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Journal of Virology, April 2005, p. 5129-5141, Vol. 79, No. 8
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.8.5129-5141.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Murine Gammaherpesvirus 68 Open Reading Frame 45 Plays an Essential Role during the Immediate-Early Phase of Viral Replication

Qingmei Jia, Vasili Chernishof, Eric Bortz, Ian Mchardy, Ting-Ting Wu, Hsiang-I Liao, and Ren Sun*

Department of Molecular and Medical Pharmacology, AIDS Institute, Jonsson Comprehensive Cancer Center, Dental Research Institute, and Molecular Biology Institute, University of California at Los Angeles, Los Angeles, California

Received 4 June 2004/ Accepted 23 November 2004


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ABSTRACT
 
Murine gammaherpesvirus 68 (MHV-68) has been developed as a model for the human gammaherpesviruses Epstein-Barr virus and human herpesvirus 8/Kaposi's sarcoma-associated herpesvirus (HHV-8/KSHV), which are associated with several types of human diseases. Open reading frame 45 (ORF45) is conserved among the members of the Gammaherpesvirinae subfamily and has been suggested to be a virion tegument protein. The repression of ORF45 expression by small interfering RNAs inhibits MHV-68 viral replication. However, the gene product of MHV-68 ORF45 and its function have not yet been well characterized. In this report, we show that MHV-68 ORF45 is a phosphorylated nuclear protein. We constructed an ORF45-null MHV-68 mutant virus (45STOP) by the insertion of translation termination codons into the portion of the gene encoding the N terminus of ORF45. We demonstrated that the ORF45 protein is essential for viral gene expression immediately after the viral genome enters the nucleus. These defects in viral replication were rescued by providing ORF45 in trans or in an ORF45-null revertant (45STOP.R) virus. Using a transcomplementation assay, we showed that the function of ORF45 in viral replication is conserved with that of its KSHV homologue. Finally, we found that the C-terminal 23 amino acids that are highly conserved among the Gammaherpesvirinae subfamily are critical for the function of ORF45 in viral replication.


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INTRODUCTION
 
Members of the Gammaherpesvirinae subfamily, including two human gammaherpesviruses, Epstein-Barr virus (EBV) and Human herpesvirus 8/Kaposi's sarcoma-associated herpesvirus (HHV-8/KSHV), have two distinct stages in their life cycle, latency and lytic replication, both of which are required for the ability to cause benign or malignant tumors in infected hosts (24). EBV is associated with Burkitt's lymphoma, nasopharyngeal carcinoma, Hodgkin's disease, and lymphoproliferative diseases in immunodeficient patients (31). KSHV is associated with Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease (7, 9, 11, 46). EBV or KSHV infection in vitro results in a latent infection in a majority of the infected population (8, 16, 26, 34). While the latency and the switch from latency to lytic replication of human gammaherpesviruses have been well documented, studies of viral genes involved in permissive infections are hampered by the lack of cell culture systems capable of supporting productive replication.

Murine gammaherpesvirus 68 (MHV-68), also referred to as {gamma}HV68, is a natural pathogen of wild rodents (2, 32, 37). The MHV-68 genome has been completely sequenced, and the virus was found to be related to KSHV and EBV (14, 35, 47, 56). Functions of some MHV-68 gene products have been observed to be similar to the corresponding gene products of human gammaherpesviruses (48, 49, 57). However, unlike KSHV and EBV, MHV-68 establishes productive infections in a variety of fibroblast, epithelial, and macrophage cell lines and is capable of infecting laboratory mice, facilitating the study of this gammaherpesvirus both in vitro and in vivo (37, 51, 52). The availability of viral mutants would significantly contribute to our understanding of viral gene functions and to evaluations of their roles in pathogenesis. MHV-68 mutants bearing site-specific alterations have been constructed for explorations of the functions of viral genes in various aspects of the viral life cycle, e.g., their requirement for infecting cultured cells, evading immune responses, establishing latent infections, and inducing tumors (10, 12, 19, 22, 33, 55). Other advantages of the MHV-68 model are the abilities to manipulate the host genome and immune system and to study the virus life cycle in different genetic backgrounds (13, 15, 28, 39, 53). Thus, MHV-68 provides a model for examining the roles of gammaherpesvirus genes in cultured cells and investigating the biology and pathogenesis of gammaherpesviruses in the host (42).

Tegument proteins of alpha- and betaherpesviruses have been found to be involved in three essential functions in viral replication: (i) the assembly and egress of virions (30, 38, 50); (ii) structural effects during the entry of virions into naïve cells, including the translocation of nucleocapsids to the nucleus; and (iii) other effects during the immediate-early phase of infection, including the transactivation of viral immediate-early genes and the possible modulation of host cell gene expression, innate immune mechanisms, and signal transduction (5, 6, 18, 50, 60). Little is known about the structure and composition of the virion teguments of the gammaherpesviruses.

