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Analysis of the orgyia pseudotsugata multiple nucleopolyhedrovirus IE1 acidic activation domain role… Pathakamuri, Joseph Aja 2004

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A N A L Y S I S OF T H E O R G Y I A P S E U D O T S U G A T A M U L T I P L E N U C L E O P O L Y H E D R O V I R U S IE1 A C I D I C A C T I V A T I O N D O M A I N R O L E I N V I R A L D N A R E P L I C A T I O N by JOSEPH AJAY PATHAKAMURI B.Sc, Sri Venkateswara University, 1992; M.Sc, Sri Venkateswara University, 1994; M.tech., Jawaharlal Nehru Technological University, 1998. A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Dept. of Plant Science) We accept this thesis as conforming to the required standard IUBCI i T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A April 2004 © Joseph Ajay Pathakamuri, 2004 ABSTRACT Baculovirus Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV) transient replication assays have previously identified six genes as essential iel, lef-1, lef-2, lef-3, DNA pol, helicase; three genes as stimulatory ie2, p34, iap-1; and two types of replication origins hrs and non-hrs. One of these essential proteins IE1, has an acidic activation domain (AAD) at the N-terminus that is required for transcriptional transactivation but its role in viral DNA replication was unknown. In this study we have determined that the AAD is also essential for DNA replication. Unlike transcriptional activation, the AAD cannot be replaced with the AcMNPV IE1 and herpesvirus VP 16 AADs for replication. Deletion analysis of the OpMNPV IE 1-AAD revealed the presence of separate sub-domains for replication and transcriptional activation. By substituting OpMNPV lef-1, -2, -3 and helicase proteins (part of the putative replisome) with corresponding AcMNPV factors, the inactive OpMNPV IE1 chimeric protein IE 1-Ac AD, which contains the AcMNPV IE1 AAD, was functional for replication activation. This suggests that AADs interact with viral replication factors in order to maintain viral specificity in replication. The IE 1-Ac AD specificity for the AcMNPV replisome was found to be similar to the native AcMNPV IE1, thus demonstrating that by changing the AAD the specificity of the protein changes. Further studies showed that the role of stimulatory factor OpMNPV IE2 in replication was to maintain the specificity between AAD and the replisome. Absence of IE2 allowed replication from non-specific AAD and replisome but presence of IE2 active replication was observed only between specific AAD and replisome. AcMNPV non-hr origin analysis indicated that it was unable to allow for replication with OpMNPV IE1 and OpMNPV replication proteins; however substituting with AcMNPV LEF-3, POL and HELICASE, resulted in replication. This result reveals the presence of specific interactions between origins and replication factors. ii Our studies on OpMNPV IEO, the only spliced gene of IE1, showed that IEO is functionally active for replication and can replace IE1. We also performed initial functional analysis of OpMNPV EXONO. To date there is no information on OpMNPV EXONO function other than the fact that it contributes 23aa to the N-terminus of IEO. Our study shows EXONO has a conserved novel ring finger and is expressed as a late gene. However, our preliminary research could not identify any function attributable to EXONO. iii Table of Contents ( ABSTRACT ii Table of Contents iv List of Tables viii List of Figures ix List of Symbols and Abbreviations xii Baculovirus abbreviations xiv Dedication xv Acknowledgements xvi CHAPTER 1. General introduction. 1 B A C U L O V I R U S 1 B A C U L O V I R U S INFECTION C Y C L E 2 G E N E R E G U L A T I O N 3 Early gene expression 4 Late gene expression 6 HOST R A N G E 7 B A C U L O V I R U S REPLICATION 9 Homologous regions ; 10 Non-/ir origins of replication 11 REPLICATION GENES 13 Genes essential for viral DNA replication... 14 I.ef-1 and Lef-2 14 Lef-3 15 DNA pol 15 Helicase 16 iel 17 Genes stimulatory for viral DNA replication 18 ie2 18 OpMNPV p34 (AcMNPV pe38) 19 lap and p35 20 Lef-7 20 llcf-1 20 iv Role of IEO and EXONO in replication 21 ieO 21 ExonO 22 STUDY OBJECTIVES 27 CHAPTER 2. The acidic activation domain of the baculovirus transactivator IE1 contains a virus specific domain essential for viral DNA replication 29 INTRODUCTION 29 M A T E R I A L S A N D M E T H O D S 32 Cell Culture 32 Plasmid and Cosmid constructs 32 Replication assay 32 CAT assay 33 RESULTS 35 DISCUSSION 38 CHAPTER 3. The OpMNPV IE1 acidic activation domain determines the specificity of the viral replisome 54 INTRODUCTION 54 M A T E R I A L S A N D M E T H O D S 56 Cell Culture 56 Plasmid and Cosmid constructs 56 Replication assay 57 RESULTS 58 Replication assay of reporter pHdNA 58 Single substitutions of AcMNPV replication factors 58 Dual combinations of substitutions of AcMNPV replication factors 59 Multiple combinations of substitutions of AcMNPV replication factors 59 Specificity of AcMNPV IE1 AAD 60 Comparison of OpMNPV IE1, AcMNPV IE1 or IEl-AcAD ability to activate replication 60 DISCUSSION 62 CHAPTER 4. Specificity of AcMNPV non-hr origin in transient replication assays 84 INTRODUCTION 84 M A T E R I A L S A N D M E T H O D S 86 Cell Culture 86 Plasmid and Cosmid constructs 86 Replication assay 86 RESULTS 87 AcMNPV non-hr does not replicate in Ld652Y cells with OpMNPV replication factors 87 V AcMNPV non-hr origin replicates in Ld652Y cells with AcMNPV lef-3, helicase, and DNA pol substitution 88 DISCUSSION 90 CHAPTER 5. The role of stimulatory factor OpMNPV IE2 in replication 105 INTRODUCTION 105 MATERIALS AND METHODS 107 Cell Culture 107 Plasmid and Cosmid constructs 107 Replication assay 107 RESULTS 108 Interaction of OpMNPV IE2 with heterologous AADs and replisomes 108 OpMNPV IE2 demonstrates cell specificity 109 DISCUSSION : HO CONCLUSIONS 122 REFERENCES .' 125 APPENDIX A. Functional analysis of IEO for viral DNA replication 147 INTRODUCTION 147 MATERIALS AND METHODS 148 Cell Culture .' 148 Plasmid and Cosmid constructs 148 Replication assay 148 Western blots 148 RESULTS 149 DISCUSSION 150 APPENDIX B. Partial characterization of exonO (orf 138) 160 INTRODUCTION 160 MATERIALS AND METHODS 161 Cell Culture 161 OpMNPV ExonO constructs 161 OpMNPV late gene reporter constructs 161 RNA isolation and Northern blots 162 Sequence analysis of the OpMNPV EXONO 162 OpMNPV replication assays 163 Western blots 163 AcMNPV late gene assay 163 OpMNPV late gene assay 164 RESULTS 165 vi OpMNPV EXONO has a novel ring finger 165 ExonO is a late gene 165 EXONO appears to augment IEO's replication function 166 EXONO might augments IEO's late gene activation 166 DISCUSSION 168 vii List of Tables Table 1. Summary of the factors in the OpMNPV replisome that need to be substituted with A c M N P V homologs to permit A c M N P V non-hr origin (pHdK) replication 102 viii List of Figures Fig. 1.1. Schematic of the IE1 protein structure and a model explaning IE Is function 23 Fig. 1.2. Schematic of the exonO and iel region showing the splicing pattern of iel and ieO, showing the contribution of exonO for the ieO formation 25 Fig. 2.1. Schematic of the O p M N P V IE1 and the chimeric constructs used for characterizing the role of A A D s in viral D N A replication 42 Fig. 2.2. Transient Dpn\ replication assays for the analysis of the IE1 chimeras ability to activate replication 44 Fig. 2.3. Schematic diagrams of deletion mutants used to map the regions of the O p M N P V IE1 A A D required for D N A replication 46 Fig. 2.4. Transient Dpn\ replication assays to map specific regions of O p M N P V IE1 A A D required for replication : 48 Fig. 2.5. Schematic diagram showing the summary of domains identified in the O p M N P V IE1 A A D 50 Fig. 2.6. Alignment of the acidic activation domains 52 Fig. 3.1. Schematic of O p M N P V IE 1, the chimeric IE1 proteins, and the replication genes of OpMNPV and A c M N P V used to study IE1 acidic activation domain interactions with viral replication factors 66 Fig. 3.2. Construction of plasmid pHdNA and replication assay to test its ability to act as a replication reporter in comparison to pHdN 68 Fig. 3.3. Ability of I E l - A c A D to support non-/ir origin replication by substituting O p M N P V essential replication proteins with single homologous A c M N P V genes 70 Fig. 3.4. Ability of I E l - A c A D to support non-/w origin replication by substituting O p M N P V core replication proteins with pairs of homologous A c M N P V genes 72 Fig. 3.5. Ability of I E l - A c A D to support won-hr origin replication by substituting O p M N P V core replication proteins with multiple homologous A c M N P V genes 74 Fig. 3.6. Replication assay to test whether A c M N P V IE1 A A D is essential for I E l - A c A D interaction with the A c M N P V replisome 76 Fig. 3.7. Comparison of the ability of OpMNPV IE1, I E l - A c A D or A c M N P V IE1 to support replication with OpMNPV and A c M N P V replication factors in Ld652Y cells 78 Fig. 3.8. Alignment of the acidic activation domains of OpMNPV IE1 and A c M N P V IE1 80 ix Fig. 3.9. Schematic diagram showing the requirement for OpMNPV and A c M N P V replication factors for both OpMNPV and A c M N P V for OpMNPV IE1, I E l - A c A D or A c M N P V IE1 to replicate the OpMNPV viral non-hr origin 82 Fig. 4.1. Schematic of O p M N P V IE1, I E l - A c A D , A c M N P V 1E1 and the replication genes of OpMNPV and A c M N P V used to study A c M N P V non-hr origin replication in heterologous system 92 Fig. 4.2. Ability of OpMNPV and A c M N P V non-hrs to replicate in Ld652Y cells in the presence of all OpMNPV factors 94 Fig. 4.3. Ability of O p M N P V and A c M N P V non-hrs to replicate by substituting O p M N P V replication factors with A c M N P V lef-1, -2 factors 96 Fig. 4.4. Ability of O p M N P V and A c M N P V non-hrs to replicate by substituting O p M N P V replication factors with A c M N P V lef-3, helicase, D N A pol 98 Fig. 4.5. Ability of O p M N P V and A c M N P V non-hr to replicate by substituting O p M N P V replication factors with A c M N P V lef-1, -2, -3,helicase,DNA pol 100 Fig. 5.1. Schematic of the IE Is and of the replication genes of OpMNPV and A c M N P V used in transient viral D N A replication assays to study the role of OpMNPV IE2 112 Fig. 5.2. Transient replication assays in Ld652Y cells 114 Fig. 5.3. Transient replication assays in Sf9 cells 116 Fig. 5.4. Flow chart summarizing the OpMNPV IE2 effect on transient replication in both Ld652Y and Sf9 cells 118 Fig. 5.5. Schematic diagram showing that OpMNPV IE2 effect on specificity in replication 120 Fig. A . l . Schematic of the exonO and iel region showing the transcription and splicing pattern of iel and ieO, and //z'wdlll restriction map of the OpMNPV genome 152 Fig. A.2. Schematic diagram of the IEO and IE1 expression constructs and Southern blot of the transient replication assays in Ld652Y to study the role of IEO in replication 154 Fig. A.3. Western blot analysis of IEO and IE1 expressed from pie 1 -IE 1, pieO-YEO, and p/e(7-IE0 (2XATG-). . . 156 Fig. A.4 Specific activity of p ie l - IE l ; p/W-IEO; p/e0-IEO(2XATG-) in replication 158 Fig. B . l . Schematic map of the OpMNPV genome, from the 5' end of me53 to the 3' end of odv-e56, showing the contribution of exonO for the ieO formation 169 Fig. B.2. Alignment of ExonO proteins from OpMNPV, CfMNPV, EppoNPV, A c M N P V , BmNPV, RoNPV, L d M N P V , SeMNPV, MacoNPV-A, MacoNPV-B, HearNPV, HzSNPV, AdhoNPV and SpltNPV.... 171 Fig. B.3. Northern blot analysis of OpMNPV exonO in Ld652Y cells from 0 to 120 hours p.i 173 x Fig. B.4. Analysis of O p M N P V EXONO role in replication and its effect on the protein expression of IEO and IE1 175 Fig. B.5. Analysis of A c M N P V EXONO function in transient late gene expression assays 177 Fig. B.6. OpMNPV transient late gene assay 179 xi List of Symbols and Abbreviations AAD acidic activation domain BV budded virus BDI&II basic domain I & II A-gal /?-galactosidase bp base pair BSA bovine serum albumin CAT chloramphenicol acetyl transferase CIP calf intestinal phosphatase DE delayed early DBD DNA binding domain dNTP deoxynucleotide triphosphate DTT dithiothreitol EMSA electrophoretic mobility shift assay EDTA ethylene-diamino-tetra-acetate GV Granulovirus HSV herpes simplex virus hrs homologous regions Hcf host cell factor Hrf host range factor hrp.i hours post infection IAP inhibitor of Apoptosis IE immediate early Kb kilobase kDa kilodalton LEF late expression factor L D 5 0 lethal dose to kill 50% MOPS morpholino propanesulfonic acid M.O.I. multiplicity of infection microgram m.u. map units nmol nanomole NPV Nucleopolyhedrovirus OBs occlusion bodies ODV occlusion derived virus OBP origin binding protein ORF open reading frame OD oligomerization domain PAGE polyacrylamide gel electrophoresis PBS phosphate-buffered saline PCR polymerase chain reaction xii pmol picomole RP-A replication protein A SV40 simian virus 40 SSB single stranded binding protein SSC Saline-Sodium Citrate SDS sodium dodecylsulfate TCID50 tissue culture infectious dose to kill 50% TE Tris (hydroxymethylaminoethane)-EDTA; TEMED tetramethylethylenediamine TBS Tris (hydroxymethylaminoethane) buffered saline Vlf very late factor xiii Baculovirus abbreviations. AcMNPV Autographa californica multiple nucleopolyhedrovirus AdhoNPV Adoxyphyes honmai nucleopolyhedrovirus AgMNPV Anticarsia gemmatalis multiple nucleopolyhedrovirus BuzuNPV Buzura suppressaria nucleopolyhedrovirus BmNPV Bombyx mori multiple nucleopolyhedrovirus CfMNPV Choristoneura fumiferana multiple nucleopolyhedrovirus HycuNPV Hyphantria cunea nucleopolyhedrovirus HzSNPV Helicoverpa zea single nucleopolyhedrovirus HearNPV Helicoverpa armigera nucleopolyhedrovirus LdMNPV Lymantria dispar multiple nucleopolyhedrovirus, MacoNPV Mamestra configurata nucleopolyhedrovirus OpMNPV Orgyia pseudotsugata multiple nucleopolyhedrovirus SeMNPV Spodoptera exigua multiple nucleopolyhedrovirus SpltNPV Spodoptera litura multiple nucleopolyhedrovirus SpliNPV Spodoptera littoralis nucleopolyhedrovirus RoMNPV Rachiplusia ou multiple nucleopolyhedrovirus CrleGV Cryptophlebia leucotreta granulovirus PlxyGV Plutella xylostella granulovirus XecnGV Xestia c-nigrum granulovirus xiv Dedication I dedicate this thesis to my family, teachers and friends. xv Acknowledgements A number of people directly or indirectly have contributed to this thesis. It is my deepest pleasure to offer my sincere thanks to all these people. Dr. David Theilmann, my doctoral research supervisor, for allowing me to pursue my doctoral studies in his lab, and for his excellent guidance and teachings. I also extend my thanks to his family for their moral support and helping me cope with the tough times in my research tenure. Dr. James Kronstad, my co-supervisor for agreeing to be my academic supervisor and for his valuable time and suggestions. Dr. Janet Chantler and Dr. Tom Grigliatti, members of my committee, for their valuable time and input into the thesis. Less Willis and my other lab mates, for their cooperation and bearing with me through my mistakes. Less, I appreciate and acknowledge your help. Your excellent organization of the lab and technical assistance contributed a lot to the progress of my research. Dr. Steve Vincent, for his kindness and helping me with funding to carry my research interests and finishing this thesis. I also extend my thanks to the staff of Dr. Vincent lab for their support. Dr. Geroge Rohrmann, for kindly providing us the clones needed in my work. PARC staff, who provided a very good atmosphere to carry out my research work. Their moral support helped me to keep up my spirits. I also want to give a special note of thanks to the security and janitorial staff who cheered us during the late night works. Plant Science staff, for their academic support. Penticton and Summerland community, for welcoming me into their lives and homes and providing the comfort of a family. My family and relatives, who even though they are miles away helped me and supported me through their thoughts and prayers and helped me at every step on my journey, I am very grateful to you mom and dad. My friends, all over the world who have sustained me through days of difficulties and who stood by me and helped me to carry out my research. Your friendship and love are priceless. xvi To the priests, sisters and church, who enriched the spiritual side of me by their constant spiritual guidance and prayers, for awakening in me the Love for God and desire to seek the truth in all directions. To the staff of Doctoral Examinations section, Grad studies for their cooperation and help. Finally for all the people whom I owe my gratitude and whose names have not been mentioned here. xvii CHAPTER 1. General introduction BACULOVIRUS Baculoviruses are large double stranded DNA viruses that are pathogenic for arthropods. Early in the 19th century baculovirus polyhedral crystals were first observed while studying wilting disease in the silk worm and in 1947 the characteristic rod-shaped virions present in the polyhedral crystals were identified. Since then Baculovirus infections have been reported in over 600 host species primarily from the order Lepidoptera, but also from the orders Hymenoptera, Diptera, possibly Coleoptera, Trichoptera and the crustacean order Decapoda (Martignoni, 1986 as cited in Herniou, et ah, 2003) (87, 219). Many of the insects infected by baculoviruses are economic pests and hence these viruses have been used as biological insecticides. However, a narrow host range and slow killing ability of target insects are limitations to their use as pesticides. Significant advances to improve baculovirus insecticides have been made by genetically engineering virus genomes so that they are more efficient in killing the insects yet remain environmentally benign. Commercial products based on genetically engineered viruses that have incorporated insect specific toxins have been developed (99). Two decades ago the molecular biology of baculoviruses became a major focus of interest because of their remarkable ability to hyperexpress heterologous eukaryotic genes under the control of the strong polyhedrin promoter (210). Because such proteins are processed in a manner similar to their native form, this system has become widely exploited for the expression of proteins for biomedical and pharmaceutical research. Other important uses of baculoviruses are as model systems to study the molecular biology of viruses as demonstrated by the discovery of the important anti-apoptotic genes tap and p35 (37, 39). 1 BACULOVIRUS INFECTION CYCLE Baculoviruses are rod-shaped, enveloped viruses with circular double-stranded DNA genomes ranging in size from 90 kb to 180 kb. The family Baculoviridae is currently subdivided into two genera Nucleopolyhedrovirus (NPV) and Granulovirus (GV) (219). The NPV genus is characterized by viruses forming polyhedral occlusion bodies (OBs), each containing rod-shaped nucleocapsids that are surrounded by a membranous envelope. These come as either single (SNPV) or multiple (MNPV) nucleocapsids per envelope (154). Viruses of the GV genus are similar to NPVs, but the virions are occluded singly in small ovoid granules (18). GVs have been described from only lepidopteran hosts, whereas NPVs have been isolated from a wider range of insects. Typically, the baculovirus life cycle involves the production of two distinct virus forms, the occlusion derived virus (ODV) and the budded virus (BV). ODV are embedded in the OB, which is composed of a crystalline viral protein called polyhedrin. The OB gives protection to these viruses from environmental degradation until ingested by a host caterpillar. After ingestion the OB dissolve immediately, in the alkaline juices of the midgut and the virions are released. The ODV then passes through the peritrophic membrane, which is a protective lining secreted by the midgut composed of chitin, glycosaminoglycans, glycoproteins and other proteins. Current evidence indicates that viral particles are associated with metalloproteinase enzymes called enhancins that facilitate localized digestion of the membrane and passage of the virus (123, 198). However, it is not known whether all the baculoviruses contain similar proteins. After crossing the peritrophic membrane, the virion envelope comes in contact with the microvillar membrane and fuses with it allowing the nucleocapsids to enter into the cell via adsorptive endocytosis (61, 234). In the cytoplasm and with the aid of microtubules, the nucleocapsids travel to the nuclear pore and then into the nucleus where they uncoat, releasing the viral DNA (235). Once the viral DNA is within the nucleus, gene expression occurs in a temporally regulated fashion that is divided into early, late, and very late phases. Early gene products are required for the replicative events and prepare the host cell for virus multiplication. Specific early genes are also essential for virus-mediated regulation of the host, including the control of larval molting and evasion of host antiviral responses such as apoptosis. During the 2 late phase of infection, the steady state levels of host cellular RNAs decline and host protein synthesis ceases (165). The switch over from early to late viral gene expression coincides with the start of D N A replication and intranuclear development of the viral replication centres called the virogenic stroma. The virogenic stroma provides the microenvironment for assembly of the progeny nucleocapsids (16, 21). B V is the first viral phenotype produced that 'bud' from infected cells acquiring a modified plasma membrane envelope to spread the infection to surrounding cells. BVs also traverse into the hemolymph where they are circulated through out the body, facilitating the rapid transmission and infection of other tissues. The fusion proteins present in the envelope allow the B V to enter the cell by adsorptive endocytosis (16, 234). After the midgut, the first tissues that show evidence of infection are hemocytes, tracheal matrix and fat body cells (11, 47). Later in infection, the viral replication phase shifts from B V to the occlusion phase, in which occlusion virions and polyhedra are produced. Cellular infection culminates in cytolysis and the release of mature OBs (80, 209). G E N E R E G U L A T I O N Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV) is pathogenic for the Douglas fir tussock moth, which is a major economic pest of forests in the Pacific northwest area of North America. OpMNPV is a well characterized baculovirus and has been extensively investigated in terms of genome replication and virion structure (2,104, 200). The genome of OpMNPV is 131,990 bp and contains 152 open reading frames of > 150 nucleotides or greater (7). OpMNPV genome organization and infection cycle are similar to the archetype Autographa californica nucleopolyhedrovirus (AcMNPV). In general, those genes that are known to be essential for A c M N P V are also found in the OpMNPV genome. The genes are expressed in a transcriptional cascade in which each successive phase is dependent on the expression of genes during the previous phase. They can be divided into three general categories early, late, and very late genes (18). The host R N A polymerase II transcribes early genes, before the onset of viral replication (56). A number of studies utilizing temperature-sensitive mutants, metabolic inhibitors, and transient assays suggest that late gene expression is 3 dependent on early gene expression (55, 69). Late and very late genes are transcribed after the onset of viral DNA replication by a virus-specific RNA polymerase that is resistant to oc-amanitin (92). Very late genes appear to be specific for the development of OBs and include the hyper expressed polyhedrin andplO genes (21). Early gene expression Transcription of early baculovirus genes initiates immediately after virus infection and they are detectable within in the first 15 to 60 min (34, 71, 92, 111, 164, 221). The host RNA polymerase II transcribes early genes, before the onset of viral replication. However the transcription of many early genes, including iel, p35, gp64, and 39k (pp31) continues late into infection. Late transcription of these early/late genes can be mediated by late promoter elements that overlap the early promoter (154, 241). Early genes have been further divided into immediate early (IE) and delayed early (DE) genes. IE genes are defined as those genes having high level of expression in the absence of viral factors and DE genes are dependent upon other viral gene products for non-basal level expression and synthesized before DNA replication (69, 221). IE and DE genes utilize host cell transcriptional machinery for their expression and their promoters resemble typical RNA polymerase type II promoters found in both insect cells and other organisms (23). A number of promoter elements have been identified that regulate early gene expression. The TATA element consists of an A/T rich motif (consensus TATAA) located 25-31 nucleotides upstream of the transcription start site. Downstream of the TATA is a conserved initiator motif CAGT or CACAGT located at or near the transcription start site. This motif resembles the RNA start sites of many insect genes and matches the consensus for arthropod transcriptional initiator elements (189). TATA and CAGT motifs have been identified in the early gene promoters of viral genes iel, ie2, gp64, p34, andp8.9K of OpMNPV (17, 221,222, 241) and iel, ie2, p35, and 39k of AcMNPV (45, 54, 68, 71, 100, 101). In plasmid transfection assays, the CAGT motif and TATA motif together contribute to the overall strength of the promoters of early genes iel, ie2, 39k, and gp64 (17, 28, 67, 101). Cg30 is an early gene that lacks a CAGT consensus sequence, as well as a consensus TATA box, however an AT rich sequence is located 30 nucleotides upstream of the transcription initiation site and 4 this appears to serve in its place (226). The TATA early promoter consensus sequence is also absent in the AcMNPV DNA polymerase gene, but it is present in the OpMNPV DNA polymerase promoter (4). It was also found that AcMNPV DNA polymerase promoter initiates transcription less efficiently than OpMNPV DNA polymerase (15). Upstream regulatory elements GAT A, CAGT, 13R, CCAAT, GC rich and enhancers have been identified in early gene promoters of OpMNPV and AcMNPV. GATA elements are found as single or multiple copies in either orientation in OpMNPV ie2 (222), gp64 (100, 110), opep-2 (206) and AcMNPV pe38 (110). Mutation of the GATA element in the gp64 of OpMNPV resulted in a significant decrease in transcriptional activity from the promoter (100), whereas no effect is observed on the AcMNPV pe38 promoter (110). A CACGTG motif present along with GATA has been shown to influence transcriptional activity of OpMNPV gp64 (100) and opep-2 promoters (207). A new positive regulatory motif called 13R that contains 13 bp direct repeats was identified on the OpMNPV opep-2 gene (207). 