JOURNAL OF VIROLOGY, June 2002, p. 5598–5604 0022-538X/02/$04.00⫹0 DOI: 10.1128/JVI.76.11.5598–5604.2002 Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Vol. 76, No. 11
The Acidic Activation Domain of the Baculovirus Transactivator IE1 Contains a Virus-Specific Domain Essential for DNA Replication† Joseph A. Pathakamuri1 and David A. Theilmann1,2* Pacific Agri-Food Research Centre, Agriculture and Agri-Food Canada, Summerland, British Columbia, Canada V0H 1Z0,2 and Department of Plant Science, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z41 Received 18 December 2001/Accepted 1 March 2002
IE1 is a potent transcriptional transactivator of the baculovirus Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV) and has been shown to be essential for viral DNA replication. IE1 contains an acidic activation domain (AAD) at the N terminus that is essential for transcriptional transactivation, but its role in viral DNA replication is unknown. In this study the role of the IE1 AAD in DNA replication is investigated. We have determined that deletion of the AAD eliminates the ability of IE1 to support DNA replication, showing that the AAD is essential for DNA replication as well as transcriptional transactivation. Replacement of the AAD with the archetype domain from herpesvirus VP16 and the evolutionarily related domain from Autographa californica MNPV (AcMNPV) IE1 produces chimeric proteins that are potent transactivators. Surprisingly, however, these chimeric proteins were unable to support DNA replication, indicating that there is a host- or virus-specific replication subdomain in the AAD that was not functionally replaced by the VP16 or AcMNPV AAD. Using N- and C-terminal deletion mutants, the region of the AAD that was essential for DNA replication was mapped to amino acids 1 to 65. AAD deletion mutants also showed that an IE1 that is functional for transcriptional transactivation is not required for viral DNA replication. The IE1 AAD therefore contains an essential replication domain that is separable from the transcriptional activation domains. Our results suggest that IE1 specifically interacts with a component of the viral replication complex, supporting the view that it acts as a nucleating factor by binding to the viral replication origins. The baculovirus Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV) has a genome of 131,990 bp and encodes approximately 150 genes of 50 amino acids (aa) or larger (5). Using transient-replication assays, it has been shown that six genes, lef-1, lef-2, lef-3, dnapol, p143, and ie1, are essential for replication and that three genes, ie2, p34, and iap-1, are stimulatory (4). Based on studies with OpMNPV and the related viruses Autographa californica MNPV (AcMNPV) and Bombyx mori NPV, data suggest that lef-1 encodes a possible primase and interacts directly with lef-2, which has an unknown function (14). lef-3 encodes a single-stranded DNA binding (SSB) protein, forms homotrimers, and is essential for transport of P143 into the nucleus (1, 15, 56). P143 is a possible helicase, and dnapol is a DNA polymerase (2). IE1 is a primary transcriptional transactivator known to activate early- and lategene expression during baculovirus infections (19, 54). The role of IE1 in baculovirus replication has been hypothesized to be transactivation of other replication genes, or it may play a role as an origin binding protein (OBP). However, Rapp et al. (45) 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 IE2 and P34 (PE38 in AcMNPV and B. mori NPV) are transcriptional activators, and IAP-1 is an inhibitor of apoptosis (4). 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. (42). IE1 forms homodimers and can transactivate genes in an enhancer-dependent or independent manner (11, 18, 27, 29, 30, 48, 49). 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 found to be composed of two subdomains, both of which were required for transactivation. In addition, it was shown that the AAD could be replaced by the archetype VP16 AAD from herpes simplex virus type 1 and the AcMNPV IE1 AAD. Chimeric proteins containing the VP16 and AcMNPV AADs appeared to be more potent activators of gene expression than the wildtype (WT) IE1 (17). A number of studies have shown that transcriptional transactivators, 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. Present evidence suggests that transcription factors appear to activate DNA replication by general mechanisms that include (i) direct recruitment of the replication machinery through protein-protein interactions (21, 32), (ii) modulation of chromatin structure (24, 31), (iii) recruitment to the nuclear matrix and replication factories, and (iv) unwinding of duplex DNA by transcriptional activation (37, 39, 44). These various mechanisms may operate in concert rather than individually.