Open reading frame 45 (ORF45) is conserved among viruses in the Gammaherpesvirinae subfamily but is not found in the alpha- or betaherpesviruses. There is no cellular homologue for ORF45. For KSHV, ORF45 was first reported to be an immediate-early gene during reactivation by chemical induction (59). Other reports indicated that KSHV ORF45 is expressed during the early phase of viral reactivation (20, 40). KSHV ORF45 has been suggested to be a component of viral tegument, which binds interferon regulatory factor 7 and interferes with the translocation of the protein to the nucleus, where it normally activates interferon response genes (60, 61). Antibodies against the ORF45 homologue (BKRF4; 217 amino acids) of EBV were found in nasopharyngeal carcinoma patient sera (17). One study of EBV gene expression during oral hairy leukoplakia detected the expression of BKRF4 in an oral hairy leukoplakia cDNA library (27). The function of EBV BKRF4 is unknown. The MHV-68 ORF45 protein is an acidic protein with a low complexity which contains a putative nuclear localization signal (NLS). The primary sequence of the predicted MHV-68 ORF45 gene product (206 amino acids) has 33.0% identity to that of KSHV ORF45 and 13.6% identity to its EBV homologue. The C-terminal region of ORF45 is highly conserved among all gammaherpesviruses. The last 23 amino acids of the MHV-68 ORF45 C terminus have identities of 58% to the C terminus of KSHV ORF45 and 50% to that of EBV BKRF4. Analyses of mRNA and protein expression kinetics have indicated that MHV-68 ORF45 is highly expressed during the early-late phase of lytic infection (21, 29). The gene product of MHV-68 ORF45 has been found to be present in purified virion particles and exhibits partial resistance to detergent treatment, consistent with its being a component of the virion tegument (3). The inhibition of ORF45 expression by RNA interference was shown to diminish MHV-68 replication (21). While this RNA interference experiment implied a critical role for ORF45 in viral replication, an analysis of an ORF45-null mutant virus would provide further insight into the function of ORF45 in viral replication.

Here we describe the generation and characterization of an ORF45-null mutant MHV-68 by use of a bacterial artificial chromosome (BAC) system. We show that the ORF45-null mutant is incapable of virion production. The defect in ORF45 can be rescued in trans by green fluorescent protein (GFP) fused to either full-length ORF45 or an ORF45 mutant with a deletion of a putative NLS, but not to an ORF45 mutant with a deletion of the last 23 amino acids at the C terminus. The functional conservation of MHV-68 ORF45 with its KSHV homologue is also discussed.


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MATERIALS AND METHODS
 
Viruses and cell culture. Wild-type (wt) MHV-68 was originally obtained from the American Type Culture Collection (VR1465). The working wt MHV-68 virus stock was generated by infecting BHK-21 cells (a baby hamster kidney fibroblast cell line; ATCC CCL-10) at a multiplicity of infection (MOI) of 0.01 PFU per cell and harvesting them at 5 days postinfection (p.i.). Extracellular wt MHV-68 virions were prepared as described previously (3). For preparation of the extracellular ORF45-null virions, BHK-21 cells were cotransfected with the ORF45-null BAC DNA plus an expression vector containing the full-length MHV-68 ORF45 fused to the C terminus of GFP. At 5 days p.i., the supernatant was harvested and cleared of large cellular debris by centrifugation (1,000 x g, 15 min, 4°C) and then by filtration through a 0.4-µm-pore-size filter. Virions were pelleted by ultracentrifugation (40,000 x g, 2 h, 4°C). BHK-21 cells were cultured in complete Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and supplemented with penicillin and streptomycin.

Molecular cloning. The plasmid pFLAG-ORF45 was constructed by inserting the full-length MHV-68 ORF45 gene into the BamHI and KpnI sites of the vector pFLAG-CMV2 (Kodak). The plasmids pGFP-ORF45, pGFP-ORF45{Delta}NLS, and pGFP-ORF45{Delta}C23 were constructed by inserting the full-length MHV-68 ORF45, MHV-68 ORF45 with a deletion of the putative NLS, or MHV-68 ORF45 with a deletion of the C-terminal 23 amino acids, respectively, into the KpnI and BamHI sites of a pEGFP-C1 vector which is used to express a protein of interest fused to the C terminus of GFP (Clontech). The plasmid pGFP-kORF45 was constructed by a similar strategy in which the full-length KSHV ORF45 was inserted into the EcoRI and BamHI sites of the pEGFP-C1 vector. The KSHV ORF45 gene was PCR amplified from the total DNA isolated from BC-1 cells (latently infected with KSHV).

The suicide shuttle vector used for BAC mutagenesis, pGS284, was a kind gift from G. Smith and L. Enquist (Princeton University) (44). The shuttle plasmid for the generation of ORF45-null MHV-68(BAC) was constructed by a strategy similar to one that was described recently (22). Briefly, a 1.0-kb DNA fragment was prepared by a two-step PCR and inserted into the NsiI and SphI sites of pGS284. The 1.0-kb PCR fragment contained an insertion of triple-ORF nonsense and frameshift codons fused to a BglII site between nucleotides (nt) 64156 and 64157 of the viral genome, with approximately 500 nt on each side of the insertion homologous to the viral genome (see Fig. 3A). The resulting plasmid, pGS284/45STOP, was used for allelic exchange to generate the ORF45-null MHV-68(BAC) (45STOP) DNA. The 45STOP BAC plasmid was screened for the stop codon insertion in ORF45 by colony PCR and BglII digestion of the PCR product. The shuttle plasmid used to generate the ORF45-null revertant was constructed by a strategy similar to that used for pGS284/45STOP, in which the wt MHV-68 ORF45 and its flanking sequence were cloned into the NsiI and SphI sites of pGS284. The resulting plasmid was designated pGS284/45WT. The inserted DNA fragment in each construct was sequenced to confirm that there were no additional mutations, deletions, or insertions in the MHV-68 coding sequences.