13R like motifs were also identified on the promoters of OpMNPV ie2 and AcMNPV gp64. These motifs are found necessary for the optimal promoter activity of OpMNPV ie2 and opep-2 genes (206, 207, 222). GATA, CACGTG and 13R motifs have also been demonstrated to bind to host-cell nuclear factors (100, 101,110, 206, 207). Analysis of the OpMNPV IE1 promoter identified a canonical CCAAT and GC rich sequences upstream of the TATA box sequence that are found to be important for maximal expression of iel promoter in Spodoptera frugiperda, Sf9 cells but not in Ld652Y cells derived from Lymantria dispar (221). Similar cell-specific effects in the transient expression of promoters have been observed for other genes, gp64, ie2, and opep-2 of OpMNPV (19, 52, 207, 222) and 39K of AcMNPV (221). A distinctive feature of all baculovirus genomes is the presence of transcriptional enhancers referred to as homologous regions (hrs) at multiple locations around the genome. Enhancers are ds-acting DNA sequence elements that increase the rate of utilization of RNA polymerase and function relatively independent of their orientation (upstream or down stream) and distance from the promoter (70, 221). In addition to their role as enhancer elements, hrs of both AcMNPV and OpMNPV have also been demonstrated to function as putative replication origins of DNA replication (104). 5 Late gene expression The transition from early to late phases of the baculovirus infection cycle is characterized by replication of viral DNA, activation of an a-amanitin resistant DNA-dependent RNA polymerase, and the apparent inhibition of host transcription (63). Viral DNA replication appears to be a necessary prerequisite for late gene transcription. Inhibitors of viral DNA replication (such as aphidicolin) also block late gene expression in infected cells (136). Concomitant with viral DNA replication, levels of host mRNAs also decline substantially, suggesting that host transcription is inhibited (170). A virus encoded RNA polymerase was purified from AcMNPV-infected Sf9 cells that supported accurate and specific transcription only from viral late and very late promoters but not early promoters (244). The complex consisted of four subunits p47, late expression factor-4 (LEF-4), LEF-8, and LEF-9. These four factors were also shown previously to be required for transient expression of viral late and very late genes (229) suggesting baculoviruses encode their own polymerase for the transcription of late and very late genes. Using a transient late expression assay system (176), 19 viral encoded late expression factors (lefs) were found to be necessary to support optimal levels of transient expression from an AcMNPV late promoter (125, 136, 191, 229). These include lefs 1-12, p47, ie-1, ie2, 39K, DNA pol, helicase andp35. Eight of these are required for early viral gene expression and DNA replication and eleven are thought to be involved more directly in late gene transcription. Four out of these eleven factors are identified as components of viral RNA polymerase. Functions of the additional factors probably include proteins that discriminate between late and very late genes, factors that are needed for termination of transcription, posttranslational processing functions (formation and methylation of 5' caps, cleavage, and polyadenylation, for example) and for the assembly of the four polymerase subunits (62, 66, 95, 135, 175). In addition to the 19 lef genes, two other factors, host cell factor (hcf)-\ and a very late factor (vlf)-1 have been identified for late gene expression. Hcf-1 is a lef required for optimal hr dependent DNA replication and transient late gene expression of AcMNPV in insect cells TN-368, derived from Trichoplusia ni (134). Vlf-1 is required for high level very late gene expression exhibited by thepolyhedrin andplO genes (148). 6 Late genes are transcribed by a viral R N A polymerase (63, 72) that recognizes distinct late promoters. Most, i f not all, late gene promoters contain the conserved sequence 5'-G/T/ATAAG-3' at the late transcription start site (199). The minimal core T A A G sequence is usually preceded by an A , and less frequently by a T or G nucleotide (158). Except for a T A A G motif, little is known regarding the sequence specificity requirements for late promoter recognition and activation. The very late phase of gene expression follows late gene expression and is characterized by hyper-expression of the very late genes polyhedrin and plO. Very late promoters appear to be similar to late promoters in that they also contain the conserved T A A G motif and flanking sequences, but differ in that they require an additional sequence called a "burst" sequence that regulates the burst of very late transcription (171). In both polyhedrin and plO genes an 8 bp TAAG-containing sequence at the transcription start site has been identified as the primary determinant of transcription (171). The exceptionally high levels of transcription from the polyhedrin and plO genes appear to be regulated or mediated by binding of viral protein VLF-1 to the "burst" D N A sequence, downstream of the transcription start site (148, 171,228,246). H O S T R A N G E The host range of any virus is determined by its ability to enter the cells and tissues of a susceptible host, and to replicate and release new infectious virus particles. The major steps in the baculovirus life cycle are the entry of virus into the cell; the viral early, late, and very late gene expression; D N A replication; budded virus assembly and release; and polyhedron formation. Blockage of productive baculovirus infection may occur at any point in the virus cycle (144, 157, 159, 240). Baculovirus genes that affect viral host range that have been identified arep35 (38, 59, I36),pl43 (helicase) (9, 97), hrf-1 (225), hcf-1 (134), ie-2 and lef-7 (30, 59, 134). The A c M N P V p35 is an apoptosis inhibitor gene that has been shown to be a host range determinant for A c M N P V infection. Apoptotic programmed cell death occurred when the 7 insect cell line Sf21, is infected with mutants of the baculovirus AcMNPV which lack a functional p35 gene. However, infection of the TN-368 cells with p35 mutants does not result in apoptosis (37, 39). AcMNPV and Bombyx mori NPV (9) genomes have 90% nucleotide sequence identity but non-overlapping host ranges. AcMNPV replicates well in Sf9 and Sfil cells, but it does not replicate in the BmN cell line, derived from B. mori. Conversely, BmNPV replicates in BmN cells, but it does not replicate in Sf9 and Sf21 cell lines. When a short DNA sequence of 572 bp within AcMNPV or/95, which encodes the viral HELICASE (PI43), is replaced with the collinear region of BmNPV, AcMNPV replicated in both Sf9 and BmN cell lines (41, 102, 140). L. dispar MNPV (LdMNPV) host range gene hrf-1 is a unique gene among the baculoviruses studied to date. The cell line Ld652Y is permissive for LdMNPV and OpMNPV but non permissive for AcMNPV. Infection of Ld652Y cells with AcMNPV results in shutoff of both viral and cellular protein synthesis (73). A recombinant AcMNPV carrying the hrf-1 gene of LdMNPV can overcome the block in protein synthesis and replicate successfully (225). The host cell-specific factor gene, hcf-1, from AcMNPV was necessary for successful infection of TN368 cells and to some extent for the enhancement of the infectivity of virus in T. ni larvae, but it was not necessary for virus replication or infectivity in Sf21 cells or Spodoptera larvae (134). The involvement of ie-2 and lef-7 in host range determination remain to be established. However, transient assays have showed that AcMNPV ie-2 and lef-7 are not essential for AcMNPV late gene expression in transient assays in TN368 cells but are required in Sf21 cells (30, 59, 134). Infection of the SL2 cell line derived from S. littoralis, with AcMNPV results in apoptosis and low yields of viral progeny (137). An AcMNPV recombinant that had a disruption in the ieO promoter yielded high titres of viral progeny in SL2 cells. The disruption of the ieO promoter also resulted in increased steady state levels of IE1 relative to IEO. AcMNPV productive infection of SL2 cell line and S. littoralis larvae was also obtained by just over expressing IE1 suggesting that a change in IE1 expression levels effects the host range. 8 BACULOVIRUS REPLICATION Viral D N A replication is a key process in the multiplication of D N A viruses that involves highly ordered protein-DNA and protein-protein interactions between cis and trans acting elements that leads to assembly of a functional replication complex (replisome) on the origin (118). A protein referred to as a replication initiator initiates replication. Initiators are typically multifunctional proteins capable of (i) binding to the origin of D N A replication, (ii) unwinding origin DNA, and (iii) recruiting additional proteins necessary for D N A replication. In baculovirus, c/s-acting elements involved in viral D N A replication have been identified by using two main strategies: (i) the analysis of defective virus genomes arising after serial undiluted passage of virus stocks in cell culture (107, 116, 117) and (ii) the characterization of replicated plasmid D N A containing cw-acting elements introduced into infected cells (3, 119, 121, 178-180, 243). Together, these studies have identified two types of origins of viral D N A replication, homologous regions (hrs) and non-homologous regions (non-hrs). Transacting replication factors were identified by the Challberg assay (29), which was originally developed to identify herpes simplex virus (HSV-1) replication genes. In this assay a set of overlapping cosmid clones representing the entire viral genome are cotransfected into insect cells with a reporter plasmid that contains a cw-acting viral origin of replication. The reporter plasmid replication is detected by the Dpnl assay, which uses Dpnl restriction enzyme to differentiate the replicated plasmid from the non-replicated one. Dpnl cleaves exclusively at fully methylated restriction sites. Since the non-replicated plasmid, which is the reporter plasmid used for transfection is produced in Darn bacteria, it contains fully methylated restriction sites (GmeATC) and can be cleaved by Dpnl. The plasmid that is replicated in insect cells are not methylated as eukaryotic cells lack the enzymatic machinery for methylation and therefore is not digested by Dpnl. Using the approach of omitting the cosmids that contain the essential sequences which result in loss of replication of reporter, a minimal set of cosmid clones were identified that are capable of supplying all the necessary viral replication factors. 9 Using cosmid and plasmid sub clones of the minimal set of essential cosmids the specific replication genes were identified. In OpMNPV six essential genes, lef-1, lef-2, lef-3, dnapol, helicase, iel; and three stimulatory ones, ie2, p34, and iap-1 were identified (6). Homologs to each of these genes have also been identified in AcMNPV (103), however the relative importance of the stimulatory genes differs in the two virus replication systems. The most profound stimulatory gene for OpMNPV is ie2 while for AcMNPV, it is p35. One more stimulatory gene, lef-7 was identified in AcMNPV but not in OpMNPV (136). Previously the AcMNPV Lef-11 gene was identified as not necessary for transient origin-dependent plasmid DNA replication assays (103, 136) but recent studies found that it is required for viral DNA replication in the context of the infection cycle (130). Homologous regions A novel feature of many baculovirus genomes is the presence of homologous regions (hrs) that are distributed throughout the genome. Hrs are shown to act as origins of replication (oris) as well as transcriptional enhancers (70, 221). Each hr contains several copies of an imperfect palindrome flanked by direct repeats. In OpMNPV five hrs (hr\-5) have been identified and are composed of a 30 bp imperfect palindrome embedded in a 66 bp repeat element that can be tandemly repeated partially or completely up to 11 times (3, 7, 223). Similarly, in AcMNPV, hrs are located in eight regions of the genome and contain 1-8 repeats of conserved 72 bp long sequence elements separated approximately by 0-131 nt of non-repepetitive DNA (40, 106). The conserved sequence element contains a 30 bp imperfect palindrome flanked on both sides by approximately 20 bp of direct repeats. The 30 bp palindrome sequence has an EcoRl at its centre. Between the AcMNPV and OpMNPV hr elements, there is 51% sequence homology (223). Similar to OpMNPV and AcMNPV, hr sequences have also been identified in the genomes of a number of other baculoviruses such as, BmNPV (142), LdMNPV (180), Choristoneurafumiferana MNPV (CfMNPV) (243), S. exigua MNPV (SeMNPV) (22) and Anticarsia gemmatalis MNPV (AgMNPV) (58), S. litura NPV (SpltNPV)(173), Hyphantria cunea NPV (HycuNPV) (51), Helicoverpa zea SNPV (HzSNPV), Helicoverpa armigera NPV (HearNPV) (31), Plutella xylostella granulovirus (PlxyGV) (78), Xestia c-nigrum granulovirus 10 (XecnGV) (81), Rachiplusia ou multiple nucleopolyhedrovirus (RoMNPV) (77), Mamestra configurata NPV-A (MacoNPV-A) and MacoNPV-B (124, 126). Functional analysis of AcMNPV hrla demonstrated that a single palindrome is sufficient for supporting plasmid DNA replication and stimulating transcription. Plasmids containing only one-half of a palindrome or a disruption in the central core either did not replicate or replicated very poorly and did not enhance transcriptional activity from the 39k promoter (119). IE1 was found to be a component of the protein complex bound to hrla. It is thought that the binding of the IEl-dimer to the hr elements positions the two half-sites of the palindromes there by creating structures that allow replication and enhancer functions (192). However, IE1 binding alone is not sufficient to allow replication and transcriptional enhancement, because fragments containing a disruption in the central core still bound to IE1 but were inactive in replication and transcription (119). Although hrs from different baculoviruses generally share similar structural features, the ability to work as origins in heterologous systems is variable. SeMNPV and AcMNPV were unable to replicate heterologous hr origin containing plasmids in Sf21 cells (22). CfMNPV hr is unable to support plasmid replication in AcMNPV-infected Sf9 cells but the AcMNPV hr origin in CfMNPV-infected Cf-124T cells could (86, 243). OpMNPV hr supported plasmid replication at barely detectable levels in AcMNPV-infected Sf9 cells (3) while AcMNPV hr origin containing plasmid replicated at fairly detectable levels in OpMNPV-infected Ld652Y cells (179). This may, in part reflect the host specificity of baculovirus replication. Non-A/* origins of replication Non-/zr origins are a second type of replication origins identified in baculoviruses and are distinctly different from hrs. In OpMNPV, a single non-hr was identified in the Hindlll-N fragment (7-0-11-3 map units (m.u)) of the OpMNPV genome. Deletion analysis from both left and right ends of 7/mdIII-N fragment showed a progressive decline in replication efficiency suggesting the origin sequences that are necessary for HindlU-N to replicate are distributed on the whole /7/odIII-N fragment. However, these deletions marked the boundaries for a 11 replication competent region, which is essential for the deletion mutants to replicate. Sequence analysis of Hindlll-N indicated the presence of 14 different categories of repeats both direct and indirect repeats. In addition, two unique palindromes in the competent region and clusters of transcription factor binding sites on both the strands were found (179). Similar to OpMNPV, only one copy of a non-hr sequence was identified in the Hindlll-K region of the AcMNPV genome but there is no obvious homology between these two origins. Hindlll-K of the AcMNPV genome supported replication of plasmids in transient replication assays (105) and was retained in defective interfering genomes (116). Analysis of the Hindlll-K fragment essential origin sequences identified five regions (I-V) of which I, II, and V contain the auxiliary sequences and regions III and/or IV contains essential sequences. Two overlapping imperfect palindromes in region III were identified that have no homology with the hr palindromes. Two other structures, A/T rich regions and direct repeats resembling replication oris of eukaryotic viruses were identified in proximity to palindromes. Non-/?™ have also been identified in the genomes of SeMNPV (85), S. littoralis NPV (SpliNPV) (91), Cryptophlebia leucotreta granulovirus (CrleGV), and Buzura suppressaria NPV (BuzuNPV) (90). Sequence analysis of the SeMNPV non-hr origin revealed a unique distribution of six different imperfect . palindromes, several polyadenylation consensus motifs, multiple direct repeats, and several putative transcription factor-binding sites (85). Similar to hr origins, species specificities have also been observed in non-hr origins. Plasmids with the AcMNPV non-hr was unable to replicate in SeMNPV infected Sf21 cells but in the reciprocal experiment those containing the SeMNPV non-hr origin replicated at low levels in AcMNPV-infected Sf21 cells (22, 85). Plasmid with the OpMNPV non-hr was able to replicate in AcMNPV virus-infected Sf9 cells (179) and it is unknown whether AcMNPV non-hr replicates in OpMNPV infected cells. Oppenheimer and Volkman (172) provided evidence that baculovirus DNA replication proceeds via the rolling circle mechanism. Plasmids containing the AcMNPV non-hr origin replicated into high molecular weight concatamers. Analysis of replicated plasmid DNA by partial digestion with a restriction enzyme that cuts the input plasmid at a single site resulted in DNA 12 fragments that were multiples of a unit length (172). In addition, A c M N P V defective genomes also appeared to consist of concatamers of the non-hr origin (116, 117). It is still unknown what the role or relative contribution of either of these two types of putative origins is in baculovirus D N A replication in vivo. Deletion of a single hr in A c M N P V (195) and BmNPV (142) did not affect the replication of these viruses suggesting that multiple hrs are redundant for replication, or the hr they tested is not involved in replication in vivo. Thus, loss of one of five possible origins of D N A replication is not deleterious to viral growth. Recently it was also shown that the SeMNPV non-hr origin can be deleted without affecting virus replication (182). It is possible that the initiation of replication may occur only once on each D N A molecule and the origin is selected at random. In such cases, the distribution of origins around the genome may increase the probability of the formation of preinitiation complexes and thus accelerate the infection cycle (2). However these are only predictions and further in vivo viral D N A replication research is required. REPLICATION GENES The Baculoviridae share a common feature with other D N A viruses such as T7, T4, Simian virus 40 (SV40) and herpesvirus in the requirement of the components essential for D N A replication. A l l these viruses require a core set of six essential genes namely: Origin binding protein (OBP), single stranded D N A binding proteins (SSB), primase, primase-associated protein, helicase, and D N A polymerase. In addition, there is often a requirement for a topoisomerase and other proteins, which have not been identified in baculovirus genomes. Baculoviruses share a number of features of D N A replication with the Herpesviridae. Both replicate in the nucleus and their genomes are circular (baculovirus) or become circular (herpesvirus) during replication. Their genomes may also replicate in a similar manner, as transfection of herpesvirus origin-containing plasmid into infected cells result in large concatamers of input plasmid D N A suggesting a rolling circle mechanism. Similar to herpesviruses (190, 218), baculovirus A c M N P V D N A replication takes place at distinct nuclear 13 domains which form large foci during DNA replication. These foci are specific viral replication centres in which the replication proteins accumulate (141, 167). Genes essential for viral DNA replication Using the Dpnl replication assay six genes encoding proteins essential for viral DNA replication and three genes whose products stimulate DNA replication but are not essential for DNA replication were identified. The six genes that are found to be essential for replication in both OpMNPV and AcMNPV are ie-1, lef-1, lef-2, lef-3, DNA pol and helicase. These six essential genes are also present in all the baculovirus genomes sequenced so far (82, 87). Lef-1 and Lef -2 The OpMNPV Lef-1 is in the Hindlll-N fragment, which is also a non-hr origin of replication. The two activities of the Hindlll-N fragment, namely, coding for an essential replication gene, and its function as an origin of replication are independent of each other. The OpMNPV lef-1 gene is homologous to the AcMNPV lef-1 gene, although the predicted nucleoside triphosphate-binding site present in the carboxy terminal region of AcMNPV LEF-1 is not conserved in OpMNPV LEF-1 (5). The AcMNPV lef-1 gene was initially recognized as an early gene important for late and very late gene expression. It was shown later that it is essential for transient DNA replication (48, 174). Analysis of the AcMNPV LEF-1 sequences revealed a putative primase domain WVVDAD. Substitution of the aspartic acid in this domain abolished transient DNA replication (48) suggesting a role for this domain in DNA replication. Recently LEF-1 was purified to homogeneity and demonstrated to have primase activity (152). Lef-2 gene was identified on the Hindlll A fragment of the OpMNPV genome and has 77.9% amino acid identity to AcMNPV LEF-2 (6). In AcMNPV, LEF-2 co-purified with LEF-1 as a fraction possessing a high affinity for single stranded DNA. Using the yeast two hybrid system, LEF-2 was also shown to interact with LEF-1 (48). Together the data suggests that LEF-2 14 might serve as an accessory factor needed for attachment of LEF-1 onto viral DNA where replication complexes are assembled. Lef-3 The Lef-3 gene has been identified in the OpMNPV genome by Dpnl transient DNA replication assays (1). The predicted protein codes for 373 amino acids with 63.4% similarity to the AcMNPV LEF-3 (75). Amino terminal region of both OpMNPV and AcMNPV LEF-3 have a motif that is similar to motifs present in SSBs. This motif contains conserved aromatic and basic amino acids separated by a variable number of unrelated residues (238). OpMNPV and AcMNPV LEF-3 have 82% amino acid similarity in the conserved motif region indicating this region is important for LEF-3 function. Affinity chromatography, yeast two-hybrid system and biochemical analysis showed AcMNPV LEF-3 interacts with itself resembling SSBs of other organisms (49). The purified LEF-3 protein preferred single stranded DNA and demonstrated cooperativity in binding DNA and no sequence specificity supporting a role for LEF-3 as a SSB. Purification of LEF-3 by ssDNA agarose chromatography found it co-eluting with HELICASE suggesting an interaction between these two proteins. AcMNPV LEF-3 has also been shown to improve the strand displacement ability of viral DNA polymerase (145) indicating a possible interaction with DNA POL. A DNA-binding protein (DBP) has been purified along with LEF-3 from nuclear lysates of BmNPV-infected BmN cells. DBP binds preferentially to ssDNA and is capable of unwinding duplex DNA whereas LEF-3 also specifically binds ssDNA but cannot unwind DNA duplexes in vitro (151, 167). A DBP homolog of AcMNPV and OpMNPV were not found to be essential or stimulatory for plasmid DNA replication by transient replication assays (6, 103). Together all the data suggests the role of LEF-3 in replication is to function as a SSB. DNA pol Sequence analysis identified the OpMNPV DNA polymerase gene on the Hindlll D fragment between the gp37 and lef-3 genes of the OpMNPV genome (4). OpMNPV DNA pol codes for a predicted protein of 985 aa and exhibited 63% aa identity to AcMNPV DNA POL. Baculovirus 15 DNA polymerase sequences revealed conserved motifs that are present in a-like DNA polymerases. These are nucleotide binding, primase interaction, pyrophosphate hydrolysis, and exonuclease motifs (76, 145, 230). Differences were found in the requirement of AcMNPV DNA pol for transient plasmid DNA replication. Kool et al (103) showed DNA pol to be essential for DNA replication, whereas Lu and Miller (136) found that DNA pol is not required in transient replication assays but stimulated plasmid replication suggesting that a cellular DNA polymerase can substitute for viral POL. Helicase An OpMNPV ORF homologous to the AcMNPV pi 43 which is the putative helicase gene was identified and subsequent Dpnl replication assays similarly confirmed this ORF as an essential replication gene (4, 103, 133). The OpMNPV DNA helicase codes for a predicted protein of 1223 aa with 59.4% aa sequence identity to the predicted AcMNPV HELICASE. Seven conserved putative helicase motifs were identified in both OpMNPV and AcMNPV HELICASE proteins supporting the hypothesis that these proteins are helicases (133). Biochemical characterization has shown that AcMNPV HELICASE has both ATPase and DNA unwinding activities and binds to double-stranded DNA (dsDNA) and ssDNA in a sequence-nonspecific manner (146, 147). Mutational analysis have also shown that the ATP binding motif and a potential helix-turn-helix region are important for HELICASE function in DNA replication (131). AcMNPV with a temperature-sensitive mutation in helicase is defective in DNA synthesis at the nonpermissive temperature (136) providing further evidence that AcMNPV helicase is necessary for replication. As described above, AcMNPV HELICASE co-purified with LEF-3 (133) suggesting they both interact which is similar to many helicases that specifically bind their cognate SSBs. In addition, this interaction has been shown to stimulate DNA unwinding (50). LEF-3 is also required for the transport of HELICASE from the cytoplasm to the nucleus suggesting the interaction of these proteins (242). The AcMNPV helicase gene has also been implicated to be involved with late gene transcription, shutoff of host protein synthesis and determination of baculovirus host range (9, 41, 136, 140). 