* Corresponding author. Mailing address: Pacific Agri-Food Research Centre Agriculture and Agri-Food Canada, 4200 Highway 97, Summerland, BC V0H 1Z0, Canada. Phone: (250) 494-6395. Fax: (250) 494-0755. E-mail:
[email protected]. † This paper is contribution no. 2150 from the Pacific Agri-Food Research Centre. 5598
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FIG. 1. Schematic of the replication genes of OpMNPV IE1 and the chimeric constructs used for characterizing the role of AADs in viral DNA replication. (a) WT OpMNPV IE1 protein showing the functional domains based on studies of OpMNPV IE1 and the homologous AcMNPV IE1 (27, 49, 51). The 560 aa of OpMNPV IE1 contains 126-aa AAD (AAD), bordered by a cluster of highly conserved basic amino acids (B); the C-terminal region contains a possible second transactivation domain (D) (51) 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 (IE1-OpAD), herpes simplex virus VP16 (IE1-VP16 AD), and AcMNPV IE1 (IE1-AcAD). The 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 VP16 and AcMNPV IE1 proteins. (c) HindIII restriction map of the OpMNPV genome showing the location of the genes used in the transient-replication assay. Cosmid 9 is used to supply lef-3 and p143 (helicase), and plasmid pHdN is used to supply the viral non-hr origin of replication and lef-1; lef-2, ie2, and ie1 are supplied by individual plasmid constructs.
The VP16 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 VP16 AAD to activate polyoma- and papillomavirus replication, it has been determined that it interacts directly with replication protein A (RP-A), which is an SSB protein, an essential component of cellular replication machinery (21, 32). In addition, AADs, when tethered to a replication origin by a heterologous DNA binding domain, such as Gal4-VP16, Gal4p53, or Gal4-BRCA1 AAD, can stimulate cellular replication in yeast and viral replication in mammalian cells (10, 13, 22, 24, 33). These data suggest that 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 subdomains that can independently or synergistically transactivate transcription (17), similar to the activation domains of VP16 (47, 52), P53 (9), GCN4 (23), and Gal4 (34). Compared to transcriptional transactivation, there is significantly less known concerning replication-specific domains in AADs. In this study, we inves-
tigated 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) that 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. MATERIALS AND METHODS Cell culture. Lymantria dispar (Ld652Y) cells were maintained in TC100 medium as described earlier (53). Plasmid and cosmid constructs. The plasmids encoding the six essential OpMNPV DNA replication genes, the stimulatory genes, and the origin-containing reporter have been described previously (Fig. 1c) (1–5). Plasmid constructs pHdN, pDNApol, pCA35, and cosmid 9 were provided by George Rohrmann. OpMNPV IE1 (pIE1-sal) and IE2 (pIE2-E2.3) clones, IE1-AD⫺, IE1 acidic domain chimeric constructs (Fig. 1a), IE1 N-terminal and C-terminal acidic domain deletion constructs (see Fig. 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 (3). Log-phase Ld652Y cells were seeded onto six-well culture plates (106 cells/well) in TC100 medium containing 50 g of the antibiotic gentamicin sulfate (Life Technologies)/ml and were incubated at 27°C overnight. The cells were transfected with DNA constructs using liposomes prepared as
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previously described (8). The optimal Lipofectin volume/DNA ratio was determined by titration. Cotransfections were performed by mixing 1 g of pHdN non-hr origin-containing reporter plasmid; 0.25 g of cosmid 9; 0.5 g of pDNApol, pCA35, and IE2; and 0.5 g of various IE1 constructs (Fig. 1). Each cotransfection was done in duplicate, and amounts of DNA were equalized with pBS⫹ plasmid. After 4 h, the DNA-liposome transfection mixture was removed and the cells were washed with 1⫻ Grace’s medium and then overlaid with TC100 media. After 62 to 65 h at 27°C, cells were removed with a rubber policeman washed once with 500 l of phosphate-buffered saline (pH 7.4) (50) and were pelleted in a microcentrifuge (5 min at 3,500 ⫻ g). Total cellular DNA was extracted from replication samples by resuspending cells in 450 l of 1⫻ Tris-EDTA buffer (10 mM Tris, pH 7.8, 0.6% sodium