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  		FIG. 3. Construction and analysis of an ORF45-null MHV-68 mutant. (A) Nucleotide sequence of the region containing the mutation in the ORF45-null MHV-68 mutant. The genome coordinates are given to the left of the nucleotide sequence and on top of the nucleotides between which mutations were introduced. The ORF45 ATG and the three inserted nonsense and frameshift mutations are boxed, and the introduced BglII site is underlined. The BglII site and stop codons are labeled on top of the nucleotides. (B) Schematic representations of the structures of ORF45 and its flanking ORFs in wild-type [wt(BAC)] or ORF45-null MHV-68(BAC) (45STOP), the position of a probe (nt 61288 to 66111), and the predicted restriction fragments detected by the probe in Southern blot analysis. (C) Southern blot analysis of wild-type and recombinant MHV-68 DNAs. Wild-type virion DNA (wt, lanes 1 and 5) was isolated from extracellular virions. Wild-type BAC DNA (lanes 2 and 6) and BAC DNAs of two clones of the 45STOP mutant (lanes 3, 4, 7, and 8) were isolated from E. coli strain DH10B. The DNAs were digested with BamHI or BglII as indicated at the top of the panel, electrophoresed, blotted, and hybridized with the [{alpha}32P]dCTP-labeled probe shown in panel B. The left column shows a 1-kb ladder, and to the right are the predicted fragments of BglII digestion of the viral DNAs.

Cell transfection. Plasmid DNAs were prepared by use of a plasmid midi kit according to the manufacturer's recommendations (QIAGEN Inc., Valencia, Calif.). For BHK-21 cell transfection, approximately 105 cells were transferred to 24-well culture plates 1 day prior to the experiment. Plasmid DNA or BAC DNA (0.4 µg per transfection) was transfected into cells by the use of Lipofectamine Plus (Gibco). The transfection of 293T cells was performed by the use of Lipofectamine 2000 according to the manufacturer's recommendations (Gibco). Cells were usually assayed at 2 days posttransfection (p.t.) unless otherwise indicated.

Construction of ORF45-null MHV-68(BAC). The ORF45-null MHV-68(BAC) plasmid was generated by allelic exchange in Escherichia coli according to a previously described procedure (22, 45). The shuttle plasmid contains a positive selection marker (an ampicillin resistance gene) and a negative selection marker (SacB) that causes toxicity to the bacteria in the presence of sucrose. The donor strain for allelic exchange was E. coli GS111 carrying the shuttle plasmid pGS284/45STOP, and the recipient strain was GS500 (recA+) harboring MHV-68(BAC). A chloramphenicol resistance marker was located within the BAC sequence residing in the MHV-68 genome. Conjugation was performed by cross-streaking the donor and the recipient strains. Cointegrates were selected by growth in the presence of chloramphenicol (34 µg/ml) and ampicillin (50 µg/ml) and were allowed to resolve by overnight growth in the presence of chloramphenicol alone to ensure the maintenance of the MHV-68(BAC). Following resolution, negative selection against the bacteria retaining the shuttle plasmid was performed by growing bacteria in the presence of 5% sucrose and chloramphenicol on Luria-Bertani plates lacking NaCl. The resulting colonies were streaked onto Luria-Bertani plates containing either chloramphenicol or ampicillin. The recombinants that had lost the integrated shuttle plasmid were chloramphenicol resistant and ampicillin sensitive. The incorporation of the stop codon in ORF45 was determined by PCR and restriction enzyme digestion screening for the insertion of the BglII site engineered next to the stop codon.

Transcomplementation assay. BHK-21 cells seeded in 24-well culture plates were grown overnight at 37°C. BAC DNA (350 ng/well) and plasmid DNA expressing GFP or a GFP fusion protein (50 ng/well) were transfected into cells by the use of Lipofectamine Plus according to the manufacturer's recommendations (Invitrogen). At the indicated times p.t., the viral DNA in a portion of the supernatant was isolated and subjected to a real-time PCR assay to determine the number of viral genome copies. Viruses in the remaining supernatant were used for further analysis. The cells were then lysed for Western blotting analysis by the use of rabbit polyclonal antisera specific for MHV-68 lytic proteins.

Dephosphorylation assay. 293T cells were transfected with an expression plasmid containing the full-length ORF45 fused to a FLAG tag at the N terminus. At 24 h p.t., cells were harvested and resuspended in dephosphorylation buffer (50 mM Tris-HCl, 10 mM MgCl2, 100 mM NaCl, 1 mM dithiothreitol, 0.5 µM phenylmethylsulfonyl fluoride, and 1 mM benzamidine). After freezing and thawing twice, the mixture was sonicated and incubated with calf intestine alkaline phosphatase (New England BioLabs) (500 U/ml) at 37°C for 30 min. Cell lysates were subjected to Western blotting analysis with a monoclonal antibody to FLAG.

Antibodies, immunoblotting, and indirect immunofluorescence assay. Rabbit polyclonal antisera specific for MHV-68 ORF26, ORF45, and ORF65 were generated in our lab (21). Mouse monoclonal antibodies to the FLAG or His epitope or to ß-actin were purchased from Sigma. For Western blotting, cell extracts were analyzed with the following primary antibodies: a rabbit polyclonal antiserum to ORF26 (1:500), ORF45 (1:500), or ORF65 (1:400) or a monoclonal antibody to FLAG tag, His tag, or actin. Anti-rabbit or anti-mouse immunoglobulin G conjugated to horseradish peroxidase (Amersham Pharmacia Biotech) was used as a secondary antibody. The proteins were detected by use of the ECL PLUS chemiluminescent detection system (Amersham Pharmacia Biotech), and the signals were detected with a STORM imaging system (Molecular Dynamics, Sunnyvale, Calif.). The indirect immunofluorescence assay was performed as described previously (22).

Fractionation of cytoplasm and nuclear DNA. BHK-21 cells grown on 24-well plates were infected with either wt or GFP-ORF45-rescued 45STOP mutant viruses for 1 h at 4°C at an MOI of 20 genome copies per cell. The inoculums were then removed, and the cells were washed twice with phosphate-buffered saline (PBS) plus 1 mM EDTA and incubated with fresh medium for 3 h at 37°C. Supernatants were harvested for the isolation of extracellular viral DNAs. The cells were briefly trypsinized, resuspended in 1 ml of PBS-EDTA, and pelleted by centrifugation at 4°C. The pelleted cells were then incubated with 100 µl of chilled NP-40 buffer (10 mM Tris-HCl [pH 7.5], 75 mM NaCl, 0.325% NP-40) on ice for 30 min. The cytoplasm and nuclear DNA were separated by centrifugation at 18,000 x g for 10 min at 4°C. The cytoplasm solutions were then carefully collected for viral DNA isolation. The nuclear pellets were washed once with 1 ml of PBS-EDTA, resuspended in 100 µl of the same buffer, and used for viral DNA isolation. Viral DNAs were then isolated from different fractions.