16 iel OpMNPV iel in transient Dpnl replication assays has been identified as an essential gene for replication of both hr and non-hr origin reporters (6). The OpMNPV IE1 gene is homologous to AcMNPV IE1, which is also an essential gene for replication. The 560 aa long OpMNPV IE1 predicted protein shares 63% sequence similarity to 582 aa long AcMNPV IE1. OpMNPV iel produces two transcripts 1.7 and 1.9 kb in size. SI nuclease and primer extension analysis has showed that the 1.9 kb mRNA is a spliced gene and the 1.6 kb mRNA is the non-spliced form (221). OpMNPV IE1 is associated with purified BV suggesting it as structural component (220). Both OpMNPV and AcMNPV IE Is have been shown by many studies to be transcriptional transactivators of both early and late viral promoters as well as transcriptional repressors of early promoters (52, 69, 221, 223). IEls have also been shown to act by both enhancer dependent and independent mechanisms (19, 52, 164, 221). For AcMNPV it was shown that IE1 binds to hr sequences as a dimer and is required for IE 1-mediated enhancer and origin specific replication functions (65, 196). Recently AcMNPV IE1 was also shown to be involved in homologous recombination (42). OpMNPV iel codes for a protein of 560 aa length with a molecular weight of 65 kDa. The N-terminal 132 aa are rich in acidic residues and carry a net charge of -13 (221). Both OpMNPV IE1 and AcMNPV IE1 acidic N-terminal regions have been shown to function as acidic activation domains (AADs) (52). Additional functional domains were identified in AcMNPV IE1. At the C-terminus they are helix-loop-helix-like domains that mediates IE1 dimerization, which is required for both nuclear import and DNA binding; basic domain II functions as the nuclear import element; and basic domain I functions as a 28-mer enhancer binding domain. In the study to identify independent transcriptional activation domains using the Escherichia coli heterologous system for protein expression an additional activation domain was identified at the N-terminus of AcMNPV IE1 located between the two basic domains (168, 208). IE1 binding to a 28-mer as a dimer is required for IEl-mediated enhancer and origin specific replication functions (35, 64, 65, 108, 119, 168,169,192, 196, 197). The role of IE1 in baculovirus replication has been hypothesized to be transactivation of other replication genes or 17 playing a role as an origin binding protein (OBP) (Fig. 1.1). However, Rapp et al. (191) expressed all late expression factors under control of the heat shock promoter and showed that IE1 transcriptional transactivation may not be required for replication. IE1 is also shown to localize to replication centres before the onset of replication. Subsequently, at the onset of replication LEF-3 (SSB) co-localizes with IE1. This supports the view that IE1 acts as an OBP and initiates assembly of viral replication factories by interaction with the other replication proteins (167). Genes stimulatory for viral DNA replication Several genes are stimulatory for viral DNA replication in both OpMNPV and AcMNPV. They are iel, p34 (pe-38 in AcMNPV), iap-l(p35 in OpMNPV), and lef-1. Unlike the essential genes, the stimulatory genes are not present in all baculovirus genomes (82, 87). For example ie2 and lef-1 genes are not present in LdMNPV but present in AcMNPV (114). ie2 OpMNPV ie2 is a stimulatory factor for viral DNA replication in the transient replication assays. In the presence of six essential replication factors, IE2 caused a 10- to 12-fold stimulation of replication signal. Out of the three OpMNPV genes (ie2, p34, iap) tested for stimulation of DNA replication, IE2 was the strongest (6). AcMNPV IE2 also stimulates transient AcMNPV DNA replication but more weaker (103). Both OpMNPV and AcMNPV IE2s are transcriptional regulators of early gene transcription. OpMNPV IE2 upregulated expression of the p8.9, iel, ie2 and opep2 promoters (28, 136, 206, 207, 222, 247) and AcMNPV IE2 transactivated expression from the iel and ie2 promoters (27, 28, 247). AcMNPV IE2 is also required for maximal levels of late gene expression (136, 176) and for efficient homologous recombination (42). The requirement of AcMNPV IE2 for replication and late gene expression is host specific since it is required in Sf21 cells but not in TN-368 cells (134,184). However, AcMNPV IE2 does block the progression of cell cycle in Sf21 and TN-368 cells. IE2 which in BmNPV functions as a ubiquitin ligase, belongs to the ring finger 18 protein family and both functions (cell cycle inhibition and ubiquitin ligase) are dependent on an intact ring finger (93,184, 186). In spite of the functional characterization of IE2 very little is known about how IE2 stimulates DNA replication. Nuclear localization studies showed IE1 and IE2 are present at replication centers and they co-localized with LEF-3 (141, 167). The role of IE2 in baculovirus DNA replication has been hypothesized (136, 176) to involve direct transactivation of replication genes or in a manner that affects replication indirectly. OpMNPV p34 (AcMNPV pe38) OpMNPV p34 stimulated transient reporter plasmid replication 1.5- to 2- fold in the presence of the six essential replication genes. However, addition of p34 to the mixture of six essential factors plus ie2 inhibited DNA replication. Similar to OpMNPV P34, its homologue PE38 stimulated AcMNPV transient DNA replication albeit weakly (6, 103). Both P34 and PE38 protein products are of different sizes (241) and both contain a RING finger and leucine zipper (109, 111, 222, 248). P34 and PE38 are transactivators stimulating expression from the OpMNPV ie2 and p8.9k promoters (241) and the AcMNPV helicase promoter (pl43) (132). The 20 kDa smaller late gene product of OpMNPV P34 was unable to transactivate the IE2 promoter. This amino-terminal truncated form lacks the ring finger but retains the glutamine-rich and leucine zipper domains, suggesting that for P34 transactivation the ring finger is important. Recently, Prikhod'ko and Miller (185) demonstrated that PE38 augments apoptosis induced by IE-1 in Sf21 cells and mutational analysis revealed that this was dependent upon the leucine zipper but not the ring finger. Nuclear localization studies showed PE38 colocalizes with IE2 and with the mammalian nuclear body protein, PML (162). Interestingly all these three proteins contain ring finger motifs. Studies on BmNPV PE38 showed that it also functions as E3 ubiquitin ligase, which is dependent on an intact ring finger similar to BmNPV IE2. Recent studies using a pe38 deletion virus showed PE38 has a direct effect on the growth and replication of AcMNPV in cell culture and on virulence in insects, but it is not essential for viral DNA replication (153). 19 lap andp35 The third gene that was identified to stimulate transient OpMNPV DNA replication 3- to 4-fold is Op-zap (6). Op-zap is the functional analog of AcMNPV p35, since both genes function as antiapoptotic genes (14, 37, 149, 236). Similar to IE2 and PE38, IAP2 of BmNPV is also ring finger dependent E3 ligase. The AcMNPV p35 gene is a stimulatory gene in AcMNPV transient replication assays. Out of the all the stimulatory factors (ie2, pe38, p35 and lef-7) tested, p35 has the most profound effect on stimulation of replication (103, 136). The function of p35 and Op-zap in transient replication assays may be associated with their ability to block apoptosis triggered by plasmid DNA replication or a product of one or more replication genes. Recently using yeast two hybrid screening, P35 was identified as interacting with a subunit of human RNA polymerase II suggesting an additional role of P35 in the regulation of cellular transcription (217). Lef-7 Lef-7 is an AcMNPV gene that stimulates transient DNA replication along with AcMNPV ie2, pe38 and p35 (136). A homologue to this gene was not identified in OpMNPV (6). Like AcMNPV IE2, LEF-7 also exhibited a cell-line-specific effect stimulating plasmid replication only in Sf21 but not in TN-368 cells (136). LEF-7 also stimulates the expression of a chloramphenicol acetyltransferase (CAT) reporter under the control of either the /?5P-capsid or polyhedrin promoters (158) and is required for optimal late reporter gene expression in Sf21 cells but not in TN368 cells (134). Deletion studies of AcMNPV lef-7 showed DNA replication occurred in the absence of LEF-7 but replication is strongly stimulated with its presence in Sf21 cells and in S. exigua UCR-SE1C cells and slightly stimulated in TN-368 cells (30). Hcf-1 The host cell-specific factor gene, hcf-1, from AcMNPV was necessary for replication in TN-368 cells and not in Sf21 cells (134). A homologue of hcf-1 has not been identified in the genome of OpMNPV (7). Hcf-1 null mutant infecting TN-368 cells was defective in viral DNA 20 replication, late gene expression and the production of BV was reduced, specific and observed only in TN-368 cells and not in Sf21 cells (134). This effect is cell-line-Role of IEO and EXONO in replication ieO The spliced form of iel is known as ieO and contains an ORF that is 130 nt longer than IE1 coding for a predicted protein of 35 additional aa at the N-terminus. Twenty three aa of these comes from ORF 138 (exonO) upstream of IE1 and the remaining 12 aa comes from the 5' untranslated region of IE1 (Fig. 1.2). ieO is expressed within 1 h p.i., peaks for 4 h p.i., and then declines but remains detectable even up to very late times p.i (220). Translation of ieO mRNA results in the production of both IE1 and IEO due to internal translation initiation of the IE1 start codon. When the second and third ATG codons are mutated to alanine codons, only IEO is produced (224). Similar to OpMNPV, IEO genes are also found in AcMNPV and in LdMNPV (109, 181). The OpMNPV IEO is a significantly (14- to 15-fold) stronger transcriptional activator of specific early genes than IE1 and could activate gene expression in an enhancer-dependent or -independent manner. In addition, OpMNPV IEO is auto-regulatory (224). Pearson and Rohrmann (181) analyzed the function of the IEO homologue in LdMNPV and showed that IEO and not IE1 was functional for both, replication and transactivation in Ld652Y cells, suggesting that splicing was a requirement for producing a functional transactivator. Kremer & Knebel-Morsdorf (113) have showed that AcMNPV IEO binds to DNA, and forms homodimers and heterodimers with IE1. Recently it was shown that IEO plays a role in the regulation of AcMNPV infection in the semi-permissive cell line SL2. To date no studies were done to test for replication of OpMNPV and AcMNPV IEO. Since IEO is functional in transcriptional activation and has all the regions of IE1 it is predicted that both OpMNPV and AcMNPV IEO can be active for replication. 21 ExonO OpMNPV exonO contributes 23 aa to the N terminus of IEO in the splicing reaction but the function of this gene is yet unknown (Fig. 1.2). OpMNPV exonO is 734 bp in length and codes for a predicted 245 aa protein. Sequence searches with the Entrez protein database showed a cysteine rich region at the C-terminal region of EXONO. This region has the consensus C 3 Y C 4 identical to the ring finger consensus C 3 H C 4 except EXONO has tyrosine instead of histidine. These features suggest that EXONO might encode a functional protein. Ring fingers are found in proteins that have diverse functions including regulation of gene expression, site-specific recombination and apoptosis (239). As described previously ring fingers have also been identified in other baculovirus proteins ie2,p34 (pe38), iapl, or/35, iap2 and cg30 (59, 112, 185, 222, 236). Ring finger proteins are involved in the ubiquitination pathway functioning as E3 ligases (53) as has been shown for BmNPV IAP2, IE2, and PE38 (93). Ubiquitination is involved in cell cycle progression, organelle biogenesis, apoptosis, transcriptional regulation, protein transport, antigen processing, virus budding and several other processes of multi vesicular body sorting (13, 98, 161, 204, 239). Since EXONO also has a conserved RING finger, it is likely that it might have a function similar to the ring finger proteins discussed above. 22 Fig. 1.1. Schematic of the IE1 protein structure and a model explaining IE Is function, a). The different domains at N- and C- terminus represented are based on studies of OpMNPV IE1 and AcMNPV IE1 (108, 197). N- terminus contains an AAD, and the C- terminus contains a cluster of highly conserved basic amino acids (B-I); a possible second trans-activation domain (D) (208), a second region rich in basic amino acids (B-II) and domains required for DNA binding (DBD), and oligomerization (OD). b). A model for IE1 function. IE1 is shown as dimer bound to origin of replication or enhancer. The smaller circle is the N-terminus of IE1 and the bigger circle is the C-terminus region. With the C-terminus IE1 is bound to origin as homodyne and with the N-terminus it interacts with transcription or replication complex. 23 Fig. 1.1 (a) AAD B-1 D DBD B-il OD N-terminus C-terminus 24 Fig. 1.2. Schematic of the exonO and iel region showing the splicing pattern of iel and ieO, showing the contribution of exonO for the ieO formation. Transcriptional start sites are indicated by vertical black lines. IEO is expressed as the spliced product of IE1 which is 130 nt longer that codes for a predicted protein of 35 additional aa at the N-terminus. 23 aa comes from exonO (orf 138) upstream of IE1 and the remaining 12aa comes from 5' untranslated regions (UTR) of IE1. The first ATG is from the exonO and the ATGs labelled as 1 ° and 2° are the first and second methionine codons of IEL 25 Fig. 1.2 me53 exonO UTR 23 12 iel 560 MM f MM ATG' ATG ATG 2° IEO (595 aa) 26 STUDY OBJECTIVES Baculovirus OpMNPV IE1 is a primary transcriptional activator of OpMNPV (221) and is essential for viral D N A replication (6). The IE1 N-terminus contains an activation domain rich in acidic amino residues and is a classic acidic activation domain (AAD) (52). AADs are the most extensively studied activation domains of eukaryotic transcription factors that are found to enhance the efficiency of D N A replication as well as transcription (32, 83, 128, 156). AADs are autonomous elements that can function independently when fused to D N A binding domains of heterologous activators (32, 187). These activation domains also have the ability to function in cells from a variety of eukaryotic species, including yeasts, plants and mammals (187). AADs are present in yeast G A L 4 (12) and GCN4 (88), mammalian P53 tumour suppressor (26) and herpes simplex virus VP 16 (231). The dual role of AADs is reminiscent of the several common features shared by both transcription and D N A replication processes (84). These include unwinding of duplex D N A (150, 160, 188), overcoming nucleosomal repression (89) and assembling a multisubunit transcriptional (187, 188) or replication complex (83, 128) at the initiation site. Another common feature is that both transcription and replication have overlapping cis acting elements that are binding sites for transcription factors (44, 84, 119). This is observed in baculovirus hr elements, which have been shown to function as replication origins and as enhancers of transcription (2, 221, 223). The various mechanisms by which transcription factors operate these common processes of replication and transcription are still not very clear. OpMNPV IE1 A A D is a very good candidate to study these important processes and gain insight into the various interactions at the initiation sites. Past studies in our laboratory have established the importance of the N-terminal acidic domain of OpMNPV IE1 in transactivation by using chimeric IE1 proteins that had foreign A A D s from herpes virus transactivator VP 16 and A c M N P V IE1. These chimeric fusion constructs showed 2-5 fold greater increase in transcriptional transactivation than the native IE1 (52). 27 The first objective of this thesis was to use these chimeric constructs and characterize the role of the IE1 acidic activation domain in OpMNPV viral DNA replication. In addition, we wanted to determine if the chimeric fusion proteins that were stronger transcriptional activators were also stronger activators of viral DNA replication. The second objective of this thesis was to study the role of IE2 in replication. OpMNPV ie2 is a stimulatory factor for viral DNA replication in the transient replication assays and also transcriptional regulator of early gene promoters (p8.9, iel, ie2, and opep2) (206, 207, 222). How IE2 stimulates DNA replication is not known. Studies showed IE1 and IE2 co-localized at replication centers suggesting they might interact (141, 167). In this thesis we wanted to study for possible interactions between IE2 and IE1 AAD, viral replication factors or cell factors. The third objective of this thesis was to analyze the role of IEO in viral DNA replication. OpMNPV IEO is the spliced form of IE1 that contains an additional 35 aa at the N-terminus. Functional analysis showed IEO is a stronger transcriptional activator of specific early genes than IE1 (224). In addition, it is unknown what role IEO, which has an extended N-terminus activation domain, plays in viral DNA replication compared to IE1. The fourth objective of this thesis was to perform a preliminary characterization of this gene in the OpMNPV life cycle. OpMNPV exonO is 734 bp in length and codes for a predicted 245 aa protein. Sequence searches with the database suggest that exonO may also encode a functional gene. Recognizable motifs within the predicted protein sequence include a possible ring finger (C3YC4) and leucine rich motif in the C-terminal region. Alignment with EXONO sequences from other baculoviruses shows this gene is very much conserved. To date no studies have been reported on exonO from any baculovirus. . 28 CHAPTER 2. The acidic activation domain of the baculovirus transactivator IE1 contains a virus specific domain essential for viral DNA replication INTRODUCTION The baculovirus Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV) has a genome of 131,990 bp and encodes approximately 150 genes of 50 aa or larger (7). By transient replication assays six genes, lef-1, lef-2, lef-3, DNA pol, helicase, iel were shown to be essential for replication and three genes, ie2, p34, and iap-1 are stimulatory (6). Based on studies with OpMNPV and the related viruses AcMNPV and BmNPV, data suggests that LEF-1 is a possible primase and interacts directly with LEF-2 which has an unknown function (48). LEF-3 encodes a SSB, forms homotrimers, and is essential for transport of P143 into the nucleus (1, 49, 242). PI 43 is a possible helicase and DNA pol is a DNA polymerase (4). IE1 is a primary transcriptional transactivator known to activate early and late gene expression during baculovirus infections (69, 221). The role of IE1 in baculovirus replication has been hypothesized to be to transactivate other replication genes or to play a role as an origin binding protein (OBP). However, Rapp et al. (191) expressed all late expression factors under control of the heat shock promoter and showed that IE1 transcriptional transactivation may not be required for replication. The stimulatory genes, IE-2 and P34 (PE38 in AcMNPV and BmNPV) are transcriptional activators, and IAP-1 is an inhibitor of apoptosis (6). Two types of replication origins have been identified as targets for the replication proteins of OpMNPV. They are the homologous repeat (hr) origins that contain tandem arrays of imperfect palindromes, and the non-hr origins that lack the hr repeats.(179). " A version of this chapter has been published (Pathakamuri, J. A., and D. A. Theilmann. 2002. The acidic activation domain of the baculovirus transactivator IE1 contains a virus-specific domain essential for D N A replication. J. Virol. 76:5598-5604)" 29 IE1 forms homodimers and can transactivate genes in an enhancer dependent or independent manner (35, 64, 108, 119, 120, 196, 197). We have shown that the N-terminus of OpMNPV IE1 contains an acidic activation domain (AAD) that is essential for transcriptional transactivation. The AAD was composed of two sub-domains, both of which are required for transactivation. In addition, OpMNPV AAD could be replaced by the archetype VP 16 AAD from herpes simplex virus type 1 (HSV-1) and the AcMNPV IE1 AAD. Chimeric proteins containing the VP 16 and AcMNPV AAD domains appeared to be more potent activators of gene expression than the wild type (WT) IE1 (52). A number of studies have shown that transcriptional transactivators, and specifically those with AADs, can play direct roles in both viral and cellular DNA replication. Replication and transcription share cis acting DNA elements that bind transcriptional factors. These elements activate transcription and also increase the efficiency of replication. The molecular basis for activation of replication and transcription therefore appears to rely on common mechanisms. Current evidence suggest that transcription factors appear to activate DNA replication by general mechanisms which include, i) direct recruitment of the replication machinery through protein-protein interactions (83, 128), ii) modulation of chromatin structure (89, 127), iii) recruitment to the nuclear matrix and replication factories, and iv) unwinding of duplex DNA by transcriptional activation (150, 160, 188). These various mechanisms may operate in concert rather than individually. The VP 16 AAD, which can functionally replace the OpMNPV IE1 AAD for transcriptional activation, has been used in several studies and its role in activation of replication appears to be well established. In studies that have used the VP 16 AAD to activate polyoma and papilloma virus replication, found VP 16 AAD to interact directly with replication protein A (RP-A), an SSB, which is an essential component of the cellular replication machinery (83, 128). In addition, AADs when tethered to a replication origin by a heterologous DNA binding domain, such as Gal4-VP16, Gal4-p53 or Gal4-BRCA1 AAD can stimulate cellular replication in yeast and viral replication in mammalian cells (32,44, 84, 89, 129). These data suggested that 30 activation of replication by AADs may be species-independent similar to the universal ability to activate transcription in a variety of eukaryotic and prokaryotic organisms. The OpMNPV IE1 AAD contains two sub-domains that can independently or synergistically transactivate transcription (52) which is similar to the activation domains of VP 16 (193, 214), P53 (26), GCN4 (88), and Gal4 (139). Compared to transcriptional transactivation there is significantly less known concerning replication specific domains in AADs. In this study, we investigated the role of the OpMNPV IE1 AAD to determine if it is essential for viral DNA replication. In addition, we determined that chimeric IE1 genes (containing heterologous AADs) which are more potent transcriptional transactivators than the native IE1 are totally inactive for DNA replication. Finally, we mapped regions of the IE1 AAD that were required for activation of replication. 31 MATERIALS AND METHODS Cell Culture. Lymantria dispar (Ld652Y) cells were maintained in TCI00 medium as described (216). Plasmid and Cosmid constructs. The plasmids encoding the six essential replication genes pDNA pol (pol) (m.u. 43.9-47.0), pCA35 (lef-2) (m.u. 5.6-6.5), cosmid 9 (lef-3, helicase) (m.u. 48.3-71.2), the stimulatory genes, and the origin-containing reporter pHdN (m.u. 8.1-11.1) which contains non-hr origin and lef-1 have been described previously (Fig. 2.1c) (1-5). Plasmid constructs pHdN, pDNA pol, pCA35, and cosmid 9 were provided by Dr. George Rohrmann. OpMNPV IE1 (pIEl-sal) and IE2 (pIE2-E2.3) clones, IE1-AD-, IE1 acidic domain chimeric constructs (Fig. 2.1a), IE1 N-terminal and C-terminal acidic domain deletion constructs (see Fig. 2.3), and the p39CAT-E3 reporter have been described previously (17). Replication assay. The replication assay used in this study has been described by Ahrens and Rohrmann (5). Log phase Ld652Y cells were seeded onto 6-well culture plates (1.0 X 106 cells/well) in TC-100 media containing 50 pg/ml of the antibiotic Gentamicin sulfate (Life Technologies) and incubated at 27°C overnight. The cells were transfected with DNA constructs using liposomes prepared as previously described (25). The optimal lipofectin volume/DNA ratio was determined by titration. Cotransfections were performed by mixing 1 p,g of pHdN non-hr origin containing reporter plasmid; 0.25 u.g of Cosmid 9; 0.5 pg of pDNA pol, pCA 35, IE2; 0.5 pg of various IE1 constructs (Fig. 2. 1). Each cotransfection was done in duplicate and amounts of DNA were equalized with pBS+ plasmid. After 4 hrs, the DNA-liposome transfection mixture was removed and the cells were washed with IX Grace's media and then overlaid with TC-100 media. After 62-65 hrs at 27°C, cells were removed with a rubber policeman washed once with 32 500 ul of phosphate-buffered saline (IX PBS is 0.14 M NaCl, 0.003 M KC1, 0.002 M KH 2 P0 4 , 0.01 M Na 2HP0 4) (pH 7.4) (202) and pelleted in a microcentrifuge (5 min at 6500 rpm). Total cellular DNA was extracted from replication samples by resuspending cells in 450 pi of lx TE buffer (10 mM Tris, pH 7.8, 0.6% SDS, 10 mM EDTA) and 50 pi of Proteinase K (20 mg/ml) and incubated at 55-65°C overnight. The samples were then extracted once with buffer saturated phenol, once with phenol-chloroform-isoamylalcohol (25:24:1), and once with choloroform-isoamyl alcohol (24:1). DNA was precipitated with 0.5 volumes of 7.5 M ammonium acetate and 2 volumes of ethanol and resuspended in 200 pi of lx TE (10 mM Tris, pH8.0, 1 mM EDTA). Five pgs of purified DNA were digested with a combination of Dpnl (10 U) and EcoKl (10 U) (New England Biolabs) in a total volume of 20 pi at 37°C overnight, followed by a 30 min digestion with 2pg of Rnase A. The digested DNA was separated in 0.7% agarose gels, alkaline blotted to BioRad zeta probe GT membrane (BioRad Inc), hybridized for 12 hrs with a 1.76 kb Hindlll-Xhol fragment of the pHdN reporter construct labelled with 3 2 P-dCTP (RadPrime labelling system; Life Technologies) in prehybridization buffer (0.25 M phosphate buffer [68.4 ml of 1 M Na 2HP0 4 plus 31.6 ml of 1 M NaH 2P0 4], 1 mM EDTA, 1% BSA and 7% SDS, pH 7.2) (36) at 65°C in a hybridization oven and washed under stringent conditions (O.lx SSC/0.1%SDS, 68°C [IX SSC is 0.15 M NaCl plus 0.015 M Na3C6H507.