dodecyl sulfate [SDS], 10 mM EDTA) and 50 l of proteinase K (20 mg/ml) and was incubated at 55 to 65°C overnight. The samples were then extracted once with buffer-saturated phenol, once with phenol-chloroform-isoamyl alcohol (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 l of 1⫻ Tris-EDTA (10 mM Tris, pH 8.0, 1 mM EDTA). Five micrograms of purified DNA was digested with a combination of DpnI (10 U) and EcoRI (10 U) (New England Biolabs) in a total volume of 20 l at 37°C overnight, followed by a 30-min digestion with 2 g of RNase A. The digested DNA was separated in 0.7% agarose gels, alkaline blotted to Bio-Rad Zeta Probe ZT membrane (BioRad), hybridized for 12 h with a 1.76-kb HindIII-XhoI fragment of the pHdN reporter construct labeled with [32P]dCTP (RadPrime labeling system; Life Technologies) in prehybridization buffer (0.25 M phosphate buffer, 1 mM EDTA, 1% bovine serum albumin, and 7% SDS, pH 7.2) (12) at 65°C in a hybridization oven, and washed under stringent conditions (0.1⫻ SSC–0.1% SDS at 68°C [1⫻ SSC is 0.15 M NaCl plus 0.015 M sodium citrate]) (6). Membranes were visualized on a Storm PhosphorImager (Molecular Dynamics). CAT assay. To compare the activation of reporter plasmids by chimeric IE1s in the presence or absence of replication factors, Ld652Y cells were transfected as described above. Cells were cotransfected with 1 g of p39CAT-E3 reporter construct, which is IE1 chimeric constructs along with all the replication factor constructs. Duplicate wells were cotransfected with p39CAT-E3 and 0.5 g of IE1 chimeric constructs alone. To assay for chloramphenicol acetyltransferase (CAT) activity, cells were scraped off dishes 48 h posttransfection and pelleted (5 min at 3,500 ⫻ g), all media were removed, and the cell pellets were resuspended in 100 l of 250 mM Tris-HCl (pH 7.8). Cells were lysed by repeated freezethawing (three cycles), and any cellular deacetylases were inactivated by incubation at 65°C for 15 min, followed by short centrifugation (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 (1 to 50 l) was added to CAT assay buffer (6.25 mM chloramphenicol, 160 mM Tris-HCl [pH 7.8], 3.2 M acetyl coenzyme A [Sigma], and 0.025 Ci [125 pmol] of [3H]acetyl coenzyme A) (CAT Assay Grade; New England Nuclear) in a total volume of 125 l (40). The mixture was overlaid with 3 ml of toluene-based scintillation fluor (Econofluor-2; Packard Bioscience Co.). All transfections were repeated a minimum of two times, each in duplicate.
RESULTS To initially investigate the role of the IE1 AAD in viral replication, we performed transient DNA replication assays using ie1 AAD chimeric constructs and the replication genes. The chimeric constructs are IE1-AD⫺, from which AAD has been deleted; IE1-OpAD, which has the WT AAD; and two chimeric constructs that had the WT AAD replaced by the VP16 AAD (IE1-VP16AD) and AcMNPV AAD (IE1-AcAD) (Fig. 1). Previous studies have shown that the two latter constructs are more potent activators of transcription than the constructs containing the WT AAD (IE1-OpAD) (17). 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 previously shown for transactivation of transcription. Figure 2 shows the results of a DpnI-based transient-replication assay using the different chi-
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FIG. 2. DpnI-based transient-replication assays for the analysis of IE1 chimeras ability to activate replication. (a) Ld652Y cells were cotransfected with five viral replication genes (lef-1, -2, and -3, p143, and dnapol), ie2, the viral non-hr origin of replication (pHdN), and different IE1 chimeras. The number to the left of the blot corresponds to the size (in kilobases) of the hybridized band of linearized pHdN. The name of the sample corresponding to each lane is shown on the top of the blot. Each sample is presented in duplicate and represents two separate transfections. The negative control lane (pBS⫹) contains ie1 replaced by pBS⫹ and lacks any replication signal, indicating that there is no background replication. The lane M is a marker lane, which is the reporter plasmid pHdN linearized with EcoRI mixed with genomic DNA cut with DpnI. C, control blank lane. (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 IE1-OpAD, which was given an arbitrary value of 100. Error bars indicate standard error.