Viral DNA isolation and analysis by quantitative real-time PCR. BHK-21 cells were transfected with BAC DNA plus an expression vector containing GFP or GFP fused to the full-length MHV-68 ORF45, one of its deletion mutants, or KSHV ORF45. Twenty-four hours after transfection, the cells were washed twice with PBS and incubated with fresh medium. At the indicated times p.t., viral DNAs were isolated from the supernatants or cell pellets by use of a DNeasy tissue kit (QIAGEN). RNAs were isolated from BHK-21 cells infected with virions harvested from the supernatants of the transfected cultures by use of an RNeasy tissue kit (QIAGEN). Ten microliters of the supernatant DNA or 100 ng of the whole cellular DNA was used for each PCR, which was performed by use of a QuantiTect SYBR green PCR kit (QIAGEN) and specific primers for ORF65 (sense primer, 5'-GTCAGGGCCCAGTCCGTA-3'; antisense primer, 5'-TGGCCCTCTACCTTCTGTTGA-3'). RNA transcripts were analyzed by the use of 100 ng of whole cellular RNA with a QuantiTec SYBR green RT-PCR kit (QIAGEN) and primers for ORF6, ORF50, ORF72, or beta-actin (sense primer, 5'-CACCCACACTGTGCCCATCTAC-3'; antisense primer, 5'-GTGAGGATCTTCATGAGGTAGTC-3') in a 25-µl reaction mixture. The sequences of the primers for ORF6, ORF50, and ORF72 were the same as those that were previously described (23, 36, 54). The numbers for the cycle threshold for beta-actin per reaction did not vary significantly, ranging from 12.7 to 13.7. All PCRs were performed in duplicate on a DNA Engine Opticon2 PCR instrument, and the results were analyzed according to the manufacturer's instructions (MJ Research).

Viral DNA analysis by Southern blotting. The bacterial strain DH10B harboring wt or ORF45-null MHV-68(BAC) DNA was inoculated and cultured in Luria-Bertani medium containing chloramphenicol at 37°C for 16 to 18 h. The BAC DNA was isolated by use of a plasmid midi kit according to a modification of the manufacturer's protocol (QIAGEN). Viral DNAs were isolated from extracellular virions as described previously (22). BAC and viral DNAs were subjected to BamHI, BglII, or HindIII digestion overnight and then separated in 0.8% agarose gels. The gels were subjected to depurination, denaturation, and neutralization. DNAs in treated gels were transferred to charged nylon membranes (Amersham Pharmacia Biotech). The membranes were UV cross-linked and prehybridized at 65°C in a buffer containing 5x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 10x Denhardt's solution, 0.5% sodium dodecyl sulfate, and denatured salmon sperm DNA (50 µg/ml). Probes were generated by the random priming method, with [{alpha}-32P]dCTP and a genomic viral DNA fragment (nt 61288 to 66111) as templates. Radioactivity was detected with a STORM imaging system (Molecular Dynamics)


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RESULTS
 
Phosphorylation of ORF45. We have observed that the ORF45 expressed from either plasmid-transfected or virus-infected cells displays two bands (21). To determine whether the doublet is due to a posttranslational modification(s) in mammalian cells, we compared the expression patterns of the ORF45 products expressed from bacteria and mammalian cells. As shown in Fig. 1A, the bacterially expressed His-tagged ORF45 product showed a single band of 48 kDa at different times postinduction (Fig. 1A, lanes 2 to 4). However, in mammalian cells, the expression of the FLAG-tagged ORF45 protein was detected as a single band of 48-kDa at 8 h p.t. (Fig. 1B, lane 3) and appeared as a doublet at 12 and 24 h p.t. (lanes 4 and 5). To test whether the upper band was a phosphorylated form of ORF45, we performed a dephosphorylation assay as described in Materials and Methods. The result showed that the upper band was eliminated by a treatment with calf intestine phosphatase (lane 6). These experiments confirmed that the upper band was a phosphorylated form of the protein. Indeed, a search for common phosphorylation motifs by the use of NetPhos 2.0 revealed multiple phosphorylation sites (25). Some of these predicted sites may be phosphorylated in mammalian cells and dephosphorylated by a calf intestinal phosphatase treatment.



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  		FIG. 1. Phosphorylation of MHV-68 ORF45. (A) Plasmid pET-30(b)/ORF45 (21) transformed into E. coli BL21(DE3) cells. Protein expression was induced by adding IPTG (isopropyl-beta-D-thiogalactopyranoside). At the indicated times postinduction (as shown at the top), cells were lysed and analyzed by Western blotting with a monoclonal antibody to the His tag. (B) 293T cells were transfected with pFLAG-ORF45. Cells were collected at the indicated hours posttransfection. For the dephosphorylation assay, cells harvested at 24 h p.t. were either left untreated (–, lane 5) or treated with calf intestine phosphatase (CIP, lane 6) as described in Materials and Methods. The cell lysates were analyzed by Western blotting with a monoclonal antibody to FLAG.

