ubiquitin is attached to the conserved cysteine of the E l ubiquitin-activating enzyme by forming an ATP dependent thiol-ester formation. Ubiquitin is transferred via the C-terminus to the E2 ubiquitin-conjugating enzyme and linked through a thiol-ester formation. The E3 ubiquitin-ligase enzyme transports the target protein (TP) to E2 enzyme-ubiquitin and forms a complex. E3 catalyzes the transfer of the C-terminus of ubiquitin to the target protein. The target protein becomes polyubiquitinated by E3, which continues to transfer ubiquitin by forming a multi-ubiquitin chain via Lysine (K48). Polyubiquitination of target proteins results in activating the target protein for a particular function or targeting to the proteasome for degradation. (Adapted from Patterson (2002)) 22 number of E2 enzymes that can also interact with the final target substrate (Kalchman et al, 1996). Saccharomyces cerevisiae contains 13 E2 enzymes and over 25 E2s have been identified in mammalian family members (Jensen et al, 1995; Rajapurohitam et al, 1999). 1.5.1.2.1 Ubiquitin E2 conjugation variant enzymes A variant of the E2 conjugating enzyme family are the ubiquitin E2 conjugating variant (UEV) proteins. Although these proteins are homologous to ubiquitin-conjugating enzymes, U E V lack the critical catalytic cysteine residue (Sancho et al, 1998; Xiao et al, 1998). U E V as an inactive enzyme usually associate with an active E2 for ubiquitination of proteins. For instance, the yeast U E V homolog Mms2 and Ubcl3 E2 enzyme have to form a heterodimer to be able to function in polyubiquitination. The yeast Mms2/Ubcl3 conjugating enzyme forms a complex with Rad5, a RING finger ubiquitin ligase. This has been shown to be required for the polyubiquitin chain assembly of the monoubiquitinated P C N A (proliferating cell nuclear antigen) (Hoege et al, 2002; Tsui et al, 2005). Rad5 recruits Mms2/Ubcl3 conjugating enzyme complex to the D N A (Ulrich and Jentsch, 2000). Several studies have suggested that polyubiquitination by Mms2/Ubcl3 and Rad5 may mediate the assembly of protein such as P C N A and D N A polymerase 8 to the sites of damaged D N A and participate in limited D N A synthesis through the undamaged sister duplex (Hofmann and Pickart, 1999). 1.5.1.3 Ubiquitin E3 ligase The ubiquitin E3 ligases recognize target substrates or proteins and subsequently interact with the E2 conjugating enzyme. The binding of E3 with the target protein occurs directly or via additional proteins. In general, E3 enzymes such as the RING finger proteins function as a 23 scaffold to join E2 and the target protein in close proximity to permit transfer of ubiquitin from the E2 enzyme to the target protein (Zheng et al, 2000). However, other E3 enzymes (HECT (homologous to E6-AP carboxy terminus) domain E3s) have been identified that accept ubiquitin from the E2 enzyme (Scheffner et al, 1995). The HECT domain enzymes form a thioester bond between ubiquitin to an internal cysteine residue followed by conjugation of ubiquitin to a lysine group in the target. 1.5.1.3.1 RING-finger E3 ligase As previously described proteins with a RING finger domain have a typical conserved pattern of cysteine and histidine residues (Borden and Freemont, 1996; Freemont, 1993). This motif makes it possible for the RING domain to interact with two zinc atoms and subsequently fold into a unique cross-brace complex. Most proteins containing RING-fingers have been identified as E3 ubiquitin ligases in ubiquitin-dependent proteolysis (Jackson et al, 2000; Joazeiro and Weissman, 2000). It has been shown that RING fingers recruit the activated E2 enzyme and then act as a scaffold to transfer ubiquitin from the E2 conjugating enzyme to the target protein (Zheng et al, 2000). RING finger E3 ligases are categorized into single or multisubunit enzymes. Single subunit E3 ligases often interact directly with their targets, whereas multisubunit E3 ligases usually require formation of protein complexes to be able to bind to the target proteins (Borden, 2000; Jackson et al, 2000). Within the complex of the multisubunit E3 ligases the RING finger protein is usually quite small but essential for activity. Three types of the multisubunit E3 ligases have been identified termed APC (Anaphase-Promoting Complex), V C B (VHL-elongin C/elongin B) and SCF (Skp-l-Cullin-F-box protein) (Deshaies, 1999; Page and Hieter, 1999; 24 Pickart, 2001; Tyers and Jorgensen, 2000). The RING finger domain of these three multi subunit complexes does not identify the target protein, however is involved in recruitment of the E2 enzyme and assembly of additional components essential for the complex formation (Skowyra et al, 1999; Tan etal., 1999). 1.5.1.3.2 HECT domain E3 ligase A number of ubiquitin E3 ligases contain a 350 amino acid region named HECT that have homology to the C-terminal domain of the E6-AP archetype (E6-associated protein) (Huibregtse et al, 1993a; Huibregtse et al, 1993b). The HECT domain consists of a conserved Cys residue that accepts the activated ubiquitin molecule from the ubiquitin E2 conjugating enzyme (Scheffner et al, 1995). The N-terminus of HECT domain E3 ligases is not highly conserved and it is assumed that this region is involved in specific substrate recognition. E6-AP was the first discovered HECT domain E3 ligase and was shown in the presence of the auxiliary Human Papilloma Virus (HPV) oncoprotein E6 to target the cellular protein p53 to the proteasome (Scheffner et al, 1993). E6-AP was also capable of targeting other cellular proteins for degradation without the HPV oncoprotein E6 as observed for Blk of the Src family of kinases (Hakak and Martin, 1999; Oda et al, 1999). 1.5.1.4 E4 and U box-containing proteins E4 proteins belong to a family that have a common modified RING finger domain named U -box, which lack the metal-chelating residues necessary for the RING finger domain (Aravind and Koonin, 2000). The cysteine residues characteristics of the RING finger are not conserved in the U-box and it is assumed that hydrogen bonds and salt bridges stabilize the structure of the U -25 box. U-box proteins rely on E l and E2 for elongation of ubiquitin chains, but can function independently of E3 enzymes (Hatakeyama et al., 2001). The U-box enzymes function by ubiquitinating ubiquitin-tagged proteins or elongate short polyubiquitin chains by mediating transfer of ubiquitin to a previously conjugated ubiquitin molecule instead of the target protein (Koegl et al., 1999). In the latter case the E3 would be required to transfer the first ubiquitin protein to the target protein. Alternatively, U-box enzymes have been shown serve as ubiquitin ligases independent of E3s (Hatakeyama et al., 2001). However, it remains uncertain i f U-box enzymes play a role in recruiting the E2 component or i f it associates with the short ubiquitin chain linked to. the target protein. 1.5.1.5 26S proteasome The 26S proteasome is a major protein complex composed of a cylinder shaped barrel called the 20S core and a 19S cap that can be located at either end of the 20S particle (Fig. 1.5). The 20S complex comprises 28 subunits, which can be classified into two families named a and /3. Both a and j3 families contain seven types of subunits. Fourteen types of a and /3 subunits are required to form four stacked heptameric rings (Fig. 1.5) (Groll et al., 1997; Lowe et al., 1995). The two rings located on the outside of the 20S subunit contain seven a-subunits that are genetically related and structurally similar, and the two internal rings consist of seven similarly proteinous conserved (3-subunits (Fig. 1.5). The proteasome channel was suggested to be gated since the N-terminus of the a-subunits blocks the entrance to the proteolytic chamber (Groll et al., 2000; Groll et al., 1997). Supposedly, the proteolytic chamber can be reached by entering via a narrow channel leading from the surface of the a-rings (Groll et al., 2000; Kohler et al., 2001). The p-subunits of the 20S subunit possesses the proteolytic activity of the proteasome, 26 which is located inside the cylinder shaped barrel and comprises of six functional threonine protease active sites (Bochtler et al, 1999; Heinemeyer and Wolf, 2000). A purified complex of the 20S subunit was shown to hydrolyse a number of unfolded proteins and small peptides, but was not able to degrade multi-ubiquitinated proteins. The 19S subunit of 26S proteasome is known to have numerous roles including target protein selection, preparation and translocating into the 20S for degradation. Recently, the 19S regulatory subunit was shown to be involved in transcriptional elongation by R N A pol II (Ferdous et al, 2001). The 19S complex consists of at least 19 subunits and can be divided into two sub-complexes called lid and base (Fig. 1.5) (Glickman et al, 1998). The base contains six subunits named in yeast as Rptl-6, which are identified as ATPases of the ATPases Associated with diverse cellular Activities (AAA) family. The role proposed for the A A A ATPases in the base was to unfold and transfer target proteins through a gated channel into the 20S core (Braun et al, 1999; Groll et al, 1997; Horwich et al, 1999; Kohler et al, 2001; Strickland et al, 2000). Furthermore, A A A ATPases are identified in many multisubunit cellular mechanisms such as translocaters, transporters, membrane fusion complexes, and proteases (Beyer, 1997; Ogura and Wilkinson, 2001; Patel and Latterich, 1998). The base also includes three non-ATPases subunits (Rpnl, -2, and -10), which have been shown to directly associate with the cc-subunits of 20S (Davy et al, 2001; Fu et al, 2001). The lid of the 400-kDa 19S subcomplex, which contains eight of the remaining non-ATPase subunits (Rpn3, -5, -6, -7, -8, -9, -11, and 12), is able to be released or reattached from the proteasome in a reversible manner under certain conditions (Fig. 1.5). The lid has been shown to be required for specific degradation of polyubiquitinated proteins but its exact function remains uncertain (Glickman et al, 1998). 27 ATPases 20S core J 19S regulator 26S proteasome Figure 1.5. The 26S proteasome. The 26S proteasome is a major protein complex composed of a cylinder shaped core called 20S and a 19S cap that can be attached at either end of 20S. The 20S complex comprises fourteen different subunits and the 19S regulatory protein complex consists of at least 19 subunits. The cylinder shaped core of 20S is formed by four stacked heptameric rings. The 19S complex can be divided into two sub-complexes called lid and base. The base contains six subunits, which are identified as ATPases of the A A A (ATPases Associated with diverse cellular Activities) family. (Adapter from Kloetzel (2001)) 1.5.1.5.1 Ubiquitin recognition by the 26S proteasome The 26S proteasome identifies substrates that are tagged with polyubiquitin. In contrast, monoubiquitinated proteins are not directed to the proteasome, but instead serve as signals for cellular targeting or localization (Hicke, 1999; Pickart, 2000; Strous and Govers, 1999). For the proteasome to recognize a target, the targeted protein ought to possess at least four ubiquition 28 molecules linked via Gly-76 of ubiquitin to Lys-48 of the former chained ubiquitin (Thrower et al, 2000). In addition, it is thought that hydrophobic residues of polyubiquitin are involved in efficient protein degradation by the proteasome (Sloper-Mould et al, 2001). In addition to polyubiquitination by Lys-48, target proteins can also be polyubiquitinated via Lys-63 mediated by U E V E2 proteins and as a consequence may not be degraded by the proteasome. These Lys-63 linked polyubiquitinated proteins have been shown to be involved in signalling and D N A repair (Deng et al, 2000; Hofmann and Pickart, 1999; Spence et al, 2000; Spence etal, 1995). 1.5.1.6 Ubiquitination and DNA repair Recent studies suggest that ubiquitination may be part of a pathway that regulates the expression of proteins as a reaction to D N A damage. D N A damage sites are believed to stall the DNA-polymerase machinery and consequently arrest cell cycle. In general, nucleotide excision repair (NER) and base excision repair removes blocking lesions before the replication complexes hit one (Hoeijmakers, 2001; Kunkel et al, 2003). In eukaryotic cells NER initiates with identifying damaged D N A and subsequently creating lesions adjacent of the damaged D N A followed by removal of the damaged D N A (Huang et al, 1992). Base excision repair is a mechanism that removes an altered base, which is filled in by D N A polymerase and D N A ligase (Lindahl and Wood, 1999). When nucleotide excision repair and base excision repair pathways fail to detect damaged D N A , alternative pathways exist that can bypass the D N A lesion. These alternative pathways include three post-DNA replication repair mechanisms and depend on the ubiquitination pathway (Prakash and Prakash, 2002). 29 Two of the three mechanisms for post-replication D N A repair include translesion synthesis, (defined as either error-prone or error-free) which utilizes the Pol f and Pol 77 D N A polymerases. The third mechanism is error-free and involves template switching with the undamaged sister chromatid. The proliferating cell nuclear antigen (PCNA) is involved at a early stage in all of three repair pathways and the Lys-164 residue of P C N A plays a central role (Haracska et al, 2004; Hoege et al, 2002; Stelter and Ulrich, 2003). Initially, Lys-K164 of P C N A is monoubiquitylated by the E2 conjugating enzyme Rad6 and the E3 ubiquitin ligase Radl8 and interacts with the translesion polymerases (Haracska et al, 2002; Hoege et al, 2002; Prakash and Prakash, 2002; Stelter and Ulrich, 2003). Additional ubiquitin proteins can be tagged on the monoubiquitinated P C N A via linkage to Lys-63 (K63) of ubiquitin using E2 ubiquitin conjugating heterodimeric complex of Ubcl3-Mms2 and the E3 ubiquitin ligase Rad5. This type of K63-linked modification of P C N A enables the error-free bypass (template switching) pathway. 1.5.1.7 Regulation of transcription by ubiquitination and the proteasome Recent studies indicate that some transcription factors are regulated by ubiquitination. For example the transcription factor MET4 that regulates the met genes, which produce enzymes to biosynthesize the sulfur-containing amino acids methionine and cysteine, has been shown to be regulated by ubiquitin. At low levels of S-adenosylmethionine (SAM) transcription of the met genes is activated by MET4. However, at increased levels of S A M , MET4 is ubiquitinated by the E3 ligase SCFMet30 and inactivated and transcription of the met genes are shut down (Kuras etal, 2002). 30 Subunits of the proteasome have also been shown to be involved in gene regulation. Using immunoprecipitation and transcription assays it has been shown that activation of the yeast Ga l l -10 gene was dependent upon recruiting the base component of the 19S proteasome complex (Gonzalez et al, 2002). This showed that the base could function independently of the larger 19S structure. Similarly the SUG1 (Rpt6) was shown to bind the activation domain (AD) of the Herpesvirus transcription factor V P 16 when fused to GAL4 and be recruited to gal4 promoter. It was also shown that the VP-16 A D is activated upon ubiquitination and subsequently targeted for degradation. This suggests a model where transcription factors are relatively stable but inactive in the absence of ubiquitination. Once ubiquitinated the transcription factor is activated but then rapidly degraded by the proteasome (Salghetti et al, 2001; Zhu et al, 2004). 1.5.2 Virus infection and the ubiquitination pathway Viruses appear to utilize the ubiquitination pathway extensively in the process of infection and replication. Cellular proteins related to the ubiquitination pathway are exploited to participate in viral infection; but certain viruses may produce and utilize their own ubiquitin-associated proteins. Viral RING and HECT E3 ubiquitin ligase families have shown to be involved in numerous regulatory pathways such as activation, immune evasion and virus budding. As indicated above the HPV E6 E3 ubiquitin ligase was shown to be involved in degradation of the cellular tumor suppressor protein p53 (Thomas et al, 1999). The human cytomegaloviruses (hCMV) use E3 ligases such as US11 and US2 to reduce antigen presentation by negative regulation of M H C class I protein by causing retrograde translocation from the endoplasmic reticulum (ER) to the cytosol. Subsequently the cytosol M H C class I molecules were polyubiquitinated and degraded 31 by the proteasome (Furman and Ploegh, 2002; Kikkert et al, 2001; Shamu et al, 2001). Recent studies demonstrated that Nedd4 or Nedd4-like proteins, which are HECT E3 ligases, are required for viral budding. The data suggests that Nedd4 or Nedd4-like proteins are able to bind the retroviral Gag protein facilitating the release of viruses (Harty et al, 2000; Kikonyogo et al, 2001; Strack et al, 2000; Yasuda et al, 2002). The herpes simplex virus type 1 regulatory protein ICP0 has been thoroughly investigated and shown to be a RING finger E3 ubiquitin ligase that mediates the degradation of the nuclear domain (ND) 10 components and centromeres and consequently supports viral transcription (Banks et al, 2003). In addition, ICP0 has been shown to contribute to viral D N A replication since this protein is able to block the proteasomal degradation of cyclin D l (Yu et al, 1998). The herpesvirus infection of human cells has been shown to significantly induce the human ubiquitin gene (UbiB) mediated by the viral transcription factor ICP4 (Kemp and Latchman, 1988). 1.5.3 Virus infection and the 26S proteasome A number of viral proteins have shown to associate with the 19S or 20S subunits of the 26S proteasome including hepatitis B virus X protein (HBX), the human T-cell leukemia virus I (HTLV-I) tax and human immunodeficiency virus type 1 (HIV-1) Tat protein (Fischer et al, 1995; Huang et al, 1996; Rousset et al, 1996; Seeger et al, 1997b). H B X was able to form an interaction with PSMC1 (proteasome (macropain) 26S subunit ATPase 1), an ATPase-like component of the 19S subunit, and has shown to affect transactivation by PSMC1 in transient assays (Zhang et al, 2000). Furthermore, H B X was suggested to act as potential inhibitor of the protease activities of cellular proteasomes (Hu et al, 1999). The viral oncoprotein E7 of the 32 human Papilloma virus appeared to be involved in degradation of the retinoblastoma protein through association with the Rpt2 ATPase of the 19S subunit, which subsequently augments ATPase activity (Berezutskaya and Bagchi, 1997; Boyer et al, 1996). HTLV-I tax protein was shown to bind with a 20S osubunit, however the effect of this protein interaction on 20S and 26S activities has not been determined thus far (Huang et al, 1996). The H r V - 1 Tat protein interacts with purified native 20S, 19S as well as 26S complexes and is able to affect the activities of the 20S and the 26S proteasomes (Seeger et al, 1997a; Seeger et al, 1997b). 1.5.3.1 Baculovirus ubiquitination related genes OpMNPV contains a number of RING finger proteins including P34, IE2, LAP 1,-2, -3 and -4, CG30, EXONO and ME53 suggesting that the ubiquitination pathways play an important role in baculovirus infections. The inhibitor of apoptosis gene IAP-3 has recently been shown to possess ubiquitin ligase activity (Wright et al, 2005). Studies have also demonstrated that a number of RING finger proteins of BmNPV might function as E3 enzymes during infection (Imai et al, 2003). BmNPV IAP2, IE2, and PE38 were able to catalyze ubiquitination in presence of the Ubc4/5 ubiquitin E2 conjugating enzymes. Furthermore, mutations of the RING finger domain of IAP2, IE2, and PE38 showed that this motif was required for their E3 activities, which has been previously observed for numerous other other RING-finger E3 ligases (Imai et al, 2003; Jackson et al, 2000; Lorick et al, 1999). Guarino (1990) discovered a baculovirus ubiquitin with a 76% similarity to cellular ubiquitin that was functional in the ubiquitin-conjugation system (L. Guarino & A . Haas, personal communication). The baculovirus ubiquitin was expressed at late time p.i. and showed to be required for viral growth (Guarino, 1990). Interestingly the ubiquitin gene is one of the most 33 highly conserved baculovirus genes suggesting it plays an important role in the baculovirus life cycle (Li et al, 2002). 1.6 Hypothesis and Objectives Insect pests are responsible for enormous global losses in both food and forest crops. As baculoviruses are capable of a high rate of infection and a low impact on non-target organisms, they are an attractive alternative to traditional chemical pesticides. Baculoviruses typically demonstrate a narrow host range, infecting either a single or a small group of insects. The underlying mechanism of host range determination by baculoviruses is unknown and to date no host proteins have been identified that regulate insect susceptibility. However, several viral proteins have been shown to be involved in host range determination at the cell culture and whole insect level (Miller and Lu, 1997). To this end it is essential to obtain detailed knowledge regarding the interaction between viral and host proteins at the molecular level and to discover the role these interactions play in determining host range and virulence. In order for viruses to effectively infect their hosts they must utilize host specific proteins for transcriptional regulation and D N A replication. Baculovirus ie genes have an intimate association with the host as they are transcribed utilizing host proteins. In addition, the LE proteins function in the absence of other viral proteins suggesting that interactions with the host are required for their function. Most IE genes have been shown to express regulatory proteins that are essential for wild type levels of viral replication and infection. It is therefore proposed that association of IE proteins with specific host proteins represent key steps in the regulation of the viral life cycle. To confirm this hypothesis it is required that host proteins that interact with 34 viral proteins be identified. Therefore for this thesis we have investigated the OpMNPV IE regulatory protein P34 P34 was chosen due to functional and structural similarities viral proteins in other eukaryotic systems that are known to bind to host proteins (Burkham, Coen, and Weller, 1998; Everett, 2001; Ishov and Maul, 1996; Lukonis and Weller, 1997; Mainz, Quadt, and Knebel-Morsdorf, 2002; Maul, 1998; Maul, Ishov, and Everett, 1996; Murges et al., 2001; Okano, Mikhailov, and Maeda, 1999; Uprichard and Knipe, 1997). P34 is the OpMNPV homolog of A c M N P V and BmNPV PE38. Both P34 and A c M N P V PE38 up regulate gene expression and augment viral D N A replication in transient assays (Ahrens and Rohrmann, 1995; Kool et al, 1994; Milks et al, 2003) but it is not known how these proteins activate transcription and D N A replication. OpMNPV p34 has been classified as an early viral gene and also has a unique transcription pattern at late times post-infection (Wu, Stewart, and Theilmann, 1993). Instead of alternate splicing an additional transcript is expressed from an internal late promoter (ATAAG), which results in a 5' truncated transcript (Wu, Stewart, and Theilmann, 1993). Translation of the alternate p34 transcript results in a 20 kDa protein named P34A that lacks a number of functional domains and has no known function. P34 contain several protein domains known to be involved in gene regulatory pathways. These include basic, RING finger, acidic, and glutamine rich and leucine zipper domains (Krappa and Knebel-Morsdorf, 1991; Wu, Stewart, and Theilmann, 1993). The functions of these domains have not yet been experimentally proven. RING finger domains have been shown to be E3 ubiquitin ligases, which are required to transfer ubiquitin from E2 conjugating enzymes to the target protein (Borden and Freemont, 1996; Lorick et al, 1999; Zheng et al, 2000). As described above the P34 homolog of Bombyx mori NPV, BmPE38, has been identified as one of 35 four baculoviral proteins to possess E3 ubiquitin-ligase activity (Imai et al, 2003). These studies and many others indicate that ubiquitin ligases are playing essential roles in animal virus life cycles (Hagglund and Roizman, 2004; Scheffner and Whitaker, 2003; Vogt, 2000). Although several studies were conducted on P34 using transient assays, the role and mechanism of action of P34 in OpMNPV infections has not been further investigated. In addition it was unknown i f P34 interacts with cellular or viral proteins during the virus life cycles. Therefore in this study we have asked specific questions about the functions of OpMNPV p34 during infection and how it interacts with host cell proteins. Based on the hypothesis mentioned above we propose the following objectives for this thesis are the following: 1. Discover i f P34 forms homodimer structures and possibly heterodimers with P34A by conducting functional analyses of the P34 and P34A domains 2. Characterize the role of P34 during OpMNPV infection of Ld652Y cells 3. Determine i f P34 possesses E3 ubiquitin ligase activity and i f it is dependent upon the RING domain 4. Identify viral or cellular proteins that also interact with P34 and to determine i f these interactions are required for the virus life cycle. This is the first baculovirus study to date that investigates the interaction between baculovirus regulatory proteins and host cell proteins. 36 1.7 Appendix In addition to the main objectives of this thesis, another study was performed on an additional baculovirus regulatory gene. The appendix in this thesis describes a study that examined the role of IEO in very late gene expression. OpMNPV and A c M N P V IE1 have been found to activate the transcription of early and late promoters that are critical for viral infection (Carson et al, 1991; Guarino and Summers, 1986a; Kremer and Knebel-Morsdorf, 1998; Lu and Carstens, 1993; Nissen and Friesen, 1989; Olson et al, 2002; Olson et al, 2003; Pathakamuri and Theilmann, 2002; Rodems et al, 1997; Slack and Blissard, 1997; Todd et al, 1995; Wu et al, 1993b). Additionally, IE1 is also known to down regulate early promoters in A c M N P V infected cells (Theilmann and Stewart, 1991). 