-2H20]) (10). Membranes were visualized on a Storm-phosphorimager (Molecular Dynamics). Al l transfections were repeated a minimum of 2 times, each in duplicate. CAT assay. To compare the activation of reporter plasmids by chimeric IE Is in the presence or absence of replication factors Ld652Y cells were transfected as described above. Cells were cotransfected with 1 pg of p39CAT-E3 reporter construct, IE1 chimeric constructs along with all the replication factor constructs. Duplicate wells were cotransfected with p39 CAT-E3 and 0.5 pg of IE1 chimeric constructs alone. To assay for chloramphenicol acetyltransferase (CAT) activity, cells were scraped off dishes 48 hours post transfection, pelleted (5min at 6,500 rpm), all media was removed, and the cell pellets were resuspended in 100 pi of 250 mM Tris-HCl (pH 7.8). Cells were lysed by repeated freeze thawing (three cycles) and any cellular deacetylases inactivated by incubation at 65°C for 15 minutes, followed by short centrifugation 33 (5 min at 6,500 rpm) to pellet cell debris. Cell extracts were titrated to determine the appropriate quantity of extract to use to ensure a linear response in the assay. To assay, the appropriate volume of cell extract (l-50pl) was added to the CAT assay buffer (6.25 mM chloramphenicol, 160 mM Tris-HCl (pH 7.8), 3.2 uM acetyl-Coenzyme A (Sigma) and 0.025 uCi (125 pmol) [3H]-acetyl-Coenzyme A (New England Nuclear, CAT Assay Grade) in a total volume of 125 pi (163). The mixture was overlaid with 3 mis of Toluene based scintillation flour (Econoflour-2, Packard Bioscience Co). All transfections were repeated a minimum of 2 times, each in duplicate. 34 RESULTS To initially investigate the role of the OpMNPV IE1 AAD in viral DNA replication we performed transient DNA replication assays using the replication genes and iel AAD chimeric constructs, IE1-AD- which has the AAD deleted, IE 1-OpAD which has the WT AAD, and two chimeric constructs which had the WT AAD replaced by the VP 16 AAD (IE 1-VP 16AD) and AcMNPV AAD (IEl-AcAD) (Fig. 2.1). Previous studies have shown that the latter two constructs are more potent activators of transcription than the constructs containing the WT AAD (IE 1-Op AD) (52). We wished to determine if stronger activators of transcription were also more effective in activation of transient DNA replication of the viral non-hr origin plasmid pHdN. In addition, the chimeric constructs would also show if the IE1 AAD is interchangeable for replication as has been shown previously for transactivation of transcription. Fig 2.2 shows the results of a Dpnl based transient replication assay using the different chimeric IE1 constructs to activate replication of the viral (non-hr) reporter plasmid, pHdN. The WT construct IE1-OpAD showed a strong Dpnl resistant replication signal of the origin plasmid. The OpIEl-AD-construct, which has the AAD completely deleted was unable to support replication and no signal was observed indicating that the acidic domain is essential for replication. Contrary to my hypothesis, for the two chimeric constructs containing heterologous AADs, IE 1-VP 16AD and IE 1-Ac AD there was no replication of the reporter plasmid. This is a significantly contrary result from transcriptional activation, where IE 1-VP 16 and IE 1-Ac A AD were shown to be strong transactivators (52). This shows that for viral DNA replication, unlike activation of transcription, the native OpMNPV IE1 AAD cannot be replaced by VP 16 AAD, or the more closely related AcMNPV IE1 AAD. To confirm that IE1 chimeric constructs were expressed and not inhibited by replication factors used in the transfection, we performed transient assays using the IE1 dependent OpMNPV enhancer containing reporter plasmid p39CAT-E3 (52). p39CAT-E3 was co-transfected with the chimeric IE 1 constructs with and without replication factor plasmids. The results show that the chimeric IE1 proteins were expressed and active in the presence or absence of replication 35 factors (Fig. 2 2b). Similar to results described by Forsythe et al (52), both IEl-AcAD and IEl-VP16AD were stronger activators of transcription. Higher expression is observed in the presence of replication factors which is likely due to the presence of IE2. These results show that the chimeric IE1 proteins containing VP 16 and AcMNPV IE1 AADs are non-functional for activation of OpMNPV DNA replication, which does not correlate with their potency in transcriptional transactivation. This indicates that an IE1 that is a strong transcriptional activator is not sufficient for supporting viral specific DNA replication. In addition, since the evolutionarily related AAD from AcMNPV was also non-functional it suggests that the OpMNPV IE1 AAD contains either host or virus specific determinants. Moreover, activation of transcription and replication appear to be separate functions based on the results with the chimeric constructs. Therefore it is possible that the AAD contains a domain specific for replication and exclusive of transcription. To identify any replication specific domains we used a series of AAD N- and C-terminal deletion clones of the OpMNPV IE1 AAD to determine the minimal sequence required for activation of DNA replication (Fig. 2.3). Previous analysis utilizing these clones have identified two domains, A (1-92 aa), and B (81-124) (Fig. 2.5), required for transcriptional activation in Ld652Y cells (52). The results of transient replication assays with WT IE1, and each N- and C-terminal AAD deletion construct is shown in Fig. 2.4. The ability of each construct to activate transcription is shown below each lane as indicated by the + or - symbol. The WT IE1 containing the intact N-terminal region of AAD activated replication, but surprisingly all deletion mutants, A14-25 through A14-143 representing 14 to 143aa of the IE1 N-terminus region (Fig. 2.3 and 2.4a) eliminated the ability to support replication of the pHdN reporter plasmid. As all the N-terminal deletions eliminated replication ability, the replication domain border could only be mapped to the first aa. Interestingly, even though all the N-terminal deletions abrogated replication many of them are still able to activate transcription (Fig. 2.4a). In particular, A14-25 and A14-27 transactivate transcription at levels equally to or greater than WT IE1 (52). This supports the results of Fig. 2.2a showing that a strong transcriptional activator is not sufficient for replication. To map the C-terminal border of the AAD domain required for replication the IE1 C-terminal deletions were used (Fig. 2.3, 2.4b). The C-terminal deletions Al 13-124 through A73-124 36 showed a strong replication signal. The largest deletion that showed a weak but reproducible replication signal was A66-124. Deletions larger than A66-124 (A51-124, A45-124, A36-124, A27-124 and A16-124) completely abrogated the replication signal (Fig. 2.4b). Therefore the C-terminus of the replication domain in the AAD maps to amino acid 65. Three C-terminal deletion clones A78-124, A73-124, and A66-124 are inactive for transcriptional activation yet are competent for DNA replication. Therefore the data from the N- and C-terminal deletions show that we can obtain IE1 constructs that are transcriptionally active and replication inactive or vice versa. This supports the view that these functions are independent of each other, that is, transcriptional activation is not required for IE1 to support DNA replication. Figure 2.5 shows a summary of the domains mapped to the OpMNPV IE1 AAD in Ld652Y cells. The replication domain contains a number of acidic and aromatic amino acids which have been shown to be involved in AAD function. No distinct homologies with known protein motifs were identified. Further In addition, comparison of predicted structures of the OpMNPV IE1 replication domain with the other baculovirus IE1 AADs did not identify anything that was consistent to all proteins. 37 DISCUSSION It has been shown that the AAD of OpMNPV IE1 is essential for transcriptional transactivation and contains at least two domains that interact synergistically (52). In this study the role of the IE1 AAD in viral DNA replication in Ld652Y cells was characterized using transient assays. Our results showed that the AAD of OpMNPV IE1 was essential for replication but quite surprisingly the heterologous AAD from VP 16 or the baculovirus AcMNPV IE1 failed to support DNA replication. These results reveal important differences between OpMNPV IE1 AAD and AADs in other systems. Previous studies have shown that AADs can be exchanged with heterologous domains for both replication and transcription (32, 44, 83, 84, 89,128, 129). Whereas, the OpMNPV IE1 AAD is not exchangeable for DNA replication, and in addition confers virus specificity. A deletion analysis mapped the minimum sequence of the OpMNPV IE1 AAD that permitted replication to amino acids 1-65. This overlaps with but is different from the Ld652Y cells minimal transcriptional activation sub-domains (A [l-92aa] and B [81-124aa], [Fig. 2.5]). The deletion analysis also identified mutants that were functional for replication and inactive for transcriptional activation. Conversely, mutants functional for transcriptional activation and inactive for replication were also identified. This indicates that transcriptional activation by IE1 is not required for transient DNA replication. Therefore the transcription and replication roles of IE1 are independent and separable. Baculovirus replication requires six essential factors, which are IE1, DNA POL, HELICASE, LEF-1, -2 and -3 (6, 103). Presumably, these factors associate to form a replication complex or replisome at the viral replication origin with or without cellular components. The nature of the interactions between these proteins is not yet clear but studies have identified some specific interactions. Using two-hybrid data and GST fusion experiments Evans et al. (48) have shown that LEF-1, a possible viral primase, interacts specifically with LEF-2 which has an unassigned function. The SSB protein LEF-3, forms homotrimers and has also been shown by 2-hybrid analysis to interact with PI43 (49, 50, 75). Co-transfection experiments have also shown that LEF-3 is required for the transport of PI43 from the cytoplasm to the nucleus (242). McDougal 38 and Guarino (145) showed that LEF-3 stimulated viral replication by improving strand displacement activity of DNA POL, which suggested a possible protein-protein interaction between these two proteins. IE1 is known to bind to baculovirus replication origins as dimers (119, 192, 196) suggesting that it is an origin binding protein. In the herpesvirus replication systems it has been shown that OBP (UL9) and SSB (ICP8) proteins interact resulting in local unwinding of the origin and stabilization of the single stranded conformation rendering it accessible to other replication proteins (20, 118, 143). Okano et al. (167) presented similar observations showing that IE1 is localized to discrete nuclear regions before the onset of replication. Subsequently, at the onset of replication LEF-3 co-localized with IE1 suggesting a possible interaction of IE1 with LEF-3 in the assembly of replication complex. This supports the view that IE1 acts as an OBP and initiates assembly of viral replication factories by interaction with the other replication proteins. Native AADs can be substituted by heterologous AADs from a variety of eukaryotic species and can remain functional in both transcription and replication activation (129, 150, 160, 187, 233) indicating that the functional mechanisms used by AADs to activate transcription and replication, are conserved across viruses, yeast and higher eukaryotes. Studies have shown that AADs activate transcription or replication by remodelling chromatin structure (89, 96, 127) or recruitment of proteins to either the replication or transcription initiation complexes (83, 128, 188). Our results show that the transcriptional transactivation activity of the IE1 AAD is not required for DNA replication. Therefore it is possible that the replication domain we mapped specifically activates replication by direct interaction with other components of the viral replication complex via the AAD domain (1-65 aa) instead of the transcriptional complex. As described above, IE1 is known to co-localize with LEF-3 suggesting a possible interaction between these two proteins. Studies with small DNA viruses have shown that the AADs of the transcription factors P53 and VP 16 activate replication by a mechanism that is dependant upon a direct interaction with the cellular SSB protein, RP-A. Mutations that abolish activation of replication also disrupt interaction between the AAD and RP-A (83, 128). OpMNPV IE1 AAD may be activating replication via similar mechanism, that is, LEF-3 or other replication proteins may directly interact with IE1 and be recruited to the replication origin. 39 Slack and Blissard (208) showed that AcMNPV contains a second activation domain C-terminal to the basic domain (Fig. 2.1a). OpMNPV IE1 contains homologous sequences but it is unknown these function as a transcriptional activating domain. The OpMNPV IE1-AD- mutant, which contains this region, does not activate early genes in the context of the native protein (Fig. 2.2b) (52). However, this second activation domain may also play a role in the replication complex possibly interacting with or recruiting other essential proteins. Our data showed that VP 16 and AcMNPV IE1 AADs are unable to replace OpMNPV IE1 AAD for replication. The specificity of the OpMNPV IE1 AAD could be due to interactions with a cellular factor provided by Ld652Y cells, or any of the OpMNPV viral replication factors provided in the transient replication assay. Past studies have shown that AcMNPV DNA replication occurs in Ld652Y cells (157). This suggests that AcMNPV IE1 AAD could interact with any required cellular factors. However, since IEl-AcAD is non-functional with OpMNPV replication factors in Ld652Y cells, it would support the notion that the AAD replication domain is interacting with a viral factor. Alternatively, in the presence of all viral proteins or alternate cell types, cellular factors or other viral proteins may also play a key role in the IE1 AAD viral DNA replication function. The amino acid sequences of the native acidic domains used for the construction of OpMNPV IE1 AAD chimeras were aligned using T-COFFEE programme (166) (Fig. 2.6). The alignment is colorized by the programme to show the correctly aligned portions. Overall there is no considerable sequence identity but the sequences were better aligned at C-terminal region of AAD in comparison to N-terminus. In summary, we have shown that the AAD of OpMNPV IE1 is essential for replication and the heterologous AADs from VP 16 or AcMNPV IE1 failed to activate DNA replication revealing important differences between OpMNPV IE1 AAD and AADs in other systems. A deletion analysis mapped OpMNPV IE1 replication domain to amino acids 1-65, which overlaps with, but is different from the minimal transcriptional activation sub-domains. We found that transcriptional activation by IE1 in transient assays is not required for replication and that the transcription and replication roles of IE1 are independent and separable. These findings will 40 help to reveal the mechanism by which IE1 stimulates viral DNA replication and suggests possible interactions with other viral proteins to form the replication complex. 41 Fig. 2.1. Schematic of the OpMNPV IE1 and the chimeric constructs used for characterizing the role of AADs in viral DNA replication, a) Wild type OpMNPV IE1 protein showing the functional domains based on studies of OpMNPV IE1 and the homologous AcMNPV IE1 (108, 197). The 560 aa of OpMNPV IE1 contains 126 aa AAD (AAD), bordered by a cluster of highly conserved basic amino acids (B-I); the C-terminal region contain a possible second trans-activation domain (D) (208), a second region rich in basic amino acids (B-II) and domains required for DNA binding (DBD), and oligomerization (OD). b) Schematic diagrams of the OpMNPV IE1 AAD chimeras. IE1-AD- has the complete AAD removed. The IE1-AD- was used to insert the acidic domains from OpMNPV IE1 (IEl-OpAD), HSV VP16 (IE1-VP16 AD), and AcMNPV IE1 (IEl-AcAD). Clone designation is given on the left and the number under each clone name represents the chimeric protein length in amino acids. The numbers under the AAD indicate the location of the amino acids from the native VP 16 and AcMNPV IE1 proteins, c) The length and the amino acid sequences of the native acidic domains used for the construction of OpMNPV IE1 AAD chimeras, d) Hindlll restriction map of the OpMNPV genome with arrows showing the location Of the genes used in the transient replication assay. Cosmid 9 is used to supply lef-3 and helicase, plasmid pHdN is used to supply viral non-hr origin of replication and lef-1, lef-2, ie2, and iel are supplied by individual plasmid constructs. 42 (a) (b) (c) WT (560aa) AA B-l D DB B-IIOD IE1-AD-(437aa) !E1-OpAD (560aa) IE1-AcAD | (589aa) OpMNPVIEl AAD (2-126 aa) AcMNPV IE1 AAD (2-151 aa) IE1-VP16AD (516aa) OpMNPV IE1 AAD (aa2-T26) NMETLQRSYMGPSTPNHNLFNNATELPDDLNFSTMDVPYDGSMPMNMSSDSLMNLLEDRSKKLACAVDTELARESTASE FVAGFSADSPQAQLAETGAETGAAGGSKRKASEVDSDSDSDDS AcMNPV IE1 AAD (33 2-151) TQINFNASYTSAS7PSRASFDNSYSEFCDKQPNDYLSYYNHPTPDGADTVISDSETAAASNFLASVNSLTDNDLVECLLKTTD NLEEAVSSAYYSESLEQPWEQPSPSSAYHAESFEHSAGVNQPSATGTKRKLDEYLDNSQGWGQFN HSV VP16AD (aa 413--49Q) PPTDVSLGDELHLDGEDVAMAHADALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG ® lef-1 «— Hindlli-N T. N. . H I R K C DNApol lef-3 helicase < < — < ^ ^ ^ L . D U i v B , P , J , G , 0 F , E M Q iel lef-2 — * ie2 < — A O p M N P V Hindlll map 43 Fig. 2.2. Transient Dpn\ replication assays for the analysis of the IE1 chimeras ability to activate replication, a) Ld652Y cells were cotransfected with five viral replication genes (lef-1, -2, -3, helicase, DNA pol), ie2, the viral non-/V origin of replication pHdN and with different IE1 chimeras. The number to the left of the blot corresponds to the size (in kb) of the hybridized band of the reporter plasmid pHdN linearized with EcolU. The name of the sample corresponding to each lane is shown on the top of the blot. Each sample is presented in duplicate and representing two separate transfections. The negative control lane (pBS+) contains iel replaced by pBS+ and lacks any replication signal indicating there is no background replication. The lane M is a marker lane which is the reporter plasmid pHdN linearized with EcoRl mixed with genomic DNA cut with Dpnl. b) Transactivation analysis of p39CAT-E3 by IE1 chimeric proteins in the presence or absence of replication factors in Ld652Y cells (as described in materials and methods). All transfections were repeated a minimum of two times, each in duplicate. All CAT activities obtained are reported relative to the reporter plasmid cotransfected with IEl-OpAD, which was given arbitrary value of 100. Error bars indicate standard error. 44 (a) IE1- IE1- IE1- IE1- w OpAD AD- AcAD VP16AD <£_ C M AD- Op A c V P 1 6 + AD- Op Ac Vp16 + AD AD AD m AD AD AD £ P39CAT-E3 P39CAT-E3 +Replication genes 45 Fig. 2.3. Schematic diagrams of deletion mutants used to map the regions of the OpMNPV IE1 AAD required for DNA replication, a) Amino acid sequence of the N-terminus of OpMNPV IE1 (aa 1- aa 150). Boxed amino acids indicate basic domain (B) (aa 147- aa 143) that borders the AAD (aa 1- aa 126). b) Diagram of AAD N-terminal and c) C-terminal deletion clones. Each deletion mutant has been designated according to the amino acids that have been deleted which are shown in a. Clone WT and WT4 are the full length IE1 AAD for N- and C- terminal deletion analysis respectively. The G in the C-terminal deletions refers to the glycine that was inserted during the construction of the deletions (52). The domain designations are the same as that shown in Fig. 2.1. The ability of each sample to activate transcription or replication is indicated by a + or - symbol towards right side of each clone. 46 (a) 10 20 20 40 50 MPKMtETLQR SKMQPSTPNH HLFNHATELP DDLNFSTMDV PYD3SMPMNM 60 70 BO 90 100 SSD&LHNLLB DRSKKIACAV DTELOREETA SEFVAGP&OD SPQAQL&E1X} 110 120 120 140 _ _ ' 150 HETGBAQGSK RXftSEVDSDE DSDDSSKGKK LVNKPKIE.QEYKKATIQHRT AAD B-1 D DBD B-11 OD AD-•1: WT • A14-3 • AT4-27 • A14-13 • A14-44 • A14-52 • A14-56 • AI4-62 • A14-66 • £14-68 • A14-74 • A14-76 • A14-B0 • A14-B4 • A14-B5 • A14-SE • A14-96 • Ali-104 • A14-1Q5 • A l i 107 • A14-121 • A14-127 • A14-127 • (A2-127) i Transcription + + + + + + 4-+ + + + + + + + + 47 Fig. 2.4. Transient Dpnl replication assays to map specific regions of OpMNPV IE1 AAD required for replication, a) N-terminal deletion clones and b) C-terminal deletion clones as described in Fig. 2.3. Ld652Y cells were co-transfected with the appropriate iel construct, plasmids with five viral replication genes (lef-1, -2, -3; helicase; DNA pol and ie2) and a reporter plasmid with non-hr viral origin of replication (pHdN). The arrow to the left of the blot corresponds to the hybridized band of linearized pHdN. The name of the sample corresponding to each lane is shown on the top of the blot. In the control lane (pBS+) iel is replaced with the plasmid pBS+. The ability of each sample to activate transcription as previously determined (52) is indicated by a + or - symbol at the bottom of the blot. Each sample is in duplicate except pBS+ and clone A14-107 for which there is only a single lane. 48 (a) N-terminal deletions Fig. 2.5. Schematic diagram showing the summary of domains identified in the OpMNPV IE1 AAD. Two domains, A (1-92) and B (81-124) required for transcriptional transactivation in Ld652Y cells were previously mapped (17). The location and amino acid sequence of the replication domain is shown below the transcription activation domains. The sequence of the minimal replication domain (aa 1-65) is shown and the symbols at the bottom refer to the charge distribution: acidic amino acid (-) and basic amino acid (+). 50 AAD B D DBD OD 1 . . rr?-7: : : : 1 Trans, domain A (1-92 aa) I eplication domain A (1-65 aa 1 65 72 MPKIVMETLQRSYMGPSTPNHNLFNNA^ + - + . . . . . . . . + + + 51 Fig. 2.6. Alignment of the acidic activation domains. The amino acid sequences of the native acidic domains used for the construction of OpMNPV IE1 AAD chimeras were aligned using T-COFFEE programme (166). OpAD refers to OpMNPV IE1 AAD, AcAD refers to AcMNPV IE1 AAD and Vpl6AD refers to HSV VP 16 AD. The alignment is colorized by the programme to show the correctly aligned portions. (http://www.ch.embnet.org/software/TCoffee.html). 52 LD A V ; OpAD AcAD V p l 6 4 9 5 2 4 2 OpAD AcAD KNMETLQR -TQINFNA Vpl6 Cons TELPD YSEFCDKQPND PPTDVSLGD- ELHL^MDVA- -MDVPYDGSMPM- -PT? - H H D T V I S OpAD AcAD Vpl6 Cons MS S D S LMN"LL EDR S KKL AC AVD TEL ARE S T ASEF VjAG F TDNDLVE CLLKTTDNLEEAVSSAYYSESLEQPVVEQP DMLGDGDSPGPGFTPHDS OpAD AcAD V p l 6 Cons SADSP - QAQLAE TG^  SPSSAYHAESFE HS AG: APYGALDMADFEF EQMFTDALG EVDSDSDSDDS LDNSQGVVGQFN 53 CHAPTER 3. The OpMNPV IE1 acidic activation domain determines the specificity of the viral replisome INTRODUCTION The acidic activation domains (AADs) of eukaryotic transcription factors are bifunctional in that they can enhance both transcription and replication. Several AADs have been shown to function independently when fused to DNA binding domains of heterologous activators (32, 187). In the case of OpMNPV, IE1 has been shown to be essential for both transcription and replication and to contain an AAD at the N-terminus (52). Previously (see chapter 2) we have characterized the OpMNPV IE1 AAD using chimeric constructs including IEl-AcAD which has AcMNPV IE1 AAD and IE1-VP16AD which has Herpes simplex virus type 1 (HSV-1) VP16 AAD. Both of these chimeric proteins were strong transcriptional activators but completely inactive for supporting viral DNA replication. This suggests that heterologous VP 16 and AcMNPV IE1 AADs cannot replace OpMNPV IE1 AAD in stimulating replication, a finding that differs from that found in other systems (32, 44, 84, 89, 129). Deletion analysis of the AAD in Ld652Y cells showed that the transcription and replication roles of OpMNPV IE1 are independent and separable. Also, a replication domain within OpMNPV IE1 AAD was mapped to amino acids 1-65, which overlaps with, but is different from the minimal transcriptional activation sub-domains (Chapter 2). These results suggest that the specificity of the OpMNPV IE1 AAD in replication could be due to its interactions with a cellular factor provided by Ld652Y cells, or any of the OpMNPV viral replication factors provided in the transient replication assay. Past studies have shown that AcMNPV DNA replication occurs in Ld652Y cells suggesting that AcMNPV IE1 AAD could interact with cellular factors available in Ld652Y cells (157). However, since IEl-AcAD is non-functional with OpMNPV replication 54 factors in Ld652Y cells, it would support the notion that the AcMNPV IE1 AAD replication domain requires AcMNPV replication factors to function. Studies with small DNA viruses have shown that the AADs of the transcription factors P53 and VP 16 activate replication by a mechanism that is dependent upon a direct interaction with the cellular SSB protein, RP-A (83, 128). OpMNPV IE1 AAD may be activating replication by a similar mechanism, that is, LEF-3 or other replication proteins may directly interact with IE1 and be recruited to the replication origin. These interactions might be specific to AAD and replication factors. It is possible that AcMNPV IE1 AAD in the IEl-AcAD chimeric protein lacks these interactions with OpMNPV factors resulting in loss of replication. In this study we investigated these possible interactions by providing AcMNPV replication factors to the IE1-AcAD chimeric protein. Transient replication assays were performed with IE 1-Ac AD substituting OpMNPV replication factors with AcMNPV factors in single, dual or multiple combinations. In addition we compared OpMNPV IE1, full length AcMNPV IE1 and IE1-AcAD replication activation function with combinations of OpMNPV and AcMNPV replication factors. Our results showed multiple substitutions with AcMNPV lef-1, -2, -3, and helicase proteins enabled IE 1-Ac AD to activate replication. This suggests that AcMNPV IE1 AAD interacts with replication factors and has specificity for the homologous essential replication factors of the replisome. Surprisingly, full length AcMNPV IE1 also has similar specificity for the homologous factors suggesting that just changing the AAD changes the specificity for the replisome. OpMNPV IE1 was found to be promiscuous or less specific than AcMNPV IE 1 AAD and it can activate replication with both AcMNPV and OpMNPV replication proteins or their combinations. Finally, these results show that the IE1 AADs plays a key role in determining replisome specificity. 