Localization of ORF45 in plasmid-transfected and virus-infected cells. As shown in Fig. 2A, the C-terminal 23 amino acids of MHV-68 ORF45 are well conserved among the C termini of ORF45 homologues in other gammaherpesviruses, e.g., EBV BKRF4 and KSHV ORF45. An analysis of the amino acid sequence of MHV-68 ORF45 revealed a putative NLS (amino acids 174 to 178 [HKKRR]). To examine the functional domain of ORF45, we constructed plasmids that expressed the full-length ORF45, ORF45 with a deletion of the NLS, or ORF45 with a deletion of the C-terminal 23 amino acids fused to the C terminus of GFP, designated pGFP-ORF45, pGFP-ORF45{Delta}NLS, and pGFP-ORF45{Delta}C23, respectively. These expression plasmids were used to transfect BHK-21 cells. At 24 h p.t., the cell lysates were analyzed by Western blotting with a monoclonal antibody to GFP. GFP-ORF45 and GFP-ORF45{Delta}NLS were expressed as approximately 85-kDa doublets, which were equivalent to the molecular mass of GFP (35 kDa) plus that of ORF45 (48 and 51 kDa) (Fig. 2B, lanes 2 to 4). GFP-ORF45{Delta}C23 was expressed as an 83-kDa doublet (lane 5). Bands equivalent to the size of GFP were detected with GFP-ORF45, GFP-ORF45{Delta}NLS, and GFP-ORF45{Delta}C23, suggesting that they were breakdown products of these proteins. The instability of these proteins was also indicated by their lower expression level than that of GFP.



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  		FIG. 2. Cellular localization of MHV-68 ORF45. (A) Comparison of the C termini of ORF45 proteins. Toward the middle is a diagram showing the position of a putative NLS, amino acids 174 to 178 (HKKRR), and the conserved part of the C terminus of MHV-68 ORF45 (black box). A comparison of the C-terminal 23 amino acids of MHV-68 ORF45 and its homologues in EBV (BKRF4, amino acids 196 to 217) and KSHV (ORF45, amino acids 385 to 407) is shown at the top. The consensus sequence is shown under the sequence alignment. Also diagramed are the two deletion mutants of MHV-68 ORF45, one with a deletion of the NLS (ORF45{Delta}NLS) and the other with a deletion of the last 23 amino acids of the C terminus (ORF45{Delta}C23). (B) Western blotting analysis of the GFP-ORF45 fusion proteins. BHK-21 cells were mock transfected (lane 1) or transfected with a plasmid expressing GFP (lane 2), GFP-ORF45 (lane 3), GFP-ORF45{Delta}NLS (lane 4), or GFP-ORF45{Delta}C23 (lane 5). At 24 h p.t., the cells were lysed and analyzed by Western blotting with a monoclonal antibody to GFP. For the bottom panel, actin was probed as a loading control. (C) Subcellular localization of ORF45. BHK-21 cells were infected with MHV-68 (panel 1) or transfected with pFLAG-ORF45 (panel 2), pEGFP-C1 (panel 3), pGFP-ORF45 (panel 4), pGFP-ORF45{Delta}NLS (panel 5), or pGFP-ORF45{Delta}C23 (panel 6). At 24 h postinfection or 9 h posttransfection, the cells were fixed in 2% paraformaldehyde. The untagged ORF45 (panel 1) or FLAG-taggedORF45 (panel 2) was detected in two steps with a polyclonal antiserum to ORF45 or a monoclonal antibody to FLAG, followed by a goat anti-rabbit or anti-mouse antibody conjugated to Cy3. No further staining was performed on cells transfected with GFP fusion protein expression plasmids. Immuno- and GFP fluorescence were then visualized by fluorescence microscopy.

To examine the cellular localization of MHV-68 ORF45 in de novo virus-infected cells, we infected BHK-21 cells with wt MHV-68 at an MOI of 1 PFU per cell. At 24 h p.i., the cells were fixed and subjected to indirect immunofluorescence staining with a rabbit antiserum to ORF45 as the primary antibody. The ORF45 protein was detected in the nuclei of the infected cells. The morphology of the infected cells showed typical signs of a cytopathic effect, i.e., distortion of the nuclei and clumping of the chromatin (Fig. 2C, panel 1). The ORF45 protein was also consistently detected with a FLAG-specific monoclonal antibody in cells transfected with pFLAG-ORF45 at 9 h p.t. and was localized in the nuclei (panel 2). However, unlike the case for virus-infected cells, the morphology of the nuclei in transfected cells was essentially normal, with a central position and dispersed chromatin. We also examined the cellular localization of the GFP-ORF45 fusion protein, which localized to the nuclei as well (panel 4). GFP alone showed a diffuse distribution pattern between the cytoplasm and the nucleus (panel 3). Deletion of the putative NLS prevented the import of ORF45 into the nuclei of plasmid-transfected cells (panel 5). Deletion of the C-terminal 23 amino acids did not change the localization of the ORF45-GFP fusion protein (panel 6).

Disruption of ORF45 in an MHV-68 BAC vector. The inhibition of ORF45 expression by RNA interference diminished MHV-68 replication (21). However, RNA interference may target several overlapping transcripts at the same time. To further study the role of ORF45 in MHV-68 lytic replication, we constructed an ORF45-null MHV-68(BAC) mutant by inserting translation termination codons and a BglII site between nt 64156 and 64157 of the viral genome (114 nt downstream of the translation start codon for ORF45) and designated the mutant 45STOP (Fig. 3A). The introduction of triple stop codons ensured that any translation would stop and would not produce a functional gene product. The incorporation of the BglII site next to the mutation allowed us to screen for positive clones in bacteria and to confirm the insertion. 45STOP clones were selected as described in Materials and Methods and were analyzed by BamHI or BglII digestion, electrophoresis, ethidium bromide staining, and Southern blot analysis with a 4.8-kb probe (nt 61288 to 66111). The probe and the predicted fragment patterns of restriction enzyme digests of ORF45 and its flanking ORFs are shown in Fig. 3B. In BamHI digests, two bands (4.0 and 4.9 kb) were detected by the probe with wt MHV-68 virion DNA (Fig. 3C, lane 1) and wt MHV-68(BAC) DNA (lane 2) as well as with two independent clones of 45STOP BAC DNAs (lanes 3 and 4). BglII digestion of the stop codon-BglII mutation in ORF45 resulted in two bands, of 4.2 and 1.5 kb, for 45STOP instead of the 5.7-kb band detected for wt MHV-68 virion and MHV-68(BAC) DNAs (lanes 5 to 8). A 2.0-kb band was detected with wt and 45STOP MHV-68(BAC) DNAs, as predicted. No other rearrangements were detected in 45STOP BAC DNAs compared to wt MHV-68(BAC). We concluded that the triple stop codon-BglII mutation was successfully inserted into MHV-68 ORF45.