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Infection by baculoviruses is characterized by three distinct phases of viral gene expression (early, late and very late), which occur in a tightly regulated sequence primarily at the level of transcription. Upon infection the cycle starts with the expression of early viral genes, which are transcribed by the host R N A polymerase II (Friesen, 1997). Early gene expression includes regulatory genes and genes required for the formation of the D N A replication complex and ultimately viral D N A replication. Among the early OpMNPV regulatory proteins that are expressed in the early phase P34 has been shown to up regulate viral gene expression and in addition augment viral D N A replication (Ahrens and Rohrmann, 1995; Wu et al., 1993a; Wu et al., 1993b). This gene is unique because it is transcribed from both early and late promoters. The early promoter produces a 1.1 kb mRNA that is expressed from 1 h p.i. to 48 h p.i. and the transcript is translated into the 34 kDa protein. A second 0.7 kb transcript initiates from a late promoter that is internal to the p34 ORF and is expressed from 18 h p.i. to 120 h p.i. (Wu et al., 1993a). Translation of this transcript results in an N-terminally truncated 20 kDa form of P34 called p34A. The A c M N P V genome contains a homolog of OpMNPV P34 known as A c M N P V PE38. In a similar manner to OpMNPV, A c M N P V PE38 can produce both 38 kDa and an N-terminal truncated protein. Immunofluorescence studies revealed that A c M N P V PE38 was able to form 59 nuclear foci (Krappa et al, 1995; Murges et al, 2001). Although the presence of the smaller protein has been verified by Western blot analysis, an R N A transcript coding for it has never been detected (Krappa et al, 1995). . Several studies have indicated that P34 and its homologs play a central role in D N A replication. Transient expression assays with OpMNPV P34 or A c M N P V PE38 demonstrated that they were able to enhance D N A replication (Ahrens and Rohrmann, 1995; Kool et al, 1994). Furthermore, comparative studies with wild type and ape38 deletion mutant revealed that the deletion of PE38 produced a significant decrease in genomic replication (Milks et al, 2003). In addition to D N A replication both P34 and PE38 have been shown to be involved in gene regulation. Studies have revealed that P34 transactivates both the OpMNPV ie2 andp8.9 promoters (Wu et al, 1993a; Wu et al, 1993b) and that A c M N P V PE38 up regulates the expression of the A c M N P V helicase gene pi43 (Lu and Carstens, 1993). However, A c M N P V PE38 expression has been shown to be negatively regulated by high levels of IE1 (Leisy et al, 1997). P34 possesses a number of distinct protein domains that have been reported to function in number of different regulatory pathways (Wu et al, 1993a). The P34 protein includes a basic, RING finger as well as the acidic, glutamine rich and leucine zipper domain. These domains were also identified in A c M N P V PE38 and Bombyx mori N P V (BmNPV) PE38. The N -terminally truncated 20 kDa P34A consists of only the acidic, glutamine rich and leucine zipper domains. The objective of this study was to examine the function of P34 and P34A domains and the roles of these proteins in the OpMNPV infection. Novel methodologies such as R N A 60 interference (RNAi) are used as well as the established techniques of confocal microscopy, yeast 2-hybrid analysis and co-immunoprecipitation. R N A i can be used to examine the roles of P34 and P34A during viral D N A replication and budded virus production. P34 was analyzed using in vitro assays to determine i f it functions as an ubiquitin E3 ligase and participates in the ubiquitination pathway. 2.2 Results 2.2.1 Oligomerization of P 3 4 , P 3 4 A and P 3 4 domain deletions in 2-hybrid assays P34 contains several domains, which include the basic, RING finger, acidic, glutamine rich and the leucine zipper domains. The N-terminally truncated P34A lacks the basic and RING finger domains. To determine i f P34 forms homodimers and to identify domains required for oligomerization, yeast 2-hybrid constructs were prepared that contained p34, p34A or p34 domain deletions cloned into either the D N A binding domain (BD) vector (pBD-GAL Cam) or the transcriptional activation domain (AD) vector (pAD-GAL4-2.1) (Fig. 2.1 A). Initial results with p34 in both the BD and A D vectors showed that P34 was able to form homodimers but the protein interactions were determined to be weak based on growth on triple drop-out plates (Fig. 2. IB). In contrast P34 was found to form a strong heterodimer with P34A. Analysis of the P34 domains indicated that deletion of the basic domain enhanced the interactions with P34 whereas deletion of the RING finger, glutamine rich and leucine zipper domains eliminated dimerization P34. These results suggest that for P34 homodimerization the RING finger, glutamine rich and leucine zipper domains are required. The basic domain however appears to inhibit homodimerizaton as its deletion resulted in increased growth for P34-P34AB compared to P34-61 Figure 2.1. Analysis of protein-protein interactions between P34, P34A and P34 deleted domains in yeast 2-hybrid system. (A) P34, P34A and P34 domain deletions cloned in the 2-hybrid yeast vectors pBD-Gal4 Cam and pAD-Gal4-2.1. The domain deletions include the basic domain (P34AB), RING finger domain (P34ARING), glutamine rich domain (P34AQ) and the leucine zipper domain (P34ALZ). The numbers above the diagram indicate the position of the amino acids that are deleted in P34 domain deletions. Each construct was cloned in frame with the Gal4 D N A binding domain (BD) or activating domain (AD) of the yeast vectors. D> Symbolizes the location of the P34A start codon. The brackets ( ) indicate the regions that have been deleted in each construct. (B) Protein-protein interactions of P34, P34A or P34 domain deletions fused to either the Gal4 BD or the Gal4 A D as described in Fig. 2.1 A . The constructs were cotransformed into YRG-2 cells and 20 /xl of cells were spotted onto Trp-Leu-His dropout plates. 62 Figure 2.1. continued. A baste RING acidic Q LZ WT i — r n r r r m k i — B f H P34AB P34ARING domain deletions A D H l GAL4 promoter BD A D H l Terminator pUC <rt chloramphenicol 2-micron on pBD GaMCam A D H l G A L 4 promoter AD vectors pAD-GaM-2.1 B B D WT domain deleltons A D WT l —I u P34 P34 P34 P34 i P34 P34A AB ARING AQ ALZ a domain deletions P34 P34A P34AB P34ARING P34AQ P34ALZ pAD-Gil4-2.1 Trp Leu His DO 63 P34. Interestingly though P34AB had reduced heterodimerization with P34A relative to P34. These observations suggest that the basic domain alters P34 homodimerization and favours heterodimerization with P34A for a more stable protein interaction (Fig. 2. IB). These results were consistent with P34 expressed in either the B D or A D vectors. P34A was able to interact with the same constructs as P34 with the exception of P34A expressed from the BD vector, which showed a weak interaction with P34ARING (Fig. 2.IB). P34A formed very strong homodimers therefore to further define the domains required for this interaction two additional N-terminal deletions oip34A were cloned into the yeast BD (pPC62) and A D (pPC86) vectors (Fig. 2.2A). The N-terminal deletions include P 3 4 A i . i 7 i , which has the first 171 amino acids of P34 removed deleting the basic, RING finger domain and most of the glutamine rich domain but leaves the leucine zipper intact. The second construct expresses P34Ai_203, which has an additional 32 amino acids deleted and disrupts the leucine zipper domain. As shown in Fig, 2.2B P34A'i-i7i formed homodimers showing that the acidic and glutamine rich domains are not required for P34A homodimerization. However, disruption of the leucine zipper eliminates this interaction showing that this domain is required for P34A homodimerization. 2.2.2 Co-immunoprecipitation analysis of P34 and P34A using HA-tagged proteins To verify homodimerization of P34, P34A and the heterodimerization of P34-P34A observed in the yeast 2-hybrid analysis co-immunoprecipitation was conducted using the hemeagglutinin (HA) antibody for pulling down protein complexes. To perform immunoprecipitation with the H A antibody, vectors were constructed expressing P34 and P34A tagged at the C- terminus with 64 Figure 2.2. Analysis of protein-protein interactions between N-terminal deletions of P34A in yeast 2 -hybrid system. (A) P34A N-terminal deletions cloned into the yeast 2-hybrid vectors pPC62 and pPC86. The construct P34Ai.ni is a deletion of the first 171 amino acids, which deletes the basic domain, RING finger domain and partially deletes the glutamine rich domain, but contains an intact leucine zipper coiled coil domain (SLMSLALL) . P34Ai_203 retains only part of the predicted leucine zipper domain (SLALL) . The numbers above the diagram indicate the position of the amino acids that are deleted in P34A. Each construct was cloned in frame with the Gal4 D N A binding domain (BD) or activating domain (AD) of the yeast vectors pPC62 and pPC86 respectively. The brackets ( ) indicate the region that has been deleted. (B) Protein-protein interactions of P34 N-terminal deletions fused to either the Gal4 BD or the Gal4 A D as described in Fig. 2.2A. The constructs were cotransformed into YRG-2 cells and 20 / i l cells were spotted onto Trp-Leu-His dropout plates. (C) Schematic overview of the domains required in P34 and P34A homodimerization and P34 heterodimerization with P34A. The start codon of P34 and P34A are symbolized by • and D> respectively. I I Symbolizes the domain or domains involved in dimerization. 65 Figure 2.2. continued. acidic P34A P34A1-171 P34A1-203 deletions JOCl i C C l promoter GALA TenrMtor m M C S ampicillin ColEl art PPC62(BD) /OC1 promoter GtIA JOC\ Icmnifu A D . M C S ARSH4 CEN6 ampic] vectors 1E1 on EE j zmmm pPC86 (AD) B Trp-Leu-His DO 66 Figure 2.2. continued. c basic 4 ---3D 39 RING 91 T T t - f OCCHCCCC acidic Q LZ 155 ---186-203 140-146 200 126 i -235 307 P34 Domains required for dim eriz ation P34 - P34 P34 - P34A P34A - P34A P34A the H A epitope (Fig. 2.3A). Fig. 2.3B shows that P34 and P34A HA-tagged proteins are expressing the correct size and can be co-immunoprecipitated from protein extracts of transfected cells. In cells containing no HA-tagged proteins P34 was not immunoprecipitated by the HA-antibody (Fig. 2.3C, P34 only). Co-immunoprecipitation of P34 and P34-HA resulted in only a single detectable band corresponding to full length P34. We were unable to resolve both P34 and P34-HA and therefore confirm P34-P34-HA interactions. However, co-immunoprecipitation of P34 and P34A-HA pulled down both proteins (Fig. 2.3C). This was further confirmed by co-immunoprecipitation of P34-HA and P34A, which also detected both proteins (Fig. 2.3D). Finally co-immunoprecipitation of P34A and P34A-HA resulted in both bands being detected by western blot with the anti-P34 antibody. These results confirm the previous observations 67 A P34 P34-HA P34A basic RING acidic Q L Z basic RING acidic Q L Z HA acidic Q L Z acidic Q L Z HA P34A-HA B - P34-HA - P34A-HA k D a 32.5 -25 HA P34A-HA kDa 32.5 -25 -Figure 2.3. Analysis of oligomerization of P34 and P34A using co-immunoprecipitation. (A) Schematic representation of the proteins used for co-immunoprecipitation, which include P34, P34A, P34-HA and P34A-HA. P34 or P34A were fused at C-terminus to the H A (hemeagglutinin) epitope. (B-D) Western blot analysis (WB) of anti-HA co-immunoprecipitation (IP) of P34-HA or P34A-HA. P34-HA or P34A-HA was co-expressed with P34 or P34A in Ld652Y cells. (B) Western blot using the anti-HA antibody confirming the expression and immunprecipitation of P34-HA and P34A-HA. (C) Interactions of P34 with P34-H A or P34A-HA. Control (P34) contains only P34 and no H A tagged protein. (D) Interactions of P34A with P34-HA or P34A-HA. The interacting proteins were pulled down with H A monoclonal antibody (a-HA) and analyzed for either expression using H A monoclonal antibody or protein interaction using the P34 (a-P34) monoclonal antibody. The sizes of the molecular weight markers (kDa) are shown on the left. 68 obtained by the yeast two-hybrid assays showing that P34 and P34A heterodimerize and P34A-P34A homodimerize. Further analyses of P34-P34-HA wil l be necessary to confirm that both proteins are co-immunoprecipitated and thus supporting the yeast 2-hybrid results. 2.2.3 Localization of P34 and P34A in infected and transfected cells No previous studies have analyzed the cellular localization of OpMNPV P34 and P34A. To address this question we used confocal microscopy. To localize P34, a clone (pP34-CFP) was constructed that expressed P34 fused at the C-terminus to cyan fluorescent protein (CFP) (Fig. 2.4A). P34-CFP localization was initially analyzed in uninfected transfected Ld652Y cells and it was found to localize in the nucleus in a punctate pattern (Fig. 2.4B). In infected cells from 12 to 18 h .p.i., P34-CFP also accumulated in the nucleus in a punctate pattern. From 24 h p.i. P34-CFP showed in addition to the punctate appearance to accumulate at the nuclear membrane (Fig. 2.4B). From 48 h p.i. P34-CFP at the nuclear membrane became less pronounced and a more diffuse nuclear pattern emerges in addition to the punctate appearance. By 72 h p.i. the majority of the fluorescence is not associated with the punctate foci but is diffusely distributed in the nucleus and to a lesser extent in the cytoplasm. Due to the p34A internal transcriptional initiation at late times p.i. the analysis of P34-CFP in Fig. 2.4B could not distinguish between P34 and P34A. To compare and contrast the localizations of P34 and P34A two additional constructs pP34 M " A -CFP and pP34A-YFP were generated (Fig. 2.4A). pP34 M " A -CFP expresses P34-CFP but the translational start site of P34A was mutated into G C G to prevent P34A-CFP translation. pP34A-YFP expresses only P34A fused at the C-terminus to yellow fluorescent protein (YFP). 69 expressed protein r P34-CFP pP34-CFP |ATG AXG 1 I + P34A-CFP p p 3 4 M ^ A C F p c~ . f 2 v r m J |ATG GCG | C1F | P34-CFP pP34A-YFP «T" \e2vrm "~** I ' lATG " I ^ I P34A-YFP PP34ACFP < gB| 31 I | A T G 1 * P34A-CFP Figure 2.4. Localization of O p M N P V P34 and P34A in uninfected and infected Ld652Y cells. (A) Schematic diagrams of the fluorescent protein (FP) constructs used for localization of P34 and P34A. P34, P 3 4 ^ A and P34A were fused at the C-terminus to the cyan fluorescence protein (CFP) and P34A was fused at the C-terminus to the yellow fluorescence protein (YFP). Ld652Y cells were transfected with (B) pP34-CFP, (C) pP34 M ^ A -CFP and pP34A-YFP or (D) pP34A-CFP. After transfection the cells were infected with OpMNPV (MOI: 5) and analyzed at 12, 18, 24, 48 and 72 h and transfected non-infected cells were also included. Expression of P34-CFP, P34 M ^ A -CFP and P34A-CFP are shown in red, P34A-YFP is shown in green and the DAPI nuclear staining is shown in cyan. The merged images in Fig. 2.4B and D include expression of the CFP fusion proteins and DAPI. In addition, merged images are of both p 3 4 M - A _ C F p m d p 3 4 A _ Y F P localization but not DAPI are illustrated in Fig. 2.4C. The cells were also visualized by light microscopy. The images of the expressed proteins in the cells were obtained sequentially using a confocal microscope (Leica). To portray a more informative image of protein localization during infection, some samples contain more than one localization series, which is indicated by a bracket (see Fig. 2.4C, 48 h p.i.). 70 71 Figure 2.4. continued. C P 3 4 M _ > A - C F P P34A-YFP DAPI Transfcctcd cells Merge 18 h p.i. 72 Figure 2.4. continued. Expression of P34 M _ > A -CFP in the cotransfected cells showed a highly punctate distribution as observed with P34-CFP (Fig. 2.4C). P34A also showed a similar punctate appearance and the merged images show that the two proteins are highly colocalized. In infected cells from 12 to 48 h p.i. surprisingly both P34 M ^ A -CFP and P34A-YFP were localized predominately in the nucleus (Fig. 2.4C). The major difference observed between infected and transfected cells was that in infected cells a significant amount of P34M~^A-CFP and P34A-YFP was not associated with the punctate foci and dispersed throughout the nucleus. To determine i f P34A depends on P34 to form punctate nuclear structures in transfected and infected cells localization studies were performed with a fourth construct expressing P34A-CFP (Fig. 2.4A). In the absence of P34 expression P34A-CFP showed a predominantly uniform distribution in both the nucleus and cytoplasm (Fig. 2.4D). In the cytoplasm however there were a number of denser diffused dots. Unlike the punctate localization previously observed when 73 P34 and P34A both are expressed in transfected cells, P34A by itself did not form foci in both the nucleus of transfected and infected cells. 2.2.4 P34 plays an important role in viral DNA replication and budded virus production in infected Ld652Y cells The function of P34 in D N A replication and budded production was explored throughout the course of infection by silencing p34 expression using R N A interference (RNAi). Prior to preparation of p34 double stranded (ds) RNA, the 3' end of the p34 ORF was amplified from Litmus28i-P34PstI and the resulting template was used to generate dsRNA (Fig. 2.5A). Time course analysis was performed in Ld652Y cells transfected withp34 dsRNA and infected by OpMNPV. Transfected and infected cells were analyzed at 12, 24, 48 and 72 h p.i. for viral D N A replication, budded virus production and expression of P34 and P34A. To confirm that P34 and P34A expression was silenced throughout infection Western blot analysis was performed to compare between a control wild type infection and infected cells treated withp34 dsRNA by Western blot (Fig. 2.5B). In OpMNPV infected cells expression of P34 and P34A was consistent with previously published results (see Fig. 1.2B, Chapter 1). In contrast in cells depleted of P34 by R N A i , P34 and P34A expression was completely shut down. To examine the function of P34 relative to viral D N A replication, cells infected with wild type OpMNPV and infected cells depleted of P34 were compared. The onset of viral D N A replication for wild type OpMNPV infected cells is approximately 18 h p.i. (Fig. 2.5C). The levels of replication for the infected cells treated with p34 dsRNA were very low and it was unclear i f the time of intiation of D N A replication was affected. Compared to wild type OpMNPV, the levels of viral D N A replication in the infected cells treated with p34 dsRNA 74 Figure 2.5. Analysis of viral DNA replication and budded virus production in untreated Ld652Y cells and in cells depleted of P34 using RNAi. (A) Schematic overview of the preparation of p34 dsRNA. M l 3 Forward and Reverse primers amplified the 3' end of the p34 ORF from Litmus28i-P34PstI, which was used as a template to generate double stranded R N A transcribed by T7 R N A polymerase. Ld652Y cells infected with wild type OpMNPV or in cells depleted of P34 by R N A i were analyzed for (B) P34 Western blot analysis, (C) viral D N A replication and (D) budded virus production. R N A i was performed by transfection of Ld652Y cells with dsRNA followed by infection with OpMNPV (MOI: 5). Infected cells and supernatant were harvested at 12, 18, 24, 48 and 72 h p.i. Total viral D N A was determined using the slot blot method followed by hybridization with a viral 3 2P-labelled probe. The signal of each sample was quantified using a phosphoimager (Molecular Dynamics). The B V titers were determined by quantitative PCR and compared to a standard curve of a previously tittered OpMNPV B V stock, which was included in every Q-PCR assay. Western blotting was performed using a P34 monoclonal antibody and the location of P34 and P34A are shown on the right. Mock represents cells that were not treated with dsRNA and OpMNPV. Each time point in figures 2.5C and D presents and average of two separate samples. 75 ure 2.5. continued. B pS4 O R F I 5'* Liz Pstl I Litmus28i-P34PstI M13F —• 5. 3-T 7 - » | — T7 •M13R 1 E 12 h p.i. 18 h p.i. 24 h p.i. 48 h p.i 72 h p.i. - P34 - P34 - P34 - P34 - P34A - P34 - P34A 76 77 showed a significant reduction of approximately 90% (Fig. 2.5C). B V production levels of the R N A i p34 depleted virus were also compared to wild type infected cells (Fig. 2.5D). Relative to wild type infection, budded virus production of infected cells treated with p34 dsRNA was reduced approximately 1 to 2 logs throughout the infection (Fig. 2.5D). These results show that OpMNPV P34 and P34A are essential for high levels of replication and have a dramatic impact on both D N A replication and B V production. 2.2.5 P34 is an E3 ubiquitin ligase and depends on the RING finger domain for activity P34 contains a RING finger domain, which suggests that it can likely function as an E3 ubiquitin ligase. Recent studies with the P34 homolog BmNPV PE38 showed that it acts as an E3 ubiquitin ligase and relies on the RING-finger domain for its activity (Imai et al, 2003). To determine i f P34 also possessed ubiquitin ligase activity glutathione-S-transferase (GST) fusion constructs (pGEX2T-P34) were used for in vitro ubiquitination assays. pGEX2T-P34 expresses GST fused to the N-terminus of P34 (Fig. 2.6A). To determine whether the RING finger domain was required for ubiquitin ligase activity, a second construct was included that contained GST fused to the C-terminus of the P34 with the deletion of the RING finger domain (pGEX2T-P34ARING). GST-P34, GST-P34APJNG and GST were expressed in E. coli and subsequently used in ubiquitination assays. After 1.5 hour of incubation the samples were examined for ubiquitin ligase activity by Western blot analysis using a monoclonal ubiquitin antibody. GST-P34 produced a smear of high molecular weight protein bands from approximately 47.5 kDa and beyond 175 kDa (Fig. 2.6B). The in vitro ubiquitination assays using GST-P34ARTNG and GST did not show any signal on the Western blot (Fig. 2.6B). These results show that P34 is also an E3 ubiquitin ligase and the RING finger domain is required for ligase activity. 78 A pGEX2T-P34 basic RING acidic Q I tacprm ^ GST • i i i n D G E X 2 T - P 3 4 A R I N G «f toe M M G S T 1 1 pGEX2T C tacprm ^_ GST B 6 2 -47.5-32.5-Figure 2.6. In vitro ubiquitination assay of P34 and P34ARING. (A) Schematic representation of pGEX2T constructs that express P34 glutathione-S-transferase (GST) fusion proteins used for ubiquitination assays. The pGEX2T constructs consist ofp34 or p34 containing a deletion of the RING finger domain (p3 4A RING). (B) Western blot analysis of ubiquitination assays with the purified GST tagged OpMNPV P34, P34ARING or GST by itself. The ubiquitinated proteins were detected using an ubiquitin monoclonal antibody. The sizes of the molecular weight markers (kDa) are shown on the left. 79 2.3 Discussion The p34 gene differs from other OpMNPV genes for the reason that it has a unique transcription pattern. In addition to transcription of the p34 mRNA in the early phase of infection from an early promoter, a 0.7 kb 5' truncated transcript is expressed from an internal late promoter at late times p.i. (Wu et al, 1993a). Translation of both transcripts results in the 34 kDa full length P34 protein and a 20 kDa N-truncated protein called P34A (Wu et al, 1993a). Although several studies were conducted on P34 using transient assays, the role and mechanism of action of P34 in OpMNPV infections has not been further investigated. This study explores the function of P34 during OpMNPV infection. We also determined i f P34 and P34A form homo- and heterodimers and identified protein domains required for those interactions. P34 possesses a number of distinct domains including a basic, RING finger, acidic, glutamine rich and leucine zipper domains (Wu et al, 1993a). The N-terminally truncated P34A consists of the acidic, glutamine rich and leucine zipper domains. In order to determine i f P34 forms homodimers yeast 2-hybrid, co-immunoprecipitation assays and colocalization assays were conducted. The results support that P34 is able to form homodimers, however the protein-protein interaction appeared weak compared to P34-P34A and P34A-P34A dimerization (Fig. 2.IB; 2.3B). This suggests that heterodimerization of P34-P34A would be favoured over P34-P34 interactions. The domain deletions indicated that for P34 homodimerization the RING, glutamine, and leucine zipper domains were required (Fig. 2. IB; 2.2C). Deletion of the basic domain enhanced homodimerization suggesting that it inhibited P34-P34 interactions. In contrast, deletion of the basic domain appeared to increase the interaction of P34-P34A suggested it might enhance heterodimerization. For formation of P34A-80 P34A homodimers the P34 N-terminal deletion constructs suggested that only the region containing the complete leucine zipper coiled coil domain (SLMSLALL) from amino acids 186 to 235 was required (Fig. 2.2C). Our analysis of P34 therefore suggests that the RING, glutamine, and leucine zipper domains are required for P34 homodimerization implying a precise tertiary structure is needed. Each of these types of domains is well known to facilitate protein-protein interactions in other systems (Chen et al, 1998; Grbavec et al, 1998; Hattori et al, 2003; Lekstrom-Himes and Xanthopoulos, 1998; Miyasaka et al, 1993; Pinto and Lobe, 1996; Ren et al, 1999). The formation of homo- and heterodimers can play a critical role in regulating the activity of proteins. For example a number of transcriptional regulatory proteins have been shown to be dominantly inhibited by heterodimerizing via leucine zipper domains with partners that lack specific sequences such as D N A binding domains (Cooper et al, 1995; Hattori et al, 2003; Krylov et al, 1995). Alternatively it has also been shown that absence of a dimerization domain such as the leucine zipper of the transcription factor NF-IL6 resulted in loss of dimerization and consequently degradation of these proteins by the ubiquitin-proteasome system (Cooper et al, 1995; Hattori et al, 2003). The P34 domain deletions suggested that the RING domain was also required for homodimer formation, which has also been shown for other proteins containing this domain. The M D M 2 proto-oncoprotein and the structurally related M D M X contain RING finger domains and have been shown to interact with the p53 tumor suppressor gene (Barak and Oren, 1992; Momand et al, 1992; Shvarts et al, 1996). Yeast 2-hybrid analysis indicated that the RING finger domain of M D M 2 was required for homodimerization and also to form a heterodimer via the comparable domain of M D M X (Tanimura et al, 1999). 