55 MATERIALS AND METHODS Cell Culture. Lymantria dispar (Ld652Y) cells were maintained in TCI00 medium as described (216). Plasmid and Cosmid constructs. OpMNPV constructs: The plasmids and cosmids encoding the replication genes of OpMNPV have been described previously in the material and methods of chapter 2 and in Fig. 3.1b (1, 3-7). Plasmid pHdN (m.u. 8.1-11.1) and pHdNA (m.u. 8.1-10.0) are the non-hr origin containing reporters. Plasmid pHdNA, the reporter without lef-l was constructed by cloning the 2.7 kb fragment of a Notl-Hindlll (m.u. 8.1-10.0) digest of pHdN into pBS+. Plasmid Op lef-l, the OpMNPV lef-l containing plasmid was constructed by cloning a 2.5 kb of the Op cosmid 1 Hindlll-Xhol (m.u. 9.5-11.1) digest into pBS+. For OpMNPV helicase, a 5.32 kb fragment of Xhol-Sstl (m.u. 61.4-65.4) digested viral DNA was isolated and cloned into pBS+. Cosmid 9 is used to supply both lef- 3 and helicase; cosmid 55 (m.u. 20.6-40.8) is used to supply only lef-3. Constructs pHdN, pDNA pol, pCA35, cosmid 55 and cosmid 9 were kindly provided by Dr. George Rohrmann and described previously (1,6). OpMNPV IE1 acidic domain chimeric constructs IEl-AcAD, IE1-VP16AD; OpMNPV IE1; IE1 acidic domain less construct (IE1-AD-) (Fig 3.1a); and IE2 (pIE2-E2.3) clones have been described previously (52). AcMNPV constructs: The plasmids encoding the replication genes of AcMNPV have been described previously (103). AcMNPV lef-3 is located on SstF and was cloned as an ^ al-^coRI fragment (m.u. 42.8-44.5) into pKS-. The construct AcMNPV IE1 has been described previously (221). All the replication genes of both OpMNPV and AcMNPV are under their respective native promoters. 56 Replication assay. As per chapter 2 except DNA is purified by using Qiagen DNeasy tissue kit. Briefly, the cell pellet from replication samples was resuspended in 200 pi PBS and total cellular DNA was extracted from replication samples by using Qiagen DNeasy tissue kit. We used the RNase free protocol for cultured Animal cells with minor modification of using 50 pi of proteinase K and incubating at 55-65°C overnight. 5 pi of purified DNA sample was ran in 1% agarose gel and quantified using ID multi-Alphalmager 3300, Image analysis software (Alpha Innotech Corporation). High DNA mass ladder (Invitrogen) was used as a standard for quantification. 2.5 pgs of purified DNA was digested with a combination of Dpnl (10 U) (New England Biolabs) and Sstl (10 U) (Invitrogen) restriction enzymes overnight. The digested DNA was separated and analyzed by southern blotting as per chapter 2. All transfections were repeated a minimum of 2 times, each in duplicate. 57 RESULTS OpMNPV IE1 with its native AAD is essential and active for both transcription and replication. Replacing the native AAD of OpMNPV IE1 with the AAD from the evolutionarily related AcMNPV IE 1 produces a chimeric protein IEl-AcAD that is a potent transactivator but is inactive for OpMNPV DNA replication (Chapter 2). This suggests that the AcMNPV AAD lacks interactions necessary for DNA replication with OpMNPV virus specific or host replication factors. In this study we determined if IEl-AcAD DNA replication function can be enabled by substituting OpMNPV essential replication factors with their counterparts in AcMNPV. Transient replication assays were performed with IEl-AcAD and by substituting OpMNPV replication factors with AcMNPV factors in single, dual or multiple combinations. Replication assay of reporter pHdNA For our previous studies, pHdN was used as an origin containing reporter for transient replication assays. This reporter contains both the OpMNPV non-hr origin and the lef-1 gene on one plasmid (179). In order to use this for substitutions with AcMNPV lef-1, a deletion clone of pHdN was made that has only the replication competent region without lef-1 (Fig. 3.2a). This reporter, which is called pHdNA, was then tested for its ability to serve as a reporter in transient DNA replication assays in Ld652Y cells (Fig.3.2b). The results demonstrated that the construct pHdNA is active in replication and the levels of replication are similar to that of pHdN. We previously showed that pHdN non-hr was inactive when the OpMNPV chimeric proteins, IE 1-Ac AD and IE1-AD- were used. These constructs were also found to be unable to replicate pHdNA (Fig. 3.1c). This shows that pHdNA acts a replication origin similar to pHdN and can be used as a functional replication reporter. Single substitutions of AcMNPV replication factors The OpMNPV chimeric protein IE 1-Ac AD, which has heterologous AcMNPV AAD is inactive in replication with OpMNPV replication factors. This suggests that there might be virus 58 specific interactions between the AAD and the remaining replication factors lef-1, -2, -3, DNA pol or helicase which would be necessary for activation of replication to occur. To test this prediction we performed transient replication assays with IE 1-Ac AD and substituting single AcMNPV factors for the corresponding OpMNPV replication factors. Fig. 3.3a shows the results of AcMNPV lef -1, -2, -3, and helicase substitution and Fig. 3.3b shows the results of the AcMNPV DNA pol substitution. None of the single factors enabled IEl-AcAD to activate replication. These results suggest that IEl-AcAD might require more than a single AcMNPV replication factor. We therefore tested with dual factor substitutions of OpMNPV with AcMNPV factors. Dual combinations of substitutions of AcMNPV replication factors Transient replication assays were performed with the chimeric protein IE 1-Ac AD and substituting AcMNPV factors in dual combinations into corresponding OpMNPV replication factors. Fig. 3.4 a&b shows the results of AcMNPV lef-1, -2; lef-2, -3; lef-1, helicase; lef-2, helicase; lef-3, helicase; and lef-3, DNA pol factor substitutions into corresponding OpMNPV factors. Lef-1, -2 and lef-3, helicase have been shown to interact and these factors may act in concert. However, none of the dual factors were able to restore IE 1-Ac AD ability to activate replication. We therefore continued the replication assays using multiple factor substitutions. Multiple combinations of substitutions of AcMNPV replication factors Transient assays were performed with the chimeric protein IE 1-Ac AD and substituting AcMNPV factors in multiple combinations for the corresponding OpMNPV replication factors. Fig. 3.5a, b, & c show the results of multiple factor substitutions. The combinations AcMNPV lef-2, -3, helicase; lef-3, DNA pol, helicase; lef-1, -2, -3 did not enable IEl-AcAD to activate replication. However lef-1, -2, -3, helicase and lef-1, -2, -3, helicase, DNA pol combinations restored the ability of IEl-AcAD ability to activate replication. These results suggested that the minimal AcMNPV replication factors required for IE 1-Ac AD to support replication of the OpMNPV non-hr origin in Ld652Y cells are lef-1, -2, -3, and helicase. It is therefore possible that the IE1 AAD requires minimum of these four factors to form a specific replisome complex 59 and initiate DNA replication. Other than IE land POL, these four factors LEF-1, -2, -3 and HELICASE represent the core factors of the viral replisome complex. Past studies also showed that DNA POL is not species specific and can be interchangeable (4, 136). Lu and Miller (136) in their transient replication assays found that viral DNA POL is not essential but stimulated plasmid replication suggesting that a cellular DNA polymerase can substitute for viral POL. Since our experiments are dealing with swapping of specific viral replication factors, taking into account the promiscuous nature of POL and for convenience of use we referred replisome to only IE1, LEF-1, -2, -3, AND HELICASE. Specificity of AcMNPV IE1 AAD IEl-AcAD was active for replication only with the AcMNPV replisome (Fig. 3.5c) and not with the OpMNPV replisome (Fig. 3.2c). To confirm that IE1 AcAD activation of replication is dependent on the AcMNPV acidic domain, transient replication assays were performed using IEl-AcAD and IE1-AD- (the AAD less construct). In addition, to also show that IEl-AcAD replication activation with the AcMNPV replisome was specific to the AcMNPV AAD, transient replication assays were also performed with IE 1-VP 16AD the chimeric construct which has Herpes virus VP 16 AAD (Fig. 3.1a). The IE1-AD- construct was unable to support replication indicating that the acidic domain is essential for IE-Ac AD to activate replication with the AcMNPV replisome (Fig. 3.6a). The chimeric construct IE 1-VP 16AD was also unable to support replication indicating that the interaction between the AAD and AcMNPV replisome is virus specific (Fig. 3.6b). Comparison of OpMNPV IE1, AcMNPV IE1 or IEl-AcAD ability to activate replication. These results show that replacing the OpMNPV IE1 AAD with the AcMNPV IE1 AAD the specificity changed from the OpMNPV to the AcMNPV replisome. Therefore just switching the AAD, changes the specificity of the protein, or in other words IE 1-AcAD appears to function similarly to AcMNPV IE1 and not OpMNPV IEL To seek further evidence in support of this result, transient replication assays were performed with IEl-AcAD, full length OpMNPV 60 IE1, and AcMNPV IE1 in the presence of all OpMNPV replication factors (lef-1, -2, -3, helicase, DNA pol, and iel) and substituted with AcMNPV factors lef-1, -2; lef-3, helicase, DNA pol; and lef-1, -2, -3, helicase, DNA pol. The experiment shown in Fig. 3.7 compares the ability of OpMNPV IE1, IE 1-AcAD and AcMNPV IE1 proteins to activate replication from the non-hr origin reporter. OpMNPV IE1 is able to activate replication with OpMNPV, AcMNPV and all the combinations of replication factors tested suggesting OpMNPV IE1 AAD is promiscuous. Further more, OpMNPV IE1 appears to be a stronger activator of replication with heterologous factors compared to homologous replication proteins. AcMNPV IE1 (similar to IEl-AcAD) is active for DNA replication only in the presence of the AcMNPV replisome. This supports our prediction that just changing AAD changed the virus specificity of the IE1 protein. Collectively our results suggest that AADs play a role in replication by interacting with viral replication factors and these interactions for AcMNPV are virus specific. 61 DISCUSSION Our previous studies (Chapter 2) showed that AcMNPV IE1 AAD in the chimeric protein IE1-AcAD is non-functional with OpMNPV replication factors in Ld652Y cells. It is predicted that the chimera, IE 1-AcAD might lack interactions with OpMNPV replication factors provided in the replication assay thus resulting in loss of replication. In our present study, we tested the possibility of enabling IE 1-AcAD to support replication by substituting OpMNPV replication factors with AcMNPV replication factors. Our results show that IE1 -AcAD is active in replication with AcMNPV lef-1, -2, -3, and helicase proteins providing evidence for AcMNPV AAD interaction with replication factors and that AAD determines the viral replisome specificity. Both IE 1-AcAD and full length AcMNPV IE1 function similarly suggesting that replacing AAD modified the chimera to function as AcMNPV IEL OpMNPV IE1 was able to interact with all AcMNPV factors suggesting that OpMNPV IE1 AAD is promiscuous or less specific than AcMNPV IE1 AAD. In baculoviruses, six proteins have been identified as essential for replication activation from hr and non-hr origins. The respective replication proteins are IE 1, DNA POL, HELICASE, LEF-1, -2, and -3 (6, 103). To date the various complexes that have been characterized are viral primase (LEF-1 and -2) (48, 152,(E) SSB and helicase complex (LEF-3 and helicase) (49, 50, 75); SSB and pol complex (LEF-3 and POL) (145). Presumably, these complexes form a replisome at the origin and initiate replication. It is not known in baculoviruses how these complexes are recruited to the origin. In the case of other eukaryotic viruses, similar complexes are recruited to the origins of replication by initiator proteins. For example, UL9 of HSV, T-antigen of SV40 virus, and El of Papillomavirus are shown to act as initiators of replication (118). Their interaction with SSB proteins, primase complex, helicase complex and polymerase complexes has been shown (74, 237). In particular UL9 has been shown to interact with at least one partner of each of the viral protein complexes required for HSV DNA replication (94). IE1 in the past has been shown to bind to the hr origin of replication as a dimer, similar to the UL9 protein of HSV, suggesting it could function as a Initiator protein (Chapter 2) (119, 192, 196). 62 Studies have also shown that activation domains interact with the cellular SSB protein RP-A (83, 128). Taken together it is possible that IE1 like other initiators described above might bind to the origin and through its AAD, interact with viral replication factors recruiting them to the origin of replication. However, there is limited data available on IE1 AAD interactions with replication factors. Our present study examines interactions between IE1 AAD and various replication proteins and their effect on replication. The OpMNPV IE1 AAD when replaced with AcMNPV IE1 AAD was inactive in replication with OpMNPV factors suggesting a possible species specific interactions between AAD and replication proteins. If this is true, then substituting AcMNPV factors for the OpMNPV replication factors, the species-specific interactions between AcMNPV IE1 AAD and replication proteins would initiate replication. This possibility was tested initially with single and pairs of replication factors (Fig. 3.3 and 3.4). The pairs were chosen according to the complexes discovered previously such as viral primase (LEF-1 and-2) (48, 152); SSB and helicase complex (LEF-3 and helicase) (49, 50, 75); SSB and pol complex (LEF-3 and DNA POL) (145). Contradictory to our previous prediction all single and pairs of replication factors tested did not enable IEl-AcAD to activate replication. This suggested that AAD might interact with more than two replication factors or the factors AAD interacts with may in turn require other homologous replication factors for the replisome complex assembly (eg AAD interacts with LEF-3 and LEF-3 binds with HELICASE and HELICASE with DNA POL). To test this, IE1-AcAD was tested with multiple combinations of OpMNPV and AcMNPV replication factors. Interestingly the ability of IE 1-Ac AD to activate replication was restored only when AcMNPV /e/-l,-2,-3, and helicase proteins were used to replace the corresponding OpMNPV replication proteins. These replication proteins constitute the minimal replisome. The results also show DNA POL is interchangeable as IE 1-Ac AD is active with both OpMNPV and AcMNPV DNA POL. This agrees with the past studies, which also showed that DNA POL is interchangeable (4,136). Our results show that the AAD requires an entire homologous core replisome to activate replication. This suggests that the AAD determines the specificity for the core virus replisome. IE1-AD- and IE1-VP16AD were inactive with the AcMNPV IE1 replisome complex, showing the dependence upon the AAD, and the specificity of the AcMNPV IE1 AAD for the AcMNPV replisome. 63 These results do not show how many of the AcMNPV factors (lef-1, -2, -3, and helicase) directly interact with the AAD. As described above, other DNA virus OBPs have independent multiple binding sites to interact with SSB, helicase and the primase sub-complex and recruit them to the replication complex (118). This supports the possibility that AcMNPV IE1 AAD has multiple determinants that can interact with all four proteins in the minimal replisome and recruit them to the origins. In addition the interactions between lef-1,-2,-3 and helicase to form the AcMNPV replisome may also be virus specific. Changing the OpMNPV AAD with the AcMNPV AAD resulted in a chimeric protein that functioned similarly to native AcMNPV IE1 (Fig. 3.7). That is, AcMNPV IE1 is only active for replication when associated with the AcMNPV replisome. Surprisingly OpMNPV IE1 was able to activate replication with OpMNPV, AcMNPV and all the combinations of replication factors tested, suggesting that OpMNPV IE1 AAD is relatively promiscuous. This is unlike AcMNPV IE1 AAD, which has strict specificity for its homologous replication factors. Past studies also showed similar results where OpMNPV IE1 was able to activate replication with AcMNPV HELICASE and the rest of the OpMNPV replication proteins. However, in the reciprocal experiment, AcMNPV IE1 was unable to activate replication with OpMNPV HELICASE and the rest of the AcMNPV replication proteins (4). Therefore the results suggest that OpMNPV IE1 AAD might have multiple determinants that can interact with both OpMNPV and AcMNPV replication proteins and hence this AAD is promiscuous. The polyomavirus SV40 replication initiator T-antigen has been shown to have specificity in binding to replication factors. T-antigen can support replication only with the human DNA polymerase alpha-primase but not with the mouse homolog. However, the human polyomavirus JC virus can support replication with both human and murine DNA polymerase alpha-primase (211, 212). In addition T-antigen of SV40 strongly interacted with human RPA (SSB) but poorly interacted with Schizosaccharomycespombe RPA (237). Further studies with the SV40 large T-antigen, suggested the J domain harbours species-specific elements required for viral DNA replication (213). 64 The amino acid sequences of the OpMNPV IE1 AAD (1-126 aa) were compared with AcMNPV IE1 AAD (1-151 aa) using T-COFFEE alignment programme (166) (Fig. 3.8). The alignment is colorized by the programme to show the correctly aligned portions. Overall there is no considerable sequence similarity however ther regions that are alighned are bettere towards C-terminal region of AAD than compared to N-terminus. Studies in chapter 2 (Fig. 2.5) identified a specific sub-domain (l-65aa) for replication. This region was predicted to interact with OpMNPV factors. Since AcMNPV IE1 AAD has very little sequence homology to this region of OpMNPV IE1 AAD, it suggests that it might not interact with OpMNPV replication factors and hence inactive for replication. Compared to N terminal region there is considerable homology between OpMNPV IE1 AAD and AcMNPV IE1 AAD at the C-terminus region. It is possible that this region in AcMNPV IE1 AAD interacts with replication factors. Since the OpMNPV IE1 has this region it is possible that it might be able to interact with AcMNPV replication factors allowing it to be active in replication with AcMNPV proteins. This would explain why OpMNPV IE1 is promiscuous in activation of replication with both OpMNPV and AcMNPV replication factors. In summary, as described in Fig. 3.8, our results have shown that the AcMNPV IE1 AAD determines specificity for the homologous replisome as both IE 1-Ac AD and full length AcMNPV IE1 functioned similarly. OpMNPV IE1 was able to utilize all AcMNPV factors suggesting the OpMNPV IE1 AAD is promiscuous or less specific than AcMNPV IE1 AAD. However, it is possible that the cell type accessory factors or origins of replication may also affect the specificity of OpMNPV IE1 and will be investigated in future studies. 65 Fig. 3.1. Schematic of OpMNPV IE1, the chimeric IE1 proteins, and the replication genes of OpMNPV and AcMNPV used to study IE1 acidic activation domain interactions with viral replication factors, a) The functional domains identified in both OpMNPV and AcMNPV IE Is are represented by the block letters above the constructs. AAD, acidic activation domain; BDI, basic domain one; AD, second activation domain; DBD, DNA binding domain; BDII, basic domain two; and OD, oligomerization domain. OpMNPV IE1 and AcMNPV IE1 are the wild type IE Is of OpMNPV and AcMNPV. IE1-AD- has the complete AAD removed. The IE1-AD- was used to insert the acidic domains from AcMNPV IE1 (IE 1-AcAD), HSV VP 16 (IE1-VP16 AD). The Clone designation is given on the left and the number under each clone name represents the chimeric protein length in amino acids, b) HindlH restriction map of the OpMNPV genome. The arrows above the map indicate the location of replication genes used in the transient replication assay. Cosmid 9 was used to supply lef-3 and helicase; Cosmid 55 is used to supply only lef-3. Hind III N is the region containing a putative non-hr origin of replication, c) Hindlll restriction map of the AcMNPV genome. The arrows above the map are the location of replication genes used in the transient replication assay. Sstl-¥ is used to supply lef-3 and DNA pol. 66 (a) A A D B D I D D B D OpMNPV IE1 (560 aa) IEl-AcAD (589 aa) IE1-VP16AD (516 aa) IE-AD-(437 aa) AcMNPV IE1 (5S2 aa) B D I I O W/ndlll restriction map of OpMNPV lef-2 lef-l tf/ndill-N (non-hr orgln) A T N S H I R K C dnapol tef-3 L D U,V helicase Cosmid 55 Cosmid 9 P J G 0 F E M Q lef IG2 —¥ •+ (c) W/ndlll restriction map of AcMNPV lef-2 lef-l dnapol lef-3 helicase le] F V T N D X J L M R E O U I B C W H S Al A 2 K Q P G F 1 1 1 1 11 1 1 1 1 1 1 1 1 1 I  H I 1 lil 1 67 Fig. 3.2. Construction of plasmid pHdNA and replication assay to test its ability to act as a replication reporter in comparison to pHdN. a) Linear maps of plasmids pHdN and pHdNA. The vertical dashed lines mark the boundaries of replication competent region located in the 557 nt between nt 1786 and 2342 (179). The position of essential replication gene lef-1 present in pHdN is indicated. pHdNA is the Notl-HindlYl deletion of pHdN constructed to knock out lef-1. b) Comparison of pHdN and pHdNA in transient replication assays, c) Transient replication analysis of pHdNA using OpMNPV IE1, IE 1-AcAD and IE1-AD-. Both pHdN and pHdNA reporters were tested for replication in Ld652Y cells in presence of OpMNPV core replication genes (lef-1, -2, -3, helicase, DNA pol) and ie2 as described in materials and methods. The arrows and the numbers represent the location and sizes of the linearized pHdN and pHdNA reporters. The name of the sample corresponding to each lane is shown on the bottom of the blot. Each sample is presented in duplicate and representing two separate transfections. The negative control lane (pBS+) lacks any replication signal indicating there is no background replication. All transfections were repeated a minimum of 2 times, each in duplicate. 68 non-hr origin 4 1 Hindi II Sol i — J | Not\ < H/ndlll Hindll! (b) pHdNA (5.64kb) pHdN pHdN A (c) 1 2 3 4 pHdNA(5,64kb) OpMNPV I El-AcAD I El-AD- pBS+ IE1 All OpMNPV replication factors (lef-1 ,-2,-3, pol, he licase, ie2) 69 Fig. 3.3. Ability of IE 1-AcAD to support non-hr origin replication by substituting OpMNPV essential replication proteins with single homologous AcMNPV genes. The individual factors lef-1, lef-2, lef-3, helicase and DNA pol were tested using the pHdNA (a) or pHdN (b) reporter constructs. Transient replication assays were performed as described in the materials methods with the AcMNPV replication gene replacing the homologous OpMNPV gene. The location and size of the linearized pHdN or pHdNA is shown on the right of the blot. The name of the sample corresponding to each lane is shown on the bottom of the blot. All transfections were repeated a minimum of 2 times, each in duplicate. 70 1 2 3 4 5 6 pHdNA-OpMNPV Substituted AcMNPV E l replication factors: IE!-AcAD lef-1 lef-2 lef-3 helicase 1 2 3 pHdN(6,9kb)-OpMNPV Substituted AcMNPV I E 1 replication factors: IE1-AcAD Pol 71 Fig. 3.4. Ability of IE 1-AcAD to support non-hr origin replication by substituting OpMNPV core replication proteins with pairs of homologous AcMNPV genes. The pairs of AcMNPV replication proteins substituted for the homologous OpMNPV proteins are indicated below the blots, (a) AcMNPV lef-1, -2; lef-2, -3; lef-1, helicase; lef-2, helicase; and lef-3, helicase; and (b) AcMNPV lef-3, DNA pol were tested using the pHdNA (a) or pHdN (b) reporter constructs. Transient replication assays were performed as described in the materials and methods with the AcMNPV replication genes replacing the homologous OpMNPV genes. The location and size of the linearized pHdN or pHdNA is shown on the right of the blot. The control lanes numbered 1 and 2 above the blots of (a) and (b) are the same as in Fig. 3.3 lanes 1 and 2 of (a) and (b). All transfections were repeated a minimum of 2 times, each in duplicate. 72 pHdNA OpMNPV 1E1 Substituted AcMNPV replication factors: IE!-AcAD lef-1 ,-2 lef-2,-3 lef-1, lef-2, lef-3, helicase helicase helicase 1 2 3 pHdN Substituted AcMNPV replication factors: 73 Fig. 3.5. Ability of IE 1-Ac AD to support non-hr origin replication by substituting OpMNPV core replication proteins with multiple homologous AcMNPV genes. The multiple combinations of AcMNPV replication proteins substituted for the homologous OpMNPV proteins are indicated below the blots, (a) AcMNPV lef -2, -3, helicase (b) AcMNPV lef-3, DNA pol, helicase and (c) AcMNPV lef -1, -2, -3; lef-1, -2, -3, helicase; lef -1, -2, -3, helicase, DNA pol were tested using the pHdNA (a and c) or pHdN (b) reporter constructs. Transient replication assays were performed as described in the materials and methods with the AcMNPV replication genes replacing the homologous OpMNPV genes. The location and size of the linearized pHdN or pHdNA is shown on the right of the blot. The control lanes numbered 1 and 2 above the blots of (a) and (b) are the same as in Fig. 3.3 lanes 1 and 2 of (a) and (b). All transfections were repeated a minimum of 2 times, each in duplicate. 