meric IE1 constructs to activate replication of the viral (nonhr) reporter plasmid pHdN. The WT construct IE1-OpAD showed a strong DpnI-resistant replication signal of the origin plasmid. The OpIE1-ADⴚ construct, which has the AAD completely deleted, was unable to support replication, and no sig-
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nal was observed, indicating that the acidic domain is essential for replication. Surprisingly, for the two chimeric constructs containing heterologous AADs, IE1-VP16AD and IE1-AcAD, there was no replication of the reporter plasmid. This is a significantly different result from that for transcriptional activation, where IE1-VP16 and IE1-AcAAD were shown to be strong transactivators (17). This shows that, for viral DNA replication, unlike activation of transcription, the native OpMNPV IE1 AAD cannot be replaced by VP16 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-replication assays using the OpMNPV enhancer containing reporter plasmid p39CAT-E3 (17). p39CAT-E3 was cotransfected with the chimeric IE1 constructs with and without replication factors. The results show that the chimeric IE1 proteins were expressed and active in the presence or absence of replication factors (Fig. 2b). Similar to results described by Forsythe et al. (17), both IE1-AcAD and IE1-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 VP16 and AcMNPV IE1 AADs are nonfunctional for activation of OpMNPV DNA replication and do not correlate with their potency in transcriptional transactivation. This indicates that an IE1 that is a strong transcriptional activator is not sufficient for supporting virus-specific DNA replication. In addition, since the evolutionarily related AAD from AcMNPV was also nonfunctional, it suggests that the OpMNPV IE1 AAD contains either host- or virus-specific determinants. 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. To identify any replicationspecific 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. 3). Previous analysis utilizing these clones has identified two domains, A (1 to 92 aa) and B (aa 81 to 124) (see Fig. 5), required for transcriptional activation in Ld652Y cells (17). The results of transient-replication assays with WT IE1, and each N- and C-terminal AAD deletion construct, are shown in Fig. 4. The ability of each construct to activate transcription is shown below each lane, indicated by a ⫹ or ⫺ symbol. The WT IE1 containing the intact N-terminal region of AAD activated replication, but surprisingly all deletion mutants, ⌬14–25 through ⌬14–143, representing 14 to 143 aa of the IE1 N terminus region (Fig. 3 and 4a), eliminated the replication signal of the pHdN reporter plasmid. As all the N-terminal deletions eliminated replication, the replication domain border could only be mapped to the first amino acid. Interestingly, even though all the N-terminal deletions abrogated replication, many of them are still able to activate transcription (Fig. 4a). In particular, ⌬14–25 and ⌬14–27 transactivate transcription at levels equal to or greater than those for WT IE1 (17). This supports the results of Fig. 2a showing that a strong transcriptional activator is not sufficient for replication. To map the C-terminal border of the AAD required for replication, the IE1 C-terminal deletions were used (Fig. 3 and
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FIG. 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-terminal OpMNPV IE1 AAD. Boxed amino acids indicate the basic domain (B) that borders the AAD. (b and c) Diagrams of AAD N-terminal (b) and C-terminal (c) deletion clones. Each deletion mutant has been designated according to the amino acids that have been deleted, which are shown in panel a. Clones WT and WT4 are the full-length IE1 AAD for N- and Cterminal deletion analysis, respectively. The G in the C-terminal deletions refers to the glycine that was inserted during the construction of the deletions (17). The domain designations are the same as those shown in Fig. 1.
4b). The C-terminal deletions ⌬113–124 through ⌬73–124 showed a strong replication signal. The largest deletion that showed a weak but repeatable replication signal was ⌬66–124. Deletions larger than ⌬66–124 (⌬51–124, ⌬45–124, ⌬36–124, ⌬27–124, and ⌬16–124) completely abrogated the replication signal (Fig. 4b). Therefore, the C terminus of the replication domain in the AAD maps to amino acid 65. Three C-terminal deletion clones, ⌬78–124, ⌬73–124, and ⌬66–124, 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
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FIG. 4. DpnI-based transient-replication assays to map specific regions of OpMNPV IE1 AAD required for replication. (a and b) Nterminal deletion clones (a) and C-terminal deletion clones (b) as described in the Fig. 3 legend. Ld652Y cells were cotransfected with the appropriate ie1 construct, five viral replication genes (lef-1, -2, and -3, p143, and dnapol), ie2, and a 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⫹), ie1 is replaced with the plasmid pBS⫹. The ability of each sample to activate transcription as previously determined (17) is indicated by a ⫹ or ⫺ symbol at the bottom of the blot. Each sample is in duplicate except pBS⫹ and clone ⌬14–107, for which there is only a single lane. The additional panel of sample ⌬66–124 highlights weak bands with enhanced contrast.
constructs that are transcriptionally active and inactive for replication or vice versa. This supports the view that these are independent functions; that is, transcriptional activation is not required for IE1 to support DNA replication. Figure 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 that have been shown to be involved in AAD function. No distinct homologies with known protein motifs were identified. 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.