81 Cellular localization of P34 and P34A This has been the first study to examine the cellular localization of OpMNPV P34 and P34A. Confocal microscopy was performed using fluorescent tags to localize P34 and P34A in both transfected and OpMNPV infected Ld652Y cells, hi transfected cells P34-CFP localized predominately in the nucleus in a distinct punctate pattern (Fig. 2.4B and C). In infected cells the P34 was distributed diffusely in both the cytoplasm and nucleus but similar to transfected cells was predominantly in punctate nuclear structures at early times post-infection. From 24 h p.i. P34 was also observed along the inner nuclear membrane and by 72 h p.i. the punctate structures had largely dissapeared. Previous studies on the P34 homo log A c M N P V PE38 observed similar but not identical results. PE38 was shown to form very fine punctate structures at early times p.i., which disappear very rapidly after replication is initiated (8 h p.i. in A c M N P V infected cells (Murges et al, 2001)). In contrast the P34 associated punctate structures remain after initiation of replication up to very late times p.i. The initial analysis of P34 could not distinguish between the cellular location of P34 and P34A. To address this problem two constructs were used for colocalization studies. In addition to a construct with P34A fused to YFP ap34-CFP mutant was used that had the internal start codon of p34A changed into G C G producing the protein P34 M _ > A -CFP . The results showed that P34 and P34A are colocalizing in uninfected and infected cells in the nucleus (Fig. 2.4C). In transfected cells in the absence of P34 it was observed that P34A was expressed in both the nucleus and cytoplasm in a uniform pattern with some diffuse spots in the cytoplasm. This result indicates that P34A depends on P34 for localization as punctate foci in the nucleus. Nuclear expression appears to contrast with previous results that were able to detect in A c M N P V infected cells the 20 kDa late PE38 protein only in the cytoplasm by subcellular fractionation instead of 82 immunofluoresence (Krappa et al, 1995). The confocal analysis of P34A-YFP may be more sensitive than cell fractionation or this result represents a functional difference between OpMNPV P34 and A c M N P V PE38. The co localization of P34-CFP and P34A-YFP supports the results obtained with the yeast 2-hybrid and co-immunoprecipitation analysis suggesting that these two proteins are interacting with each other forming heterodimers. The reason for the heterodimerization remains to be determined but similarities with other regulatory proteins would suggest that P34A is potentially negatively regulating the activity of P34. R N A i experiments that knocked out P34 function dramatically reduced viral D N A replication and budded virus production relative to OpMNPV wild type infection (Fig. 2.5C and 2.5D). This would agree with previous transient assays, which have shown that P34 is not essential but significantly enhanced D N A replication (Ahrens and Rohrmann, 1995). These results suggest that P34 and its homolog A c M N P V PE38 serve similar functions. Previously Milks et al. (2003) showed using a pe38 deletion virus that D N A and B V production were also significantly reduced. In addition, studies indicated that P34 transactivates the OpMNPV ie2 promoter (Wu et al, 1993a; Wu et al, 1993b) and A c M N P V PE38 up regulates the expression of the A c M N P V helicase genepl43 (Lu and Carstens, 1993). Therefore the loss of P34 expression could perhaps reduce IE2 and PI43 helicase expression and as a consequence negatively affect viral D N A replication. It is unclear at the moment i f loss of P34 function delays the onset of viral replication as the levels of viral D N A replication were too low at early times to distinguish time of initiation (Fig. 2.5C). However, in A c M N P V infected cells the loss of PE38 function dramatically decrease replication but did not affect time of onset (Milks et al, 2003). In vitro ubiquitination assays performed in this study have also determined that P34 has 83 ubiquitin ligase activity that depends on the RING finger for activity (Fig. 2.6B). Recently it was shown that the P34 homolog Bombyx mori NPV PE38 also functions as an E3 ubiquitin ligase (Imai et al, 2003). Ubiquitination is involved in many regulatory pathways. This study and others are suggesting that P34 appears to have a significant impact on many viral processes including D N A replication, transcriptional activation, and budded virus production (Wu et al, 1993a; Wu et al, 1993b). A c M N P V and BmNPV PE38 were showed to colocalize with the RING finger proteins IE2 and promyelocytic nuclear bodies (PML) in insect and mammalian cells in NDlO-like structures (Mainz et al, 2002; Murges et al, 2001; Okano et al, 1999). Several studies with herpesviruses have indicated that ND10 forms an interaction with parental viral genomes, which becomes a site for viral immediate-early (IE) gene transcription and are subsequently assembled into D N A replication compartments (Burkham et al, 1998; Everett, 2001; Ishov and Maul, 1996; Lukonis and Weller, 1997; Maul, 1998; Maul et al, 1996; Uprichard and Knipe, 1997). Everett et al. (2001) suggested that the immediate-early RING finger regulatory proteins ICP4 and ICF27 of herpes simplex virus type 1 (HSV-1) are recruited into the viral nucleoprotein complex very rapidly after the onset of infection. Subsequently, the ICP4 and ICF27 nucleoprotein complex were shown to induce the formation with associated ND10 bodies. BmNPV PE38 and IE2 in confocal microscopy studies have also been shown to colocalize with the replication related proteins LEI, D N A binding protein (DBP) and LEF-3 (Imai et al, 2005; Mainz et al, 2002; Okano et al, 1999). Therefore, it may be possible that P34 is initiating or facilitating the assembly of the transcription or replication complex potentially in partnership with LE2. In summary, the results obtained in this study support the conclusion that OpMNPV P34 is a predominantly nuclear protein that functions as an E3 ubiquitin ligase, forms homodimers and 84 heterodimers with P34A, is involved in viral D N A replication and potentially budded virus production. Additional studies are necessary to determine the associations of P34 with other viral and cellular factors such as those in the ubiquitination pathway to identify the role they play in viral replication. 85 2.4 Materials and Methods 2.4.1 Cells and virus OpMNPV-WT virus was propagated in Lymantria dispar 652Y cells, which were maintained in TCI00 medium (Invitrogen) as previously described (Summers and Smith, 1978). Ld652Y cells used for the R N A i time course experiment and colocalization studies were infected with OpMNPV using a multiplicity of infection (MOI) of 5. Virus titers were determined by end point dilution (Summers and Smith, 1978) and quantitative PCR (Lo and Chao, 2004). 2.4.2 Yeast 2-hybrid assay A yeast 2-hybrid assay was used to determine the domains involved in P34 homodimerization. Full-lengthp34 ORF was amplified using plasmid P34SalI and 5' primers p373 ( 3 ' - G A A T A G T G T C G A C G T T T G A C A A A T G T A T - 5 ' ) and 3' p533 (5'-T A A G A A T T C A A C A T G T C C T C A A G A T A T T - 3 ' ) . The 958 bp PCR product was digested with Sail and EcoRl and subsequently cloned into both pBD-GAL4 Cam (GAL4 DNA-binding domain vector) and pAD-GAL4-2.1 (GAL4 activating domain vector) (Stratagene). The resulting constructs were named pBD-P34 and pAD-P34 (Fig. 2.1 A). Two p34A constructs (pBD-P34A and pAD-P34A) were produced using the 5' primer p789 (5'-G T G T G A A T T C T T T A T G T T G G C G C T C A A - 3 ' ) and p533. Primer p789 anneals downstream of the RING finger domain and produces a 584 bp fragment that lacks the basic and RING finger domains. After EcoRl and Sail digestion the fragment was subsequently cloned into both the 86 pBD-GAL4 Cam and pAD-GAL4-2.1 vectors. The resulting constructs were named pBD-P34A and pAD-P34A (Fig. 2.1 A) A series of clones were constructed that contained single domain deletions across the P34 protein (Fig. 2.1 A). The basic (P34AB) domain in pBD-P34 was deleted by PCR using the template pBD-P34 with the primers p729 (5 ' -CCGGTCACGGCAGTGGCGGCGGTTTG-3 ' ) and p730 (3 ' - T A T C G C C G G A A T T C A A C A T G T C C T C A - 5 ' ) . The P34 RING finger domain (P34ARLNG) was deleted by PCR using the template pBD-P34 with primers p727 (5'-A A C A A A A A A G T T G G T A C C T G G C A A G C - 3 ' ) andp728 (3 ' -GGCAGTGGCGGCGGTT-5 ' ) . The glutamine rich (P34AQ) domain was deleted by PCR using the template pBD-P34 with primers p725 ( 5 ' - C T G G A C A G G T C G G A A G C G T - 3 ' andp726 (3'-G T C G T T C G T G A T A A A G A A C C T G - 5 ' ) . The leucine zipper coiled-coil (P34ALZ) domain was deleted by PCR using the template pBD-P34 with primers p748 ( 5 ' - G A A G C G G C G C A A A G C -3') and p759 (3 ' - C A G A C C A C A T T G G C G G A C - 5 ' ) . A l l four amplified products were religated to generate the constructs pBD-P34AB, pBD-P34ARLNG, pBD-P34AQ and pBD-P34ALZ (Fig. 2.1 A). The modifiedp34 deletion ORF from each construct was then digested with EcdRl and Sail, and cloned into pAD-GAL4-2.1. The clones that were obtained include pAD-P34AB, pAD-P34ARING, pAD-P34AQ and pAD-P34ALZ (Fig. 2.1 A). A p34 5' deletion series was also produced in the vectors pPC62 and pPC86, which contain the GAL4 binding domain (BD) and the activating domain (AD) (Chevray and Nathans, 1992) (Fig. 2.2A). A construct containing a 171 amino acid deletion from the N-terminus of P34 was produced by digesting p34NRev with Ncol, polishing with T4 polymerase followed by digestion with Xbal. The .Y&al-blunt fragment was subsequently cloned into pPC62 to produce pPC62-P34Ai.i7i. A similar approach was used to clone the same fragment into pPC86, that is, 87 p34NRev was digested with Ncol, polished with T4 polymerase and then digested with Sstl. The SM-blunt fragment was subsequently cloned into pPC86 to produce pPC86-P34Ai.ni. A construct containing a 203-residue deletion from the N-terminus of P34 was produced by digesting Op47Nsi with Sstl and Pvull. The SM-blunt fragment was subsequently cloned into pPC86 to produce pPC86-P34Ai_203. Similarly, pPC62-P34Ai.203 was created by digestion of p34NsiN with Pvull and Xbal and insertion into pPC62. D N A binding domain constructs were cotransformed with activating domain plasmids into the yeast strain YRG-2 according to the Hybrizap-2.1 X R library protocol (Stratagene). Transformed cells with equal densities for each sample were applied (20 ul) as spots onto Trp-Leu-His dropout (DO) SD-media (Amersham Biosciences) and the cotransformants were screened for high stringency interactions. 2.4.3 Western blot analysis For Western blot analysis, transfected and/or infected cells were harvested, collected in an eppendorf tube and pelleted for 5 minutes at 1300 g. The supernatant was removed and the cells were washed gently with 1 ml of I X Tris-buffered saline ( IX TBS; 137 m M NaCl, 2.7 m M KC1, 25 m M Tris, pH 7.4) and centrifuged for 5 min at 1300 g. This wash procedure was repeated twice and the cells were lysed by freeze-thawing (respectively at -80°C and 37°C) three times. Protein sample buffer (5x; 0.5 M Tris pH 6.8, 10% glycerol, 10% SDS (w/v), 5% 2-/3 mercaptoethanol, 0.05% (w/v) bromophenol blue) was added to a final concentration of I X and D N A was sheared with a lOcc syringe equipped with 24-gauge needle. Samples were heated to 95°C for 5 minutes. 88 Protein samples were separated by denaturing SDS polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, 1970) and transferred onto Immobilon-P polyvinylidene difluoride membranes (Millipore) using a mini-blotter (Biorad) according to the manufacturers recommended protocol. The blots were initially incubated for 1 hour with 5% blotto (5% milkpowder in I X phosphate-buffered saline (PBS; 137 m M NaCl, 2.7 m M KC1, 10.14 m M N a 2 H P 0 4 , 1.76mM K 2 H P 0 4 , pH 7.4)-0.02% Tween followed by incubation for 1 hour with 5% blotto-0.02% Tween containing a 1:750 dilution of the an ubiquitin monoclonal antibody (P4D1, sc-8017; Santa Cruz Biotechnology), 1:1000 H A monoclonal antibody ( H A . l l , MMS-101R; CRPinc) or 1:1000 dilution of P34 monoclonal antibody (BI 1B10). Blots were washed 3 times in I X PBS and then incubated for 1 hour with a 1:10,000 dilution of a goat anti-mouse horseradish peroxidase secondary antibody (315-035-044; Jackson Laboratories). Finally, the blots were washed 3 times in I X PBS and exposed using E C L chemiluminescence reagents (Amersham Biosciences). 2.4.4 Preparation of dsRNA To obtain dsRNA ofp34 for R N A i experiments the ORF was cloned into the multiple cloning site (MCS) of Litmus28i, which is flanked by T7 promoters located (NEB). The primers p679 (5' - C C G G G A T C C A A C A T G T C C T C AAGAT-3 ' ) and p680 (3'-T A G T G A G T C Q A A T T C C T G C A G C C A A - 5 ' ) were used to amplify the entire p34 ORF from pBD-P34. The 980 bp fragment was digested with BamHl and EcoRl and cloned into Litmus28i, which generated pLitmus28i-P34. pLitmus28i-P34 was digested with PstI and BamHl, which deleted 580 nucleotides from the 5' end of p34, and the remaining vector was 89 religated to generate Litmus28i-P34PstI which was the final clone used to generatep34 dsRNA (Fig. 2.5A). Prior to in vitro transcription of dsRNA, the template was prepared by PCR according to the manufacturer's protocol of the HiScribe R N A i transcription kit (NEB). Briefly, for PCR amplification 100 17 g of Litmus28i-P34PstI was used as template and 625 pmol of M l 3 forward and M13 reverse primers. The amplified product was purified and a 0.5 /xg aliquot was used as the template to obtain dsRNA using T7 R N A polymerase. To establish stable dsRNA complexes the prepared dsRNA was denatured for 20 minutes at 65 °C and renatured slowly to room temperature. 2.4.5 R N A i time-course experiment Ld652Y cells were seeded onto 6-well or 24-well culture plates with a density of 1 x 106 or 0.75 x 106 cells/well respectively and incubated at 27 °C 12 hours prior to transfection. The liposomes, which are used to transfer the D N A into the cell, were prepared as previously described (Campbell, 1995). Transfection efficiency was determined using plasmid D N A , constitutively the green fluorescent protein (GFP). Six-well or 24-well culture plates of Ld652Y cells were transfected with 30 pig or 7.5 \ig dsRNA and then infected with OpMNPV-WT virus at an MOI of 5. In addition to the transfected cells (0 h p.i.) the infected cells and supernatant were collected at 12, 18, 24, 48 and 72 h p.i. The time course experiment was performed two times. 90 2.4.6 Budded virus titration by quantitative PCR (Q-PCR) Titers were determined using the protocol of Lo and Chao (2004). Briefly, the infected and uninfected cells of the R N A i time course experiment were collected into an eppendorf tube and centrifuged for 5 min at 2,650 g. The supernatant was removed from the cells and the viral D N A was extracted using the viral nucleic acid isolation kit (High Pure, Roche Mannheim, Germany). An aliquot of 100 fil of supernatant was added to 50 fil Proteinase K (20 mg/ml), 300 fil working buffer (200 fig poly (A) carrier R N A + 6 M guanidine-HCl, 10 m M urea, 10 m M Tris-HCl, 20% Triton X-100 (v/v), pH 4.4). Subsequently the samples were incubated overnight at 52 °C and 50 fil of viral D N A was eluted for each sample according to the manufacturer's protocol. To determine the number of budded viruses present in each sample, 8 fil of extracted viral nucleic acids was used in a total volume of 20 fil, which also contained 0.5 fiM of the primers P846 (5 ' - C G C T G A C G C A A T G G T A C T G T - 3 ' ) andp847 (3 ' -TTTACTCGTCGTGCGTTTGG-5') and I X S Y B R Green I master mix (Dynamo HS Sybr Green, NEB). Q-PCR was performed using the Mx4000 multiplex quantitative PCR system (Stratagene) using the PCR cycle profile of 1 cycle of 15 seconds at 95 °C, 40 cycles of 30 seconds at 95 °C, 24 seconds at 52 °C and 20 seconds at 72 °C. To determine the dissociation curve for each Q-PCR experiment, the temperature was increased by 1 °C from 55 °C up to 95 °C for 41 cycles with 30 seconds per cycle. The resulting data was analyzed using the software provided by Mx4000 multiplex quantitative PCR system. A standard curve was included in every Q-PCR assay consisting of a dilution series of an OpMNPV B V stock that previously was tittered by end point dilution. 91 2.4.7 Analysis of DNA replication using slot blot procedure The cells of the R N A i time course experiment were harvested and pelleted for 5 min at 2,650 g. To release the viral D N A from the cells the pellets were resuspended in 0.4 M NaOH and 0.125 m M E D T A at a density of 1,000 cells per /xl and incubated at 95 °C for 10 minutes. A total of 30,000 cells for each sample were applied into a slot of the Schleicher & Schuell slot blot apparatus using vacuum conditions and directly transferred onto the Zeta-Probe GT membrane (Bio-Rad). The membranes were hybridized with a 32P-probed OpMNPV P34NsiN plasmid (RadPrime; Invitrogen Life Technologies) according to the Zeta-Probe membrane protocol (Bio-Rad). Hybridized membranes were exposed to Kodak Phosphorscreens and scanned using the Storm Phosphorimager (Amersham). To quantify the signal of each sample on these scanned membranes ImageQuant TL v2003.02 software was used (Amersham). 2.4.8 Localization of P34 and P34A To localize P34 and P34A in Ld652Y cells the ORFs were cloned in frame and upstream of with the genes encoding the cyan or yellow fluorescent protein (CFP or YFP respectively) (Clontech). Furthermore, the fusion genes were subcloned into p2Zop2E downstream of the OpMNPV iel promoter, which allows transcription of these genes in absence of any viral factors. The p34 ORF was amplified by PCR of pBD-P34 with the primers p773 (5'-C C G A A G C T T A A C A T G T C C T C A A G A T - 3 ' ) and p774 (3'-G A A T G G G A T C C A C T C T A G A G C C C T A - 5 ' ) producing a fragment of 955 bp. The resulting fragment containing p34 was digested with Hindlll and BamHl and cloned into pECFP (Clontech), which generated pCFP-P34. 92 A p34 mutant was constructed by amplifying two fragments that contain the 5' or 3' region of p34. A 387 bp fragment was amplified by PCR using the primers p773 and p799 (3'-CCCCGGCGTGTTTGCAGTTT-5 ' ) and contains the 5' end of the p34 ORF. The second fragment was amplified using the primers p774 (3 ' - G A A T G G G A T C C A C T C T A G A G C C C T A -5') and p798 (5 ' - G C G T T G G C G C T C A A A A C G A T T - 3 ' ) and obtained 568 bp PCR product. This 568 bp fragment contains the 3' region of the p34 ORF with a mutation of the translational start site of P34A into GCG. The 5' and 3' fragments ofp34 were ligated and subsequently amplified with the primers p773 and p774 to produce p34M^A. The p34M~*A fragment was digested with Hindlll and BamHl and cloned into pECFP (Clontech) to generate pCFP-P34 M " A . The p34A ORF was obtained by digestion of pCFP-P34 with Kpnl, which was polished by T7 D N A polymerase, followed by a BamHl digestion. The p34A fragment was cloned into BamHl and the Sphl modified blunt-end site of pEYFP to generate pYFP-P34A. pEYFP was derived by PCR of pEYFP-Peroxi (Clontech) with the primers p737 (5'-C T A G C G C T A C C G G A C T C A G A T C T C G A G C - 3 ' ) andp738 (3'-G A G C T G T A C A A G T A A A A G A A T T C G C G G C C G C A A C T - 5 ' ) . Subsequently pYFP-P34A was digested with BamHl and Hindlll and the resulting p34A fragment was inserted into pECFP, which produced pCFP-P34A. pCFP-P34, pCFP-P34A M " A , pYFP-P34A and pCFP-P34A were digested with Hindlll and EcoRl and the fusion genes were cloned into p2Zop2E, which produced the clones pP34-CFP, pP34 M " A -CFP, pP34A-YFP and pP34A-CFP (Fig. 2.5A). Prior to transfection Ld652Y cells (106) were seeded onto the wells of the 6-wells culture plate and incubated at 27 °C for 1-2 hours. The cells were transfected with 2 fig of pP34-CFP or pP34A-CFP or 1 fig of pP34 M " A -CFP and 1 \ig pP34A-YFP using liposomes, which were prepared as previously described (Campell, 1995). After 24 h p.t. OpMNPV (MOI: 5) was 93 added to the cells, which were then transferred onto a dish (Willco Wells) for confocal microscopy. The transfected cells were examined for fusion protein localization at 12, 18, 24, 48 and 72 h p.i. At each time point the cells were fixed with 2% formaldehyde-0.2% glutaraldehyde in I X PBS at 4 °C for 10 minutes. The cells were washed using I X PBS (phosphate-buffered saline) and subsequently stained with 4'-6-Diamidino-2-phenylindole (DAPI; 1 /ig/ml in PBS per dish; Sigma) for localizing the nucleus. The images showing expression of CFP (A ex.: 458) or Y F P (A ex.: 514) and DAPI colouring (A ex.: 405) were obtained sequentially using a confocal microscope (Leica). 2.4.9 In vitro ubiquitination assay A glutathione-S-transferase (GST)-P34 fusion construct was designed in order to purify P34 for in vitro ubiquitination assays. To obtain this construct a 980 bp fragment was amplified from pBD-P34 using primers p679 and p680. The 980 bp p34 fragment was digested with BamHI and EcoRI and cloned in frame downstream of GST in pGEX2T (Amersham Biosciences), which generated pGEX2T-P34 (Fig. 2.6A). Primers p679 and p680 were also used to amplify 821 bp fragment containing the p34ARING ORF from pBD-P34ARTNG. The synthesizedp34ARING was digested with BamHI and £coRI and cloned in frame downstream of the GST in pGEX2T generating pGEX2T-P34ARTNG (Fig. 2.6A). The GST constructs were transformed into BL21 cells and then induced with isopropyl-/3-D-thiogalactopyranoside (LPTG) and the expressed proteins were purified using glutathione Sepharose 4B (Amersham Biosciences) (Boutell et al., 2002). Ubiquitination assays were performed as previously described (Nerenberg et al., 2005). Briefly, 0.5 l i M of purified GST-P34, GST-P34ARING or GST (control) were added to 15 [il of 94 reaction buffer (50 m M Tris-HCl (pH 7.5), 100 m M NaCl, 0.1 m M ZnCl 2 , l m M DTT, 6.7 m M ATP and 3 /xg of ubiquitin (Sigma); E l ubiquitin activating enzyme (8.3 n M ; Calbiochem) and E2 ubiquitin conjugating enzyme (41.6 m M UbcH5a; Calbiochem). The samples were incubated for 90 minutes at 30 °C and the reactions stopped by the addition of 1/5 of a volume of 5X SDS loading dye and heating to 95 °C for 5 minutes. The reactions were analyzed by SDS-PAGE and Western blot as described above. 2.4.10 Immunoprecipitation Several plasmids were constructed for this experiment, which contain the early OpMNPV ie2 promoter for the expression of genes. p2Zop2E-P34 was constructed by digestion of the vector pP34-CFP with Xbal and BamHl, which deleted the cfp ORF and was subsequently religated (Fig. 2.3A). In addition, yfp of pP34-AYFP was deleted by digestion with BamHl and the modified vector was religated, which generated p2Zop2E-P34A (Fig. 2.3A). Several vectors were constructed, which contain a hemagglutinin (HA) fused to the C-terminus of either P34 or P34A. To construct p2Zop2E-P34-HA, pBD-OpP34 was digested with EcoRl and Xbal and the p34 fragment was used as template for PCR ofp34-HK with the primers p533 and p829 (3'-G T T T G A C G A A T G G G A T C C A T A C C C C T A C G A C G T G C C C G A C T A C G C C T G A - 5 ' ) . The PCRed product was phosphorylated, digested with -EcoRl and ligated with p2Zop2E (Fig. 2.3A). p2Zop2E-P34A was digested with Hindlll and BamHl to obtain the p34A fragment. In addition, the p34 ORF was deleted from p2Zop2E-P34-HA by digestion with Hindlll and BamHl and was ligated with the Hindlll-BamHl fragment that contained p34A to generate p2Zop2E-P34A-HA (Fig. 2.3A). 95 Ld652Y cells (106) were cotransfected with 1 fig of p2Zop2E-P34 or p2Zop2E-P34A and 1 ug p2Zop2E-P34-HA or p2Zop2E-P34A-HA. The control in this assay include Ld652Y cells that were transfected with 1 iig of p2Zop2E-P34 and 1 tig pBS+. The transfections, including the control, were incubated for 48 hours at 27 °C. Generally co-immunoprecipitation was performed according to the protocol described by Adams et al. (Adams et al., 2002). Briefly, the cells that are attached on the 6-wells plates were washed twice with 1.5 ml of pre-chilled I X PBS. Subsequently 1 ml of cold I X PBS was added to each well, the cells were harvested and transferred into 1.5 ml eppendorf. The samples were centrifuged 500 g at 4 °C for 5 minutes. After removal of I X PBS, 200 pA of EBC buffer (50 m M Tris-HCl, (pH 8), 120 m M NaCl and 0.5% NP40) with protease inhibitors (P-8340; Sigma) was added and incubated on ice for 5 minutes. To disrupt the nuclear membranes 70 ill of pre-chilled 2 M NaCl was added to each sample, which was subsequently incubated on ice for 10 minutes. EBC buffer was added to each sample to a total volume of 770 pA and then 100 pA (13%>) was removed for analysis of total protein expression. The samples with the remaining 670 pi were centrifuged at 10,000 g for 10 minutes at 4 °C. 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J Gen Virol 74, 1591-1598. 