74 (a) (b) pHdNA > OpMNPV Substituted AcMNPV 1 E 1 replication factors: IEl-AcAD lef-2,-3, helicase pHdN * Substituted AcMNPV replication factors: OpMNPV El IEl-AcAD lef-3,pol helicase (c) p H d N A Substituted AcMNPV replication factors: OpMNPV IE! IE 1-AcAD lef-1,-2,-3 lef-1,-2,-3, lef-1,-2,-3, helicase, helicase,pol 75 Fig. 3.6. Replication assay to test whether AcMNPV IE1 AAD is essential for IEl-AcAD interaction with the AcMNPV replisome. The chimeric proteins (a) IE1-AD- and (b) IE1-VP16AD along with IE 1-AcAD were tested for their ability to activate replication from pHdNA reporter construct in the presence of AcMNPV replisome. Replication assays were performed as described in materials and methods with the indicated AcMNPV factors replacing the homologous OpMNPV factors. The negative control lane (pBS+) contains iel replaced by pBS+ and lacks any replication signal indicating there is no background replication. The location and size of the linearized pHdNA is shown on the left of the blot. All transfections were repeated a minimum of 2 times, each in duplicate. 76 (b) pHdNA Substituted AcMNPV replication factors: IEl-AcAD IE1-VP16AD lef-1,-2,-3, helicase 77 Fig. 3.7. Comparison of the ability of OpMNPV IE1, IEl-AcAD or AcMNPV IE1 to support replication with OpMNPV and AcMNPV replication factors in Ld652Y cells. Replication assays were performed as described in materials and methods with the indicated AcMNPV factors replacing the core OpMNPV replication genes. The location and size of the linearized pHdN or pHdNA is shown on the left of the blot. The name of the sample corresponding to each lane is shown on the bottom of the blot. The lanes numbered 1,2,5 and 11 are same as in lane 1,2 of Fig. 3.3b; lane 3 of Fig. 3.5b; and lane 5 of Fig. 3.5c. All transfections were repeated a minimum of 2 times, each in duplicate. 78 pHdN • 10 11 12 p H d N A — » lEl Substituted AcMNPV replication factors; IE1- AcMNPV OpMNPV 1 El - AcMNPV OpMNPV IE1- AcMNPV OpMNPV IE1- AcMNPV AcAD IE! lEl AcAD lEl lEl AcAD IE1 IE1 AcAD IE1 lef-3,helicase,pol lef-1,-2 lef-1,-2,-3,helicase,pol 79 Fig. 3.8. Alignment of the acidic activation domains of OpMNPV IE1 and AcMNPV IE1. The amino acid sequences acidic activation were aligned using T-COFFEE programme (166). OpAD refers to OpMNPV IE1 AAD, AcAD refers to AcMNPV IE1 AAD. The alignment is colorized by the programme to show the correctly aligned portions. (http://www.ch.embnet.org/software/TCoffee.html). The dashed lines show the region of replication sub-domain mapped in OpMNPV IE1 AAD (Fig. 2.5). 80 81 Fig. 3.9. Schematic diagram showing the requirement for OpMNPV and AcMNPV replication factors for both OpMNPV and AcMNPV for OpMNPV IE1, IE 1-AcAD or AcMNPV IE1 to replicate the OpMNPV viral non-hr origin. 82 83 CHAPTER 4. Specificity of AcMNPV non-hr origin in transient replication assays INTRODUCTION Viral DNA replication involves complex interactions between transacting replication factors and cw-acting replication origins that leads to the assembly of a replication complex and initiation of replication from the origin (118). Studies of baculovirus DNA replication have identified two types of cw-acting elements that are putative origins of DNA replication: homologous regions (hrs) and nonhomologous regions (non-hrs). OpMNPV contains five hrs and AcMNPV contains eight hrs. In general, hrs from different baculoviruses share similar structural features. They contain several copies of repeat elements with a characteristic imperfect palindrome embedded in the repeat element. Hrs also acts as enhancers of early gene expression when placed in cis to immediate-early and delayed-early promoters (69, 221). It is unknown whether only one hr or multiple hrs act as in vivo putative origins in baculovirus DNA replication. However in AcMNPV a single hr ori can be deleted without affecting the replication of the virus (142, 182, 195). Non-hr origins are the second type of putative replication origins identified in baculoviruses that are distinctly different from origins in hrs. Only a single non-hr has been identified both in OpMNPV and AcMNPV. Non-hr origins have been shown to contain direct repeats, indirect repeats, /V-unrelated unique palindromes, and A/T rich regions resembling eukaryotic origins (44, 105). Similar to hr, studies conducted in SeA/NPV showed that a non-hr can be deleted without affecting the virus replication (142,182,195). Using transient replication assays, six essential trans-acting factors lef-1, lef-2, lef-3, DNA pol, helicase, iel and three stimulatory trans-acting factors ie2, p43, and iup-1 have been identified for replication of OpMNPV origins (6, 103). Similar to higher eukaryotes (44, 118) it is predicted that six essential baculoviruses replication proteins (IE1, DNA POL, HELICASE, and LEF-1,-2,-3) assemble at the origin and initiate replication from hr and non-hr origins. IE1 is 84 predicted to play the role of initiator of replication and to facilitate the unwinding and assembly of the replication complex (Chapter 3) (6, 167, 177). In other systems assembly of the replication complex involves highly ordered protein-DNA and protein-protein interactions (44, 118). Our studies identified virus-specific interactions between AAD and replication proteins suggesting AAD determines the specificity for viral replisomes (Chapter 3). However, little is known about species-specific protein-DNA interactions that occur between replication proteins and the origin. Past studies showed that the non-hr origin of OpMNPV was able to replicate in AcMNPV virus-infected Sf9 cells (179). Our studies have shown similar results in transient replication assays in Ld652Y cells using AcMNPV replication factors (Chapter 3). This suggests that AcMNPV essential replication factors can interact with the OpMNPV non-hr origin. It is still not known whether the AcMNPV non-hr origin is active with OpMNPV factors. The non-hr origin of another baculovirus, SeMNPV, similar to that of OpMNPV replicated but at low levels in AcMNPV-infected Sf21 cells. However the reciprocal AcMNPV non-hr did not replicate in SeMNPV-infected Sf21 cells suggesting virus specificity of the non-hr origin (22, 85). Similarly it is possible that the AcMNPV non-hr origin might exhibit specificity in Ld652Y cells with OpMNPV factors. In this study we investigated the specificity of AcMNPV non-hr origin replication with OpMNPV and AcMNPV replication factors using AcMNPV IE1, OpMNPV IE land the chimeric protein IE 1-AcAD (Fig. 4.1). The AcMNPV non-hr origin was completely inactive with OpMNPV IE1 and OpMNPV factors. However substitution of AcMNPV lef-3, helicase, and DNA pol for the homologous OpMNPV replication proteins permitted replication to occur, suggesting specificity of AcMNPV non-hr origin for AcMNPV replication factor homologues of lef-3, helicase and DNA pol. These results give insight into the specific interactions between origins and replication proteins. 85 MATERIALS AND METHODS Cell Culture. Lymantria dispar (Ld652Y) cells were maintained in TCI00 medium as described (216). Plasmid and Cosmid constructs. OpMNPV constructs: OPMNPV IE1 acidic domain chimeric constructs IE 1-AcAD, IE1-VP16AD, OpMNPV IE1 (IEl-OpAD) (Fig. 4.1a); and IE2 (pIE2-E2.3) clones have been described previously (52). The plasmids and cosmids encoding the replication genes of OpMNPV have been described previously (Fig. 4.1b) (1, 3-7). Plasmid pHdN and pHdNA are the non-hr origin containing reporters. Plasmid pHdNA is the reporter without lef-l. Plasmid lef-1, pCA35 (lef-2), DNA pol, Cosmid 55 (lef-3), Cosmid 9 (lef-3 and helicase), ie2 have been described previously (Chapter 3) (177). Constructs pHdN, pDNApol, pCA35, Cosmid 55 and cosmid 9 were kindly provided by Dr. George Rohrmann. AcMNPV constructs: The plasmids encoding the replication genes lef-1, lef-2, lef-3, DNA pol, helicase, and iel of AcMNPV have been described previously (Fig. 4.1c) (Chapter 3)(103). Plasmid pHdK, the AcMNPV non-hr reporter was constructed by cloning 2.9 kb of Hindlll K fragment (m.u. 84.9-87.2) of the AcMNPV cosmid 58 #wdIII digest into pBS+ (103). Replication assay. As per chapter 3 except for analyzing the replication of the Hindlll-K origin containing plasmid the blots are probed with Hindlll-K fragment of pHdNK plasmid. 86 RESULTS Previously in chapter 3 transient replication assays were performed to compare OpMNPV IE1, AcMNPV IE1, and IE 1-AcAD (the chimeric protein with OpMNPV DNA binding domain (DBD) and AcMNPV IE1 AAD) ability to activate replication (chapter 3, Fig 3.7). Ld652Y cells were cotransfected with both reporters pHdN (plasmid with OpMNPV non-hr origin) and pHdK (plasmid with AcMNPV non-hr origin), with different IE Is, with all OpMNPV replication factors (lef-1, -2, -3, helicase, DNA pol, and iel) and substituted with AcMNPV factors lef-1, -2; lef-3, helicase, DNA pol; and lef-1, -2, -3, helicase, DNA pol. Only plasmid pHdN replication was analyzed in chapter 3 and the results showed that pHdN replicated with the essential heterologous AcMNPV replication factors. It is unknown whether the reciprocal situation holds, that is, if the AcMNPV non-hr origin containing plasmid, will replicate with OpMNPV replication factors. To study this, previous blots from chapter 3, (Fig 3.7) were analyzed for the replication of reporter plasmid pHdK and compared with the blots from chapter 3. All transfections were only done once, with duplicate of each sample. AcMNPV non-hr does not replicate in Ld652Y cells with OpMNPV replication factors. The previous blots from chapter 3 of the replication assays performed in Ld652Y cells cotransfected with OpMNPV viral replication genes lef-1, -2, -3, helicase, DNA pol, ie2; OpMNPV iel or ie 1 -AcAD or AcMNPV iel; and the reporters pHdN and pHdK were probed with Hindlll-K fragment. The three lanes in Fig. 4.2a are same as lanes 1,2, 3 of Fig. 3.7. The OpMNPV IE1 was ineffective with the heterologous AcMNPV non-hr origin (pHdK) but active with homologous origin (pHdN) (Fig. 4.2a,b). This suggests two possibilities, one is that the OpMNPV IE1 DNA binding domain (DBD) is unable to bind to the AcMNPV non-hr origin and hence OpMNPV replication proteins cannot be recruited, or OpMNPV replication proteins cannot interact with the AcMNPV non-hr origin or both. IE 1-AcAD and AcMNPV IE1 were inactive with both origins and this could be due to inability of AcMNPV IE1 AAD to interact with OpMNPV replication proteins as hypothesized in chapter 3. 87 AcMNPV non-hr origin replicates in Ld652Y cells with AcMNPV lef-3, helicase, and DNA pol substitution. If the AcMNPV non-hr origin is inactive due to its inability to interact with OpMNPV replication factors, then by substituting OpMNPV factors with the homologous AcMNPV factors, replication should occur. To investigate this, the previous blots from chapter 3 of the replication assays carried in Ld652Y cells substituting OpMNPV lef-3, helicase, DNA pol with AcMNPV lef-1, -2; lef-3, helicase, DNA pol; and lef-1, -2, -3, helicase, DNA pol viral replication genes were analyzed for pHdK replication. Fig. 4.3 shows the results of AcMNPV lef-1, -2 substitutions with the AcMNPV homologs. Fig. 4.3a, which shows the results of the OpMNPV non-hr (pHdN) origin, is same as Fig. 3.7 (lanes 7, 8, 9) and Fig. 4.3b shows results of AcMNPV non-hr (pHdK) origin. All three, OpMNPV IE1, AcMNPV IE1, IE 1-AcAD were unable to activate AcMNPV non-hr replication. This suggests that AcMNPV lef-1, -2 are not sufficient to enable interaction with the heterologous origin. Fig. 4.4 shows the results of OpMNPV lef-3, helicase and pol substitutions with the AcMNPV homologs. Fig. 4.4a is same as Fig. 3.7 (lanes 4, 5, 6) shows the results of the OpMNPV non-hr (pHdN) origin and 4.4b shows results of AcMNPV non-hr (pHdK) origin. With these substitutions OpMNPV IE1 was now able to replicate the AcMNPV non-hr origin. Previous studies showed DNA pol is interchangeable for the replication of AcMNPV and OpMNPV origins (4). Therefore this result suggests that lef-3 and helicase are the specific factors required for recognition and replication of the AcMNPV pHdK origin by OpMNPV IE1. Interestingly, these factors are not sufficient for AcMNPV IE1 or IE 1-AcAD to replicate the pHdK origin. Fig. 4.5 shows the results of OpMNPV lef-1, -2, -3, helicase, DNA pol substitutions with the AcMNPV homologs. Fig. 4.5a is same as Fig. 3.7 (lanes 10, 11, 12) shows the results of the OpMNPV non-hr (pHdN) origin and 4.4b shows results of AcMNPV non-hr (pHdK) origin. Similar to the result with pHdN origin high levels of replication were obtained with OpMNPV 88 IE1 for pHdK origin and in addition both IE 1-AcAD and AcMNPV IE1 were also able to replicate pHdK origin. These results show that in Ld652Y cells IE1 proteins containing the AcMNPV AAD require all five AcMNPV replication factors to replicate either the AcMNPV or OpMNPV non-hr origins. In contrast OpMNPV IE1 AAD only requires lef-3 and helicase of AcMNPV origin to replicate AcMNPV non-hr origin and lef-1, -2 can be of either OpMNPV or AcMNPV origin. Together our results suggest that AcMNPV non-hr has species-specific interactions with lef-3, helicase homologs (Table 1). However these results are from only a single transfection done in duplicate. 89 DISCUSSION Viral DNA replication involves highly ordered interactions that lead to assembly of a functional replication complex (94, 155). Initiators of replication bind to the origin and recruit core replication proteins, and examples include UL9, Zta (57, 74, 94, 118, 203, 237), E l , and T antigen (57, 74, 94, 118, 203, 237). Our previous studies suggested that IE1 might play the role of an initiator with the N-terminal AAD interacting with the replication proteins recruiting them to the origin. The OpMNPV IE1 AAD was able to interact with both OpMNPV and AcMNPV replication proteins whereas AcMNPV IE1 AAD was able to interact with only AcMNPV replication proteins. The origin used in these assays, OpMNPV non-hr was found to be active with both OpMNPV and AcMNPV virus replication proteins. In our present study we found that the AcMNPV non-hr is inactive in the presence of OpMNPV IE1 AAD and all OpMNPV replication proteins. However, if AcMNPV lef-3 and helicase are used in place of the OpMNPV homologs, OpMNPV IE1 was able to replicate the AcMNPV non-hr origin. This suggests that the AcMNPV non-hr origin has specificity for homologous AcMNPV lef-3 and helicase, which is in contrast to OpMNPV non-hr that can replicate with heterologous AcMNPV replication proteins in Ld652Y cells. Specificity of baculovirus origins has been observed in other studies. The AcMNPV non-hr origin did not replicate in SeMNPV-infected Sf21 cells. But in the reciprocal experiment the SeMNPV non-hr origin replicated at low levels in AcMNPV-infected Sf21 cells (22, 85). This is similar to our results and based upon our finding it can be predicted that the AcMNPV non-hr will replicate with SeMNPV if supplied with AcMNPV lef-3 and helicase. The baculovirus hr origins have also been shown to have species specificities. CfMNPV hr is unable to replicate in AcMNPV-infected Sf21 cells but the AcMNPV hr origin replicated in CfMNPV-infected Cf-124T cells (86,243). OpMNPV hr replicated at barely detectable levels in AcMNPV-infected Sf9 cells (3) while AcMNPV hr origin replicated at detectable levels in OpMNPV-infected Ld-652Y cells(179). In contrast to these results, in our study the AcMNPV non-hr origin did not replicate with OpMNPV factors in Ld652Y cells. In summary, our results show that the 90 baculovirus non-hr origin can exhibit specificity in regard to the viral origins of the essential replication proteins. Replication origins that show viral specificity could have significant advantages in field situations where co-infections can occur. This study is based on results from only a single transfection done in duplicate. Further repetitions have to be done to support our conclusions. 91 Fig. 4.1. Schematic of OpMNPV IE1, IE 1-AcAD, AcMNPV IE1 and the replication genes of OpMNPV and AcMNPV used to study AcMNPV non-hr origin replication in heterologous system, a) The N-terminal acidic domains are shown filled with a cross-hatched bar pattern and the C-terminal region are shown by solid fill. AAD refers to the acidic activation domain. OpMNPV IE1 is wild type OpMNPV IE1 backbone; IEl-AcAD is the chimeric construct which contains AcMNPV IE1 AAD replaced in the OpMNPV IE1; AcMNPV IE1 is the full length wild type AcMNPV IE1 b) Hindlll restriction map of the OpMNPV genome. The arrows above the map are the location of replication genes used in the transient replication assay. Cosmid 9 is used to supply lef-3 and helicase; Cosmid 55 is used to supply only lef-3; Hindlll N is the region containing a putative non-hr origin of replication. C) Hindlll restriction map of the AcMNPV genome. The arrows above the map are the location of replication genes used in the transient replication assay. Sstl-F is used to supply lef-3 and DNA pol. Hindlll K is the region containing non-hr origin of replication. All transfections were only done once, with duplicate of each sample. 92 (a) OpMNPV-IE1 (560 aa) I OulEI AAD IE1 -AoAO (589 aa) Ai: :n -AA0 A 0 M N P V - I E 1 (582 aa) ACIE1-AAD (b) H/fidlll restriction map of OpMNPV lef-2 lef-1 Hlll -N (non-hr orgln) A T N S H I R K dnapd kf-3 i— «_ helicase , Cosmid 55 Cosmid 9 D U , V B P J G O F E M Q " I 1 I I I I I M Iel m w H/ndlll restriction map of AcMNPV lef-2 lef-l F VTN D X J L M R E OU t i l l 1  M i l Ml dnapol lef-3 , Sstl-F helicase w H s Al Iel -* mndlll-K (non-ftr orgln) A2 K Q P G F 93 Fig. 4.2. Ability of OpMNPV and AcMNPV non-hrs to replicate in Ld652Y cells in the presence of all OpMNPV factors. The previous blots from chapter 3 of the replication assays performed in Ld652Y cells with OpMNPV viral replication genes lef-2, -3, helicase, DNA pol, ie2; OpMNPV IE1 or IE 1-AcAD or AcMNPV IE1; and the reporters pHdN and pHdK were probed with Hindlll-K fragment of plasmid pHdK. a) The blot was probed with OpMNPV non-hr origin Hindlll N fragment. The three lanes are same as lanes 1, 2, 3 of Fig. 3.7. b) The blot was probed with AcMNPV non-hr origin Hindlll K fragment. The location and size of the linearized pHdN or pHdK is shown on the left of the blot. The name of the sample corresponding to each lane is shown on the bottom of the blot. The replication of the reporter is indicated by a + or - symbol at the top of the blot. All transfections were done only once, each in duplicate 94 (a) pHdN (6.9kb) Op H/ndlll N probe + OpMNPV IE 1 - AcMNPV IEJ AcAD |E1 All OpMNPV Factors (b) Ac H/ndlH K probe pHdK (6.17kb) OpMNPV IE! - AcMNPV IE! AcAD IE1 All OpMNPV Factors 95 Fig. 4.3. Ability of OpMNPV and AcMNPV non-/Vs to replicate by substituting OpMNPV replication factors with AcMNPV lef-1, -2 factors. The previous blots from chapter 3 of the replication assays carried in Ld652Y cells substituting OpMNPV lef-1, -2 with AcMNPV lef-1, -2; were probed with Hindlll-K fragment of plasmid pHdK. a) The blot was probed with OpMNPV non-hr origin Hindlll N fragment. This figure is same as Fig. 3.7 (lanes 7, 8, 9). b) The blot was probed with AcMNPV non-hr origin Hindlll K fragment. The location and size of the linearized pHdNA or pHdK is shown on the left of the blot. The name of the sample corresponding to each lane is shown on the bottom of the blot. The replication of the reporter is indicated by a + or - symbol at the top of the blot. All transfections were only done once, with duplicate of each sample. 96 97 Fig. 4.4. Ability of OpMNPV and AcMNPV non-hrs to replicate by substituting OpMNPV replication factors with AcMNPV lef-3, helicase, DNA pol. The previous blots from chapter 3 of the replication assays carried in Ld652Y cells substituting OpMNPV lef-3, helicase, DNA pol with AcMNPV lef-3, helicase, DNA pol; were probed with Hindlll-K fragment of plasmid pHdK. a) The blot was probed with OpMNPV non-hr origin Hindlll N fragment. This figure is same as Fig. 3.7 (lanes 4, 5, 6). b) The blot was probed with AcMNPV non-hr origin Hindlll K fragment. The location and size of the linearized pHdN or pHdK is shown on the left of the blot. The name of the sample corresponding to each lane is shown on the bottom of the blot. The replication of the reporter is indicated by a + or - symbol at the top of the blot. All transfections were only done once, with duplicate of each sample. 98 (a) Cb) OpH/ndlllN probe AcH/ndlll K probe pHdN Substituted AcMNPV replication factors: OpMNPV IE1-IE1 AcAD AcMNPV IE1 lef-3, helicase, pol pHdK OpMNPV IE1- AcMNPV IE1 AcAD jEl lef-3, helicase, pol 99 Fig. 4.5. Ability of OpMNPV and AcMNPV non-hr to replicate by substituting OpMNPV replication factors with AcMNPV lef-1, -2, -3,helicase,DNA pol. The previous blots from chapter 3 of the replication assays carried in Ld652Y cells substituting OpMNPV lef-1, -2, -3, helicase, DNA pol with AcMNPV lef-1, -2; lef-3, helicase, DNA pol; were probed with Hindlll-K fragment of plasmid pHdK. a) The blot was probed with OpMNPV non-hr origin Hindlll N fragment. This figure is same as Fig. 3.7 (lanes 10,11, 12). b) The blot was probed with AcMNPV non-hr origin Hindlll K fragment. The location and size of the linearized pHdNA or pHdK is shown on the left of the blot. The name of the sample corresponding to each lane is shown on the bottom of the blot. The replication of the reporter is indicated by a + or - symbol at the top of the blot. All transfections were only done once, with duplicate of each sample. 100 (a) pHdNA Substituted AcMNPV OpHfod l l lN probe pHdK OpMNPV 1E1- AcMNPV IE! AcAD |E1 replication factors: lef-1 ,-2,-3, hellcase,pol Ac H/ndlll K probe + + + OpMNPV IE1- AcMNPV IE1 AcAD |E1 lef-1,-2,-3, hellcase,pol 101 Table 1. Summary of the factors in the OpMNPV replisome that need to be substituted with AcMNPV homologs to permit AcMNPV non-hr origin (pHdK) replication. The results from each figure are represented in the table. 102 Figure Virus Source of Replication Genes IF. 1 and Reporter Replication | Virus lef-l | lef-2 | lef-3 11,1 /ml I F.I Reporter Op + + + + • + OpIEl pHdN + Ac AAD 4.2 Op + + + + AcIEl pHdN -Ac AAD Op + + + + + OpIEl pHdK -Ac AAD Op + + + + AcIEl pHdK -Ac AAD Op + + + OpIEl pHdNA + Ac + + AAD Op + + + AcIEl pHdNA -4.3 Ac + + AAD Op + + • OpIEl pHdK -Ac + • AAD Op + + AcIEl pHdK -Ac + • + AAD Op + + OpIEl pHdN + Ac + + + AAD Op + + AcIEl pHdN -4.3 Ac + + • + AAD Op + + OpIEl pHdK + Ac + + + • AAD Op + + AcIEl pHdK -Ac + + + AAD Op OpIEl pHdNA + Ac + + + + AAD Op AcIEl pHdNA + 4.4 Ac - + + + + + AAD Op OpIEl pHdK + Ac + + + + + AAD Op AcIEl pHdK + Ac + + + + . AAD 103 Acidic activation domain OpMNPV replication factors AcMNPV replication factors Replication OpMNPV Non-Ar AcMNPV Non-hr OpMNPV IE1 A A D Lef-1, -2, -3,helicase,UNA pol (ie2) + Lef-3, helicase, D N A pol, (ie2) Lef-1, -2 + Lef-1, -2 (ie2) Lef-3, helicase, D N A pol + + (ie2) Lef-1, -2, -3, helicase, D N A pol + + A c M N P V IE1 A A D Lef-1, -2 ,-3, helicase, D N A pol (ie2) Lef-3, helicase, D N A pol, (ie2) Lef-1, -2 Lef-1, -2 (iel) Lef-3, helicase, D N A pol (ie2) Lef-1, -2, -3, helicase, D N A pol + + 104 CHAPTER 5. The role of stimulatory factor OpMNPV IE2 in replication INTRODUCTION The ie2 gene has been identified as a stimulatory gene for both OpMNPV and AcMNPV viral DNA replication in the transient Dpnl replication assays but OpMNPV IE2 is a stronger stimulatory factor than AcMNPV IE2 (6, 103). The role of IE2 in baculovirus replication is not known but plays a role in several other functions. Both OpMNPV and AcMNPV IE2 are transcriptional regulators of early gene transcription. OpMNPV IE2 upregulates expression of thep8.9, iel, ie2 and opep2 promoters (28, 136, 206, 207, 222, 247) and AcMNPV IE2 transactivates expression from the iel and ie2 promoters (27, 28, 247). Besides regulating early gene expression, AcMNPV IE2 is also required for maximal levels of late gene expression (136, 176), for efficient homologous recombination (42) and is involved in cell division arrest of Sf21, TN-368 and H. Zea-AMl cells. Recent studies on BmNPV showed IE2 to function as an E3 ubiquitin ligase. IE2 along with the baculovirus proteins PE38, IAP1, ORF35, and CG30 (59, 112, 184, 222, 236) contain a ring finger motif, which is commonly associated with ubiquitin modifying proteins. Interestingly, only the cell cycle inhibition and ubiquitin ligase functions of IE2 are dependent on an intact ring finger but transcription activation is not (93, 184, 186). Based upon the known multiple functions of IE2, its role in replication has been hypothesized to be transactivation or stabilization of other replication genes, or a function that affects replication indirectly (136,176). Nuclear localization studies showed that IE1 and IE2 co-localize with LEF-3 at replication centers (126, 150) suggesting an association with IEL Recent studies on other viruses and acidic transactivators showed E3 ubiquitin ligase interacts with acidic activation domains (AADs) and activates them for transcriptional transactivation and degradation (46, 79, 161, 201). The OpMNPV IE1 N-terminal acidic activation domain (AAD) is essential for transcriptional 105 transactivation and DNA replication (52) (Chapter 2). This suggests that IE2, which is an E3 ligase, might have a direct interaction with IE1 AAD. The objective of this study was to determine if IE2 interacts with the IE1 AAD or replication factors in stimulating transient DNA replication. The requirement of AcMNPV IE2 for replication and late gene expression is host specific as it has been shown to be required in Sf21 cells but not in TN-368 cells (134, 184). This suggests that IE2 might interact with a cellular factor. The second objective was to study cell specific affects of IE2 stimulation of viral DNA replication. Our results found that OpMNPV IE2 determines the specificity of the AAD for the core replication proteins. In addition, depending upon the presence of a compatible replication proteins, host cell or AAD it activates or inhibits replication. Our results also suggest that OpMNPV IE2 has a possible interaction with a cell specific factor for its role in replication. 