Deficiency of and complementation for viral replication in ORF45-null mutant. To generate an ORF45-null MHV-68(BAC) virus, we transfected 45STOP BAC DNA into BHK-21 cells and used wt MHV-68(BAC) as a positive control. The transfected cells were cultured for 5 days, and both of the independent clones of 45STOP failed to replicate in BHK-21 cells. Two more rounds of blinded passage of the supernatants harvested from 45STOP-transfected cultures did not result in the development of a cytopathic effect, suggesting that no viruses were produced in the supernatants (data not shown). Next, we used the cotransfection of 45STOP BAC DNA and a GFP-ORF45 fusion protein expression plasmid to complement the mutant virus. BHK-21 cells cotransfected with wt MHV-68(BAC) and the GFP expression vector were used as a control. The transfected cells were observed daily for up to 5 days. Cells transfected with wt MHV-68(BAC) DNA plus the GFP expression vector showed a severe cytopathic effect at 3 days and cell death at 5 days p.t. (Fig. 4A, panels 1, 4, 1', and 4'). No obvious cytopathic effect was detected in 45STOP- plus-GFP-transfected cells at 3 or 5 days p.t. (panels 2, 5, 2', and 5'). Providing the GFP-ORF45 fusion protein in trans rescued the viral replication of the null mutant, as shown by the cytopathic effect and plaques of GFP expression in cells cotransfected with 45STOP plus GFP-ORF45 (panels 3, 6, 3', and 6'). To further examine viral protein expression in the ORF45-null mutant, we subjected the transfected cell lysates to Western blotting with polyclonal antibodies to the viral capsid (ORF26 and ORF65) and tegument (ORF45) proteins. As shown in Fig. 4B, the late viral proteins tested were expressed in cells transfected with wt MHV-68(BAC) DNA plus GFP, but not in cells transfected with 45STOP plus GFP (top panel, lanes 2 and 3). The cotransfection of BHK-21 cells with 45STOP plus the GFP-ORF45 expression plasmid rescued the expression of the late viral proteins ORF26 and ORF65. Importantly, wild-type ORF45 expression was not detected in these cells (top panel, lane 4), which confirmed that no endogenous ORF45 protein was produced in 45STOP-transfected cells. Reprobing the membrane with a monoclonal antibody against GFP demonstrated the expression of GFP or the GFP-ORF45 fusion protein. Although the GFP-ORF45 fusion protein was not as highly expressed as GFP (Fig. 4B, middle panel, lanes 2, 3, and 4), it was still capable of rescuing the 45STOP virus. As a control, actin was expressed evenly in all groups of transfected cells (bottom panel).



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  		FIG. 4. Deficiency in and complementation of viral replication for the ORF45-null mutant. (A) BHK-21 cells were transfected with wt MHV-68(BAC) plus plasmid pEGFP-C1 (panels 1, 4, 1', and 4'), 45STOP plus pEGFP-C1 (panels 2, 5, 2', and 5'), or 45STOP plus pGFP-ORF45 (panels 3, 6, 3', and 6'). The cell morphology and GFP fluorescence were visualized under a fluorescence microscope at 3 or 5 days p.t., as indicated to the left. (B) BHK-21 cells were transfected with various BAC DNAs plus expression plasmids, as shown at the top. At 5 days p.t., the cells were lysed and analyzed by Western blotting with a mixture of polyclonal antibodies to the lytic viral proteins ORF45, ORF26, and ORF65 (upperpanel). The membrane was stripped and reprobed with a monoclonal antibody against GFP (middle panel). Reprobing with a monoclonal antibody to actin provided a loading control (lower panel). (C) BHK-21 cells were cotransfected with wt MHV-68(BAC) plus pEGFP-C1 or 45STOP plus pGFP-ORF45. At 5 days p.t., viral DNAs were isolated from either extracellular virions or the infected cell pellet. The numbers of viral genome copies were determined by a real-time PCR assay using primers specific for ORF65, as described in Materials and Methods. The copy number was normalized to the total amount of viral or cellular DNA harvested per transfection. Errors generated in determining viral genome copies are not visible. (D) BHK-21 cells were cotransfected with wt, 45STOP, or 45STOP.R BAC DNA plus an expression plasmid containing GFP or GFP fused to the full-length MHV-68 ORF45 (GFP-ORF45) or KSHV ORF45 (GFP-kORF45). At the indicated times p.t., viral DNAs were isolated from the supernatants and analyzed by a real-time PCR assay using primers specific to ORF65. The copy number was normalized and expressed per 10 µl of supernatant. The data shown are averages of three independent experiments. Error bars represent standard deviations.