101 Chapter 3: Orgyia pseudotsugata MNPV P34 interacts with LdUEV, a ubiquitin E2 conjugating enzyme variant 3.1 Introduction Baculovirus infection has a tightly regulated cascade of viral expression that consists of three distinct phases called early, late and very late. Infection begins with the expression of early phase viral genes, which are transcribed from an early promoter (CAGT) by the host R N A polymerase II (Friesen, 1997). Late genes are typically expressed from a late promoter (A/G/TTAAG) and depend on the viral R N A polymerase for transcription. The Orgyia pseudotsugata multiple nucleopolyhedrosis virus (OpMNPV) is a member of the Baculoviridae family and contains a double stranded (ds) circular 132 kb genome comprising 152 predicted open reading frames (ORFs) with 50 or more amino acids. P34 is an early gene expressed as a 1.1 kb transcript from 1 h p.i. to 48 h p.i. and is translated as a 34 kDa protein (Wu et al, 1993a). From 18 h p.i. to 120 h p.i. an alternate transcript of 0.7 kb is transcribed by a late internal promoter (ATAAG). This transcript encodes a 5' truncation of the p34 mRNA, which is translated as a 20 kDa protein named P34A (Wu et al, 1993a). P34 is a regulatory protein that augments D N A replication and transactivates the OpMNPV iel andp8.9 promoters in transient assays (Ahrens and Rohrmann, 1995; Wu et al, 1993a; Wu et al, 1993b). In chapter 2 it was shown that P34 forms homodimers as well as heterodimers with P34A suggesting that this smaller late protein is regulating the function of P34. In addition, R N A i silencing showed that p34 is required for high-level D N A replication and wild type levels of B V production, which is similar to previous studies performed with the A c M N P V 102 homologous protein PE38 (Ahrens and Rohrmann, 1995; Kool et al, 1994; Milks et al, 2003). Cellular localization analysis showed that P34 is found in punctate foci in the nucleus (Chapter 2). A c M N P V PE38 has also been shown to form nuclear foci and in addition colocalize with the cellular protein P M L . In vitro ubiquitination assays (Chapter 2) also revealed that P34 posses E3 ubiquitin ligase activity. These data indicate that P34 is likely to be interacting with viral and cellular proteins. In the ubiquitination pathway one or multiple ubiquitin proteins are transferred from the ATP dependent E l ubiquitin-activating enzyme via the E2 ubiquitin-conjugating enzyme and the E3 ubiquitin-ligase enzyme to the target protein. Although a single E l activates the ubiquitin conjugation machinery, a large number of E2 conjugating enzymes and E3 ligases are now known to exist. This study explores interactions of OpMNPV P34 with other viral and cellular proteins. The yeast 2-hybrid system was used to identify novel interactions of P34 with viral and host proteins by screening a cDNA library containing both host and viral proteins. A Lymantria dispar cellular protein called Ubiquitin E2 conjugating Variant (LdUEV) was identified to interact with P34. The U E V family of proteins are variants of the E2 conjugating enzyme family. E2 conjugating enzymes are generally small proteins (14-32 kDa) and contain a conserved catalytic cysteine, which receives ubiquitin from the E l activating enzyme (Glickman et al, 1998; Jackson et al, 2000). However, U E V proteins lack the critical catalytic cysteine residue for ubiquitin transfer. Studies were conducted to verify the protein-protein interactions and also to examine the function of LdUEV and P34 using R N A i methodologies and confocal microscopy. It was shown that LdUEV is critical for a wild type productive infection. 103 3.2 Results 3.2.1 Yeast 2-hybrid screen of a cDNA library from OpMNPV infected Ld652Y cells with P34 identified an interaction between P34 and the cellular protein LdUEV As shown in Chapter 2, P34 is functioning as an E3 ubiquitin ligase, which suggests that the cellular ubiquitination pathway plays a role in the OpMNPV infection cycle. Very little is known concerning the roles of host proteins in baculovirus infections. Therefore to further characterize the interactions of P34 with host proteins and specifically those involved in the ubiquitination pathway yeast 2-hybrid analysis was conducted. P34 was used as a bait to screen a cDNA library derived from OpMNPV infected Ld652Y cells at 12 h p.i. Yeast colonies that showed P34 - cDNA interaction were selected; the cDNA fragments were sequenced and analyzed and compared to the GenBank translated nucleotide sequence database using the tblastX algorithim. The blast search identified a rare protein that showed homology to an ubiquitin conjugating enzyme (E2) called the Ubiquitin E2 Variant (UEV) protein. We have called this U E V homolog Lymantria dispar U E V (LdUEV) and the predicted cDNA ORF consists of 435 nucleotides and translated into a 144 amino acid protein with a molecular weight of 16 kDa. Due to its known role in the ubiquitination pathway in other systems we characterized this gene to determine i f it served a function in the OpMNPV infection cycle (Garrus et al, 2001; Hofmann and Pickart, 1999; Sancho et al, 1998). 3.2.2 Comparative analysis of LdUEV and homologous proteins Comparison of LdUEV with homologs in GenBank revealed that the LdUEV was most similar to the dipteran UEVs ranging in identity from approximately 74-79%. A protein 104 alignment of the predicated amino acids of nine U E V homologs relative to LdUEV is shown in Fig. 3.1 A . The similarity between LdUEV and the insect related homologous U E V proteins of Anopheles gambiae, Drosophila pseudoobscura, D. menalogaster and D. yakuba U E V are noticeably higher than the other species used for this alignment (Fig. 3.1 A). LdUEV is predicted to contain an ubiquitin-conjugating (UBC) domain (14-132; see Fig. 3.1 A). However the hallmark of E2 proteins is missing; the conserved cysteine located in the U B C domain, which is essential for ubiquitination (the location is shown by the arrow in Fig. 3.1 A). Phylogenetic analysis was also conducted on the predicted amino acids of LdUEV and the nine U E V homologous proteins using the neighbour-joining method (Saitou and Nei, 1987). The resulting phylogenetic tree shows that the insect related LdUEVs cluster and the other species are closely related (Fig. 3.IB). 3.2.3 LdUEV forms dimers with P34 but not with P34A To confirm the protein interaction between P34 and LdUEV, which was identified in the yeast 2-hybrid screen, we performed co-immunoprecipitation using C-terminally H A epitope tagged LdUEV in combination with P34 or P34A (Fig. 3.2A). The interactions of L d U E V - H A and P34 or P34A were compared with the previously observed interactions between P34-HA or P34AHA and P34 or P34A (Fig. 3.2A). Co-immunoprecipitation showed that L d U E V - H A was able to pull down P34 to levels similar to that observed with P34-HA and P34 (Fig. 3.2C). A similar experiment with L d U E V - H A and P34A did not detect co-immunoprecipitation (Fig. 3.2D). This suggests that P34A and LdUEV do not form protein complexes. 105 A i 100 1 1 1 Gl 1 1 : ; 1 I Gl 1 1 3! 1 I 1 : 1 1 ii 1 \ s •ESP. YNLL T T i! HNIV IE|P IEGR Y|LS YQL! LdUEV ( 1 0 0 ) AgUEV ( 1 0 0 ) DpUEV ( 1 0 1 ) DmUEV (93) DyUEV HsUEV RnUEV CeUEV ScUEV (94) QT-DFHTLRDW AtUEV ( 1 0 1 ) |S|KFG|L|N> Figure 3.1. Comparative analysis of L d U E V and U E V homologous proteins. (A) Protein alignment of 9 homologs relative to LdUEV. The U B C domain of LdUEV is indicated below the alignments by a stretch o f+ . The pink arrow indicates the position (D or Q) where normally the conserved catalytic cysteine of E2 conjugating enzymes is located. Alignments were done using Align X (Vector NTI Advance; Invitrogen). The yellow regions present the amino acids that are 100% identical, green regions represent residues with an occurrence of > 50%, blue indicates a consensus residue derived from a block of aligned similar amino acids, green letter residues represent weakly similar amino acids and the black letters indicate non-similar amino acids. (B) Phylogenetic analysis of LdUEV and 9 U E V homologs. The U E V homologs used for the protein and phylogenetic alignments include LdUEV (L. dispar, Douglas fir tussock moth); AgUEV (A. gambiae; mosquito), DpUEV (D. pseudoobscura; fruit fly), DmUEV (D. menalogaster; fruit fly), DyUEV (D. yakuba; fruit fly), HsUEV (Homo sapiens; human), RnUEV (Rattus norvegicus; rat), CeUEV (Caenorhabditis elegans; soil nematode), ScUEV (Saccharomyces cerevisiae; bakers yeast) and AtUEV (Arabidopsis thaliana; plant). Phylogenetic analyses were conducted using neighbor-joining method and branch numbers represent bootstrap scores (%) of 1000 replicates with a bootstrap of 1000 using M E G A version 3.1 (Kumar et al, 2004). Scale represents 0.05 substitutions per site for the trees' branch length. 106 Figure 3.1. continued. B UEV •50-99 96: • 1. dispar A. gambiae — D: pseudbobscura . D yakuba 96 L-D. melanogaster H: sapiens 100 '160 { K norvegicus - C: clegaus • A. thaliana -iSf cerevisiaei 005 3.2.4 Localization of P34 and P34A with LdUEV in infected and transfected cells Previous studies have shown that U E V E2 proteins interact with RING finger E3 ubiquitin ligases, usually in complexes (Suzuki et al, 2002; Tsui et al., 2005; Ulrich, 2003; Ulrich and Jentsch, 2000; Wooff et al, 2004). To further analyze the association between LdUEV and P34, cellular localization studies using confocal microscopy were conducted in transfected and infected cells. The plasmid pP34M~*ACFP was used to localize only full length P34-CFP fusion protein in cells. This construct contains a P34 mutation that has the internal start codon of P34A changed into GCG, which enables the analysis of full length P34 M H " A in absence of P34A 107 A baaicKIMU acidic Q 1,/. FP a-HA WB a-HA Figure 3.2. Analysis of protein-protein interactions of P34, P34A and with L d U E V using co-immunoprecipitation. (A) Schematic representation of the proteins used for co-immunoprecipitation with the H A epitope tagged LdUEV, which includes P34, P34A, P34-HA, P34A-HA and L d U E V - H A . (B-D) Western blot analysis (WB) of anti-HA co-immunoprecipitation (IP) of P34, P34A with the H A fusion proteins. (B) Western blot to verify H A expression and co-immunoprecipitation of LdUEV-HA with P34, P34A-HA with P34, and P34HA and P34 using the anti-HA monoclonal antibody. (C) Western blot analysis of co-immunoprecipitated P34 (control), P34-HA and P34, and L d U E V - H A with P34 using anti-P34 monoclonal antibody. (D) Western blot of co-immunoprecipitation of P34A-HA and P34A, and L d U E V - H A with P34A using anti-P34 monoclonal antibody. Proteins were expressed in Ld652Y cells and the interacting proteins were pulled down with H A monoclonal antibody (a-HA) and analyzed for either expression using H A monoclonal antibody or protein interaction using the P34 (a-P34) monoclonal antibody. The sizes of the molecular weight markers (kDa) are shown on the left. 108 expression (Fig. 3.3A). LdUEV was fused at the C-terminus to YFP and the resulting clone was named pLdUEV-YFP (Fig. 3.3A). The constructs pP34 M " A -CFP and pLdUEV-YFP were cotransfected into Ld652Y cells followed by infection with OpMNPV. As previously shown in Chapter 2, P34M"* A -CFP was expressed in the nucleus in punctate foci in both transfected and infected cells (Fig. 3.3B). In transfected cells LdUEV-YFP was predominantly expressed in the nucleus with regions of higher density and at lower levels in the cytoplasm near the plasma membrane. The distinct punctate nuclear structures of P34 M ^ A -CFP are colocalized with the more diffuse LdUEV-YFP nuclear bodies (Fig. 3.3B). Similar colocalization was observed in infected cells from 12 to 24 h p.i. From 24 h p.i. P34 M _ > A still forms nuclear foci, but sometimes is associated with the nuclear membrane and subsequently does not colocalize with LdUEV-YPF (Fig. 3.3B). This suggests that at late times p.i. there is a change in function of either P34 or LdUEV. P34A was also analyzed for colocalization with LdUEV in infected Ld652Y cells and transfected cells that lack P34 expression. Expression of P34A-CFP (P34A tagged at the C-terminus by fusion to CFP; Chapter 2) and LdUEV-YFP in cotransfected cells was observed in a dispersed pattern throughout the cytoplasm and nucleus with no obvious colocalization (Fig. 3.3C). Similar results were observed in infected cells up to 24 h p.i. Small P34A-CFP nuclear foci appear by 48 h p.i, which appears to colocalize with LdUEV-YFP. However, the majority of both proteins remained diffusely distributed across the nucleus and cytoplasm (Fig. 3.3C). This result supports the immunoprecipitation results indicating that P34A does not directly interact with LdUEV. 109 A expressed protein pP34M * A - C F P [ATG oco E P34-CFP pP34A-CFP p L d U E V - Y F P < • 3 P34A-CFP L d U E V Y F P Figure 3.3. Localization of OpMNPV P34, P34A and LdUEV in uninfected and infected Ld652Y cells. (A) Schematics of the fluorescent protein (FP) constructs used for localization of P34, P34A and LdUEV. P34AM~*A and P34A were fused at the C-terminus to the cyan fluorescence protein (CFP) and LdUEV was fused at the C-terminus to the yellow fluorescence protein (YFP). The fused genes were cloned downstream of the OpMNPV ie2 promoter. Ld652Y cells were cotransfected with (B) pP34A M _ < A -CFP and pLdUEV-YFP or (C) pP34A-CFP and pLdUEV-YFP. After transfection the cells were infected with OpMNPV (MOI: 5) and analyzed at 12, 18, 24 (two series are shown) and 48 h p.i. Transfected (non-infected) cells were also analyzed. Red color represents the CFP fusion proteins, green indicate the YFP fused proteins and cyan is specific for the DAPI nuclear staining. Merged images are obtained by combining localizations of P34 M ^ A -CFP and P34A-YFP. The cells were also visualized by light microscopy. The images of the expressed proteins in the cells were obtained sequentially using a confocal microscope (Leica). 110 18 h p.i. 24 h p.i. 48 h p.i. I l l Figure 3.3. continued. 112 3.2.5 P34 and LdUEV play significant roles in viral DNA replication and budded virus production in infected Ld652Y cells The isolation of Lduev from a yeast 2-hybrid library screened with p34 and the colocalization in infected cells suggests this cellular protein is required for P34 function and plays a role in the viral infection cycle. Therefore the roles of P34 and LdUEV were compared at various times post-infection by silencing p34, Lduev or both transcripts using R N A i . The 3' ends of both p34 and Lduev ORFs were amplified from Litmus28i-P34PstI and Litmus28i-LdUEVRsaI respectively and the PCR fragments were used to obtain dsRNA (Fig. 3.4A). Time course analysis was performed in Ld652Y cells treated with dsRNA homologous to p34, Lduev or both followed by infection with OpMNPV. Infected cells were analyzed at 12, 24, 48 and 72 h p.i. for viral D N A replication, budded virus production, transcription of Lduev, and expression of P34 and P34A. Northern blot analysis was conducted to verify the reduction of Lduev transcription by R N A i . Ld652Y cells treated with Lduev dsRNA were compared with uninfected cells (Mock) and cells infected at 23 h p.i. In uninfected cells a transcript of approximately 2.4 kb was detected. In infected cells the steady state levels of Lduev increased approximately 2 fold by 23 h p.i. (Fig. 3.4B). In uninfected cells treated with Lduev dsRNA the 2.4 kb transcript was also detected, however levels were significantly reduced relative to non-RNAi treated cells (Fig. 3.4B). Western blots were performed to confirm that P34 and P34A expression was silenced throughout infection. The protein levels were compared between a wild type OpMNPV infection and infected cells that had been treated with p34, Lduev or both dsRNA. The results show that in cells treated withp34 dsRNA no P34 or P34A was detected indicating that expression had been completely shut down (Fig. 3.4C). In infected cells treated with Lduev dsRNA P34 and P34A 113 Figure 3.4. Analysis of viral DNA replication and budded virus production in untreated Ld652Y cells and in cells depleted of LdUEV or P34 using RNAi. (A) Schematic overview of the preparation ofp34 and Lduev dsRNA. M l 3 Forward and Reverse primers amplified the 3' end of the ORFs p34 and Lduev from Litmus28i-P34PstI and Litmus28i-LdUEVRsaI respectively. The fragments were amplified by PCR and used as templates to generate dsRNA transcribed by T7 R N A polymerase. Ld652Y cells infected with wild type OpMNPV or in infected cells that were depleted of P34, LdUEV or both proteins by R N A i . (B) Northern blot analysis of Lduev mRNA expression, (C) Western blot analysis of P34 and P34A, (D) Slot blot analysis of viral D N A replication and (E) budded virus production. R N A i was performed by transfection of Ld652Y cells with dsRNA followed by infection with OpMNPV (MOI: 5). Infected cells and supernatant were harvested at 12, 18, 24, 48 and 72 h p.i. Northern blot analysis of Lduev transcription was performed with total R N A isolated from untreated cells (Mock), non-infected cells treated with Lduev dsRNA, or infected cells at 23 h p.i. and detected using a 3 2P-labelled probe. The sizes of R N A ladder (kb) are shown on the left. Western blot analysis of P34 and P34A expression in infected cells using a P34 monoclonal antibody. The position of P34 and P34A (kDa) is shown on the right. Uninfected cells (Mock) are included in each blot. Each time point in Figures 3.4D and E presents and average of two separate samples. 114 Figure 3 . 4 . continued. I 5 ' ' I r ~ PstI y p34 0RF 1 M13F-*> 5. 3. Lihnus28i-P34PstI T 7 - » | |<-T7 4- M13R Lduev O R F _ J I I i M 1 3 F - 4 . s . 3. Litiniis28i-Ldl KNRsal JZllCZLtllL 4- M13R B kb 2.37 -1.35 -• Lduev 12 h p.i. 18 h p.i. M 7 1 *« QC 3 i % kDa - P34 - P34 24 h p.i. P34 48 h p.i. 72 h p.i. - P34 - P34A | - P34 • P34A 115 Figure 3.4. continued. D Time post infection (Ii) 116 , were considerably affected at all stages of infection (Fig. 3.4C). From 12 up to 24 h p.i. significantly lower levels of P34 expression were observed. The levels of P34 at 48 h p.i. and 72 h p.i. in cells depleted of L d U E V resembled the same expression pattern as observed at wild type infection at 24 h p.i. suggesting that viral infection was delayed (Fig. 3.4C). Viral D N A replication was analyzed in OpMNPV infected cells depleted of P34, LdUEV or both proteins and compared to a wild type infection. D N A replication of the infected cells treated withp34 dsRNA was significantly reduced throughout infection (Fig. 3.4D) as previously shown in Chapter 2. Similarly infected cells depleted of LdUEV, or P34 and LdUEV, showed very low levels of D N A replication up to 48 h p.i. but increased at the same rate as wild type between 48 and 72 h p.i. (Fig. 3.4D). Nevertheless the overall steady state levels of D N A replication remains significantly reduced at 72 h p.i. in comparison to wild type infection. Budded virus production was also analyzed relative to a wild type infection in cells depleted of inp34, Lduev or both transcripts by R N A i . It was observed that budded virus production in infected cells depleted P34, LdUEV or both proteins was reduced approximately 1 or 2 logs at all stages of infection relative to wild type OpMNPV (Fig. 3.4E). The results show that LdUEV appears to be essential cellular component that is required for wild type levels of budded virus. 117 3.3 Discussion OpMNPV regulates its infection cycle in an ordered cascade that requires detailed control by viral encoded regulatory proteins. There have been significant advances in defining the function of OpMNPV and other baculovirus regulatory genes but very little is known about how they interact with the host cell and specifically with which host proteins. The OpMNPV p34 gene was shown by transient assays to have a number of functions including transcriptional activation and augmenting viral D N A replication (Ahrens and Rohrmann, 1995; Wu et al, 1993b). In addition, previous analyses indicated that P34 has the domain and enzymatic activity associated with E3 ligases of the ubiquitination pathway (Chapter 2). Therefore in this study we were interested in identifying cellular host proteins involved with P34 function and specifically those associated with the ubiquitination pathway. Yeast 2-hybrid analysis using P34 as bait was conducted to screen a cDNA library prepared from OpMNPV infected Ld652Y cells at 12 h p.i. A cDNA ORF was identified coding for a protein that showed homology to an Ubiquitin E2 conjugating Variant (UEV) protein. The L. dispar U E V homolog has been named Lduev and contains an ORF of 435 nucleotides coding for a predicted protein of 144 amino acid protein of 16 kDa. The first U E V protein discovered was human UEV-1 (or CROC-1), which showed similarity in sequence and structure with the ubiquitin E2 conjugating enzymes (Chen et al, 1993; Jentsch, 1992; Sancho et al, 1998). Ubiquitin E2 conjugating enzymes form a thiol-ester binding with ubiquitin at the catalytic conserved cysteine residue when ubiquitin is donated by the ubiquitin E l activating enzyme. Even though UEV-1 was highly similar to E2 conjugating enzymes, UEV-1 lacks the conserved cysteine necessary to catalyze the transfer of ubiquitin to the target 118 proteins (Sancho et al, 1998). Subsequently a family of U E V homologous proteins was revealed with a highly conserved phylogeny from yeast to mammals. LdUEV also shows a high degree of similarity with insect UEVs as well as the more distantly proteins related mammalian, yeast, and nematodes (Fig. 3.1 A). LdUEV is consistent with the other proteins in that it lacks the catalytic cysteine normally associated with the catalytic site of E2 ubiquitin conjugation enzymes (Fig. 3.1 A). To confirm the P34-LdUEV interactions co-immunoprecipitation was able to show an interaction in vivo between these two proteins. In addition, colocalization of P34 and LdUEV in both transfected and infected Ld652Y cells was revealed by confocal microscopy using FP-tagged proteins (Fig. 3.3B). Using R N A i to knock-out uev function also showed that infected cells exhibited a similar phenotype to a P34 knock-out having reduced viral D N A replication and budded virus production. These results indicate that U E V is required for P34 function in OpMNPV infected Ld652Y cells. U E V proteins are an interesting group of the ubiquitination pathway as they have been implicated in a number of different cellular functions. U E V proteins are inactive E2 ubiquitin conjugating enzymes but appear to function as cofactors for Lys63 ubiquitination. Polyubiquitination of proteins involves adding successive ubiquitin molecules by forming isopeptide bonds at specific lysine residues of ubiquitin. Polyubiquitin chains that form at Lys45 appear to be normally directed to proteosomal degradation. Polyubiquitination via Lys63 on the other hand have been observed to be involved in various functions that include post-replicative D N A repair (Hofmann and Pickart, 1999; Spence et al, 1995), retrovirus budding (Amit et al, 2004; Garrus et al, 2001; Harty et al, 2000; Kikonyogo et al, 2001; Strack et al, 2000; Yasuda et al., 2002), activation of k B a kinase (Deng et al., 2000) or NF-kB (Andersen et al., 2005), translational regulation (Spence et al, 2000), and ubiquitin-dependent endocytosis (Galan and 119 Haguenauer-Tsapis, 1997). Lys63 chains were shown to be catalyzed by the E2 conjugation enzyme Ubcl3 but a number of studies have shown that U E V homologs bind to Ubcl3 and function as co-factors influencing the specificity of the reaction. RING finger proteins that have E3 ligase activity like P34 have been shown to interact with U E V homologs (Schreader et al, 2003; Suzuki et al, 2002; Ulrich, 2003; Ulrich and Jentsch, 2000; Wooff et al, 2004). For example tumor necrosis factor receptor associated factor 6 (TRAF6) is required for Lys63 polyubiquitination by binding to Ubcl3-UEV complexes and the RING domain is required for the interaction (Hofmann and Pickart, 1999; Wooff et al, 2004). However, using a 2-hybrid system a direct interaction between TRAF6 and U E V independent of Ubcl3 was not observed. Similarly, the E3 protein Rad5 recruits the Ubcl3-Mms2 (UEV homolog) heterodimer through its RING domain (Tsui et al, 2005). In the plant Arabidopsis, COP 10, a U E V homolog, interacts directly with the RING domain of COP1 and is predicted to be part of a signalosome in the ubiquitination pathway required for seedling development (Suzuki et al, 2002). Further analyses have to be performed to determine i f the RING domain of P34 is responsible for the interaction with LdUEV. Confocal studies indicated that P34-CFP and LdUEV-YFP are colocalized in punctate nuclear foci in both transfected and infected cells up to 24 h p.i. (Fig. 3.3B). However, at 24-48 h p.i. P34 is translocated towards the periphery of the nuclear membrane and most of LdUEV remains in the core of the nucleus (Fig. 3.3B). These findings are showing that colocalization of P34-CFP with LdUEV-YFP appears to be transient and corresponds to temporal expression of P34. In addition to localizing with P34, LdUEV showed dispersed expression throughout the cytoplasm and nucleus with higher levels in the nucleus. Analyses of other UEVs have been described in both nuclear and cytoplasmic localization. For example the human U E V 1 A was 120 expressed with a uniform distribution in the nucleus of unstressed cells whereas the yeast Mms2 is found predominately in the cytoplasm but migrates to the nucleus after treatment with agents that damage D N A (Andersen et al, 2005; Thomson et al, 2000; Ulrich and Jentsch, 2000). Similar results have been observed with the complex of the yeast Ubcl3-Mms2 and the RING finger ubiquitin ligase D N A repair enzyme Rad5, which catalyzes the formation of Lys63 polyubiquitin chains (Tsui et al, 2005). Colocalization of Mms2 and Ubcl3 is observed in the cytoplasm in healthy cells but not with Rad5. However after treatment of the yeast cells with alkylating agent M M S or U V irradiation both Mms2 and Ubcl3 were detected in the nucleus in punctate structures (Ulrich and Jentsch, 2000). The tumor susceptibility gene 101 (TsglOl) protein contains a N-terminus domain that is homologous to full-length U E V . Cellular localization of TsglOl has been shown to be cell cycle dependent. In mouse fibroblastic cells, TsglOl was shown to be present predominantly in the nucleus during GO and G l phase associated with microtubules, but becomes cytoplasmic as the cells progresses to S phase (Xie et al, 1998). In addition to mouse TsglOl, the human homolog was also observed in both the nucleus and cytoplasm of other mammalian cells such as COS7 cells (Muromoto et al, 2004; Xie et al, 1998). These findings show that cellular location of U E V homologs can vary depending on the environmental conditions of the cell. In agreement with this, the expression of LdUEV in infected cells also showed variable subcellular localization patterns becoming predominately nuclear with the progression of the viral infection cycle (Fig. 3.3B and C). Colocalization of LdUEV and P34A was also explored by expressing LdUEV-YFP and P34A-CFP fusion protein (Fig. 3.3C). Both P34A-CFP and LdUEV-YFP in transfected cells and at 12-24 h p.i. are expressed in both the cytoplasm and nucleus in a uniform pattern with occasional small foci, which seems to suggest that P34A and LdUEV are not interacting with each other. 