106 MATERIALS AND METHODS Cell Culture. Lymantria dispar (Ld652Y) cells were maintained in TCI00 medium as described (216). Sf9 cells were propagated in TC-100 media as previously described (215) Plasmid and Cosmid constructs. OpMNPV constructs: The plasmids and cosmid encoding the replication genes of OpMNPV have been described previously (Fig. 5.1b) (1, 3-7). Plasmid pHdNA is the non-hr origin containing reporter (Chapter 3, Fig 3.2). OpMNPV lef-l is located on OpMNPV cosmid 1 and was cloned as a Hindlll-Xhol fragment into pBS+. For OpMNPV helicase, a 5.32 k bp fragment of Xhol-Sstl digested viral DNA was isolated and cloned into pBS+. OpMNPV IE1 wild type acidic domain construct (OpIEl) chimeric constructs IEl-AcAD, IE1-VP16AD; IE1 acidic domain less construct (IE1-AD-) (Fig 5.1a); and IE2 (pIE2-E2.3) clones have been described previously (52). AcMNPV constructs: The plasmids encoding the replication genes of AcMNPV have been described previously (Fig. 5.1c) (103). Plasmid Ssrl-F was used to supply lef-3 and DNA pol genes. The construct AcMNPV IE1 has been described previously (221). Replication assay. The replication assay used in this study is as per Chapter 2 except the resuspended DNA form Phenol choloform extraction was quantified using ID multi-Alphaimager 3300, Image analysis software (Alpha Innotech Corporation). High DNA mass ladder (Invitrogen) was used as a standard for quantification. All transfections were repeated a minimum of 2 times, each in duplicate. 107 RESULTS E3 ubiquitin ligases might have a direct interaction with the AAD of transcription factors (77, 157, 194). IE2, which has been shown to act as an E3 ligase, might have a similar interaction with the AAD of the OpMNPV IE1 and stimulate replication. To examine this we performed transient replication assays in Ld652Y and Sf9 cells with OpMNPV IE1, IE 1-AcAD, IE1-VP16AD; with OpMNPV or AcMNPV core replication factors supplied by individual plasmid or cosmid clones (Fig. 5.1) and in the presence or absence of OpMNPV IE2. As explained in chapter 3, in this study we referred to the core replication proteins (LEF-1, -2, -3, HELICASE and POL) as replisome. Interaction of OpMNPV IE2 with heterologous AADs and replisomes. As an initial approach, using different IE Is (OpMNPV IE1, IE 1-AcAD, IE 1-VP 16 AD) transient DNA replication assays were performed in Ld652Y cells with OpMNPV core replication genes and in presence and absence of OpMNPV IE2. OpMNPV IE2 augmented replication with OpMNPV IE1 as reported previously (Fig. 5.2a) (6). The two chimeric proteins IE 1-AcAD and IE 1-VP 16AD that contain heterologous AADs were inactive for replication in the presence of OpMNPV IE2 as we reported previously (Chapter 2). Surprisingly, in the absence of OpMNPV IE2 both IE 1-AcAD and IE 1-VP 16AD were able to support replication albeit weakly. The replication signal was extremely weak with IE 1-VP 16AD. These results demonstrate that OpMNPV IE2 inhibits activation of replication by IE1 proteins that have heterologous AADs. This suggests that OpMNPV IE2 may interact with AADs and help maintain their specificity. To determine if OpMNPV IE2 has any effect on the heterologous replisomes we performed transient DNA replication assays with different IEls (OpMNPV IE1, IEl-AcAD, IE1-VP16AD) and the AcMNPV core replication genes in the presence and absence of OpMNPV IE2 in Ld652Y cells. OpMNPV IE1, IE 1-AcAD and IE1-VP16AD supported viral DNA replication with the AcMNPV replisome in the absence of OpMNPV IE2 (Fig. 5.2b). In contrast, the 108 presence of OpMNPV IE2 completely inhibited IE 1-VP 16AD replication function. This is similar to the results in chapter 2, suggesting IEl-Vpl6AD is not compatible with both OpMNPV and AcMNPV core replication proteins. OpMNPV IE2 demonstrates cell specificity Previous studies have shown cell line-specific effects of IE2 stimulation of replication (134, 184). We therefore wanted to determine if the role of OpMNPV IE2 in stimulating or inhibiting replication in Ld652Y cells was cell type dependent. To study this we performed transient DNA replication assays in Sf9 cells, which is non-permissive for OpMNPV, with different IE Is (OpMNPV IE1, IE 1-AcAD, IE 1-VP 16AD) and the OpMNPV replication genes in the presence or absence of OpMNPV IE2. In the presence of the OpMNPV replication proteins, OpMNPV IE2 inhibited replication with all three IE1 proteins (Fig. 5.3a). This is opposite to the result in Ld652Y cells where OpMNPV IE2 augmented replication by OpMNPV IE1 (Fig. 5.2a). Additional transient DNA replication assays were performed in Sf9 cells with the AcMNPV replication genes (Fig. 5.3b). The results showed that OpMNPV IE2 inhibited replication with each IE Is as was observed with the OpMNPV replication proteins. Fig. 5.3 summarizes the results of transient replication assays with OpMNPV IE2. In absence of IE2, non-specific interaction occurs between AAD and replisome complex leading to active replication. In its presence IE2 appears to maintain specific interaction between AAD and replisome complex and stimulate replication. The results show that OpMNPV IE2 also inhibits replication in the non-permissive or heterologous cell type, revealing cell specificity of OpMNPV IE2 suggesting a possible interaction with a cell factor. 109 DISCUSSION Both IE1 and IE2 are involved in transcription, replication and homologous recombination (28, 42, 112, 136, 176, 206, 222, 247). Nothing is known about whether IE2 has any direct interaction with IE1 for these functions. In our present study we wanted to determine if OpMNPV IE2 interacts with the AAD of OpMNPV IE1, AcMNPV IE1, or herpes virus VP 16 in stimulating the replication of the OpMNPV non- hr origin in Ld652Y cells. The results of chapter 2 showed that OpMNPV IE1 chimeric proteins, IE 1-Ac AD and IEl-Vpl6AD were not active for replication with the OpMNPV replisome in the presence of OpMNPV IE2. The chimeric protein IE 1-AcAD was then shown to be able to support DNA replication if the essential replication factors were replaced with homologous AcMNPV proteins (Chapter 3). This showed specificity between the AAD and the viral replisome. In this study we showed that the chimeric proteins IE 1-AcAD and IE 1-VP 16AD can also support replication with the OpMNPV replisome if the OpMNPV stimulatory factor IE2 is removed from the assay. This suggests that IE2 dictates the specificity of the AAD for the viral replisome. For OpMNPV IE2 to stimulate and not inhibit replication our results suggest two criteria must be met which is summarized in Fig. 5.5. The first is that IE2 must be compatible with the cell type. Secondly, IE2 must also be compatible with both the IE1 AAD and the associated replisome (lef-1, -2, -3, helicase). Therefore in Sf9 cells, which are non-permissive, OpMNPV IE2 only inhibits replication. Whereas in the permissive Ld652Y cells, OpMNPV IE2 will stimulate replication if the AAD is specific for the replisome. Therefore IE2 only stimulates replication by IE 1-AcAD and AcMNPV IE1 when associated with the AcMNPV replisome. OpMNPV IE1, which replicates with either replisome is stimulated by IE2 in both cases. IE1-VP16AD, which has no specificity for either replisome is inhibited by IE2. A possible explanation of these results is the recent discovery showing that BmNPV IE2 has E3 ligase activity in the ubiquitin pathway and that AADs have been shown to act as substrates for E3 ligases (46, 79, 93, 161, 201). E3 ligase complex binds to substrates and positions them for 110 ubiquitination. This positioning of the substrate creates a rigid spatial constraint (43, 204, 239, 249). Based upon these studies and our results, IE2 acts as E3 ubiquitin ligase and forms with its cognate partners a ligase complex (Fig. 5.5). This complex binds to AADs and imposes a spatial restraint that generates specificity towards the replisome. IE2 also needs the correct cellular factor for active replication. In the absence of IE2 the spatial constraint and cell specificity is lost and replication occurs irrespective of the virus specific AAD, replisome and cell factor (Fig. 5.3). IE2s stimulatory role in replication has been postulated as indirect, involving stimulation of other viral essential genes for replication (136). For the first time our studies suggest that IE2 might have a direct role in replication by interacting with the IE1 N-terminal domain and a cell factor, maintaining specificity for cell type, AAD and replisome. I l l Fig. 5.1. Schematic of the IE Is and of the replication genes of OpMNPV and AcMNPV used in transient viral DNA replication assays to study the role of OpMNPV IE2. a) The N-terminal acidic domains are shown filled with a cross hatched bar pattern and the C-terminal regions are shown by a solid fill. AAD refers to the acidic activation domain, OpMNPV IE1 is the wild type IE1 of OpMNPV, IE 1-AcAD and VP 16 AAD are the chimeric constructs which contain AcMNPV IE1 AAD and HSV VP 16 AAD replacing the OpMNPV IE1 AAD respectively, b) Hindlll restriction map of the OpMNPV genome. The arrow heads above the map are the location of replication genes used in the transient replication assay. Cosmid 9 was used to supply lef-3 and helicase; Hindlll N is the region containing the putative non-hr origin of replication, c) Hindlll restriction map of the AcMNPV genome. The arrows above the map are the location of replication genes used in the transient replication assay. Sstl-F is used to supply lef-3 and DNA pol. 112 (a) OpMNPV-IEI I OpIEl-AAD~ (560 aa) (589 aa) 1 IE1-VP1SAD \ (516 aa) (b) W/ndlll restriction map of OpMNPV lef-1 dnapoj m helicase Mndlll-N [non-hr orgln) ^ ^ ^ ^ A T N S H I R K C L D U , V B P J G O F E M Q Iel feJ (c) H/ndlll restriction map of AcMNPV lef-2 lef-1 dnapol lef-3 helicase F VTN D X J L MR E OU I B C W H S A l A2 K Q P G F 1 1 1 1 II II 1 I H I I 113 Fig. 5.2. Transient replication assays in Ld652Y cells. L4652Y cells were cotransfected with (a) plasmids carrying OpMNPV core replication genes (lef -I, -2, -3, helicase, DNA pol) or (b) the AcMNPV core replication genes (lef -1, -2, -3, helicase, DNA pol), using different IE Is (OpMNPV IE1, IE 1-AcAD or IE 1-VP 16AD), with or without OpMNPV IE2. The reporter construct is the OpMNPV viral non-hr origin reporter, pHdNA. The number to the left of the blot corresponds to the size (in kb) of the hybridized band of linearized pHdNA. The name of the sample corresponding to each lane is shown on the top of the blot. The + or - symbol at the bottom of the blot indicates presence or absence of OpMNPV IE2 in the replication cocktail. All transfections were repeated a minimum of 2 times, each in duplicate. 114 (a) OpMNPV IE1- IE1--IE1 AcAD VP16AD pHdNA OpMNPV 1E2 (b) OpMNPV core replication factors (lef-1 ,-2,-3,poi,heiic8se) OpMNPV -IE1 AcAD VP 16 AD pHdNA OpMNPV IE2 AcMNPV core replication factors (ief-i,-2,-3,poi,heiicase) 115 Fig. 5.3. Transient replication assays in Sf9 cells. Sf9 cells were cotransfected with (a) plasmids carrying OpMNPV core replication genes (lef -1, -2, -3, helicase, DNA pot) or (b) the AcMNPV core replication genes (lef -1, -2, -3, helicase, DNA pot), using different IEls (OpMNPV IE1, IE 1-AcAD or IE 1-VP 16AD), with or without OpMNPV IE2 and in the presence of viral non-hr origin reporter, pHdNA. The number to the left of the blot corresponds to the size (in kb) of the hybridized band of linearized pHdNA. The name of the sample corresponding to each lane is shown on the top of the blot. The + or - symbol at the bottom of the blot indicates presence or absence of OpMNPV IE2 in the replication cocktail. All transfections were repeated a minimum of 2 times, each in duplicate. 116 (a) OpMNPV IE1- IE1--IE1 AcAD VP 16 AD pHdNA OpMNPV IE2 - + - + - + OpMNPV core replication factors (lef-1 ,-2,-3,p0l,rieiicase} OpMNPV IE1- IE1--IE1 AcAD VP 16 AD pHdNA OpMNPV IE2 - + - + - + AcMNPV core replication factors (lef-1 ,-2,-3^0), helicase) 117 Fig. 5.4. Flow chart summarizing the OpMNPV IE2 effect on transient replication in both Ld652Y and Sf9 cells. All the data with OpMNPV and AcMNPV replisome and with different IE Is is presented. Active indicates replication remained the same or increased in the presence of IE2. Inhibited means that replication levels were completely inhibited or declined significantly. 118 • OplE2 OpMNPV replisome LD652YceHs ^Sk-'" Active I E 1 - A e A D inhibited t K V E ™ P ! ^inhibited;: - OplE2 -9el*_ Active' M M * Active CplE1 + Op_IE2 AcMNPV replisome Active W*m Active •iEi-vPieAD inhibited • OpiEq -9*!*- Active S * ' Active ji£i.^m'eife-.,>vjtctivie: + OplE2 OpMNPV replisome OpIEl inhibited •M&gr inhibited *T«VW«fr inhibited. 'dpi eii - OplE2 Active fe*P Active Aft Active OpIEl-; + OplE2 AcMNPV replisome inhibited IE1-VP16ABV inhibited •Op|E2 OplE1 . .. Active i l i ^ ? Active IE1-VR16AD/ Active 119 Fig. 5.5. Schematic diagram showing that OpMNPV IE2 effect on specificity in replication. 120 Specificity 121 CONCLUSIONS This thesis investigated the mechanism by which IE1 stimulates viral DNA replication and suggests possible interactions with viral replication proteins to form the replication complex or replisome. The N-terminus of IE 1 is essential for both transcription and replication. OpMNPV IE1 AAD was found to be replaceable by both heterologous VP 16 AAD and AcMNPV IE1 AAD for transcription but not for replication, revealing important differences between OpMNPV IE1 AAD and AADs in other systems. We found that transcriptional activation by IE1 in transient assays is not required for replication and that the transcription and replication roles of IE1 are independent and separable. This is attributed to two independent domains that are specific and separate for transcription and replication. A number of studies have shown eukaryotic transcription factors, specifically those that contain AADs, have a dual role and are found to enhance the efficiency of DNA replication as well as transcription. Native AADs can be substituted with heterologous AADs from a variety of eukaryotic species and can retain both transcription and replication activation. Our results reveal important differences between OpMNPV IE1 AAD and AADs in other systems suggesting that there is specificity in AADs and not all AADs are interchangeable. AADs have been shown to interact with complexes involved in both transcription and replication. However it is unknown whether the whole AAD domain or just the sub-domains within AAD interact with these complexes. Our results suggest that there are independent and separable sub-domains that are specific and separate for transcription and replication. These findings suggest that baculoviruses are good candidates to study the various interactions at the initiation stages of the two important processes, gene regulation and replication. Our data will be useful to understand the function of important proteins like mammalian p53 tumour suppressor, and breast cancer BRCA1, which also are shown to contain AADs. Using the clones that are 122 transcription positive and replication negative and vice versa, which we obtained in our studies the mechanisms of replication and transcription can be studied in more detail. A OpMNPV IE1 chimeric construct with AcMNPV IE1 AAD was active for replication when the OpMNPV replisome was substituted with the AcMNPV lef-1,-2,-3, and helicase proteins indicating that AcMNPV IE1 AAD has specificity towards homologous (AcMNPV) replication proteins. Both IE 1-AcAD and full length AcMNPV IE1 behave similarly providing the evidence that replacing the AAD modifies the function of the protein to that from which the AAD originates, in this case to AcMNPV IE1. OpMNPV IE1 was able to interact with both OpMNPV and AcMNPV factors suggesting that OpMNPV IE1 AAD is promiscuous or less specific than AcMNPV IE1. This data suggests that IE1 binds to a replication origin and with AAD it interacts with replication factors facilitating their assembly onto the origin. This is similar to initiator-replication protein Interactions observed in other viruses. Species specificities were also observed with the OpMNPV and AcMNPV non-hr origins. To replicate the AcMNPV non-hr origin, OpMNPV IE1 AAD required at least AcMNPV lef-3, helicase and pol. This shows that apart from specificities between initiator and replication proteins there is also specificity between the origin and replication proteins. Our data can be applied to understand the specificities of viruses and their replication, which could have significant advantages in field situations where co-infections of insect viruses occur. It also helps to understand the molecular interaction between initiator-replication proteins in the replication of other eukaryotic DNA viruses. In the absence of OpMNPV IE2, the IE1-VP16 AD and IEl-AcAD chimeric proteins that were inactive with OpMNPV replisome were active for replication function suggesting that the presence of OpMNPV IE2 determine specificity of the AAD to the replisome. OpMNPV IE2 activated replication with only compatible replisome-AAD, otherwise it inhibited replication indicating an interaction with AAD and replication proteins. Further OpMNPV IE2 activated replication only in Ld652Y cells and it inhibited in heterologous Sf9 cells suggesting it interacts with a cellular factor(s). Together our results suggest that IE2 maintains specificity between AAD, replisome and cell type and stimulates replication when compatible proteins are present. 123 Studies with VP 16, E2F-1 and Myc activation domains show a common link between transcriptional activation and Ub-mediated proteolysis of transcription factors. Both these processes require ubiquitination of activation domains by E3 ligases. IE2 has been shown to be an E3 ubiquitin ligase and our studies for the first time show that E3 ligases will also play a role in replication. Our results can be used in to further study the binding partners of IE2 in replication, the domains of IE2 involved in replication and the mechanism it uses for creating specificity between AAD and replisome. Our data will be useful to study the roles of other E3 ligases in replication. IEO is the spliced form of IE1 and contains 35 additional aa at the N-terminus. The exact function if IEO is not clear but it is better transcriptional transactivator of specific early genes. IE1 is essential for replication but it was unknown whether IEO is active in replication or has any functional differences compared to IE1. Our studies conclude IEO is functionally active for replication in transient assays and can replace IE1. 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J. Elledge, M. Pagano, R. C. Conaway, J. W. Conaway, J. W. Harper, and N. P. Pavletich. 2002. Structure of the Cull-Rbxl-Skpl-F boxSkp2 SCF ubiquitin ligase complex. Nature 416:703-709. 146 APPENDIX A. Functional analysis of IEO for viral DNA replication INTRODUCTION IEO is the only viral protein shown to be produced by splicing in baculovirus infected cells. OpMNPV IEO contains 35 additional aa at the N-terminus compared to IE1. Out of the 35 aa, 23 aa comes from exonO (orf 138) upstream of iel and the remaining 12 aa comes from the 5' untranslated regions (UTR) of IE1 (Fig. 6.1a). IEO expression peaks at early times and IE1 continues to increase up to very late times. Functional analysis of IEO showed that it is a significantly stronger transcriptional activator of specific early genes than IE1 and could activate gene expression in an enhancer-dependent or -independent manner, and in addition, was shown to be auto regulatory. This study and previous studies from our laboratory characterized role of the OpMNPV IE1 N-terminus acidic activation domain (AAD) in transcription and replication (52). The AAD was found to be necessary for both these functions and has separate domains for transcription and replication (Chapter 2). The AAD was also shown to be specific for replication proteins suggesting that IE1 acts as an initiator in assembling the replication complex at the replication origin. Since IEO has the same C-terminal regions as IE1 that are required for DNA binding and oligomerization, it is possible that IEO binds to DNA similar to IE1 and also works as an initiator. Indeed Kremer & Knebel-Morsdorf (113) showed that AcMNPV IEO binds to DNA. It is also possible that similar to transcription activation, the additional 35 aa will make IEO a stronger activator of replication than IEL To date no studies were done to show whether IEO is active for replication or has any functional differences compared to IE1. In this study we investigated the role of OpMNPV IEO in replication by using transient DNA replication assays. Our results show that IEO is active in replication and can replace IE1. This is the first report to show that IEO can replace IE1 for replication. 147 MATERIALS AND METHODS Cell Culture. Lymantria dispar (Ld652Y) cells were maintained in TCI00 medium as described (216). Plasmid and Cosmid constructs. The plasmids encoding the OpMNPV replication genes pDNA pol (pol), pCA35 (lef-1), cosmid 9 (lef-3, helicase) and the origin containing reporter pHdN (which contains origin and lef-1), have been described previously (Fig. 6.1b) (1-5). Plasmids pIEl-sal (OpMNPV IE1), pIE2-E2.3 (IE2), p/eO-IEO (translates as both IEO, IE1) and pJe0-IEO(2XATG-) (translates as only IEO) are described previously (52, 224). Replication assay. As per chapter 2. Western blots. Transfected cells were prepared for calf intestinal phosphatase (CIP, Sigma) treatment as described by Slack and Blissard (208). Proteins were separated using denaturing gel electrophoresis (SDS-PAGE) (115) and transferred to Immobilon membranes (Millipore) using standard techniques (202). To detect OpMNPV IE1, blots were incubated with the mouse monoclonal antibody IE 1-10 (220). Bound antibody was then detected using goat anti-mouse peroxidase-linked antibody (The Jackson Laboratories) and the ECL (Amersham) substrate using the manufacturer's specifications. 148 RESULTS To investigate the role of IEO in replication, transient replication assays were performed. The ieO constructs used were p/eO-IEO which is translated as both IE1 and IEO; and /e0-IE0(2XATG-) that has the second and third ATGs mutated to glycine and is translated as only IEO (Fig. A.2a). Ld652Y cells were cotransfected with OpMNPV viral replication genes lef-2,-3, helicase, DNA pol, ie2; the reporters pHdN and ielox ieO or ieO (2xATG-) or pBS+. All three constructs activated replication from the reporter demonstrating that IEO is functionally active in replication and can replace IE1. There was no replication signal in the pBS+ lane indicating absence of any background replication. To compare the effectiveness of IEO and IE1 in activating DNA replication western blots were performed to determine the levels of expression from each construct. Phosphorylated and dephosphorylated protein samples from the replication assays were analyzed by Western blot (A.3a). As has been previously shown translation of pzeO-IEO produced both IEO and IE1 with the predominant protein being IE1, p/'e0-IEO(2XATG-) produced only IEO and p/ei-IEl only IE 1(224). The blots were quantified by densitometry to determine relative amounts. Both the Southern and western blots were quantified and the specific activity of p/ei-IEl; piel-IEO; pie0-IE0(2XATG-) in replication were calculated. As shown in Fig. 6.4 the construct pieO-IE0(2XATG-) which produces only IEO appears to be 1.6 fold more active in replication. This preliminary analysis suggests that IEO is better activator of replication. 149 DISCUSSION IEO is the spliced form of IE1 and contains 35 additional aa at the N-terminus and functional analysis of IEO in transcription showed that it is a stronger transcriptional activator than IE1. IE1 is known to be essential for replication but it was unknown whether IEO is active for replication and has any functional differences compared to IE1. Here we showed IEO is functionally active for replication in transient assays and can replace IE1. The N-terminal IE1 AAD is necessary for both transcription and replication (52)(Chapter 2). In IEO the extra 35 aa extends the AAD domain. Previous studies showed the extra 35aa also increased the activation potential for early genes (224). Our initial results show that similar to transcription; IEO appears to be 1.6 times better activator of replication than IE1 suggesting that 35aa may have an effect in replication. The 35aa are not acidic in nature suggesting it not a domain extension of AAD (224). It is possible that these additional aa might create a stronger interaction of the AAD with the replication complex or it might interact with an unknown factor. However, these results are mostly preliminary and have to be extensively repeated before it can be concluded that IEO activates replication to higher levels than IE1. Both IE1 and IEO are active in replication but in the virus life cycle IEO is expressed at early time points which is prior to the onset of replication and then declines whereas IE1 is expressed from early to very late times p.i. (220, 221). OpMNPV virus replication begins atl2 hrs p.i and then proceeds into late and very late time points (21). It is possible that IEO is required at the onset of viral DNA replication and at late time points IE1 is required. AcMNPV IE1 is shown to bind as homodimers to enhancer regions through helix-loop-helix domain (HLH) and oligomerization domain (35, 169, 196). In the same study they provided evidence for homo- and heterodimers of IE1 and IEO. Several studies showed multiple dimer combinations of HLH proteins is an efficient mechanism for regulation of gene expression since different dimers are likely to target different sets of genes (194). Therefore it is also possible 150 that the homo and heterodimers of IEO and IE1 (IE0-IE0, IE0-IE1, and IE1-IE1) could have a different function in both transcription and replication. Recent studies using BmNPV IE1 showed an N-terminal truncated IE1 has an antagonist effect on full length IE1. This is attributed to the heterodimerization between truncated and full length IE1. Similarly IE1 -IEO heterodimerization might prevent one of the partners from functioning (245). Finally our results show IEO alone can activate replication without IE1 but more experiments are required to determine the different role IEO might play compared to IE1. 151 Fig. A . l . Schematic of the exonO and iel region showing the transcription and splicing pattern of iel and ieO, and Hindlll restriction map of the OpMNPV genome, a) Schematic map of the OpMNPV genome, from the 5' end of me53 to the 3' end of odvp-6e, showing the transcription and splicing pattern of iel and ieO. Names of the genes in this region are shown below the ORF arrows. Transcriptional start sites are indicated by vertical black lines. The sequence shown below the schematic is of ieO cDNA. Transcriptional start sites are indicated by vertical black lines and the splice site is indicated by the arrowhead. The four-amino-acid minicistron in the 5' end of the ieO mRNA is boxed 7 bp upstream of the ieO start codon. The sequences that originate from the iel gene are underlined. The amino acid sequence of both the minicistron and IEO is shown below the nucleic acid sequence. The first and second methionine codons of IE1 are labelled l°and 2° respectively. Figure a is from Theilmann et al, 2001 (224). b) The arrows above the map are the location of replication genes used in the transient replication assay. Cosmid 9 is used to supply lef-3 and helicase; //mdlll-N is the region containing putative non-hr origin of replication. 