To quantitate the complementation of the ORF45-null mutant by the GFP-ORF45 fusion protein, we assayed the numbers of viral genome copies of extracellular and cell-associated viruses produced from BHK-21 cells cotransfected with 45STOP plus a GFP-ORF45 expression plasmid. As shown in Fig. 4C, the numbers of intracellular viral genome copies were similar between 45STOP/GFP-ORF45 and wt MHV-68(BAC)/GFP. The number of genome copies of extracellular virions for 45STOP/GFP-ORF45 was 24-fold lower than that for wt MHV-68(BAC)/GFP. FLAG-tagged ORF45 or nontagged ORF45 from an expression plasmid was also tested for the ability to rescue the ORF45-null mutant. FLAG-tagged ORF45 showed a result similar to that with the GFP-ORF45 fusion protein. Interestingly, nontagged ORF45 driven by a human cytomegalovirus immediate-early promoter more efficiently rescued the viral replication of the ORF45-null mutant, as demonstrated by the appearance of a severe cytopathic effect and similar viral genome copy numbers to those of wt MHV-68(BAC), as analyzed by real-time PCR (data not shown). We concluded that the ORF45-null MHV-68(BAC) mutant is deficient for viral replication and can be rescued by providing nontagged ORF45 as well as ORF45 fused to the FLAG tag or GFP.

Complementation for viral replication by KSHV ORF45. An amino acid sequence alignment showed a 33% identity between MHV-68 ORF45 and KSHV ORF45 (56). We therefore tested whether KSHV ORF45 was able to rescue the 45STOP mutant. Full-length KSHV ORF45 was cloned into the pEGFP-C1 vector to generate a GFP-KSHV ORF45 fusion protein. This construct, pGFP-kORF45, was cotransfected into BHK-21 cells with the 45STOP BAC DNA. At the indicated times p.t., virion DNAs were isolated from the supernatants and analyzed by a real-time PCR assay using primers specific for the viral genome (ORF65). The fluorescence from GFP-kORF45 was localized to the cytoplasm of the transfected cells (data not shown), consistent with the results of a previous report (60). Surprisingly, the cotransfection of pGFP-kORF45 and 45STOP partially restored the viral replication of the ORF45-null mutant, as indicated by a 10-fold increase in the number of viral genome copies compared to cotransfection with 45STOP plus GFP (Fig. 4D). Although GFP-kORF45 rescued the production of extracellular virion DNAs 10-fold less efficiently than GFP-ORF45, these results strongly indicate that the ORF45 function in viral replication is conserved between MHV-68 and KSHV.

Generation of ORF45-null and null revertant viruses. We have shown that the cotransfection of BHK-21 cells with 45STOP and a GFP-ORF45 fusion protein rescued the lytic protein expression and DNA replication of the null mutant. It is formally possible that a revertant arose during the transcomplementation assay. To test this possibility, we used viruses from the supernatants generated in the transcomplementation assay to infect fresh BHK-21 cells at an MOI of 50 or 500 genome copies per cell. BHK-21 cells infected with wt MHV-68(BAC)/GFP developed a cytopathic effect at 2 days p.i. (Fig. 5A, top row). However, BHK-21 cells infected with 45STOP/GFP-ORF45 did not show any cytopathic effect at the end of the infection (5 days p.i.) (bottom row). We also analyzed extracellular virions harvested from the culture supernatants by Western blotting with a rabbit antiserum to ORF26 or ORF45. As shown in Fig. 5B, the ORF45 protein was abundantly packaged into wt MHV-68 virions (top panel, lane 2). However, no significant amount of ORF45 was detected in 45STOP mutant virions (top panel, lane 3), although the virion lysate was intentionally overloaded, as indicated by the higher level of ORF26 in the null mutant than that in the wt MHV-68 virion lysate (middle panel, lanes 2 and 3). Trace amounts of the ORF45 protein signal for the ORF45-null mutant virions may have been due to a breakdown product of the GFP-ORF45 fusion protein, which was inefficiently packaged into maturing virions (top panel, lane 3). Cellular protein (beta-actin) was not detected in the virion samples (bottom panel). These results indicated that revertants of the ORF45-null mutant are not detectable by the method described here in extracellular virions produced from the cotransfection of 45STOP and pGFP-ORF45.



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  		FIG. 5. Analyses of ORF45-null virus and its revertant BAC DNA. (A) Extracellular viruses of wt(BAC)/GFP and 45STOP/GFP-ORF45 were prepared in a transcomplementation assay. Viruses were quantitated for viral genome copies by a real-time PCR using primers to ORF65. Known amounts of viruses (50 to 500 genome copies per cell) were used to infect fresh BHK-21 cells. The infected cells were examined daily under an inverted fluorescence microscope. Pictures were taken at 2 days p.i. (B) Extracellular virions of the wt or the 45STOP mutant were prepared as described for panel A. Lysates of mock-infected BHK-21 cells (lane 1) and wt (lane 2) and 45STOP/GFP-ORF45 (lane 3) virions were analyzed by Western blotting with a rabbit polyclonal antiserum to ORF45 (upper panel) or to the capsid protein ORF26 (middle panel). Actin was reprobed to evaluate the cellular protein contamination in the virion samples. The protein in the virions that was nonspecifically recognized by the ORF26 antibody is indicated by an asterisk. (C) BAC DNA of the wild-type (B), ORF45-null (C), or null-revertant (D) virus and virion DNA of the wild-type (A) or ORF45-null virus (complemented by providing GFP-ORF45) (E) were digested with BamHI (lanes 1 to 5), BglII (lanes 6 to 10), or HindIII (lanes 11 to 15), as indicated in the panel. The predicted DNA fragment patterns are depicted in Fig. 3B. The digested DNAs were separated in a 0.8% agarose gel and transferred to a nylon membrane. The blot was hybridized with a [{alpha}32P]dCTP-labeled probe for ORF45 and its flanking sequence (nt 61288 to 66111). The image was analyzed in a Storm phosphorimager scanner. The left column shows a 1-kb marker.