121 This finding agrees with the observation obtained by co-immunoprecipitation that also did not show interaction between P34A and LdUEV. Colocalization only becomes evident at 48 h p.i. LdUEV and P34A appeared in a more obvious punctate pattern in the nucleus, suggesting that both proteins colocalize with untagged P34 expressed from the viral genome (Fig. 2.3B and Fig 3.3B). P34 expression from the viral genome may be lower than observed in plasmid transfected cells thus making smaller and fewer punctate foci. Previous studies on A c M N P V PE38 encountered difficulties with the visualization of PE38 in A c M N P V infected cells and cells transfected with plasmid DNAs expressing PE38 as higher levels were required (Krappa et al, 1995; Murges et al, 2001). To provide additional support for involvement of LdUEV in the OpMNPV infection cycle R N A i time course experiments were conducted that shutdown Lduev function. Western blot analysis confirmed that P34 and P34A expression was not observed when infected cells were depleted of P34, LdUEV or both proteins. Silencing of Lduev in infected cells revealed a significant reduction and delay of P34 and P34A expression. This finding implies that LdUEV augments P34 expression at the transcriptional or translation level. Previous studies showed that the human UEV-1 mediates transcriptional activation of the human FOS promoter (Rothofsky and Lin, 1997). The human TsglOl N-terminus, which includes the U E V domain has also been shown to activate transcription in transient assays (Sun et al, 1999). It is therefore possible that LdUEV is involved in activating or enhancing transcription ofp34 in infected cells. R N A i of p34, Lduev or both genes in infected cells resulted in a significant reduction of viral D N A replication and budded virus production relative to OpMNPV wild type infection (Fig. 3.4D and E). If LdUEV is required for P34 function this would agree with previous results in transient replication assays showing that P34 enhanced OpMNPV D N A replication but was not 122 essential (Ahrens and Rohrmann, 1995). In addition, the reduction of viral D N A replication and B V production by p34 R N A i is consistent with the phenotype of the A c M N P V PE38 knockout virus, which also showed a significant reduction in viral D N A replication (Milks et al, 2003). As indicated above the U E V homolog Mms2 has been shown to be involved in post-replication D N A repair pathway. In addition the yeast Ubcl3-Mms2 conjugating enzyme forms a complex with the RING finger ubiquitin ligase Rad5, and is required for the Lys63 polyubiquitin chain assembly of the monoubiquitinated P C N A (proliferating cell nuclear antigen) (Tsui et al, 2005). Rad5 recruits Mms2/Ubcl3 conjugating enzyme complex to the D N A (Ulrich and Jentsch, 2000). Several studies have suggested that polyubiquitination by Mms2/Ubcl3 and Rad5 may mediate the assembly of proteins such as P C N A and D N A polymerase 8 to the sites of damaged D N A and participate in limited D N A synthesis through the undamaged sister duplex (Hofmann and Pickart, 1999). Recent studies showed that the cellular P C N A of Sf9 cells associates with the A c M N P V D N A replication sites and it was suggested that both viral and cellular P C N A are involved in A c M N P V D N A replication. It is possible that P34 has a role similar to the ubiquitin ligase Rad5 to augment D N A replication. That is, P34 may interact with LdUEV to assemble polyubiquitin chains on P C N A or other replication factors to recruit proteins to OpMNPV viral replication centres. Similar to P34 the E3 ubiquitin ligase Rad5, which associates with the Ubcl3-Mms2 heterodimer through its RING domain, was expressed in a punctate pattern and also showed to colocalize with chromatin (Tsui et al, 2005; Ulrich and Jentsch, 2000). Although localization of P34 with the viral D N A replication centers has not been reported, A c M N P V PE38 was shown to colocalize with LE2, which has been demonstrated to interact with viral D N A replication centers (Mainz et al, 2002). In addition to A c M N P V IE2, the cellular protein P M L was also observed 123 to be located juxtaposed to the viral D N A replication centers (Mainz et al, 2002). A recent study reported that silencing ofpml by siRNA prevented the recruitment of single-stranded D N A into the nuclear foci, suggesting a role of P M L in post-replication D N A processing (Jul-Larsen et al., 2004). Budded virus production of infected cells depleted of P34, L d U E V or both proteins also showed a reduction relative to wild type infection (Fig. 3.4E). Studies have revealed that several TsglOl homologous proteins are required for budding of viruses via the vacuolar sorting pathway (vsp) (Amit et al, 2004; Bouamr et al, 2003; Garrus et al, 2001). However budding of baculoviruses using the vsp pathway has not been observed. In summary, this is one of the first studies to identify a cellular protein that interacts with a baculovirus regulatory protein. The results are suggesting that the OpMNPV E3 ubiquitin ligase P34 interacts with LdUEV during infection. Loss of Lduev function dramatically affects OpMNPV replication in Ld652Y cells highlighting its importance. U E V homologs function in diverse cellular processes through physical interactions and alternative polyubiquitination (Andersen et al, 2005). Therefore identifying the functional targets for P34 and LdUEV interactions wil l provide valuable insights into baculovirus-host cell interactions and the molecular basis for viral virulence. 124 3.4 Materials and Methods 3.4.1 Cells and virus OpMNPV-WT virus was propagated in Lymantria dispar Ld652Y cells, which were maintained in TCI00 medium (Invitrogen) as previously described (Summers and Smith, 1978). To construct the 12 h p.i. OpMNPV cDNA library, Ld652Y cells (9.0 x 107) were infected with the OpMNPV using a multiplicity of infection (MOI) of 10. In addition, for the R N A i time course experiment and colocalization studies cells were infected with a MOI of 5. Virus titers were determined by end point dilution (Summers and Smith, 1978) and quantitative PCR (Lo and Chao, 2004). 3.4.2 Construction of the 12 h p.i. O p M N P V cDNA library The OpMNPV cDNA library was obtained using standard methods. Briefly, infected Ld652Y cells were harvested at 12 h p.i. and total R N A was obtained using the guanidine isothiocyanate (GIT)-ACLD phenol isolation protocol (Chomczynski and Sacchi, 1987). Transfer R N A was removed from the total R N A using the RNeasy midi kit (Qiagen) and polyA was isolated by Oligotex mRNA spin-columns. The cDNA library was generated using the Hybrizap-2.1 X R cDNA synthesis and hybrizap-2.1 X R library construction kit from Stratagene using the manufacturers' protocols. 125 3.4.3 Yeast transformation of OpMNPV P34 and 12 h p.i. OpMNPV cDNA library and yeast 2-hybrid assay The 12 h p.i. OpMNPV cDNA plasmid library was transformed into the yeast strain YRG-2 with pBD-P34 according to the Hybrizap-2.1 X R library protocol (Stratagene). Transformed cells were plated onto Trp-Leu-His dropout SD-media (Amersham Biosciences) and the cotransformants were screened for high stringent interactions with P34. Yeast 2-hybrid assays were used to confirm the identified cellular proteins interacted with P34. After the YRG-2 cotransformants were screened on Trp-Leu-His dropout SD-media pAD plasmid containing a 1505 bp cDNA fragment with a 435 bp LdUEV ORF was identified to interact with BD-P34 fused protein. This plasmid named pAD-LdUEVcDNA was isolated by D N A extraction from the yeast clone and transformed into Xl l -Blue M R F ' bacterial cells (Stratagene). The transformed cells were plated on an ampicillin selection plates and pAD-LdUEVcDNA was isolated using the Qiaprep spin miniprep kit (Qiagen). The Lduev ORFwas amplified from pAD-LdUEVcDNA using the primers p693 (5'-G T G G A A T T C G T A A T C A T G G C G A - 3 ' ) and p694 ( 3 ' - T T A C T C T C G A G C A T A G A C C C A A - 5 ' ) . The resulting 485 bp fragment was digested with EcoRl and Xhol and inserted into pBD-GAL4 Cam to generate pBD-LdUEV. The vector pBD-P34 cloned, which contains the binding domain (BD) was previously described in chapter 2. 3.4.4 Sequence analysis of clones with P34 - cDNA interaction To identify the insert (target) in the positive interacting P34 cotransformant, the cDNA fragment was amplified by PCR using primers p514 (5 ' -AGGGATGTTTAATACCACTAC-3 ' ) and p504 (3 ' - C C C T A T A G T G A G T C G T A T T A - 5 ' ) , which are flanking the M S C of pAD-GAL4-126 2.1. After analysis of the PCR samples by gel electrophoresis the amplified fragments were purified using the Qiaquick purification kit. The nucleotide sequence was obtained by primer p600 (5 ' - A T G A T G A A G A T A C C C C A C C A - 3 ' ) , which localizes several nucleotides downstream of the primer p514 binding site nevertheless recognizes the 5' flanking region of the M S C in pAD-GAL4-2.1. The nucleotide sequence was screened for homologous protein alignments using the translated query vs. translated database (tblastx). The U E V protein alignments were obtained using Align X (Vector NTI Advance, Invitrogen), which is a program based on ClustalW (Thompson et al, 1994) (Fig. 3.1 A). Phylogenetic analyses were conducted using neighbor-joining method and branch numbers represent bootstrap scores (%) of 1000 replicates with a bootstrap of 1000 using M E G A version 3.1 (Kumar et al, 2004). Scale represents 0.05 substitutions per site for the trees' branch length (Fig. 3.IB). 3.4.5 Western blot analysis Western blot analysis was previously described in chapter 2. In this study we used P34 (BI 1B10) or H A (HA. l 1, MMS-101R; CRPinc) monoclonal antibodies as primary antibody with a dilution of 1:1000 dilution and as secondary antibody with used a 1:10,000 dilution of a goat anti-mouse horseradish peroxidase secondary antibody (315-035-044; Jackson Laboratories). 3.4.6 Preparation of dsRNA To obtain dsRNA of p34 and Lduev the ORFs were cloned into the multiple cloning site (MCS) of Litmus28i, which flanks the T7 promoter region located at opposite ends (NEB). The construction of Litmus28i-P34PstI has been previously described in chapter 2 (Fig. 3.4A). The 127 Lduev ORF was obtained using the primers p677 (5' - G T G G G A T C C G T A A T C A T G G C G A - 3 ' ) and p678 ( 3 ' - T T A C T G A A T T C C A T A G A C C C A A - 5 ' ) and pAD-LdUEV cDNA as template and amplified a 485 bp product. Digestion of this PCR product with Rsal and EcoRl resulted in a fragment encoding the 146 nucleotides located at the 3'end of the Lduev ORF. Litmus28i was digested with BamHI, polished with T7 polymerase and then cut with EcoRI. The vector was ligated with the EcoBl- Rsal Lduev fragment and generated the clone Litmus28i-LdUEVRsaI (Fig. 3.4A). Preparation of dsRNA was previously described in chapter 2. 3.4.7 RNAi time-course experiment The R N A i time-course experiments were conducted as described in chapter 2. 3.4.8 Total RNA isolation and Northern blots R N A i time course experiment the supernatant was removed from the six wells plates and washed one time with I X PBS. Total R N A of each sample was extracted according to the manufacturer's protocol of the RNeasy mini kit (Qiagen). The Northern blots were obtained using methods previously described by Thomas (Thomas, 1983). Blots were incubated overnight at 60 °C with the hybridization buffer, which included an 3 2P-labeled ssRNA probe in 5X SSC (0.15 M NaCl, 0.015 M CeHsOyNaa^HbO, pH 7.0), 0.5% SDS, 5X Denhardt's solution (0.02 polyvinylpyrolidone, 0.02% BSA, 0.02% Ficoll 400), 50% formamide and 100 jig/ml denatured salmon sperm D N A . The radioactive labeled ssRNA probe were prepared according to Sambrook et al. (Sambrook et al, 2001), which included the T7 R N A polymerase (NEB), 50 i iCi 128 J 2 P-UTP and Litmus28i-LdUEV as template. Blots were washed at 60°C with 2x SSC and 0.1% SDS followed by additional washes of several dilutions of SSC buffer to a final dilution of 0.1X SSC and 0.1 % SDS. The Northern blots were exposed onto the Kodak Phosphorscreens and scanned using a Storm Phospholmager (Molecular Dynamics). 3.4.9 Budded virus titration by quantitative PCR Titrations were performed as described in Chapter 2. 3.4.10 Analysis of DNA replication using slot blot procedure Slot blot analysis of viral D N A replication was performed as described in Chapter 2. 3.4.11 Localization of P34 and P34A with LdUEV To localize P34 and P34A with L d S U G l in Ld652Y cells the ORFs were cloned in frame and upstream of the genes encoding the cyan or yellow fluorescent protein (CFP or YFP). Furthermore, the fusion genes were subcloned into p2Zop2E downstream of the OpMNPV ie2 promoter, which allows transcription of these genes in absence of any viral factors. The construction of pP34 M " A -CFP and pP34A-CFP were described in chapter 2 (Fig. 3.3A). The primers p775 ( 5 ' - A A A A A G C T T A T C A T G G C G A A T C - 3 ' ) andp776 (3'-T C T G G G A T C C C A C T C C G A T T A C - 5 ' ) and pBD-LdUEV as template were used to amplify Lduev. The 464 bp PCR product was digested with HindlLI and BamHI and cloned into pEYFP, which resulted in pYFP-LdUEV. Subsequently pYFP-LdUEV was digested with Hindlll and 129 EcoRl and the fusion gene was cloned into p2Zop2E, which generated pLdUEV-YFP (Fig. 3.3A). Cotransfections of the plasmids into Ld652Y cells and infections as well as the staining and confocal microscopic handling were described in the previous chapter 2. 3.4.12 Immunoprecipitation The construction of p2Zop2E-P34, p2Zop2E-P34A, p2Zop2E-P34-HA and p2Zop2E-P34A-H A was previously described in chapter 2 (Fig. 4.3A). To obtain the Lduev ORF and the p2Zop2E vector with the H A tag, pLdUEV-YFP and p2Zop2E-P34-HA were digested with Hindlll and BamHl. The digested p2Zop2E-HA vector was ligated with the Lduev Hindlll -BamHl fragment and generated p2Zop2E-LdUEV-HA (Fig. 4.3A). Ld652Y cells (106) were cotransfected with 1 /xg of p2Zop2E-P34 or p2Zop2E-P34A and 1 p,g p2Zop2E-LdUEV-HA. The control in this assay includes Ld652Y cells that were cotransfected with 1 /xg of p2Zop2E-P34 and 1 [ig of pBS+. The transfections, including the controls, were incubated for 48 hours at 27 °C. Co-immunoprecipitation was performed as previously described in chapter 2 followed by SDS-PAGE and Western blot analysis as described above. 130 3.5 References Ahrens, C. H. , and Rohrmann, G. F. (1995). Replication of Orgyia pseudotsugata baculovirus D N A : lef-2 and ie-1 are essential and ie-2, p34, and Op-iap are stimulatory genes. Virology 212, 650-662. Amit, I., Yakir, L. , Katz, M . , Zwang, Y . , Marmor, M . 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Yasuda, J., Hunter, E., Nakao, M . , and Shida, H . (2002). Functional involvement of a novel Nedd4-like ubiquitin ligase on retrovirus budding. EMBO Rep 3, 636-640. 134 Chapter 4: Orgyia pseudotsugata MNPV P34 interacts with the 26S proteasome ATPase homolog LdSUGl 4.1 Introduction The viral gene p34 is one of the 152 open reading frames (ORFs) identified on the double stranded (ds) circular 132 kb genome of the Baculoviridae group I Orgyia pseudotsugata multiple nucleopolyhedrosis virus (OpMNPV). P34 is an early gene that in addition to an early transcript has an alternate late product expressed from a late internal promoter. Subsequently the p34 transcript is expressed as a 34 kDa protein and the second N-terminal truncated transcript translates into a 20 kDa protein known as P34A. P34 comprises several domains characteristic for gene regulation, which include a basic, RING finger, acidic, glutamine rich, and a leucine zipper domains (Wu et al, 1993). These domains were also identified in A c M N P V PE38 and the P34 homolog Bombyx mori N P V (BmNPV) PE38. However, the N-terminally truncated 20 kDa P34A consists of the acidic, glutamine rich and leucine zipper domains. The results obtained from chapter 2 and 3 revealed that P34 possesses E3 ubiquitin ligase activity and associates with the cellular E2 ubiquitin conjugating enzyme variant named LdUEV, suggesting a function for P34 in Lys63-ubiquitination during infection. The ubiquitination pathway is a cascade of reactions by three different enzymes (E l activating enzyme, E2 conjugating enzyme an E3 ligase) that eventually result into the assembly of multiple ubiquitins to the target protein (Finley and Chau, 1991; Glickman et al, 1998a; Jackson et al, 2000; Pickart, 2001). Ubiquitination of proteins has been shown in a diverse array of cellular functions from gene activation to protein degradation and P34 appears to be interacting with the cellular pathways to enhance viral replication. 135 Polyubiquitination of target proteins can cause translocation to the proteasome for degradation (Chau et al, 1989). The 26S proteasome is a major proteolytic complex assembled from a barrel shaped core called 20S and a 19S cap located at either end of the 20S (Glickman et al, 1998a; Groll et al, 1997; Lowe et al, 1995). The cylinder shaped proteolytic 20S core is formed by four stacked heptameric rings and the 19S regulatory cap contains two sub-complexes called lid and base (Glickman et al, 1998a; Groll et al, 1997; Lowe et al, 1995). The base is composed of six subunits, which are identified as ATPases of the A A A (ATPases Associated with diverse cellular Activities) family (Davy et al, 2001; Fu et al, 2001). Based on chapters 2 and 3, P34 should be considered an important but not an essential factor for D N A replication. These results are consistent with previous reports on P34 using transient assays, which showed stimulation of viral D N A replication (Ahrens and Rohrmann, 1995; Kool et al, 1994; Milks et al, 2003). In addition, chapter 3 showed that when Lduev was silenced using R N A i viral D N A replication was significantly reduced similar to the p34 knock-out phenotypes. This finding indicates that the cellular protein LdUEV is required for a wild type infection. Viral E3 ubiquitin ligases are known to have pleiotropic affects during infection. Therefore additional interactions of P34 with other cellular host proteins related to the ubiquitination pathway were investigated. In this study a second cellular protein was identified by a yeast 2-hybrid screen to interact with P34 and has been named L d S U G l . Proteins homologous to L d S U G l have been identified as a component of the 26S proteasome. Specifically L d S U G l is predicted to be one of six ATPase subunits of the proteasome 19S regulatory cap. The function of L d S U G l and its interactions with P34 were investigated using the yeast 2-hybrid system, co-immunoprecipitation, R N A i and confocal microscopy. The results support the conclusion that 136 P34 is interacting with L d S U G l and suggest this cellular protein is required for a productive viral infection. This study therefore provides new and novel insights into how baculoviruses usurp the cellular machinery to support viral replication. 4.2 Results 4.2.1 Yeast 2-hybrid analysis screen of a cDNA library of OpMNPV infected Ld652Y cells with P34 identified an interaction with the cellular protein LdSUGl We have shown in Chapter 2 and 3 that P34 has E3 ubiquitination ligase activity and potentially interacts with an E2 ubiquitin conjugating variant (UEV) protein. However it is well known that E3 proteins such as the human papilloma virus (HPV) E6 and Herpes simplex virus 1 (HSV) ICPO have multiple potential interactions with proteins involved in the ubiquitination pathway (Banks et al, 2003; Galinier et al, 2002; Harty et al, 2001; Winberg et al, 2000). To build upon our previous results identifying cellular proteins that form complexes with P34 (Chapter 3) additional interactions were investigated. This was performed using P34 as the bait to screen a yeast 2-hybrid cDNA library derived from 12 h p.i. OpMNPV infected Ld652Y cells. Yeast colonies were selected that indicated a positive interaction followed by sequencing the cDNA fragment and analyzing any predicted proteins using tblastx of the Genbank database (Altschul et al, 1990). A protein was identified showing homology to a protein named Suppressor for G a l l or SUG1 and the L. dispar homolog has been named L d S U G l . The Ldsugl cDNA encodes an ORF of 1209 nucleotides, which codes for a predicted protein of 402 amino acids with a molecular weight of 45 kDa. Homologs of LdSUGl are known by various names including RPT6, TAT-binding protein TBP-1, CIM3, CRL3, SCB68, Pros45 and MSS1 (Cheng 137 et al, 1998; Gerlinger et al, 1997; Ghislain et al, 1993; Glickman et al, 1998b; Swaffield et al, 1992). Homologs of L d S U G l are known as an ATPase subunit of the base of the 19S regulatory cap of the 26S proteasome. 4.2.2 Comparative analysis of L d S U G l and homologous proteins LdSUGl is predicted to have a coiled coil domain (17-74) and an ATPase domain (178-368) (Fig. 4.1 A). Protein alignment was conducted on the predicated amino acids often SUG1 homologs and compared relative to L d S U G l . The similarity among the SUG1 homologous proteins is very high indicating that this is a highly conserved protein (Fig. 4.1 A). The identity between LdSUGl and the homologous proteins varies from 74% (Saccharomyces cerevisieae) to 99.9 % (Manduca sexto). Remarkably the Lepidopteran derived M. sexta M s S U G l only had a 3 amino difference with L d S U G l of which two were conservative changes. Phylogenetic analysis on the predicted amino acids of LdSUGl and ten homologs shows that it clusters with the insect homologs separate from the vertebrate and yeast proteins (Fig. 4.IB). 4.2.3 A l l P34 domains are required the interaction with L d S U G l To map the domains of P34 that are required for binding to L d S U G l yeast 2-hybrid assay was performed using P34, P34A, and P34 domain deletions and LdSUGl (Fig. 4.2A). The interaction between BD-P34 and A D - L d S U G l was obviously very strong, but the reciprocal interactions were reduced. P34A did not interact with L d S U G l suggesting that this late gene product is not required for binding to L d S U G l during infection. Interactions of L d S U G l were not detected when the basic, RING, glutamine, and leucine zipper domains deletion constructs 138 Figure 4.1. Comparative analysis of LdSUGl and SUG1 homologous proteins. (A) Protein alignment of 10 homologs relative to L d S U G l . Alignments were done using Align X (Vector NTI Advance; Invitrogen). The coiled coil and ATPase domains of L d S U G l are illustrated below the alignments. The coiled coil domain is indicated by + and the ATPase domain is presented by a stretch of • . The yellow regions present the amino acids that are 100% identical, green regions represent residues with an occurrence of >50%, blue indicates a consensus residue derived from a block of aligned similar amino acids, green letter residues represent weakly similar amino acids and the black letters indicate non-similar amino acids. (B) Phylogenetic analysis of LdSUGl and 10 SUG1 homologs. The SUG1 homologs used for the protein and phylogenetic alignments include L d S U G l (Lymantria dispar; Douglas fir tussock moth); M s S U G l (M. sexta; tobacco hornworm), A g S U G l (Anopheles gambiae; mosquito), A m S U G l (Apis mellifera; honeybee), DpSUGl (Drosophila pseudoobscura; fruit fly), DmSUGl (D. menalogaster; fruit fly), TcSUGl (Toxoptera citricida; brown citrus aphid), X1SUG1 (Xenopus laevis; South African clawed frog), HsSUGl (H. sapiens; human), ScSUGl (S. cerevisiae; bakers yeast) and SpSUGl (Schizosaccharomyces pombe; fission yeast). Phylogenetic analyses were conducted using neighbor-joining method and branch numbers represent bootstrap scores (%) of 1000 replicates with a bootstrap of 1000 using M E G A version 3.1 (Kumar et al., 2004). Scale represents 0.05 substitutions per site for the trees' branch length. 139 Figure 4.1. continued. A LdSUGl MsSUGl AgSUGl AmSUGl DpSUGl DmSUGl TcSUGl X1SUG1 HsSUGl ScSUGl SpSUGl (88 (88 (89 (91 (94 LdSUGl MsSUGl AgSUGl AmSUGl (91 DpSUGl (91 DmSUGl TcSUGl X1SUG1(101 HsSUGl (92 ScSUGl (91 SpSUGl (88 LdSUGl(188 MsSUGl(188 AgSUGl(189 AmSUGl(191 DpSUGl(191 DmSUGl(191 TcSUGl(194 X1SUG1(201 HsSUGl{192 ScSUGl(191 SpSUGl(188 LdSUGl MsSUGl AgSUGl AmSUGl DpSUGl DmSUGl TcSUGl X1SUG1 HsSUGl ScSUGl SpSUGl (287 (287 (288 (290 (290 (290 (293 (300 (291 (290 (288 1 - M R £ T | T B - M T L T N K M ; - M T V T N R M E M T A Y — M T V T N R M E H S A Y H | M T P N P P E F | E V D Q N S | M A P P G E Y K A A A A D G M E Q M E M D H R G I I M A L D G P E Q M E L E | G K A | S M T A A V T | S N I | L E T H E S ^ E V L K T N V L Q S 101 R R L A Q R N LI R R L A Q R N LI R R L A Q R N Li N|RR1 A Q R N Li R R L A Q R N Li R R L A Q R N Li R R L A Q R N LI R R L A Q R N LI R R L | A Q R N | L I R L E A Q R N A L N R R L E A Q R N G L 1 LLQE GSYVGEV LLQE GSYVGEV LLQE GSYVGEV |LLQE GSYVGEV LLQE GSYVGEV LLQE GSYVGEV LLQE GSYVGEV LLQE GSYVGEV LLQE GSYVGEV LLQEPGSYVGEV LLQE PGSYVGEV +++++++++++++++++++++++++++++++++++++++ 100 KVLVKV P KVLVKV P KVLVKV P KVLVKV P KVLVKV P KVLVKV P KVLVKV P KVLVKV P KVLVKV P IKVLVKVQP JG(NKVLVKV|P NKBDPLVSLMMVEKBPDSTYBMBGGL JNKBDPLVSLMMVEKBPDSTYBMBGGL IJNKBDPLVSLMMVEKBPDSTYMMBGGL IMNKBDPLVSLMMVEK«PDSTYMMBGGL INK D L I N K D L I N K DPLVSLM L I N K DPLVS L I N K DPLVS ENKADPLVSLMMVEKBPDSTY|M|GGLT| L | N K § D PLVSLMMVE K | P D S T Y | M § G G PLVSLMMVEK PDSTY M GGL PLVSLMMVEK PDSTY M GGL EK PDSTY M GGL LMMVEK PDSTY M GGL LMMVEK PDSTY M GGL 200 LGI QPKC • L Y G ? LGI QPKC • LGI QPKC LGI QPKC • L G I QPKC HLYGP L G I QPKC HLYGP L G I QPKC • L G I QPKC H L Y G P LGI QPKC H L Y G P LGI QPKC H L Y G P LGIPQPKC H L Y G P 201 PGTGKTLIAPAVAHHT!C|FIRVSCJ|ELVQK|IGEC1RMWELFVMAREHAPSI PGTGKTLLARAVAHHT C FIRVSG ELVQK IGEG RMVRELFVMAREHAPSI PGTGKTLLARAVAHHT C FIRVSG ELVQK IGEG RMVRELFVMAREHAPSI PGTGKTLLARAVAHHT C FIRVSG ELVQK IGEG RMVRELFVMAREHAPSI PGTGKTLLARAVAHHT C FIRVSG ELVQK IGEG RMVRELFVMAREHAPSI PGTGKTLLARAVAHHT C FIRVSG ELVQK IGEG RMVRELFVMAREHAPSI PGTGKTLLARAVAHHT C FIRVSG ELVQK IGEG RMVRELFVMAREHAPSI FIRVSG ELVQK IGEG RMVRELFVMAREHAPSI FIRVSG ELVQK IGEG RMVRELFVMAREHAPSI CKFIRVSG ELVQK IGEG RMVRELFVMAREHAPSI PGTGKTLLARAVAHHT PGTGKTLLARAVAHHT PGTGKTLLARAVAHHT PGTGKTLLARAVAHHTBCKFIRVSMELVQKBIGEMRMTOELFVMAREHAPSI : K E ; K E V : E L ? V K H P E ; K| IKEIKEVIELPVKHPELF IKEIKEVIELPVKHPELF IKEIKEVIELPVKHPELF IKEIKEVIELPVKHPELF IKEIKEVIELPVKHPELF IKEIKEVIELPVKHPELF IKEIKEVIELPVKHPELF IKEIKEVIELPVKHPELF IKEIKEVIELPVKHPELF IKEIKEVIELPVKHPELF 3-GDSEVQRTMLELLNQLDGFE 3-GDSEVQRTMLELLNQLDGFE G-GDSEVQRTMLELLNQLDGFE G-GDSEVQRTMLELLNQLDGFE G-GDSEVQRTMLELLNQLDGFE G-GDSEVQRTMLELLNQLDGFI G-GDSEVQRTMLELLNQLDGFE G-GDSEVQRTMLELLNQLDGFE G-GDSEVQRTMLELLNQLDGFE |GSGG-GDSEVQRTMLELLNQLDGFE: ISGGSGDSEVQRTMLELLNQLDGFI 300 KNIK KNIK KNIK KNIK KNIK KNIK KNIK KNIK KNIK KNIK KNIK 301 IMATNRiDI IMATNR DI IMATNR DI IMATNR DI IMATNR DI IMATNR DI IMATNR DI IMATNR DI IMATNR DI IMATNR DI IMATNR DI LLRPGRI LRPGRI LRPGR LRPGRI LRPGRI LRPGRI LDTALLRPGRI LDSALLRPGRI LDSALLRPGRI LDlALLRPGRI LDIALLRPGRI DRKIEFPP DRKIEFPP DRKIEFPP DRKIEFPP DRKIEFPP DRKIEFPP DRKIEFPP DRKIEFPP DRKIEFPP! DRKIEFPPPSVAA1 DRKIEFPPPSA|A IHSR MNLTRG IHSR MNLTRGI I HSR MNLTRGI ILlLHSR MNLTRGI I l l l H S R MNLTRGI ILl lHSR MNLTRGI i J l H S R MNLTRGI IHSR MNLTRGI IIIHSR MNLTRGI IHSRfMNLTRGI IHSRSMNLTRGI DLlSl [KGVCTEAGMBALRERR GVCTEAGMIALRERR GVCTEAGMBALRERR KGVCTEAGMBALRERRMHVTu KGVCTEAGMBALRERRl GVCTEAGN ALRERR HVTQ DFE AV KV |KGVCTEAGM ALRERR HVTQ DFE AV KV iGVCTEAGM ALRERR HVTQ DFE AV KV [KGVCTEAGM ALRERR HVTQ DFE AV KV GVCTEAGM ALRERR HVTQ DFE AV KV KGVCTEAGX ALRERR HVTQ DFE AV§KV 400 HVTQlDFElAVlKV H V T Q | D F F ^ V I K V HVTd HVTQBD FEMAVMKV 401 416 LdSUGl(387) \ 1 I • MsSUGl(387) I 1 AgSUGl(388) 1 1 IKL AmSUGl(390) | 1K L DpSUGl(390) i IK_ -DmSUGl (390) | j IK:. • TcSUGl(393) j IKL • X1SUG1(400) 1 • HsSUGl(391) I ScSUGl(390) ii CNQ|T3 | j EKL SpSUGl(388) LN C G D S G E i g QKL 140 Figure 4.1. continued. B SUG1 100 100 [~ L. dispar sir 74 .100 •tS: pombe M. sexta A. meilifera' D. melanqgaster D. pseudoobscura; • A. gambiae T. citricida. -II. sapiens - X. laevis; •S. cerevisiae: 0.05 were cloned in the BD vector. However, weak interactions between LdSSUGl were observed with the basic, RING, and leucine zipper deletions when cloned into the A D domain vector (Fig. 4.2B). No interactions were observed with the P34 glutamine domain deletion. These results suggest that all the P34 domains affect the 2-hybrid formation but the glutamine domain has the greatest influence. 