152 (a) 130 nt 1 T T C A A C A A G T C A O C T A G G C G C T T T G C G C A C G G C C C C ^ C m T A T C G C A A C A C ^ ^ 100 1 0 1 CTMMBiKSflC6M3BEQCftG(MnKffCK^ | ATO ACC GOG GCG TGA|CGC 1 9 4 1 H T A a * 5 E p i l o g sits 1 9 5 AGAC A T G ATA AAA GGC A C C C A T TGG CCC A A T T T A GTT T O G AAG AGC T A C A C C GAC GOG T G C GAG A C C AGC AAG T T G 2 7 0 1 • T I K G T H W E N I i V S K S Y T D A C B T S K L 2 4 1D i ° 2 7 1 CAA GTT GGC TGC GGC GCG CGC AGC ACC T T T GCO ATG CCC AAG AAT ATS GAA ACT CTA C A G CGT TOG TAT ATQ QGC 3 4 5 2 5 Q V G C G ? . R S T E A H P K J f H B T L Q R S Y M G 49 346 CCG T C C ACG CCA AAC CAC A A T T T G T T C A A C A A T GCC A C C GAG CTQ COS GAC GAC CTA A A C T T T A G C A C A A T G GAC 5 0 P S T P H H N L F N N A T B L F D D L IT F S T U D 421 G T C OCC T A C GAC GGC AGT A T G OCC ATG A A C AUG AGC AGC G 7 5 V P I D G S M P M N M S S 4 2 Q 7 4 4E0 8 7 (b) Hindlll restriction map of OpMNPV m 'f2 'f dnapolief-3 heScase EmfiQiel ie2 Hindll l-N (non-hrongin) m^m^^mmmmmmamm Cosmid 9 A T N S H I R K C L D U V B P J G O F E M Q A 153 Fig. A.2. Schematic diagram of the IEO and IE1 expression constructs and Southern blot of the transient replication assays in Ld652Y to study the role of IEO in replication, a) 1° and 2° refer to the first and second ATG of IE1 and IEO respectively. The name of each plasmid is shown on the left. Plasmid p/eO-IEO (2XATG-) is a mutant of p/eO-IEO in which the 1° and 2° ATGs have been mutated. Mutations in the methionine codons are shown in bold (224). b) Ld652Y cells were cotransfected with OpMNPV replication factors (lef -1, -2, -3, helicase, DNA pol) the viral non-hr origin of replication pHdN and with p/'ei-IEl or p/eO-IEO or p/'eO-IEO (2XATG-) or pBS+. The number to the left of the blot corresponds to the size (in kb) of the hybridized band of linearized pHdN. The name of the sample corresponding to each lane is shown on the top of the blot. 154 (a) p*W-IEl ( i e l prm 1° 2° lATG ATG I TGA| p/eO-IEO I minicistron 1 0 2 ° // lATG TGAI lATG ATG ATG // TGA | pi>0-IEO(2XATG-) I 1 ATG TGAI lATG GGG GGG j} TGA| (b) / # # & « pHdN(6.9kb) OpMNPV replication factors {Iet1, 2, 3, pol, helicase, ie2) 155 Fig. A.3. Western blot analysis of IEO and IE1 expressed from piel-IE 1, pieO-IEO, and pieO-IEO (2XATG-). Ld652Y cells from replication assays transfected with replication genes and p/e7-IEl; piel-IEO; p/e0-IEO(2XATG-) and pBS+ were harvested, one set was dephosphorylated (+ CIP) and the other set was left untreated (-CIP). Samples were separated by 8% SDS-PAGE, transferred to membranes and probed with a IE1 monoclonal antibody (221). The locations of the IEO and IE1 bands for the samples are shown by arrow heads, a) Untreated (-CIP). b) Dephosphorylated (+ CIP). 156 tf tf IE0(68.0) & IE1(64.2) - C I P # 0 Si cr t IEO (68.0)_ IE1 (64.2)~ + C I P 157 Fig. A.4 Specific activity of p/el-IEl; pzei-IEO; p/e0-IEO(2XATG-) in replication. The southern and western blots were quantified and specific activity was calculated by dividing the levels of replication with levels of protein. The name of the each sample is shown on X-axis. Y-axis represents the specific activity. 158 0.33 0.32 0.55 p/e/-IE1 p/eO-lEO p;eO-IE0(2XATG-) 159 APPENDIX B. Partial characterization of exonO (orfl38) INTRODUCTION OpMNPV exonO (orfl38) is 734 bp in length, located upstream of iel and codes for a predicted 245 aa protein. The importance of this gene comes from its contribution of 23 aa to the N-terminus of IEO by splicing. To date no function has been assigned to this gene. Initial sequence searches of the OpMNPV EXONO predicted protein with the database suggest that exonO may also encode a functional gene. Recognizable motifs within the predicted protein sequence include a possible ring finger and a leucine rich motif in the C-terminal region Ring fingers are found in proteins that have diverse functions including regulation of gene expression, site-specific recombination, apoptosis and ubiquitination (239). Ring fingers have also been identified in other baculovirus proteins ie2,pe38, iapl, orf35, iap2 and cg30 (59, 112, 184, 222, 236). Leucine zippers are also common to transcription factors and play a role in dimerization (24, 193). EXONO having similar structures might play a role in the baculovirus life cycle. This study was performed in an attempt to characterize and identify the possible function of EXONO. Analysis of OpMNPV exonO expression showed exonO to be a late gene. Experiments were done to determine if EXONO augments replication and late gene expression. However these results were not consistent and further analyses are needed to determine EXONO function. 160 MATERIALS AND METHODS Cell Culture. Lymantria dispar (Ld652Y) cells were maintained in TCI00 medium as described (216). Sf9 cells were propagated in TC-100 media as previously described (215) OpMNPV ExonO constructs. Using two primers, 5' ATAGGAGCTCCCGTACAACACAATA3' and 5'AATCGAATTCCAAAATGTCTACAGT3', OpMNPV ExonO was amplified from Op cosmid 47 with Sad and BamHl generated ends. The fragment was cloned into pBS+ and referred to as pOpExonO. Plasmid pOpExonO was digested with BstBI and EcoNl, filled with T4 and blunt end ligated to obtain a in frame deletion of 560 bp. The deletion mutant is called pOpExonOA. To add the poly A signals to OpExonO and pOpExonOA, their respective constructs were amplified with universal reverse primer (5'AGCGGATAACAATTTCACACAGG3') and primer 5' CTCATGGATCCCACTTACTATGACT3'. The Mwdlll and BamHl amplified fragments were blunt ended at Hindlll end and cloned into pOp27/233 digested with Smal and BamRl. The clones are called as pOpExonOPA and pOpExonOAPA. For northern blot analysis a clone was made with only the exonO sequence excluding the ieO and orfl39 overlaps. Two primers 5' GC AAGGATCCGG ACTTTAATTTTAT and GCAGAATTCGTAGCACAGCGAGTAC3'were used to amplify this region with BamHl and EcoRl sites and later cloned into pBS+ referred to as pOpExonO (5'&3'A). OpMNPV late gene reporter constructs. OpMNPV polyhedrin promoter and termination signals were selected for studying late gene assays in OpMNPV. Using two primers 5'ATGAAGCTTCGCAATACCCCGAACAC3'and 161 5'CATGGATCCAGGAAATTTTTTTACAA3' the 5' promoter sequence of the polyhedrin gene was amplified from Op cosmid 64 and cloned into pBS+ as Hindlll and BamRl fragments, the construct is called pOppH(5'p). Using two primers 5' CATGAGCTCCGTTACAATTGATACGA3' and 5'AAAGGACCGGCTCGATAGAATTCCAA3' the termination region was amplified from Op cosmid 64 and cloned into pOppH(5'p) as Sacl and EcoRI fragments. The final construct is called pOppH5'p&3't. Chloramphenicol acetyltransferase (CAT) and Green fluorescent protein (GFP) fragments were separately cloned into pOppH5'p&3't as BamRl fragments to obtain two clones called pOppH5'p&3't-CAT and pOppH5'p&3't-GFP. RNA isolation and Northern blots. Total RNA from OpMNPV-infected Ld652Y cells for Northern blots was prepared as described (33, 232). For Northern blotting, total RNAs (5 ug per lane) were separated by electrophoresis in 1.25% agarose gels containing 6% formaldehyde, lx MOPS buffer [20 mMMOPS (3-[N-morpholinojpropanesulfonic acid, pH 8.0), 5 mM sodium acetate, 1 mMEDTA] (227). Separated RNAs were transferred to BioRad zeta probe GT membrane (BioRad Inc) by capillary blotting. Hybridization was carried out at 60°C in 6X SSC (lx is 0.15 MNaCl, 0.015 Msodium citrate, pH 7.0), 10X Denhardt's solution (IX is 0.02% polyvinylpyrolidone, 0.02% BSA, 0.02% Ficoll 400), 0.1% SDS, 100 pg/ml denatured DNA from salmon sperm, 100 pg/ml yeast RNA. Single-stranded RNA probes complementary to OpMNPV ExonO mRNA were synthesized using T7 RNA polymerase. After hybridization, blots were washed twice for 15 min in 2X SSC, 0.1% SDS at, 60°C (IX SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and twice for 15 min in 0.1X SSC, 0.1%> SDS at 75°C and membranes were visualized on a Storm-phosphorimager (Molecular Dynamics) (10). Sequence analysis of the OpMNPV EXONO The predicted protein sequence of OpMNPV EXONO was searched against protein database using BLAST network service (8) and SMART search (205) (http://smart.embl-162 heidelberg.de/smart'). The alignments were done using chroma program (http://www.lg.ndirect.co.uk/chroma) (60). OpMNPV replication assays. The plasmids encoding the OpMNPV replication genes pDNA pol (pol), pCA35 (lef-1), cosmid 9 (lef-3, helicase) and the origin containing reporter pHdN (which contains origin and lef-1), have been described previously (Chapter 2. Fig. 2.1) (1, 3-7). Plasmids pIEl-Sal (OpMNPV IE1), pIE2-E2.3 (IE2), pieO-IEO are described previously (52). To study the effect of exonO on replication, Dpnl replication assays were performed as described previously (Chapter 2) with replication factors, IE1, IEO, pOpExonOPA and pOpExonOAPA constructs. Western blots. The cells from the replication assay were used to test the levels of IE1 and IEO protein expression. Cells were prepared for calf intestinal phosphatase (CIP, Sigma) treatment as described by Slack and Blissard (208). Proteins were separated using denaturing gel electrophoresis (SDS-PAGE) (115) and transferred to Immobilon membranes (Millipore) using standard techniques (202). To detect OpMNPV IE1, IEO blots were incubated with the mouse monoclonal antibody IE1-10 (220). Bound antibody was then detected using goat anti-mouse peroxidase-linked antibody (The Jackson Laboratories) and the ECL (Amersham) substrate using the manufacturer's specifications. AcMNPV late gene assay. AcMNPV cosmids 1, 10, 58 and 59, plasmid clones containing ie2 (pAcIE2), /W(pAcIEl), lef-1 (pielef7), andp35 (piep35), ieO (pzop2eIE0) and exonO (pzop2ExonO), reporter plasmid pVL941-CAT have been described previously (125, 138, 221, 224) (Fig. 7.5a). To study the effect of ExonO in late gene assay transient assays were performed in SJ9 cells as described previously (125). The cells were harvested and assayed for CAT activity (177). 163 OpMNPV late gene assay. OpMNPV cosmids 1,13,54,41,9,5,27 were kindly provided by Dr. George Rohrmann and are described previously (122). Plasmids pIEl-sal (OpMNPV IE1), pIE2-E2.3 (IE2), p/eO-IEO are described previously (52, 224) (Fig. 7.6). Transient late gene assays were performed in Ld652Y cells as described previously (125) with the reporters pOppH 5'p&3't-CAT and pOppH 5'p&3't-GFP. The cells were harvested and assayed for CAT activity (177). 164 RESULTS OpMNPV exonO is 734 bp and codes for a predicted protein of 245 aa. It gains importance in the splicing event of IE1 (Fig. B.l). Apart from its involvement in the formation of IEO, it is unknown if EXONO by itself has any function. To gain insight into EXONO function the predicted protein was first searched with BLAST network. OpMNPV EXONO has a novel ring finger Searches against the BLAST protein database showed sequences homologous to OpMNPV ExonO in baculoviruses CfMNPV, EppoNPV, AcMNPV, BmNPV, RoNPV, LdMNPV, SeMNPV, MacoNPV-A, MacoNPV-B, HearNPV, HzSNPV, AdhoNPV and SpltNPV. Alignment of the predicted amino acid of these proteins revealed a cysteine rich region with a Ring finger like domain at the carboxy terminus. The ring finger differs from the consensus C 3 H C 4 in that it contains tyrosine instead of histidine ( C 3 Y C 4 ) (Fig. B.2). All the predicted EXONO proteins have tyrosine in the ring finger except the SpliNPV homolog which has phenylalanine. In the search for motifs using SMART, the C-terminal region also showed homology to ZZ type cysteine rich putative Zn4-1" binding proteins that have tyrosine interspersed (183). Another interesting feature found is the repeats of leucines suggesting the presence of a leucine zipper or coiled coil domain. Both ring finger and leucine rich regions are present in other OpMNPV transcription factors IE2 and P34. ExonO is a late gene Temporal expression of exonO was analyzed by Northern blot of total RNAs extracted from OpMNPV-infected Ld652Y cells at various time post-infection and hybridized to a strand specific RNA probe homologous to the exonO as shown in Fig. B.3. A transcript of 3.5 kb was detected at 18 hr pi to 120 hr pi, indicating exonO is a late gene. At 24 pi another transcript of 6.4 kb was detected suggesting there are multiple termination sites. Since exonO is a late gene we studied its role for two late functions, DNA replication and late gene transcription. 165 EXONO appears to augment IEO's replication function To determine if OpMNPV EXONO influenced viral DNA replication, transient replication assays were performed. Ld652Y cells were cotransfected with the viral origin containing reporters pHdN; OpMNPV viral replication genes lef-2, -3, helicase, DNA pol, ie2; OpMNPV iel, ieO and exonO. Fig. B.4a shows the results of the replication assay. No significant difference in levels of replication was observed in the presence or absence of EXONO with IE1 or IEO. Similarly no differences in the levels of IE1 or IEO were observed as shown by Western blot (Fig. 7.4b). EXONO might augments IEO's late gene activation Since a working late AcMNPV gene expression system was available in our laboratory we investigated the influence of AcMMPV exonO on late gene expression. Fig. B.5 shows the result of the late gene assay. AcMNPV EXONO appeared to enhance IEO transactivation of a late gene reporter. However, these results were not reproducible and no conclusion could be made on EXONO gene function. To study the effects of OpMNPV exonO on OpMNPV late gene expression a transient system was established. Two reporters CAT and GFP were made, driven by OpMNPV very late polyhedrin promoter. The reporters were tested in Ld652Y cells for expression with OpMNPV DNA, overlapping cosmid clones representing the entire genome of OpMNPV, and with all the overlapping cosmids except 27. Cosmid 27 containing the Hindlll A, T and N fragments was substituted with iel, ieO, ie2, p34 and OpMNPV ExonO. The results are shown in Fig. B.6b and c. Both GFP and CAT constructs were expressed suggesting they are functional and active as late gene reporters. However, the expression levels were low when cosmid 27 is substituted with iel, ieO, ie2, p34 and exonO. It is possible that there are more genes required on cosmid 27 that we did not supply. This suggest the better way to study effect of EXONO in late gene assays is by making a deletion mutant of cosmid 27 166 knocking out exonO. However this is the first time a late gene assay was developed for OpMNPV and our results suggest that the reporter we made is functional with all the overlapping cosmids. 167 DISCUSSION OpMNPV exonO is a highly conserved baculovirus ORF coding for a predicted protein of 245 amino acids. To date it is unknown of exonO has any function in the baculovirus life cycle. ExonO shares the same early gene promoter as ieO suggesting that it may be expressed as an early gene. However, northern blot transcriptional analysis showed that exonO is expressed as a late gene and not an early gene. This also means that 100% of the early transcripts from the exonO/ieO promoter are spliced. Using transient DNA replication assays and late gene expression assays for both OpMNPV and AcMNPV we were unable to show any function for EXONO. The sequence alignments indicated that EXONO is conserved and has some identifiable motifs. The cysteine rich region aligns with RING finger motifs except it has tyrosine instead of a histidine. Either this is a novel ring finger or it is just a cysteine rich region interspersed with tyrosines. In the motif scans the C-terminal region also showed homology to ZZ type cysteine rich putative Zn** binding proteins that have tyrosine interspersed (183) (http://smart.embl-heidelberg.de/smart). Taking into account the conserved nature of EXONO and also the presence of important domains suggests it has an important function in the virus life cycle. Our studies cannot conclude any function to EXONO and more studies are required to determine the exact role. 168 Fig. B.l. Schematic map of the OpMNPV genome, from the 5' end of me53 to the 3' end of odv-e56, showing the contribution of exonO for the ieO formation. Names of the genes in this region are shown below the OPvF arrows. IE1 is expressed as 1.7 kb transcript. IEO is expressed as the spliced product of IE1 which is 130 nt longer that codes for a predicted protein of 35 additional aa at the N-terminus. 23 aa comes from exonO (orf 138) upstream of IE1 and the remaining 12aa comes from 5' untranslated regions (UTR) of IEL 169 (ORF m) i e 1 mRNA 1. 7 Kb > ieO mRNA 1.8 kb 130 nt (35 aa) 35aa:23aa from Exon0+12aa from 5' untranslated region of IE1 170 Fig. B.2. Alignment of ExonO proteins from OpMNPV, CfMNPV, EppoNPV, A c M N P V , BmNPV, RoNPV, LdMNPV, SeMNPV, MacoNPV-A, MacoNPV-B, HearNPV, HzSNPV, AdhoNPV and SpltNPV. The ring finger motif is shown below the consensus sequence in bold letters. The Leucine rich region is shown by the dashed line. The alignments were done using chroma program (http://www.lg.ndirect.co.uk/chroma) (60). 171 OpMNPV CfMNPV EppoNPV AcMNPV BmMNPV RoNPV LdMNPV SeMNPV MacoNPV-A MacoNPV-B HearNPV HzSNPV AdhoNPV SpltNPV Consensus/90% OpMNPV CfMNPV EppoNPV AcMNPV BmMNPV RoNPV LdMNPV SeMNPV MacoNPV-A MacoNPV-B HearNPV HzSNPV AdhoNPV SpltNPV Consensus/90% OpMNPV CfMNPV EppoNPV AcMNPV BmMNPV RoNPV LdMNPV SeMNPV MacoNPV-A MacoNPV-B HearNPV HzSNPV AdhoNPV SpltNPV Consensus/90% R i n g f i n g e r OpMNPV CfMNPV EppoNPV AcMNPV BmMNPV RoNPV LdMNPV SeMNPV MacoNPV-A MacoNPV-B HearNPV HzSNPV AdhohNPV SpltNPV Consensus/90% -CETSKVMDFtfilFAHMy--CADMSTDGKlaA£^Ag«aBr;T»nnK -MIKGTHWPNL V S K S Y T D A -M1KGTYWPNV V S E S Y I E S C E I N K V M D I J ^ J I F D H M Y - - C D D | L V D A K A Q A G V R T ^ J A M I D A K -MIKSGYWQNV LENCGRSD LTEANKMDFNgVFAHMY--CAD|LVDSKVHRDVRG^g V IXDDK - M I R T S S H V L N VQENIMTSNCASSPYSCEA—TSACAEAQQVMI D N § V F F H M Y —NADIQIDAKLQCGVRS^JAMIDDK - M I R T S S H V L N V Q E N I M T S N C A S S P Y S C E A — T S A C A E A Q Q V M I D N Q V F F H M Y — T A D I Q I D A K V Q C G V R S ^ ^ A I I D D K -MIRTSNHVLN VQENIMTSNCASSPYSCEA—TSACAEAOOVMTDNBVFFHMY—NADlOTnAKLOCGVRsSSlAMIDDK M D Q T S S S T Q I L V S LMENAQPDPTAGQPVAQVILDN(JCLSSMY—SPDVLRNPRAQHTIKTgvgQWIDEH MLGEYKM-SNYDIELCTQVFSNgLFPNLY—TSD|ALNIKAHHNVRM^jKIIQDT M -QKYNNELSTQVFSNglLHDLHNDTNN«SKCPKAQFAVKL^jKIIQDM M-QKYNNELS IQVF S N g l LHDLHNDSNNVGKCPKAQFSVKLJJSJEK 1 1 Q D M M S G T L K R I L - Y D I [SDDSI JAKLFRYNSEMQPPXSQCMNTAVDYE I D V E V T K C G K L K N M Y — S S V T T N A R A Q Y I W K L ^ J S L T V L D E MSGTLKP : L-YDDISDDSDJ AKLFRYNSEM0JPPASQC3MNTAVDYEIDVEVIKCFLKLKMMY--SSDVITKARAQYNVKLBB^ I V L D E M N F T V S S E H M G E E E Y G F Q S N V M N N Y I N E V I T D Q V F S D K Y - - A P A L L Y N S K L R N D L K I L ^ N I I N D A MILHQSDNEINKPLSQVFKHLREMGSMYDDTEMAAIYAAATTTATDEKCNKNDVSSYH-DHQVLSN§MLNGMY—SDDLKVNLKAQYNVKL^ S S^IVEHK p . . . . h . . s F . h . p h a . - s s s l . . s . + h p . s l + . A A F . h i . c . HFELYK-PJlIENALFS-YRDQCDGNAAPPARLSDNG -Dgcj^FWIWu^i^SJKSJSEATSV- Gim'IVI^Bl J < '2Q I A L K M L S D A F RS A K' :' I G HIELYK-RRlENGFFS-YRDRCDNASALPQRLF^E -cSc^FVSDAARVIECWKSSETASA GHNVVVLL^LKl4jQVALKVLSDAJ7\g3AKVIS HFELYK-RRJENNFFH-YYDPCDDMA-FPKHLLNND-V^C^HFINDAVCVVEC^KSyEKASV GVDI IVLL^LKijjQLILKMLNnAFV^EKSLG HZiEMYK-HRjENKFFY-YYDQCADIA-KPDRLPDDDGASc^HFlFDAQRIIQCIKE^ESAYG—VRDRGNVIVFY^LK^RDALKLIKNSFA^r'Kl IN HLEMYK-YRIENKFFY-YYDQCADIA-KPDRLPDDDGA^C^HFIFDAQRIIQCiKEIEGAYG—VRDRGNVIVFY^SLKCJJRDALKLIKNSFA^FKNIN HiEMFK-HRIENKFFY-YYDQCADIT-KPDRLPDDDGASajHFIFDAQPvIIQCIKEIESAYG—VRDRGNVIVFY^^kK^RDALKLIKNSFAJSJjjFKI IN HRKMYD-CPIE--PPLRFTDD AHLSAD-Rg»|S|HLJGKLERWSVtRAMRA MSRFEHNVFVFLggcKSJjRALVELFRHDY-SSQSTVA YCQSYN-CDLD--EL1VFRENDDDVS SS IPRD-RgvjjYLINDIKNyLDViEHLKS QPKFQYNMYIFM^VKOIMVINDMFKNDF-ggTAIVK YQQSYD-NAij>--SLLAYRESISE AVLPRD-sBvjJJYLlNDIKNVIDVlGHiNN OPKFOYNMYIFlJgiVRdlROINTLFINDY-SSISK 1VK YQQSYD-NACD--SLLAYRESISE AVLPRD-S^V^YLINEIKNVIDVIGHLNN OPKFOYNMYTFlJBnVRfjlROTNTT.FTjND V-IS73sKTVT< YKKQYK-NNBDKHSVLYYKETSES-V ITLDED-Q^HJ^TLLPI IQQJjLKTTCYIJ^FSDDEVNYVKQKFIFI^jLKrjNKILKLFQYDK-^SAKLTK YKKQYK-NNW3KQSVIYYKETSES-V ITLDED-cgH|]]TI.LPI IQQLLKTICYLMNFSDDEVNYVKQKFIFLggLKrgNKILKLFQYDK-^AKLTK FLRAHN-EDIP—NQilYRENDED MKFCKD-GglflFLIRKINDJSNIWE JVN SPKYKNSQYIFll^VKCJlKLAIYYFVNDY-SjNKLVN YNEDHHDSGtDKTSPIYYGEDESV VLCGED-K^YJJJDLIAETADLSKIIEDLY'D ASPVYRLNYFTFvgBVKBJlLQI ICMFTNDV-ggKRSVR a b p . a p . p . l - . . . . b . a . - p s p - . .ChHhhl. . h . p i . p h i . . 1 . 8 p.hlhhPYl+bL. .h.phh. .sb .CC.p.h. GLQMYf^DIj§SHCLLYADRfEAAGRAl QVXSlgr.N.:::. i LQE@Dl^KEA.*: AD: RFI^gjKJ^gjfl; L g l A g C / A l g T A S r—HAj<B|Agsj5sFiJs G L Q L Y V Q f l l J L S H C L L C A E K j F ^ G R T L C ^ I N l j j L D T N - S l ^ ^ L g K E A S T D —HAKgjAgSJJSFJJS R L Q M Y V N E L L S H C L L C A E K t E A A S R T L Q V M S j g V N T G - T I ^ ^ l g ^ SMOMYVNEIJSNCLLFIEKlETI>ntTVKgMNl3^NL-VliteiaNvl»^ SMQMYVNELISNCLLFIEKlETINKWKVMNigVDNS - VI^giaVgKE IS TDERFlQjKJ^gEQAlBNAgCVTMjJjTAT- T - - HAKgjAgRJJSYJJ S M Q M Y V N E L J S N C L L F I E K i j ^ T I O T C T V K V M N i Q v D N L - V I ^ ^ V ^ K E I S T D E R F I ^ ^ K ^ ^ E ^ GCARALDETIADAQRHLLVVRSMSERAAVMLVJjjSDWV-RVQC^'I^DSSM TNSNAIjreLCF,RGEKYMHVIKTl^RMOIINVBTNP--KVBc^ SNSAAtoT,LiAHCnKYLHVlASMNERMOLINVBTEP--Kia^ll»bDTSVEEHFtja3N^ SNGAAXDTLXAHCDKYLHVYASMNERMQLINVgTEP— KIjjO^NlSQDTSvEEHFT^gNgy^C^NI^NMSYANL^FCN-L—YPC^Bv^E^SE^jGSKQV QLQAQI^TIiTQSADSCKHIHAINRQSCiVLTV2LEN--Pi^^^l2PJDTFNDEFJi l23N^22c^^I^l5YANL22YST -V- -FPTgJ.'gKJjSFUsSEYS 0!,OA01^TIXTOSADSCKHIHAINROSOVI,TVBLEN--PLgteiaNll»toTFNDFRHlE^^ F .TTF,RIJIT,II^SNKYIJIYYRIXHERVQVM(f^DF,T-Vljai^l l« taTSIXKSFI^^ S T I A R J U E T C L K R G N E K L E S V R R L N K T L N V M S V i j L E Q — N y ^ ^ l E K D V S N D E R F l 4 g ^ j ^ g G F R l g N I^ANI^ F S N-TPHNPV33 v S K i l s Y R V E K Y E l p . h l . p s . . . h e . 1 . . h s c p . p l h s l F . p l Y p C s l C p - h . s - . p a l K P p E C C . Y . I C . h c h s . h W K h s s . h . . a s . C T s C p T t a K C C C . Y . C C C C KRLQK—ADDPATL RNSAP—SVEL VEREI VEQEL SFKQVYTADTTDNI SFKQVYTADTTDNI SSSPt 172 Fig. B.3. Northern blot analysis of OpMNPV exonO in Ld652Y cells from 0 to 120 hours p.i. The arrow marks and numbers to the right represent the length of the exonO transcripts. The numbers on the top of the blot are the different time points post-infection the total RNAs were isolated from OpMNPV-infected Ld652Y cells. The schematic above the blot is the OpMNPV genome showing the exonO region. The RNA probe homologous to exonO sequence excluding the ieO and orfl39 overlapping region is shown by a solid bar and labelled as ExonO probe. AATAAA are the poly A termination signals present in this region. The dashed lines represent the possible location of the beginning and the end sites for major transcripts 3.5 kb and 6.4 kb detected in the northern blot. 173 174 Fig. B.4. Analysis of OpMNPV EXONO role in replication and its effect on the protein expression of IEO and IE1. a) Ld652Y cells were co-transfected with OpMNPV replication factors (lef-1, -2, -3, helicase, DNA pol) the viral non-/zr origin of replication pHdN and with pz'e/-IEl or pieO-lEO and pOpExonO and pOpExonOts constructs. The negative control lane (pBS+) contains pBS+ replaced into p/e/-IEl or pieO-lEO. The number to the right of the blot corresponds to the size (in kb) of the hybridized band of linearized pHdN. The name of the sample corresponding to each lane is shown on top of the blot, b) Western blot analysis of IEO and IE1 expressed from p/ei-IE 1 and pzei-IEO. Ld652Y cells from replication assays transfected with replication genes and pzW-IEl; pz'e/-IE0 and pBS+ and with pOpExonO and pOpExonOA were harvested. Samples were separated by 8%SDS-PAGE, transferred to membranes and probed with an IE1 monoclonal antibody. The location of the IE0/IE1 bands for the samples is shown by arrow head. 175 (a) IEO IEO IEO IE1 pBS+ + + ExonO ExonOA ft - •• • - < P HdN(6 .9kb) (b) , IE0(68.0) & 161(64.2) 176 Fig. B.5. Analysis of AcMNPV EXONO function in transient late gene expression assays, a) Schematic of AcMNPV genome map. This figure is from Li et,al. 1999 (125). The arrows above the map are the location of genes required for transient late gene expression assays in Sf9 cells. Cosmids 10, 59, 1 and 58 carried all the genes reported previously for the late gene assay, b) Analysis of AcMNPV transient late gene expression with exonO in Sf9 cells. The reporter plasmid pPH-CAT which contains the CAT gene under the control of polyhedrin promoter was co-transfected with Cosmids 10,59,1 and the late genes + lef-7, ie2, p35 along with iel or ieO and with exonO. The Y axis represents the rate of CAT activity as counts per minute. 177 266S5 5 4 7 9 8 COS01O 62S8T 1 1 I c o s # 5 9 lots 3 S « f ) ) w i u 5 >eW telID o loO a eos#1 8 9 7 6 4 t I i kiU hoi KH5 I I, cos#S8 I I kilobase 20 40 60 80 100 Hirti\\\ restiction map of A cMNPV 120 178 Fig. B.6. OpMNPV transient late gene assay, (a) Schematic of OpMNPV genome map. The boxes are the location of OpMNPV Hindlll cosmids the overlap to cover the entire length of genome. The arrow heads above the map are the location of iel, ieO, exonO, iel and p34 genes, (b and c) Analysis of OpMNPV transient late gene expression with exonO in Ld652Y cells. The reporter plasmids pOppH5'p&3't-CAT and pOppH5'p&3't-GFP which contains CAT and GFP genes under the control of polyhedrin promoter were co-transfected with viral DNA or all the overlapping cosmids (1,13,54,41,9,5,27); or all the overlapping cosmids except 27 and the late genes lef-7, iel,p34, iel, ieO, and exonO. b) Graph represents the CAT activity, c) Micrographs of cells expressing GFP. 179 (a) COS 47 Cos 13 CM I A T N S H I R K C ExonO »' p34Sma L D U V B P J G O F E M Q m Hindlll restriction map of OpMNPV pOpPh(5p&3't)-CAT All cos Allcos(-cos27S47) AI COS (-cos 27S47) (1,13,54,41,9,5,47,27) |El,IE0,IE2,Exon0 pBS+ (O pOpPh(5'p&3't)-GFP OpMNPV DNA AH cos (1,13,54,41,9,5,27) Allcos(-cos27&47) IEl,IEO,IE2,ExonO All cos (-cos 27*47) pBS+ 180 

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