DISCUSSION It has been previously shown that the AAD of OpMNPV IE1 is essential for transcriptional transactivation and contains at least two domains that interact synergistically (17). In our present study we characterized the role of the IE1 AAD in viral DNA replication in Ld652Y cells using transient-replication assays. Our results show that the AAD of OpMNPV IE1 is essential for replication, but quite surprisingly, the heterologous AAD from VP16 or the baculovirus AcMNPV IE1 failed to activate replication. These results reveal important differences between OpMNPV IE1 AAD and AADs in other systems. Previous studies have shown that AADs can be ex-
FIG. 5. Schematic diagram showing the summary of domains identified in the OpMNPV IE1 AAD. Two domains, A (aa 1 to 92) and B (aa 81 to 124), required for transcriptional transactivation in Ld652Y cells were previously mapped (17). The location and amino acid sequence of the replication domain are shown below the transcription activation domains. The sequence of the minimal replication domain (aa 1 to 65) is shown, and the symbols at the bottom refer to the charge distribution: acidic amino acid (⫺) and basic amino acid (⫹).
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changed with heterologous domains for both replication and transcription (10, 13, 21, 22, 24, 32, 33). However, our data show that for replication the OpMNPV IE1 AAD is not exchangeable and in addition confers virus specificity. A deletion analysis mapped the minimum sequence of the OpMNPV IE1 AAD that permitted replication to aa 1 to 65. This overlaps with but is different from the Ld652Y cell minimal transcriptional activation subdomains (A [1 to 92 aa] and B [81 to 124 aa] [Fig. 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 in transientreplication assays is not required. Therefore, the transcription and replication roles of IE1 are independent and separable. Baculovirus replication requires six essential genes, which are IE1, DNApol, P143, and LEF-1, -2, and -3 (4, 26). 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 glutathione transferase fusion experiments, Evans et al. (14) 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 twohybrid analysis to interact with P143 (15, 16, 20). Cotransfection experiments have also shown that LEF-3 is required for the transport of P143 from the cytoplasm to the nucleus (56). McDougal and Guarino (36) showed that LEF-3 stimulated viral replication by improving the strand displacement activity of DNApol, which suggested a possible protein-protein interaction between these two proteins. IE1 is known to bind to baculovirus replication origins as dimers (29, 46, 48), suggesting that it is an OBP. In 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 (7, 28, 35). Okano et al. (41) presented similar observations; they showed that IE1 is localized to discrete nuclear regions before the onset of replication. Subsequently, at the onset of replication LEF-3 colocalizes 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. Native AADs can be replaced by heterologous AADs from a variety of eukaryotic species and can retain both transcription and replication activation (33, 37, 39, 43, 55), 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 remodeling chromatin structure (24, 25, 31) or by recruiting proteins to either the replication or transcription initiation complexes (21, 32, 44). 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 that we mapped specifically activates replication by direct interaction with other components of the viral replication complex via the AAD (aa 1 to 65) instead of the transcriptional complex. As
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described above, IE1 is known to colocalize 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 VP16 activate replication by a mechanism that is dependent 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 (21, 32). OpMNPV IE1 AAD may be activating replication in a similar mechanism; that is, LEF-3 or other replication proteins may directly interact with IE1 and be recruited to the replication origin. Slack and Blissard (51) showed that AcMNPV contains a second activation domain C-terminal to the basic domain (Fig. 1a). OpMNPV IE1 contains homologous sequences, but it is unknown if it actively functions 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. 2b) (17). 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 VP16 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 with 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 (38). This suggests that the AcMNPV IE1 AAD could interact with any required cellular factors. However, since IE1-AcAD is nonfunctional 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. In summary, we have shown that the AAD of OpMNPV IE1 is essential for replication and that the heterologous AADs from VP16 or AcMNPV IE1 failed to activate replication, revealing important differences between the OpMNPV IE1 AAD and AADs in other systems. A deletion analysis mapped the OpMNPV IE1 replication domain to amino acids 1 to 65, which overlaps with but is different from the minimal transcriptional activation subdomains. We found that transcriptional activation by IE1 in transient-replication assays is not required for replication and that the transcription and replication roles of IE1 are independent and separable. These findings will help to reveal the mechanism by which IE1 stimulates viral DNA replication and suggest possible interactions with other viral proteins to form the replication complex. ACKNOWLEDGMENTS We are very thankful to George F. Rohrmann, Oregon State University, for kindly providing us the cosmid 9, DNApol, pHdN, and pCA35 clones. We also thank Leslie Willis for his expert technical assistance and comments on the manuscript. This work was supported in part by a Research Grant awarded to D.A.T. from the Natural Sciences and Engineering Research Council of Canada.
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