4.2.4 Co-immunoprecipitation of LdSUGl with P34 and P34A Co-immunoprecipitation was conducted to verify the interactions of L d S U G l with P34 and P34A observed in the yeast 2-hybrid assays. To perform co-immunoprecipitation LdSUGl was tagged with the H A epitope fused to the C-terminus (Fig. 4.3A). P34 or P34A were co-expressed 141 Figure 4.2. Analysis of protein-protein interactions of P34, P34A and P34 deletions with LdSUGl. (A) Schematic diagram of full length P34, P34A, P34 domain deletions and LdSUGl cloned in the 2-hybrid yeast vectors pJ3D-Gal4 Cam and pAD-Gal4-2.1. The numbers above the diagram indicate the positions of the identified P34 domains. Each construct was cloned in frame with the Gal4 D N A binding domain (BD) or activating domain (AD) of the vectors. \> Symbolizes the location of the P34A start codon. A A A represents the ATPase domain of L d S U G l . The brackets ( ) indicate the regions that have been deleted in P34. (B) Protein-protein interactions of P34, P34A and P34 domain deletions with L d S U G l . These proteins were fused to the D N A binding domain (BD) of the vector pBD-Gal4 Cam and the activating domain (AD) of the vector pAD-Gal4-2.1 and cotransformed into YRG-2. After transformation 20 il l of cells were spotted onto Trp-Leu-His dropout media. The vectors pBD-L d S U G l and pAD-LdSUGl are presented on the left and the P34 constructs either in the BD or A D constructs are shown on the top of the figure. 142 Figure 4.2. continued. bask RING -3039 I —14 acidic WT domain deletions coiled coil AAA L d S U G l AD III GAL4 ADH1 promoter BD Terminator ; , r . , , k M £ S n o r t pPCori chloramphenicol 2-raicron on ADH1 GAL4 ADH1 promoter AD M Q S Terminator H a i vectors ampicillin 2-micron ori pAD-Gal4-2.1 WT domain deletions i r P34 P34A P34 P34 A R I N G P34 A Q P34 AL2 B D - L d S U G l Trp- Leu- Ills DO 143 with L d S U G l - H A in transfected Ld652Y cells and immune-precipitated with anti-HA monoclonal antibody. Comparative analysis was performed between the co-immunoprecipitations of L d S U G l - H A with P34 or P34A and the previously observed interactions between P34-HA and P34 or P34AHA and P34A. Co-immunoprecipitation of L d S U G l - H A with P34 showed it was capable of pulling down P34 (Fig.4.3C). In the absence of L d S U G l - H A no P34 was immunoprecipitated. Interestingly L d S U G l appears to be more efficient at co-immunoprecipitating P34 then P34-HA as indicated by the band intensity. However additional analyses wil l be necessary to assess the relative strength of these interactions. LdSUGl did not pull down P34A by co-immunoprecipitation (Fig. 4.3D). These observations indicate that LdSUGl binds to P34 and not with P34A, which is consistent with the results obtained for yeast 2-hybrid assays (Fig. 4.2B). 4.2.5 Localization of P34 and P34A with LdSUGl in infected and transfected cells To investigate the localization of P34 and L d S U G l in infected and transfected cells, localization studies were performed using confocal microscopy. The plasmid pP34M~> A - C F P (Chapter 3) was previously constructed to express P34 without producing P34A. The p34 ORF of pP34M^ A -CFP has the internal start codon of P34A mutated to G C G to prevent translation of P34A-CFP at late times post-infection (Fig. 4.4A). To observe cellular localization of L d S U G l the plasmid pLdSUGl-YFP was constructed and has YFP fused to the L d S U G l C-terminus (Fig. 4.4A). The constructs expressing P 3 4 M " A - C F P and L d S U G l - Y F P were cotransfected into Ld652Y cells followed by infection with OpMNPV. Similar to previously shown in Chapter 2 and 3, P34 is detected in the nucleus forming punctate foci in both transfected cells and infected cells from 144 A nasi. RIM, acidic Q LZ P34 P34-HA basic RING acidic Q LZ HA M I i m P34A acidic Q LZ P34A-HA acidic Q LZ HA I.dSUGl-HA coiled coil n — r HA ] B kDa 47.5-32.5 • 25 g B 2 - LdSUGl HA - P34-HA kDa 32,5-- P34A-HA IP a-HA WB a-HA D Ml i + g a 3 I a. 3 - P34-HA - P34 IP a-HA WB a-P34 kDa 32.5 • 25 - - P34A-HA Figure 4.3. Analysis of protein-protein interactions of P34, P34A and with LdSUGl using immunoprecipitation. (A) Schematic representation of the proteins used for co-immunoprecipitation, which include P34, P34A, P34-HA, P34A-HA and L d S U G l - H A . P34, P34A and LdSUGl were fused at C-terminus to the H A epitope. (B-D) Western blot (WB) analysis of H A tagged proteins co-immunoprecipitated (IP) with of P34 and P34A. (B) Western blot of the expressed H A fusion protein used for co-immunoprecipitation using H A monoclonal antibody. (C) Western blot of P34 co-immunoprecipition with P34-HA or L d S U G l - H A using P34 monoclonal antibody. (D) Western blot of P34A co-immunoprecipited with P34A-HA or L d S U G l - H A using P34 monoclonal antibody. Ld652Y cells were cotransfected with plasmids expressing proteins, which are indicated above each lane. The interacting proteins were pulled down with H A monoclonal antibody (a-HA) and analyzed by western blot using H A monoclonal antibody or the P34 (a-P34) monoclonal antibody. The sizes of the molecular weight markers (kDa) are shown on the left. 145 12-24 h p.i. (Fig. 4.4B). A dispersed pattern was observed for P34 from 24 h p.i., which seems to translocate towards the nuclear membrane. In cotransfected cells L d S U G l - Y F P was expressed in the cytoplasm and in addition within the nucleus forming punctate foci similar to P34. Merged images show that in transfected cells L d S U G l and P34 colocalize in the nuclear foci. In infected cells the overall distribution of these proteins did not change from 12 to 24 h p.i. and both continued to colocalize in the nuclear bodies. From 24 h p.i. both P34 and LdSUGl were observed close to the nuclear membrane where they colocalize however are more dispersed. In addition to the general expression of L d S U G l - Y F P throughout the cytoplasm discrete cytoplasmic spots were observed suggesting local concentration (Fig. 4.4B). At 48 h p.i there does not appear to be any significant colocalization of P34 and L d S U G l . To determine if the late gene product P34A interacts with L d S U G l in Ld652Y cells the plasmid pP34A-CFP was used, which expresses P34A fused at the C-terminus to CFP (Chapter 2). The plasmid pP34A-CFP and pLdSUGl-YFP were both transfected into Ld652Y cells followed by infection with OpMNPV. In transfected cells P34A-CFP is evenly distributed throughout the nucleus and cytoplasm similar to that previously observed (Chapter 3). In contrast LdSUGl -YFP was predominantly expressed in the cytoplasm (Fig. 4.4C). This is significantly different to the nuclear punctate foci that are observed when P34-CFP is cotransfected with L d S U G l . This strongly supports the conclusion that P34 and not P34A interacts with LdSUGl and recruits it to nuclear foci. These results agree with the yeast 2-hybrid and co-immunoprecipitation assays, which also showed that P34A did not directly interact with L d S U G l . In infected cells P34A-CFP increases in nuclear localization from 12-24 hours p.i. and forms small nuclear foci by 48 h p.i. L d S U G l - Y F P from 12-48 h p.i. remains predominantly 146 expressed protein pP34M*-CFP PP34A-CFP pLdSUGl-YFP < P34-CFP P34A-CFP LdSUGl-YFP Figure 4.4. Localization of O p M N P V P34, P34A and L d S U G l in uninfected and infected Ld652Y cells. (A) Schematic representation of the fluorescent protein (FP) constructs used for localization of P34, P34A and L d S U G l . P 3 4 M " A and P34A were fused at C-terminus to the cyan fluorescence protein (CFP) and LdSUGl was fused at C-terminus to the yellow fluorescence protein (YFP). The fused proteins are expressed under the control of the OpMNPV iel promoter. Ld652Y cells were cotransfected with (B) pP34 M ^ A -CFP and pLdSUGl-YFP or (C) pP34A-CFP and pLdSUGl-YFP. After transfection the cells were infected with OpMNPV (MOI: 5) and analyzed at 12, 18, 24 and 48 h and non-infected transfected cells were also included. Expression of the CFP fusion proteins are shown in red, YFP fused proteins are indicated in green and the DAPI nuclear staining is shown in cyan. Merged images contain P34 M ^ A _CFP and P34A-YFP, but not DAPI. The cells were also visualized by light microscopy. The images of the expressed proteins in the cells were obtained sequentially using a confocal microscope (Leica). 147 Figure 4.4. continued. 148 Figure 4.4. continued. 149 cytoplasmic, however at 48 h p.i small nuclear foci are observed and a few are colocalized with P34A. In infected cells P34 is expressed from only the viral genome instead of the transfected plasmids as in Fig. 4.4B. Therefore the smaller foci in Fig. 4.4C are likely due to lower expression of P34. 4.2.6 LdSUGl play significant roles in viral DNA replication and budded virus production in infected Ld652Y cells The function of both P34 and L d S U G l in D N A replication and budded virus production was examined during infection using R N A i . To silence transcription ofp34 and Ldsugl dsRNA was generated from the 3' end of each ORF (Fig. 4.5 A). Wild type OpMNPV infected Ld652Y cells that hadp34, Ldsugl or both genes silenced were analyzed at 12, 24, 48 and 72 h p.i. Northern blot analysis of Ldsugl detected a 2.0 kbp transcript in both uninfected and infected cells (Fig. 4.5B). Interestingly the Ldsugl transcript increases in steady state levels during infection. Analysis of uninfected cells showed that treatment with dsRNA to Ldsugl significantly reduces the level of expression. The levels of P34 and P34A were compared by Western blot between wild type infection and infected cells treated with p34, Ldsugl or both dsRNA templates. The cells depleted of p3'4, and bothp34 and Ldsugl did not show expression of P34 or P34A from 12-72 h p.i. (Fig. 4.5C). Infected cells depleted of Ldsugl showed reduced or delayed expression levels of P34 and P34A expression from 12 to 72 h p.i. (Fig. 4.5C). That is, the P34 expression pattern from 48 to 72 h p.i. was similar as observed for wild type infection at 24 h p.i. and P34A was only observed at very low levels from 48 h p.i. and 72 h p.i. (Fig. 4.5C). Viral D N A replication was analyzed in OpMNPV infected cells depleted ofp34, Ldsugl or 150 Figure 4.5. Analysis of viral DNA replication and budded virus production in Ld652Y cells depleted of P34 or LdSUGl using RNAi. (A) Schematic overview of the preparation of p34 and Ldsugl dsRNA. M l 3 Forward and Reverse primers amplified the 3' end of the p34 ORF from Litmus28i-P34PstI and Litmus28i- LdSUGl c348. The fragments were amplified by PCR and used as a template to generate dsRNA transcribed by T7 R N A polymerase. (B) Northern blot analysis of Ldsugl was performed with uninfected cells (Mock), uninfected cells with Ldsugl dsRNA and infected cells at 23 h p.i. and subsequently detected by Northern blotting using a 3 2P-labelled probe. The sizes of R N A ladder (kb) are shown on the left. (C-E) Uninfected (Mock) or infected cells treated with p34, Ldsugl or p34 and Ldsugl dsRNA and analysed at 12, 18, 24, 48 and 72 h p.i. for P34 exppression, D N A replication and budded virus production. (C) Western blot analysis of P34 and P34A expression using a P34 monoclonal antibody. The localization of P34 and P34A (kDa) are shown on the right. (D) Viral D N A replication. Total viral D N A was determined using the slot blot method followed by hybridization with a viral P-labelled probe. The signal of each sample was quantified using a phosphoimager (Molecular Dynamics). (E) Budded virus production. Supernatants were harvested at 12, 18, 24, 48 and 72 h p.i. and the B V titers were determined by quantitative PCR. R N A i was performed by transfection of Ld652Y cells with dsRNA followed by infection with OpMNPV (MOI: 5). Each time point in figures 4.5C and D presents and average of two separate samples. 151 Figure 4.5. continued. A B p34 ORF Litmus28i-P34PstI I 5* ' Pstl 1 M 1 3 F - * 5> 3> T7 -»~ 1«- T7 « - M13R Ldsugl ORF Lh Litmus28i-LdSlGlc304 4 - M13R kb 2.37-1.35-*- Ldsugl 12 h p.i. 18 h p.i. -< HE 3 i ' a * I 1 + kDa - P34 I- P34 24 h p.i - P34 48 h p.i. 72 h p.i. - P34 - P34A - P34 - P34A 152 Figure 4.5. continued. D 0 10 20 30 40 50 60 70 80 Time;postinfection (h) 153 both transcripts and compared to wild type infection. In contrast to wild type infection, D N A replication was dramatically decreased for infected cells depleted of p34 or Ldsugl at all stages of infection analyzed (Fig. 4.5D). Furthermore, levels of viral D N A replication cells treated with both p34 and Ldsugl dsRNA templates were reduced to even lower levels than cells treated with either p34 ox Ldsugl separately (Fig. 4.5D). Budded virus production was also examined in cells depleted of p34, Ldsugl or both transcripts and compared to a wild type infection. Budded virus production was severely affected for all treatments and levels were 2 logs lower by 24 h p.i. and 1 log lower at 48 h p.i relative to wild type OpMNPV (Fig. 4.5E). Therefore depletion of Ldsugl results in a phenotype that appears to be the same asp34 depleted cells, which suggest that L d S U G l is required for P34 function. 154 4.3 Discussion OpMNPV P34 is a multifunctional regulatory protein that activates transcription from viral promoters and augments D N A replication. In addition, P34 has been identified as a RING finger E3 ubiquitination ligase, which belongs to a class of proteins known to regulate many diverse cellular processes. To understand the role of P34 in the baculovirus life cycle this study has continued the investigation into identifying cellular proteins that are either required for its function or are targets of its activity. P34 was used as bait to screen a cDNA library derived from 12 h p.i. OpMNPV infected Ld652Y cells to identify interacting proteins. A cDNA fragment was isolated containing an ORF of 1209 nucleotides encoding a 402 amino acids (45 kDa) predicted protein that is highly homologous to SUG1 and was therefore called L d S U G l (Fig. 4.1 A and B). LdSUGl homologs have been shown to be a subunit of the 26S proteasome complex, which is a major protein complex that recognizes polyubiquinated protein targets and degrades them. The proteasome is composed of a 20S 28-subunit cylinder with a 19S cap. The 19S cap recognizes ubiquitin tagged proteins and the 20S proteolytic subcomplex that directs their degradation (see Fig. 1.5, Chap 1) (Baumeister et al, 1998; Tanaka and Ichihara, 1990). The 19S cap consists of at least 18 components, which are divided into two parts named base and lid. SUG1 has been identified as being part of the base, which contains six subunits that are ATPases of the "ATPases Associated with diverse cellular Activities" (AAA) family (Glickman et al, 1999; Swaffield et al, 1992). This family of proteins contain one or more copies of the A A A motif, which is a conserved region of 230 amino acids (Patel and Latterich, 1998). The base of the 19S subcomplex containing SUG1 has also been shown to be recruited to PolII promoters 155 and be involved in transcriptional activation independent of the rest of the proteasome (Swaffield et al, 1992; Swaffield et al, 1995). The protein interaction between P34 and L d S U G l was also observed by co-immunoprecipitation and indirectly by immunofluorescence confocal microscopy (Fig. 4.3B and 4.4B). Confocal studies also showed that P34 was expressed primarily in the nucleus and distributed in a punctate pattern in both cotransfected and infected cells similar to that previously observed in Chapter 2 and 3 (Fig. 4.4B). In the absence of P34, L d S U G l is observed at low levels in a diffuse pattern in the nucleus but is predominately cytoplasmic. However when coexpressed with P34-CFP, L d S U G l is recruited to punctate nuclear foci. Previous studies have localized the yeast SUG1 homolog predominantly to the nucleus in a uniform pattern (Russell et al, 1999). The D. melanogaster SUG1 (DUG1) was observed to be evenly distributed across the nucleus of differentiating cells of the male germ line (Mounkes and Fuller, 1998). In contrast, in cell culture DUG1 is observed in the cytoplasm in punctate patches. The expression of L d S U G l in the cytoplasm is therefore consistent with the yeast and fruit fly SUG1 homologs; however the formation of punctate foci in the nucleus have not been previously reported and suggests another potential role of this protein during viral infection. Cellular redistribution and nuclear punctate foci formation has however been observed for the 20S proteasome subunits and ubiquitinated proteins in herpes simplex virus type I (HSV-1) infected cells (Burch and Weller, 2004). The results of this study including the yeast 2-hybrid analysis, co-immunoprecipitation, colocalization and R N A i analysis support a strong protein interaction between P34 and L d S U G l . Analysis of P34 showed that deletion of any domain (Basic, RING, glutamine, or leucine zipper) inhibited the interaction with L d S U G l . The P34 yeast 2-hybrid results suggest that the 156 glutamine domain may play a more significant role. However, late gene product P34A which contains the glutamine domain was found not to interact with L d S U G l (Fig. 4.2B and 4.3B). In order for P34 to bind L d S U G l these results suggests that a very specific tertiary structure or protein complex is required. Other E3 ubiquitin ligases like P34 have also been shown to directly interact with SUG1. The yeast S. cerevisiae E3 ubiquitin ligase UFD4 has been reported to bind the SUG1 homolog RPT6 in a complex with E2 enzymes. UFD4 binding required the N -terminal 714 amino acids half of the protein but no specific domain was identified (Xie and Varshavsky, 2002). Several studies have identified domains on SUG1 to be required for protein interactions. Furthermore specific regions on cellular regulatory proteins have been shown to be essential for binding to SUG1. GAL4 was shown to bind SUG1 via its acidic activation domain (Chang et al, 2001; Gonzalez et al, 2002; Melcher and Johnston, 1995). In vitro and in vivo studies with human SUG1 revealed an interaction with the transcription factor Spl , which was mediated by the ATPase domain of SUG1 (Su et al, 2000). Yeast 2-hybrid assays indicated that the coiled coil domain of mammalian SUG1 interacts with the bZLP segment of c-Fos (Wang et al, 1996). The glutamine rich domain of P34, in addition to the leucine zipper, is also predicted to form a coiled-coil domain and could be a potential candidate for binding to L d S U G l . However, additional experiments such as using point mutations in this region will be necessary to determine i f the P34 glutamine rich/coiled-coil domain is required for a specific binding to L d S U G l . Interestingly homologs of L d S U G l have been shown to be targets of other viral regulatory proteins including herpesvirus VP 16, Adenovirus E l A , TAT binding protein and SV40 T antigen (Barak and Oren, 1992; Grand et al, 1999; Lee et al, 1995; Melcher and Johnston, 1995; Rasti 157 et al, 2005; Swaffield et al, 1995; Tumell et al, 2000). Adenovirus E1A binds directly to mammalian SUG1 and regulates proteasome activity affecting degradation of target proteins (Grand et al, 1999; Rasti et al, 2005; Turnell et al, 2000). The Hepatitis protein HBx involved in cellular transformation binds a SUG1 homolog and it has been suggested that it affects transcriptional activation activity of the 19S lid independent of the proteasome (Barak and Oren, 1992). Similarly the potent acidic activation domain of herpesvirus V P 16 binds the 19S regulatory subunit of the proteasome and specifically SUG1. Binding to the 19S subunit was dependent upon ubiquitination of the acidic activation domain which enabled transcriptional activation but also results in proteasomal degradation (Zhu et al, 2004). Ubiquitination of the V P 16 acidic activation domain also increased the interaction with the transcription elongation factor b (P-TEF-b) and enhanced the elongation rate (Kurosu and Peterlin, 2004). These findings show that ubiquitin may be involved in establishing an interaction via SUG1 when attached to 19S and transcriptional activators. OpMNPV IE1 contains a potent acidic activation domain functional similar to the VP 16 domain (Dai et al, 2004; Pathakamuri and Theilmann, 2002). P34 as an ubiquitin ligase could potentially form complexes with the 19S regulatory subunit containing LdSUGl and viral transcriptional activators such as IE1 to establish an interaction and attach ubiquitin moieties resulting in increased transcriptional activation at baculovirus promoters. Additional evidence of the role of SUG1 in transcriptional activation comes from the analysis of cellular genes. Yeast SUG1 and the human homolog thyroid-hormone-receptor interacting (TRTP1) protein have been previously identified to be involved in activating transcription of yeast Gal4 (Lee et al, 1995; Swaffield et al, 1992; Swaffield et al, 1995). Recent studies showed, however, that the complete 19S complex including SUG1 is recruited to activate the 158 yeast Gall-10 promoter (Gonzalez et al, 2002). The SUG1 protein was also identified in the purified yeast holoenzyme. This multiprotein complex was shown to interact with the transcriptional activator GCN4 and is directly associated with the transcription factor T A T A binding protein (TBP) (Hengartner et al, 1995; Kim et al, 1994; Swaffield et al, 1995). In addition to TBP, interactions of SUG1 with several other transcription factors have been reported as well (Lee et al, 1995; Swaffield et al, 1992; vom Baur et al, 1996; Weeda et al, 1997). Studies have also shown that the mammalian SUG1 homologs are involved in degradation and activation of the transcriptional activators c-Fos and the nuclear retinoic acid receptor (RARy2) induced by retinoic acid (Gianni et al, 2002; Wang et al, 1996). Viral D N A replication in infected cells depleted of P34, LdSUGl or both proteins by R N A i resulted in a significant decrease in viral D N A replication and budded virus production relative to C p M N P V wild type infection (Fig. 4.5D and E). The role of P34 in viral D N A replication is still unclear but P34 and LdSUGl could potentially cooperate to activate viral transcriptional activators such as IE1 to initiate expression of other early viral proteins required for D N A replication. Alternatively the interaction with L d S U G l could be directly involved in the formation of replication complexes which are known to form nuclear foci in baculovirus infected cells. In summary, the results obtained in this study have shown that P34 interacts with the cellular protein L d S U G l and both genes are required for high-level viral D N A replication and budded virus production. Recruitment of LdSUGl to nuclear foci suggests an intimate association between the proteasome subcomplexes and viral replication and transcription. Further studies wil l be required to characterize the complexes associated with these proteins and what role they play in enhancing viral replication. 159 4.4 Materials and Methods 4.4.1 Cells and virus The propagation of OpMNPV-WT virus and maintaining of Lymantria dispar 652Y cells was described in chapters 2 and 3 as well as the MOI of OpMNPV-WT used for the R N A i time course experiment. 4.4.2 Construction of the 12 h p.i. OpMNPV cDNA library Construction of the 12 h p.i. OpMNPV cDNA library was previously described in chapter 3. 3.4.3 Yeast transformation of OpMNPV P34 and 12 h p.i. OpMNPV cDNA library and yeast 2-hybrid assay The yeast transformations of OpMNPV P34 and 12 h p.i. OpMNPV cDNA library was similar as described in chapter 3. 4.4.4 Sequence analysis of clones with P34 - cDNA interaction The sequence of the cDNA fragment that showed interaction with P34 in yeast was obtained as previously described in chapter3. The nucleotide sequence was screened for homologous protein alignments using the translated query vs. translated database (tblastx). The SUG1 protein alignments were obtained using Align X (Vector NTI Advance, Invitrogen), which is a program based on ClustalW (Thompson et al, 1994) (Fig. 4.1 A). Phylogenetic analyses were conducted using neighbor-joining method and branch numbers represent bootstrap scores (%) of 1000 160 replicates with a bootstrap of 1000 using M E G A version 3.1 (Kumar et al, 2004). Scale represents 0.05 substitutions per site for the trees' branch length (Fig. 4. IB). 4.4.5 Yeast 2-hybrid assays of P34, P34A and P34 deletion domains with LdSUGl Yeast 2-hybrid assay was used to confirm the identified cellular protein that interacts with P34. After the YRG-2 cotransformants were screened on Trp-Leu-His dropout SD-media pAD plasmid (pAD-LdSUGlcDNA) with a cDNA fragment of 1.4 kb encoding LdSUGl ORF was identified for the interaction with BD-P34 fused protein. pAD-LdSUGlcDNA was isolated by D N A extraction from the yeast clone and transformed into Xl l -Blue M R F ' bacterial cells (Stratagene). The transformed cells were plated on an ampicillin selection plate and pAD-L d S U G l c D N A was obtained from the ampicillin resistant colonies using Qiaprep spin miniprep kit (Qiagen). The cDNA fragment of pAD-LdSUGlcDNA was amplified with the primers p504 and p514 followed by digestion of the PCR product with EcoRl and Xbal. This fragment was cloned into pBS+ and generated pBS-LdSUGlcDNA. The Ldsugl ORF was obtained by PCR with pBS-LdSUGlcDNA as template and the primers p695 (5'-T A A G A A T T C A A G A T G A C T G T A A C A A - 3 ' ) and p696 (3'-A T T A G A C T C G A G A T A A G T G C A C T T T A - 5 ' ) . The resulting 1273 bp product was cloned as an EcoRl-Xhol fragment into pBD-GAL4 Cam and pAD-GAL-2.1, which generated pBD-L d S U G l andpAD-LdSUGl (Fig. 4.2A). The P34, P34A and P34 domain deletions cloned in either the binding domain (BD) or activating domain (AD) vectors were previously described in chapter 2 (Fig. 4.2A). D N A binding domain constructs were cotransformed with activating domain plasmids into the yeast strain YRG-2 according to the Hybrizap-2.1 X R library protocol (Stratagene). 161 Transformed cells with equal densities for each sample were applied (20 ul) as spots onto Trp-Leu-His dropout (DO) SD-media (Amersham Biosciences) and the cotransformants were screened for high stringency interactions. 4.4.6 Western blot analysis Western blot analysis was previously described in chapter 2. In this study we used P34 (BI IB 10) and H A ( H A . l 1, MMS-101R; CRPinc) monoclonal antibodies as primary antibody with a dilution of 1:1000 dilution and as secondary antibody with used a 1:10,000 dilution of a goat anti-mouse horseradish peroxidase secondary antibody (315-035-044; Jackson Laboratories). 4.4.7 Preparation of d s R N A To obtain dsRNA of p34 and Ldsugl the ORFs were cloned into the multiple cloning site (MCS) of Litmus28i, which flanks the T7 promoter region located at opposite ends (NEB). The construction of Litmus28i-P34PstI has been previously described in chapter 2 (Fig. 4.5A). The primers p697 ( 5 ' - T A A G G A T C C A A G A T G A C T G T A A C A A - 3 1 and p698 (3'-A T T A G A G A A T T C A T A A G T G C A C T T T A - 5 ' ) and the template pBS-LdSUGlcDNA were used to PCR Ldsugl and produced a fragment of 1273 bp. The synthesized Ldsugl fragment was digested with BamHl and EcoRl and cloned into Litmus28i (Fig. 4.4A), which resulted in Litmus28i-LdSUGl. A fragment was amplified using Litmus28i-LdSUGl as template and the primers p682 (5 ' -TAGGCCTGGTCGTAT-3 ' ) and p707 (3'-TTTTATGTATTAGAGAATTCATCGTG-5 ' ) . The PCR product contains the 348 nucleotides 162 located at 3'end of Ldsugl. After digestion with EcoRI the fragment was cloned into Litmus28i, which was cut with BamHI, blunted by T7 polymerase and then digested with EcoKI. The resulting construct was named Litmus28i-LdSUGlc348 (Fig. 4.5A). Preparation of dsRNA was previously described in chapter 2. 4.4.8 RNAi time-course experiment The R N A i time-course experiments were conducted as described in chapter 2. 4.4.9 Total RNA isolation and Northern blots The total R N A isolation and Northern blotting are similar as described in chapter 3. However in this chapter the radioactive labelled ssRNA probe that was prepared included Litmus28i-L d S U G l as template. 4.4.10 Budded virus titration by quantitative PCR Titrations were performed as described in Chapter 2. 4.4.11 Analysis of DNA replication using slot blot procedure Slot blot analysis of viral D N A replication was performed as described in Chapter 2. 163 4.4.12 Colocalization of P34 and P34A with LdSUGl To localize P34 and P34A with L d S U G l in Ld652Y cells the ORFs were cloned in frame and upstream of with the genes encoding the cyan or yellow fluorescent protein (CFP or YFP). Furthermore, the fusion genes were subcloned into p2Zop2E downstream of the OpMNPV ie2 promoter, which allows transcription of these genes in absence of any viral factors. The construction of pP34 M " A -CFP and pP34A-CFP were described in chapter 2 (Fig. 4.3A). The primers p777 ( 5 ' - T A A A A G C T T A A G A T G A C T G T A A C A A A - 3 ' ) and p778 (3'-A A A G G A T C C T A T T A A T A T T A A T T G T T T T - 5 ' ) and pBS-LdSUGlcDNA as template were used to amplify Ldsugl. The 1243 bp PCR product was digested with Hindlll and BamHI and cloned into pEYFP, which resulted in pYFP-pLdSUGl. pYFP-pLdSUGl was digested with Hindlll and EcoRI and the fusion gene was cloned into p2Zop2E, which generated pLdSUGl -YFP (Fig. 4.4A). Cotransfections of the plasmids into Ld652Y cells and infections as well as the staining and confocal microscopic handling were described in the previous chapter 2. 3.4.13 Immunoprecipitation The construction of p2Zop2E-P34A, p2Zop2E-P34-HA and p2Zop2E-P34A-HA was previously described in chapter 2. The Ldsugl ORF and the p2Zop2E vector containing the H A tag were obtained by digestion of pLdSUGl-YFP and p2Zop2E-P34-HA with Hindlll and BamHI. 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Latent membrane protein 2A of Epstein-Barr virus binds WW domain E3 protein-ubiquitin ligases that ubiquitinate B-cell tyrosine kinases. Mol Cell Biol 20, 8526-8535. 169 Wu, X . , Stewart, S., and Theilmann, D. A . (1993). Alternative transcriptional initiation as a novel mechanism for regulating expression of a baculovirus trans activator. J Virol 67, 5833-5842. Xie, Y . , and Varshavskiy, A . (2002). UFD4 lacking the proteasome-binding region catalyses ubiquitination but is impaired in proteolysis. Nat Cell Biol 4, 1003-1007. Zhu, Q., Yao, J., Wani, G., Chen, J., Wang, Q. E., and Wani, A . A . (2004). The ubiquitin-proteasome pathway is required for the function of the viral V P 16 transcriptional activation domain. FEBS Lett 556, 19-25. 170 Chapter 5: General discussion and future prospectives Understanding how baculoviruses interact with the host cell is essential to elucidate the molecular basis for virulence and host range. The narrow host range observed for baculovirus infections is an important trait to have as a biocontrol agent for managing pests of specific insect species. The baculovirus protein OpMNPV P34 is a regulatory protein that activates viral transcription and viral D N A replication. This study presents novel data about the function and mode of action of P34 during viral replicaton. It is also the first study to identify cellular proteins, L d U E V and L d S U G l , which specifically interact with P34. The results show that host genes that are part of the ubiquitin-proteasome pathways. Host genes also play a major role during baculovirus replication, which has not been previously shown, and represent a potentially exciting new area of research for this group of viruses. Many RING finger proteins have been shown to function as ubiquitin ligases and the RING domain is essential for the ligase activity as it catalyzes the transfer of ubiquitin to the target protein. In this study in vitro ubiquitination assays showed that P34 possesses ubiquitin ligase activity (Chapter 2) and depends on the RING finger for its activity similar to the P34 homolog Bombyx mori N P V PE38 (Imai et al., 2003). In addition, these studies have shown that P34 forms both homodimers and heterodimers with P34A and the preferred dimers appear to be P34-P34A over P34-P34 interactions. The basic domain may play a role in this interaction as it inhibited P34 homodimerization and enhanced P34-P34A dimerization. The RING, glutamine rich and leucine zipper domains are required for both P34 homodimerization and P34-P34A heterodimerization whereas only the leucine zipper is essential P34A-P34A formations. This result would suggest that P34-P34 homodimerization is more complex and is dependent on 171 multiple interactions since all the domain motifs in P34 were required. Each of the P34 domains are well known for their ability to establish protein-protein interactions (Chen et al, 1998; Grbavec et al, 1998; Hattori et al, 2003; Lekstrom-Himes and Xanthopoulos, 1998; Miyasaka et al, 1993; Nikolay et al, 2004; Pinto and Lobe, 1996; Ren et al, 1999; Tanimura et al, 1999). Defining those interactions including the ones with LdUEV and LdSUGl will require more precise analyses using point mutations. The function of P34A still remains to be resolved but the results of this study have provided some possible suggestions. P34A forms nuclear punctate foci in presence of P34 but is predominately cytoplasmic in the absence of P34. This result implies that at late times p.i. when P34A is expressed at higher levels most i f not all P34 molecules are likely to form heterodimers with P34A. Studies have shown that this kind of interaction can play a critical role in regulating the activity of the full-length protein. For example the transcriptional regulatory protein C/EBP has been shown to be dominantly inhibited by heterodimerization via leucine zipper domains with partners that lack specific sequences such as D N A binding domains (Cooper et al, 1995; Hattori et al, 2003; Krylov et al, 1995). In the ubiquitination pathway the E3 ubiquitin ligase CHIP has been shown to require homodimerizaton for its ligase activity (Nikolay et al, 2004). Therefore it is possible that preventing homodimerization of P34 could inhibit its ubiquitin ligase activity and potentially its interaction with LdUEV and SUG1. A possible function for P34A during late times post-infection could be to inhibit the transcriptional activation, or ubiquitin ligase activity of P34 by heterodimer formation. To determine i f P34A inhibits P34 ubiquitin ligase activity in vitro ubiquitination assays could be performed (Chapter 2). Furthermore the effect of P34A on the viral D N A replication activity of P34 could be assessed by performing transient assays (Pathakamuri and Theilmann, 2002). Previous studies showed that P34 172 transactivates both the OpMNPV ie2 andp8.9 promoters (Wu et al, 1993a; Wu et al, 1993b), but initial competition studies have failed to show any P34A inhibition of transcriptional activation [D. A . Theilmann, unpublished results]. If P34A inhibits P34 it may therefore be only affecting viral D N A replication or the ubiquitin ligase pathways. One possible way to test this hypothesis would be to generate a recombinant virus expressing P34A under control of an early promoter, which we would expect subsequently to inhibit P34 activity and therefore viral D N A replication. This study showed that the cellular proteins LdUEV and L d S U G l interact with P34 and are required for wild type levels of viral D N A replication and budded virus production. In addition cellular localization studies indicated that P34 colocalized with LdUEV or LdSUGl in the nucleus in punctate foci. This observation was similar to A c M N P V PE38 which was shown to form punctate foci with IE2 and the cellular protein P M L , which also appeared to be located juxtaposed to the viral D N A replication centers (Krappa et al, 1995; Mainz et al, 2002). Several studies with herpesviruses have indicated that the P M L associated ND10 bodies form an interaction with parental viral genomes, which becomes a site for viral immediate-early (IE) gene transcription and subsequently are assembled into D N A replication compartments (Burkham et al, 1998; Everett, 2001; Ishov and Maul, 1996; Lukonis and Weller, 1997; Maul, 1998; Maul et al, 1996; Uprichard and Knipe, 1997). Based on these results it could be assumed that LdUEV or L d S U G l when associated with P34 have roles in viral D N A replication. However it is not yet known i f that function is due to transcriptional activation of genes required for D N A replication or direct participation in the replication complex. When associated with P34 the function of LdUEV or L d S U G l is probably different during infection. E3 ubiquitin ligases have been shown in several studies to possess multiple functions 173 and interact with various proteins. Studies on E3 ubiquitination ligases such as the human papilloma virus (HPV) E6 and herpes simplex virus 1 (HSV) ICPO revealed multiple interactions with target proteins and proteins involved in the ubiquitination pathway (Banks et al, 2003; Galinier et al, 2002; Harty et al, 2001; Winberg et al, 2000). Therefore we can predict that P34 also acts as a multifunctional E3 ubiquitin ligase. Many studies have identified a role for LdSUGl homologous proteins in transcriptional regulation by showing interactions with transcription related factors and activation domains as either individual proteins or in a complex with the 19S proteasome subunit (Chang et al, 2001; Gonzalez et al, 2002; Melcher and Johnston, 1995; Su et al, 2000). This interaction regulates both the activation and proteolysis of viral transcription factors (Barak and Oren, 1992; Grand et al, 1999; Rasti et al, 2005; Turnell et al, 2000; Zhu et al, 2004). U E V homologs have also previously been shown to be associated with transcriptional activation (Rothofsky and Lin, 1997; Sun et al, 1999). The proteasome 19S regulatory subunit containing SUG1 interacts with the acidic activation domain of herpesvirus VP 16, is dependent upon ubiquitination of the acidic activation domain and enables transcriptional activation and proteasomal degradation (Zhu et al, 2004). OpMNPV IE1 contains a potent acidic activation domain that is functionally similar to the VP 16 domain (Dai et al, 2004; Forsythe et al, 1998; Pathakamuri and Theilmann, 2002). The potential target of OpMNPV P34-LdSUGl could therefore be the major baculovirus transcriptional transactivator LEI, which is known to be essential for both transcriptional activation of early genes and viral D N A replication. A scenario could be envisioned whereby P34 is required to activate IE1 via ubiquitination. The use of ubiquitin tagged IE1 similar to that described by Zhu et al. (2004) in D N A replication assays could be used to determine i f this alleviates the need for P34 augmentation of D N A replication. In the absence of P34 activation 174 IE1 may have to depend on cellular enzymes and the process could be significantly slower. To distinguish between the function of L d S U G l and LdUEV it will be necessary to separate their interactions with P34. One possible way to do that will be to precisely identify the amino acid residues responsible for binding both L d S U G l and LdUEV. Inactivation of either site should enable us to study independently the roles of these two cellular proteins in the baculovirus life cycle. In conclusion this is the first study to identify interactions between a baculovirus protein and cellular proteins that furthermore are required for a wild type infection. Homologs to both these proteins have been found to be key players in a number of other viral and cellular systems. Therefore future studies defining the role of L d S U G l and LdUEV in viral replication promise to create a rich new area of research and to provide valuable insights into the molecular mechanisms of baculovirus replication. 175 5.1 References Banks, L. , Pim, D., and Thomas, M . (2003). Viruses and the 26S proteasome: hacking into destruction. Trends Biochem Sci 28, 452-459. Barak, Y . , and Oren, M . (1992). 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The ubiquitin-proteasome pathway is required for the function of the viral VP 16 transcriptional activation domain. FEBS Lett 556, 19-25. 179 Appendix: Role of AcMNPV IEO in baculovirus very late gene activation* Abstract IEO is the only known baculovirus protein that is produced by splicing. In this study we have explored the role of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) IEO and its interaction with LEI in the activation of very late gene expression from the polyhedrin promoter using transient assays. IEO is co-expressed with IE1 throughout infection up to late times post-infection but shows peak levels of expression at early times. Significant changes in the ratios of the relative levels of IEO to LEI were observed throughout the course of infection. To study IEO in the absence of IE1 we constructed a plasmid pAc-IE0 M _ > A that expressed only IEO. This was due to a mutation of the internal A U G that prevented translation of LEI from the ieO mRNA. Both IEO and LE0M"*A were able to replace IE1 in transient assays, showing that IEO is functional for very late gene activation and should be considered the twentieth late gene expression factor (lef). In transient assays, IEO showed that maximum very late gene expression is achieved at very low relative levels of protein. In contrast, IE1 requires higher levels of protein to obtain maximum very late gene expression. Furthermore, when the levels of IEO become too high, very late gene expression rapidly declines. Interestingly, co-expression of LEO and IE1 results in a mutually antagonistic effect on very late gene expression. * A version of this paper has been published in Virology: Use Huijskens, Lulin Li , Leslie G. Willis, David A. Theilmann, 2004. Role of A c M N P V LEO in baculovirus very late gene activation. Virology 323 ,120-30. 180 Introduction Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the most intensively studied member of the baculoviridae (Ayres et al., 1994; Blissard and Rohrmann, 1990). The 134 kbp circular D N A genome of A c M N P V is predicted to contain 154 open reading frames (ORFs) of at least 150 or more nucleotides. Baculovirus gene expression occurs in three phases; early, late and very late. Each phase is dependent on viral or cellular factors expressed or present in the previous phase. Late gene expression occurs concomitantly with, or after, the onset of viral D N A replication and requires a virally encoded R N A polymerase (Lu et al., 1997; Rice and Miller, 1986; Thiem and Miller, 1989). Using transient assays nineteen baculovirus genes have been identified being either essential or stimulatory for late gene expression and have been called late expression factors (lefs) (Fig. 1 A) (Li et al., 1999; Rapp et al., 1998). In addition to late gene expression, the majority of the lef genes are known to be involved in other processes during infection. Five lefs have been shown to be required in transient viral D N A replication assays in Sf21 cells (Kool et al., 1994; Lu and Miller, 1995), including ie-1 (transcription factor, binds origin of replication) (Choi and Guarino, 1995a; Guarino and Dong, 1991; Pathakamuri and Theilmann, 2002; Rodems et al., 1997), lef-1 (primase) (Barrett et al., 1996; Evans et al., 1997), lef-2 (primase associated factor) (Mikhailov and Rohrmann, 2002; Passarelli and Miller, 1993), lef-3 (single stranded D N A binding protein) (Hang et a l , 1995), andpl43 (helicase), (Lu and Carstens, 1991; Tomalski et al., 1988). Recently, lef-11 has been reported to be essential for D N A replication in virus infected Sf9 cells (Lin and Blissard, 2002b). In addition, four lef genes were reported to be stimulatory for D N A replication: dnapol (DNA polymerase) (Lu and Miller, 1995), ie2 (transcriptional factor and cell cycle arrest gene) (Carson et al., 1988; Carson et al., 1991; 181 Prikhod'ko and Miller, 1998), pe38 (transcription factor and possible ubiquitin ligase (Imai et al., 2003), p35 (apoptosis inhibitor) (Clem et al., 1991). Four lef genes (p47, lef-4, lef-8, and lef-9) have been shown to co-purify with the viral late R N A polymerase (Guarino et al., 1998). Low sequence homology with vaccinia virus R N A polymerase was identified for lef-6 and has been shown to accelerate late gene transcription (Lin and Blissard, 2002a; Passarelli and Miller, 1994). Guarino et al. (2002) suggested that lef-5 may play a role as initiation factor. The lef gene pp31 is suggested to be a phosphoprotein associated with the virogenic stroma (Fraser, 1986; Guarino et al., 1992; Okano et al., 1999; Wilson and Price, 1988) and hcf-l is required for /zr-dependent D N A replication in TN-368 cells (Lu and Miller, 1995; Lu and Miller, 1996). The functions for lef-7 and lef-10 however, remain unknown. A c M N P V IE1 is a multifunctional transcription factor that in addition to be required for late gene expression is also essential for D N A replication, enhancer dependent and independent transcription and negative gene regulation (Blissard et al., 1992; Choi and Guarino, 1995b; Guarino and Summers, 1986a; 1986b; 1988; Passarelli and Miller, 1993; Pathakamuri and Theilmann, 2002; Rodems and Friesen, 1995; Theilmann and Stewart, 1991). Detailed analysis of A c M N P V and Orgyia pseudotsugata M N P V (OpMNPV) IE1 identified the following functional domains: N-terminal acidic activation domain, required for both transcription and replication; basic domain I, essential for enhancer binding; a second acidic domain, also potentially involved in activation of transcription; a D N A binding domain; basic domain II, required for nuclear entry; and an oligomerization domain (Olson et al., 2002; Olson et al., 2003; Pathakamuri and Theilmann, 2002; Rodems et al., 1997; Slack and Blissard, 1997). The iel locus is the only baculovirus gene that is known to produce a second protein, IEO, due to splicing. The A c M N P V ieO mRNA initiates from the ieO-exonO promoter located 4344 bp 182 Figure 1. The nineteen late expression factors (lef) located on cosmids and plasmids used in the AcMNPV transient very late gene expression system. (A) Schematic map of the A c M N P V genome showing the location of the cosmids 1, 10, 59 and cosmid 58 (Li et al., 1999) and 5 8A. The numbers above the diagrams are the coordinates on the A c M N P V genome determined by sequence analysis (Ayres et al., 1994). The scale on the bottom shows the size in kilobases and the locations of the homologous repeat (hr) regions. The position and nomenclature of the nineteen lefs, identified as genes involved in transient late gene expression, are indicated below the cosmid library. (B) Schematic map of the A c M N P V cosmid 58A. The black arrows indicate the location of the ORFs in this cosmid. The grey part of cosmid 58A indicates the region with the ORFs deleted from cosmid 58. (C) Diagrams of the plasmid clones, p Ac-IE 1 (Theilmann and Stewart, 1991), pAc-IEO and pAc-IE0M _ > A under control of the iel and ieO promoters. (D) Western blot analysis of Sj9 cells transfected with pAc-IEl , pAc-IEO and pAc-IE0M^A. Protein samples were dephosphorylated prior to western blotting. LEO and IE1 specific proteins were detected using a primary mouse monoclonal antibody (IE1 4B7) specific for the N-terminus of IE1 (Ross and Guarino, 1997). 183 i.o un M r kilo base 547W 62982 I I I I cos 59 cos 10 , J.kV IrfTI lu-2 I 20 40 60 897W WSM I • cos 58 cos 58A 1J2JS6 I cos 1 lrf4 M kB <?MJ> 80 100 ie-0 *•, ie-1 120 cos 58A sua R e S I i i I J ii 1 i& a. i i i « i l i i i i ie-0 ie-1 p r o m pAc-IEl t V-J l * T C — I I E 1 . IEO pAc-IEO < M I 1 ' " c | A T C pAc-IEO" * i 1 I E w W • l E a pAc-IEl pAc-IEO"* pAc-IEO - IEO (72 kDa) - IE1 (67 kDa) 184 upstream of the IE l transcriptional start site. A total of 189 nucleotides from exonO are spliced to the 5' end of the iel mRNA resulting in a 2.1 kb ieO transcript (Chisholm and Henner, 1988; Kovacs et al., 1991a; Kovacs et al., 1991b). Transcription of ieO occurs at early times post-infection (p.i.), peaking at 4 h p.i. followed by a decline to lower steady state levels that remain detectable up to very late times p.i.. In contrast, transcription of iel also initiates at very early times p.i., but continuously increases in steady state levels until very late times p.i.. Translation of ieO mRNA produces a 72 kDa protein, comprised of the entire IE l protein and an additional 54 amino acids added to the N-terminus. The additional 54 amino acids include 38 amino acids from EXONO and 16 amino acids from the region immediately upstream of the iel ORF (Chisholm and Henner, 1988). Theilmann et al. (2001) recently showed that a 66 Kda protein was translated from the ieO mRNA in addition to the predicted 72 kDa IEO. This smaller protein was identified as I E l , which was initiated from the internal iel start codon. The exact function of IEO is still not known, however several previous studies have suggested different roles for IEO and I E l during infection (Lu et al., 2003; Pearson and Rohrmann, 1997; Theilmann et al., 2001). In this study we used transient very late gene expression assays to investigate the role of IEO in comparison to IE l in the activation of very late gene expression. Expression of A c M N P V IEO and IE l was analyzed during infection and showed significant changes in the relative levels of these two regulatory proteins. We investigated how the varying levels of IEO to BEl can influence the level of very late gene expression. Our results indicate that A c M N P V IEO is functional for the activation of very late gene expression and should be considered as the twentieth LEF. Surprisingly however, IEO and IE l were found to be mutually antagonistic for the activation of very late gene expression. 185 Results IEl and IEO expression and their relative ratios during the life cycle of AcMNPV Previous northern blot and primer extension analyses have described the transcription pattern of iel and ieO during A c M N P V infection (Choi and Guarino, 1995b; Kovacs et al, 1991a; Kovacs et al., 1991b; Lu et al, 2003). However the relative protein levels of IE l and IEO throughout the course of infection have not been well characterized. Therefore, a time course analysis was performed using protein samples prepared from Sf9 cells infected with A c M N P V at 0,2, 4, 8, 12, 24, and 48 h p.i. (Fig. 2A). The 72 kDa IEO had maximum steady state levels of expression at 4 h p.i., followed by a slow decrease to low, but detectable levels until at least 48 h p.i. Expression of IE l initiated at 2 h p.i. and increased in steady state levels up to very late times p.i. Densiometric analysis of the relative levels of IEO and I E l (Fig. 2B) shows that at early times prior to the initiation of replication (2 and 4 h p.i.) IEO is expressed at higher or equal levels than I E l . After the onset of replication, I E l becomes the dominant protein such that by 48 h p.i. IE l is 12 fold more abundant than IEO. These results clearly show that IEO is observed at all stages during the virus infection but the levels relative to IE l change dramatically. IEO activation of AcMNPV very late gene expression To examine the role of A c M N P V IEO in very late gene expression, a plasmid clone (pAc-IEO) was constructed expressing IEO under control of ieO promoter (Fig. 1C). Recent studies 186 Fig. 2 M 12 24 48 - IEO (72 kDa) - IE1 (67 kDa) Figure 2. Time course analysis of IEO and IE1 expression in AcMNPV infected Sf9 cells. (A) Western blot analysis of IEO and IE1. Sf9 cells were infected with A c M N P V using a MOI of 10 and samples were collected after 0, 2, 4, 8, 12, 24 and 48 h p.i. The positions of IEO and IE1 are shown on the right along with the molecular weight. (B) Relative levels of IEO and IE1. IEO and IE1 levels were measured by densitometry and compared at each time point (left Y -axis). The ratio of IE1/IE0 is shown by the line graph (right Y-axis). 187 performed in our laboratory demonstrated translation of ieO mRNA produces both IEO and IE l (Theilmann et al., 2001). Transfection of pAc-IEO also resulted in the translation of both IEO and IE l (Fig. ID). To enable the analysis of IEO in the absence of IEl an ieO plasmid pAc-IE0M _ > A was constructed with the internal start codon of IEl mutated into GCG. This prevented the translation of IE l from the ieO mRNA as demonstrated by western blots showing the expressed products from pAc-IEl , pAcIEO and pAc-IE0M^ A (Fig. ID). To determine i f IEO and IE0M _ > A are functional for the activation of very late gene expression transient very late gene expression assays were performed. pAc-IEl , pAc-IEO or pAc-±E0M _ > A were cotranfected with the very late gene reporter plasmid pVL941-CAT, cosmids 1,10, 59, and plasmids containing ie2,p35, and lef/ (Fig. 3A, columns 1, 3, and 5). These results showed that both IEO and IE0M^ A activated very late gene expression and higher levels were obtained than with I E l . These results are therefore the first to show that IEO in the absence of IE l expression is fully functional for supporting transient very late gene expression and should be considered a lef. We noted that replacing cosmid 58 with the individual plasmids for iel, ieO or ieOu^A, ie2, p35, and lef7 resulted in very late gene expression levels that were approximately 40-75% of the levels using four cosmids to supply all the lefs (Fig. 3A, lane 1,3,5). Therefore in order to improve very late gene expression levels for the analysis of IE l and IEO, cosmid58A was generated to replace the plasmid clones containing p35 and lep. Cosmid58A has a 14 Kb region (orfs 136-152) deleted from cosmid 58, including exonO, iel and ie2, however retainsp35 and lef7 (Fig. IB). Using cosmid58A, very late gene expression levels from the polyhedrin promoter showed over 200 % increase with I E l and approximately 50% increase with LEO and IE0M^ A (Fig. 3A, compare columns 1 and 2, 3 and 4, 5 and 6). In addition, western blot analysis 188 Fig. 3 B 400 350 300 250 - 200 h 150 100 10 M pAc-IF.I 1.0 M pAc-IEO 1 0 « pAc-IEO" - -Could 1.10,99 + + + + + + + + < 'innud M ("nsmid S E A - + - + - • - • -|>Ar-IE2 + + + 1 + + + + • -ptopU + pitkf7 + - + - + - + 1 1 3 4 5 6 7 ( 9 Norn, n*. control Control Control Po.. Control <-.»ikI 1.10,50 + + + + + + • + f ,»„!»! ?» + C O S M M 9 B A - + - + - + — + -H A C - I E S + + + + + + + + -pirp>5 * pwloCT + - + - + - + - -1 0 M pAc-IF.I 1.0 ug pAc-IF.0 1.0 HC pAc-IFO- * -«-IF,fl •-IE1 Figure 3. Analysis and optimization of A c M N P V IE1, IEO and I E 0 M _ * A activation of very late gene expression. (A) Activation of very late gene expression using pAc-IEl , pAc-IEO or pAc-IE0M^A. A l l transfections contained cosmids 1, 10, 59, pAc-IE2 and pVL941-CAT as the polyhedrin very late gene reporter plasmid. In addition, cells were cotransfected with either p35 and hp plasmids (piep35 and pielef?) or cosmid 5 8A. + or - indicates the presence or absence respectively of plasmids and/or cosmids. Positive control; Sf9 cells cotransfected with the four cosmids 1, 10, 58 and 59, expressing all lefs including iel and ieO. Error bars represent the standard error. (B) Western blot analysis of expressed IE1, IEO and IE0 M ^ A proteins in the very late gene expression assays. 189 indicates that using cosmid58A also increase the expression levels of LEI, IEO and IE0 M _ > A (Fig. 3B). Due to the higher level of very late gene activation all subsequent assays were performed using cosmid 5 8A. The increased very late gene activation with cosmid58A may be due to more precise regulation of lefs from cosmids as compared to plasmid clones. Alternatively, there maybe one or more ORFs located on cosmid 58A that could potentially be stimulatory for very late gene expression. ORFs in addition top35 and lep on cosmid 58A include orfs 117-119, orf 121, orf 122, orf 124, orf 132,pk-2, chinitase, v-cath, gp64,p24, gpl6,pp34, alk-exo and 94k (Fig. IB). Orf 121 has been shown to stimulate expression from the iel promoter (Gong et al., 1998) which would be in agreement with our western blot results (Fig. 3B). However, L i et al. (1999) suggested that this orf was not involved in transient late gene expression. Additional experiments will be necessary to determine why higher levels of very late gene expression are obtained with cosmid 5 8A. Activation of very late gene expression with increasing levels of IE1, IEO and IE0 M _ > A As shown in Fig. 2 the relative IEO and LEI expression levels change during the course of A c M N P V infection. It was unknown if the varying levels of IEO and LEI expression would have differential affects on very late gene activation. We therefore investigated the role of varying IEO and IE1 levels on the activation of very late gene expression cotransfecting increasing amounts of pAc-IEl , pAc-IEO or pAc-LE0M^ A (0.1, 0.5, 1.0, 2.0 3.0 and 4.0 ug) with 0.2 ug cosmid 1, 10, 58A, 59, 0.1 ug pAc-IE2 and 1.0 ug pVL941-CAT (Fig. 4). Protein samples were analyzed initially by western blot to confirm the increasing levels of IEO and LEI for each of the treatments (Fig. 4A). For all samples, increasing amounts of pAc-IEl , pAc-IEO or pAc-IE0 M _ > A 190 Figure 4. Analysis of the effect of increasing levels of AcMNPV IEl, IEO and IE0 M _ > A on transient very late gene expression. (A) Western blot analysis of I E l , IEO or IE0M^ A transient very late gene expression assays with increasing levels of pAc-IE 1, pAc-IEO and pAcIE0M~> A (0.1, 0.5, 1.0, 2.0, 3.0 and 4.0 jug). (B) Transient very late gene expression levels with increasing levels of pAc-EEl, pAc-IEO or pAc-IE0 M ^ A (0.1, 0.5, 1.0, 2.0, 3.0 and 4.0 \ig (A)). The negative control (-) does not contain pAc-IEl , pAc-IEO or pAc-IE0M _ > A . (C) Comparison of transient very late gene expression relative to I E l , IEO and ±E0M _ > A expression levels. The levels of I E l , IEO and IE0M~*A were determined by densitometric analysis and normalized relative to IE l levels in cells transfected with 1.0 u.g pAc-IEl (X-axis). (D) Western blot comparison of IEO and I E l expression levels in transient very late gene expression assays when pAc-IEl (1.0 u,g), and pAc-IE0M _ > A(1.0 p.g) were transfected individually or together. Protein samples were dephosphorylated prior to blotting. 191 Fig. 4 250-E U m4 loo-H 0.0 0.5 1.(1 1.5 2.0 2.5 Relative protein density 3.0 MtM-k 1.0 fig pAc- IE l — + - + I . O m p A c - l E O 1 " * _ — + 192 plasmid D N A resulted in increased IE l or IEO levels. Polyhedrin-CAT activation by IEl showed maximum levels with 0.5 u.g of plasmid and started to decline in activation levels with 3 (j.g of plasmid (Fig. 4B). Expression from pAc-IEO resulted in a significant increase in very late gene expression levels up to 1.0 u,g of plasmid D N A followed by a steep decline in expression as the levels of IEO increased. Similarly, IE0M~>A obtained maximum very late gene activation levels with 1.0 u.g of plasmid D N A , however activation levels were lower than using IEO or I E l . The results of this transient assay show that i f levels of I E l and IEO reach too high a level very late gene expression will decrease. To compare the relative abilities of IEO and IE l to activate very late genes the expression levels were analyzed by western blot, densitometrically measured and normalized relative to 1.0 p.g of IE l (Fig. 4C). Analysis of the relative activities of the proteins expressed from each plasmid construct reveals different activation profiles for both IEO and IE l (Fig. 4C). The results show that IE0M^ A is most active at very low protein levels and very late expression levels decline rapidly at relatively small increases in protein levels. IE l only reaches maximum activation at levels that are inhibitory to r£0 M ^ A . In addition, after maximum expression is achieved, relatively large increases in protein levels results in only minimal decline in expression. Activation by IEO, which produces both IEO and I E l , appears to be an average between the two proteins. Therefore IEO appears to a stronger activator at low concentrations, whereas DEI requires higher levels for maximum expression and is more active over a wider range of densities. Co-transfections were performed to determine i f co-expressing IEO and IE l in transient very late gene expression assays affects the expression levels of both proteins (Fig. 4D). IEO and IE l levels were compared in Sf9 cells co-transfected with all lef factors and pAc- IE0M~*A, or pAc-193 I E l , or both pAc-IE 1 and pAc- IE0 M ^ \ The western blots indicated that both IEO and IE l levels increased upon co-transfection showing that they did not inhibit each other's expression. Interaction between IEl and IEO and its effect on very late gene expression IEO and IE l are co-expressed throughout the baculovirus infection cycle with relative ratios of the two proteins changing significantly (Fig. 2). To determine the effects of varying ratios of IE l and IEO on very late gene expression we performed two sets of cotransfections changing the relative levels of IE l and D30M"*A. The very late gene assays were performed using 0.05 ug and 0.5 ug of pAc-IEl cotransfected with 0.0, 0.05, 0.1, 0.5, 1.0, 2.0, 3.0 or 4.0 ug of p A c - I E O ^ . Very late gene expression was measured for each combination and compared to cotransfections containing only pAc-IE 1 or pAc-IE0M _ > A . In the assay using 0.05 ug of pAc-IEl , two cotransfections with the two lowest amounts of IE0M _ > A (0.05 and 0.1 ug) resulted in synergistic activation of very late gene expression (Fig. 5 A). The remaining combined transfections reduced expression relative to pAc-IE0 M _ > A alone, indicating that even at low levels of I E l , co-expression can be antagonistic to IEO activation of very late gene expression. Cotransfections of 0.5 ug pAc-IEl (Fig. 5B) with all the varying levels of pAc-IE0M~*A resulted in a decrease in very late gene expression relative to pAc-IE 1 alone. Interestingly, the lowest concentrations of pAc-IE0M^ A (0.05 and 0.1 ug) caused minimal activation of very late gene expression. However, when cotransfected with 0.5 ug of pAc-IEl , a 40% reduction in expression is observed relative to pAc-IE 1 alone. These results indicate that, at nearly all combinations of iel and ieOM~^ there is a mutually antagonistic affect on the activation of very late gene expression. 194 Fig. 5 A B Figure 5. Analysis of the effect of co-expression of IE1 and I E 0 M _ > A o n the activation of very late gene expression. Sf9 cells cotransfected with (A) 0.05 ug or (B) 0.5 jig of pAc-IEl and 0.05, 0.1, 0.5, 1.0, 2.0, 3.0 or 4.0 ug pAc-IE0 M _ > \ Each cotransfection was compared to very late expression activated by either pAc-IEl or pAc-IE0M^A alone. A l l transfections contained remaining eighteen lef genes provided by cosmids 1, 10, 58A and 59 and the plasmid pAc-IE2. The reporter plasmid used was the very late polyhedrin promoter reporter plasmid pVL941-CAT. 195 Discussion IEO is the only baculovirus protein that is known to arise from a spliced mRNA and differs from the transcription factor IE l by the addition of 54 amino acids to the IE l N-terminus (Chisholm and Henner, 1988). To date the role of IEO in baculovirus infections has not been completely defined. Previous studies have however shown that IEO has different properties which includes its ability to transactivate early genes and also to alter host range (Kovacs et al., 1991b; Lu et al., 2003; Theilmann et al., 2001). In the present study we have investigated the role of IEO and its interactions with IEl in the activation of baculovirus very late gene expression. Western blot analysis of IEl and IEO in A c M N P V infected cells revealed that the relative levels of the two proteins change dramatically throughout infection (Fig. 2). IEO is expressed at a higher level than IE l during very early times p.i., but showed lower levels than I E l from the onset of D N A replication to very late times p.i. Using transient very late gene assays with a polyhedrin promoter reporter plasmid, we have demonstrated that ieO is able to replace iel, which is essential for the activation of late gene expression. Nineteen A c M N P V genes have been previously identified as lefs (Li et al., 1999; Rapp et al., 1998; Todd et al., 1995) and based on the results of this study IEO should be considered the twentieth A c M N P V lef gene. Maximum activation of very late gene expression occurred at significantly lower levels of IEO in comparison to I E l . I E l was able to activate very late gene expression to a greater maximum than IEO but higher levels of expression were required. For both proteins i f the levels became too high, very late gene expression decreased but the decline in activation by IE l occurred over a much wider range of protein levels than observed with IEO (Fig. 4B and 4C). In contrast to results by Kovacs et al. (1991b), co-expression of both IEO and IE l resulted in up regulation of 196 both proteins (Fig. 4D). Polyhedrin is a late promoter that becomes hyper-expressed at very late times p.i. It is possible that other promoters which are not hyper-expressed, such as vp39 or p6.9, may be activated differently by IEO and IEL As described above, the ratio of IEO to IE1 varies throughout A c M N P V infection. The effect of these ratios on the activation of very late gene expression was studied using a transient assay by expressing different levels of IE1 or IE0M^A. In the majority of assays co-expression of IE1 and LE0 M _ > A resulted in lower levels of very late gene activation than was obtained using either LEI or IEO alone (Fig. 5A and 5B). These results suggest that IEO and LEI are non-cooperative or antagonistic for the activation of very late gene expression. Possibly, very high IEO and IE1 levels may saturate specific promoters and cause down regulation, which result in an antagonistic effect similar to the squelching effect described by Ptashne and Gann (1990). Our transactivation analysis however, would predict that maximal very late gene expression in infected cells would be achieved by having low IEO levels and high levels of LEI. This is in agreement with the conditions that exist in infected cells at very late times p.i. when there are peak levels of very late gene expression (Blissard and Rohrmann, 1990). Our western blot analysis showed that at 48 h p.i. LEI levels are 12 times higher than IEO, which is at its minimum level (Fig. 2). However in infected cells, as opposed to transient assays, the relative effects of IEO and IE1 may be different to what we have observed. A study by Kremer and Knebel-Morsdorf (1998) reported that IE1 and IEO formed homodimers and heterodimers when bound to hr elements. Thus, the variable levels suggest that the relative proportion of LEO and IE1 homodimers and LEO-IE 1 heterodimers would change during the course of infection. The changing ratios could be a potential global regulatory mechanism that affects the patterns of viral gene expression and viral D N A replication during 197 infection. The formation of heterodimers between related transcription factors that result in complexes with modified regulatory functions has been observed in a number of other eukaryotic systems. These include the interferon regulatory factor and Nuclear Factor-kappa B families (Barnes et al., 2003; Verma et al., 1995). The inhibitory effect of IEO on IE l during very late gene expression and the higher levels of IEO relative to IEl prior to viral D N A replication would suggest that the primary role of IEO is during early times p.i. In support of this data is the analysis of OpMNPV IEO, which was shown to preferentially activate two early promoters (gp64 and iel) (Theilmann et al., 2001). Recent results by Lu et al. (2003) showed that a reduction in IEO levels permitted A c M N P V replication in SL2 cells. We could speculate that the decline of IEO in SL2 cells reduces the antagonistic effects on I E l gene function permitting increased very late gene expression. IEO therefore appears to be playing a pivotal role in the baculovirus infection cycle and further studies will be necessary to define the additional roles of this protein and the function of the 54 additional N -terminal amino acids in IEO relative to I E l . 198 Materials and Methods Cells and Virus A c M N P V - W T virus (strain E2) was propagated in Spodoptera frugiperda (Sf9) cells and maintained in TCI00 medium (Invitrogen) as previously described (Summers and Smith, 1978). For the time course analysis, Sf9 cells were infected with the AcMNPV-E2 strain using a multiplicity of infection (MOI) of 10. Viral titres were determined by end point dilution (Summers and Smith, 1978). Cosmid and Plasmid constructs The nineteen A c M N P V lef genes required for transient late gene expression in Sf9 cells are comprised by cosmids 1, 10, 58 and 59 (Fig. 1A and IB) (Li et al., 1999). Cosmid 58A was constructed by deleting a 14 Kb Agel fragment from cosmid 58, which removed exonO, iel and ie2, but retainedp35 and lef7 (Fig. IB). Plasmid clones containing ie2 (pAc-IE2), iel (pAc-IEl), lef7 (pielef7) andp35 (piep35) have been previously described (Li et al., 1999; Theilmann and Stewart, 1991). The plasmid pAc-IEO (Fig. 1C) expressing A c M N P V ieO under the control of the ieO promoter was constructed by PCR amplification of the ieO ORF and OpMNPV ie2 polyadenylation signal from p2Zop2EIE0/IEl using the primers p288 (5'-C T C A T G C G C G T G A C C G G A C A C - 3 ' ) and p338 (3'-T C A C T C A A A G G C G G T A A T A C G G A G C T C C A - 5 ' ) . This insert was cloned as a Hindlll - Sstl fragment into pBS+, generating the plasmid pBS-IEO. The ieO promoter (365 bp) was PCR amplified using A c M N P V genomic D N A as template with the primers p335 (5'-199 C G A A A G C T T G G C A G G C G C T G G C A A A G A T T -3') and p336 (3'-G T A A T T G A T A A C G G G C A A C G A A G C T T G A T - 5 ' ) . The ieO promoter was inserted as a Hindlll fragment into pBS-LEO resulting in pAc-IEO. pAc-IEO transcribes ieO mRNA which is translated as both IEO and LEI (Fig. 1C and ID). A second ieO plasmid, pAc-IE0M _ > A , was constructed, which expresses ieO, but only translates IEO due to the mutation of the second A T G codon to G C G (Fig. 1C and ID). The ieOM^A ORF containing the OpMNPV ie2 polyadenylation signal was synthesized by PCR using the primers p288 and p338 and template p2Zop2EIE0/IEl(2°ATG-), which has the 2 n d A T G of the ieO ORF substituted for G C G by site-directed mutagenesis. The amplified product was cloned as a Hindlll - Sstl fragment into pBS+, generating pBS-LE0 M ^\ The ieO promoter was inserted as a Hindlll fragment into pBS-LE0M _ > A, resulting in pAc-IE0M^ A . The reporter plasmid, pVL941-CAT (Luckow and Summers, 1989) contains the chloramphenicol acetyltransferase (CAT) ORF under control of the polyhedrin promoter. Transfections The very late gene expression assay used in this study has been previously described by L i et al. (1999). Sf9 cells (106/well) were seeded onto six-wells culture plates and incubated at 27 °C 16-24 hours prior to transfection. The liposomes, required for transfection of plasmid and cosmid D N A into cells, were prepared as previously described (Campbell, 1995). Transfection efficiency was optimized using plasmid D N A , constitutively expressing the green fluorescent protein (GFP). Two transient very late gene expression assays were used in this study; the first assay consisted of 0.2 u,g of cosmid 1, 10, and 59, 1.0 ug pVL941-CAT, 0.1 ug of the plasmid clones 200 pAc-IE2, pielefT, piep35 and 1.0 ug pAc-IEl , pAc-IEO or pAc-IE0M^ A cotransfected into Sf9 cells (106/well) (Fig. 1A). This very late gene expression assay was modified by substituting cosmid 58A for pielef7 and piep35, resulting in the basic set of eighteen lef genes consisting of 0.2 ug of cosmid 1, 10, 58A, 59, 0.1 ugpAc-IE2, and 1.0 ug pVL941-CAT cotransfected with 1.0 ug pAc-IEl , pAc-IEO or pAc-IE0M _ > A . To determine the effects of increasing amounts of I E l , IEO or IE0M~*A on very late gene expression, the basic set of eighteen lef genes were cotransfected with various amounts (0.1 ug, 0.5 ug, 1.0 ug, 2.0 ug, 3.0 ug or 4.0 ug) of pAc-IEl , pAc-IEO or pAc-IE0M^ A (Fig. 1C). To study the effects of co-expression of IEl and IE0 M _ > A in very late gene expression, 0.05 ug or 0.5 ug of pAc-IEl were cotransfected with 0.1 ug, 0.5 ug, 1.0 ug, 2.0 ug, 3.0 ug or 4.0 ug of pAc-IE0 M _ > A and the basic set of cosmids and plasmids. In all experiments the total amount of D N A per transfection was equalized with pBS+. The positive control for all experiments consisted of 0.2 ug cosmid 1,10, 58, 59 and 1.0 u.g pVL941-CAT, the negative control was comprised of the basic set of cosmids and plasmids, containing the eighteen lef genes, excluding the plasmid clones pAc-IEl , pAc-IEO, or pAc-iE0 M _ > A . C A T assay In transient very late gene expression experiments, all C A T samples were analyzed in duplicate. The C A T assay is based on the method described by Neumann et al. (1987). Briefly, the transfected cells were harvested after 48 h post-transfection, transferred into an eppendorf tube and pelleted for 2 minutes at 10,600 g. The supernatant was discarded and the pellet was resuspended in 100 ul 0.25 m M Tris-HCl (pH 7.8). To lyse the cells, each sample was freeze-thawed (respectively at -80°C and 37°C) three times for 5 minutes, and transferred to a 65°C 201 water bath for 15 minutes to inactivate cellular deacetylases. Cell debris were pelleted and the supernatant was used for diffusion CAT-assay with 3H-Acetyl-CoA. The cell extract was added to a cocktail containing 9 m M chloramphenicol, 60mM Tris-HCl (pH 7.8), 188 uM acetyl-coenzyme A (Sigma), 0.025 uCi [3H] acetyl-coenzyme A (New England Nuclear, C A T Assay Grade) and dEbO up to a total volume of 85 ul. Each sample was overlaid with 3 ml of toluene-based scintillilation fluor (Econofluor-2; Packard Bioscience Co.) and the enzymatic reaction was measured by the scintillation counter (Beckman; LS 6500). C A T expression was titered to determine the appropriate quantity of cell extract for a linear response in the assay. Western blots For western blot analysis, transfected cells were harvested at 48 h post-transfection, collected in an eppendorf tube and pelleted for 5 minutes at 1300 g. The supernatant was removed and the cells were washed gently with 1 ml of I X Tris-buffered saline (TBS) and centrifuged for 5 min at 1300 g. This procedure was repeated twice and the cell pellet was resuspended in 100 ul phosphatase inhibitor buffer (Slack and Blissard, 1997). Cells of each sample were freeze-thawed (respectively at -80°C and 37°C) three times, followed by shearing of cellular D N A with a 10cc syringe. In some cotransfection experiments protein samples were dephosphorylated prior to western blot analysis. Cells were resuspended in 0.1% SDS-2.5 m M dithiothreitol to an appropriate volume of protein sample and overlayed with mineral oil. The samples were heated to 95°C for 5 minutes, cooled to 4°C followed by addition of 20 units calf intestinal phosphatase (CLP; Biolabs, New England) and incubated overnight at 37°C. Protein samples were separated by denaturing SDS polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, 1970) and transferred onto Immobilon-P polyvinylidene difluoride membranes (Millipore) using a mini-202 blotter (Biorad) according to the manufacturers recommended protocol. The blots were initially incubated for 1 hour with 5% blotto-0.02% tween followed by an incubation with 5% blotto-0.02% tween and a 1:4,000 - 10,000 dilution of primary antibody, mouse monoclonal IE l 4B7 (Ross and Guarino, 1997). Bound IE l antibody was detected by a 1:10,000 - 20,000 dilution of the secondary antibody, goat anti-mouse horseradish peroxidase (Jackson Laboratories). Proteins were detected using the E C L substrate chemiluminescence (Amersham Biosciences) and transferred on high performance chemiluminescence film (Amersham Biosciences). Protein densities were scanned and quantified using ImageQuant software (Molecular Dynamics Inc.). The relative protein density measurements were normalized to levels of IE l expressed in Sf9 cells transfected with 1 ug pAc-IEl , which was included on each western blot. 203 Acknowledgements We thank Xiaojiang Dai, Taryn Stewart, Kevin Wanner, and Les Willis for critical reading and helpful comments on the manuscript. Cosmid 1, 10, 58 and 59, plasmid clones pielef7 and piep35 were kindly provided by George Rohrmann. This study was supported in part by a Discovery grant to D. A. T. from the Natural Sciences and Engineering Research Council of Canada. 204 References Ayres, M . D., Howard, S. C , Kuzio, J., Lopez-Ferber, M . , and Possee, R. D., 1994. 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