Southern blot analysis was performed to exclude the possibility of homologous recombination between the 45STOP BAC DNA and the GFP-ORF45 expression plasmid. The ORF45-null mutant virion DNA was isolated from extracellular virions, digested with BamHI, BglII, or HindIII, and analyzed by Southern blotting with a 4.8-kb probe for ORF45 and its flanking sequences (nt 61288 to 66111). Wild-type and 45STOP BAC DNAs and wt virion DNA were used as controls in this experiment. The predicted DNA fragment patterns of the BamHI, BglII, and HindIII digestions are shown in Fig. 3B, and the results are shown in Fig. 5C. For BamHI digestion, two DNA fragments, of 4.0 and 4.9 kb, were detected by the probe with all samples, including wt MHV-68 virion (A, lane 1), wt MHV-68(BAC) (B, lane 2), 45STOP BAC (C, lane 3), and 45STOP virion (E, lane 5) DNAs, as predicted. For BglII digestion, the 45STOP virion DNA had a fragment pattern similar to that of the 45STOP BAC DNA (three bands, of 4.2, 2.0, and 1.5 kb), without any unexpected fragments, in contrast to wt MHV-68 virion and wt BAC DNAs (two bands, of 5.7 and 2.0 kb) (lanes 6, 7, 8, and 10). For HindIII digestion, all DNAs demonstrated the predicted fragment patterns (lanes 11, 12, 13, and 15). These results suggested that the ORF45-null mutant virions were generated from protein complementation but not from DNA recombination.

To further confirm that the ORF45-null mutant phenotype was due to the insertion of translation termination codons in the N terminus of ORF45, we generated a revertant of the ORF45-null mutant. The null-revertant BAC DNA (45STOP.R) was generated by the allelic exchange method by use of the shuttle plasmid pGS284/45WT and 45STOP BAC DNA. The 45STOP.R mutant was confirmed by Southern blotting analysis, which showed the same restriction enzyme digestion patterns as the wt MHV-68(BAC) DNA (Fig. 5C, lanes 4, 9, and 14). The null-revertant virus stock was prepared by the transfection of BHK-21 cells with 45STOP.R BAC DNA. The virus titer was similar to that of the wt, as determined by a plaque assay and real-time PCR (Fig. 6D). The null-revertant mutant was then used for further analysis.



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  		FIG. 6. Viral gene expression and DNA replication of the ORF45-null mutant. (A and B) BHK-21 cells were infected with the wt or ORF45-null (45STOP) virus at an MOI of 500 genome copies per cell. Cellular RNAs were isolated at the indicated times postinfection and analyzed by a one-step real-time PCR using primers to ORF50 (A) or ORF72 (B). Data were normalized and expressed in gene copy numbers per 100 ng of total RNA. (C) Cellular RNAs were isolated from cells infected with the wt, 45STOP.R, or 45STOP virus at an MOI of 50 genome copies per cell. Total RNAs were isolated at 0 or 48 h p.i. and analyzed by a one-step real-time PCR using primers to ORF6. The data are fold increases in gene copy numbers per 100 ng of total RNA from time zero to 48 h p.i. (D) Extracellular viral DNA or the whole cellular DNA was isolated from the infected culture supernatant or cell pellet at 0 or 48 h p.i. and analyzed by a real-time PCR assay using primers to ORF65. The data are fold increases in viral genome copies per 10 µl of supernatant or per 100 ng of cellular DNA from time zero to 48 h p.i. (E) Amounts of viral genome copies in different fractions. BHK-21 cells were infected with either the wt or 45STOP virus at an MOI of 20 genome copies per cell. At 3 h p.t., DNAs were isolated from the supernatant, cytoplasm, or nuclear fraction of the infected cells. The numbers of viral genome copies were determined by a real-time PCR assay using primers to ORF72. Copy numbers were normalized and expressed as total amounts per transfection. The errors generated in determining viral genome copies are not visible.

The ORF45-null mutant is deficient in viral gene transcription and viral DNA replication. To gain insight into the basis of the replication defect of the ORF45-null mutant, we isolated DNAs from extracellular virions or the cytoplasm or nuclear fraction of cells infected with the wt or 45STOP virus. DNAs were then quantitated by a real-time PCR using primers specific for the viral genome. As shown in Fig. 6E, there were only 1.5- and 2.2-fold decreases in nuclear and total DNAs, respectively, for the 45STOP virus compared to the wt virus. The cycle numbers for the beta-actin gene were determined for nuclear DNAs, with similar results between wt- and 45STOP-infected cells (data not shown). These results indicated that the infection efficiency in the entry stage was not significantly affected by the ORF45 expression deficiency. We next assayed viral gene expression in BHK-21 cells infected with the wt, 45STOP, or 45STOP.R virus. BHK-21 cells were infected with different viruses at an MOI of 500 copies per cell, and total RNAs were isolated from cells at 6, 12, or 24 h p.i. and analyzed by real-time reverse transcription-PCR (RT-PCR) using primers specific for immediate-early (RTA) or latent (ORF72) gene expression. No product was observed for RT-negative reactions (data not shown). At 6 h p.i., there were 4 x 105 copies of the RTA transcript, which reached a peak at 24 h p.i. in cells infected with wt MHV-68(BAC) or 45STOP.R. Notably, RTA transcripts in 45STOP-infected cells maintained a basal level during the course of infection (Fig. 6A). A similar result was observed for ORF72 transcripts (Fig. 6B). We also examined the early gene (ORF6) expression of wt MHV-68(BAC) and the ORF45-null null-revertant mutants. The total cellular RNA was isolated from cells infected with the wt, 45STOP, or 45STOP.R virus (MOI, 50 copies per cell) at 0 or 48 h p.i. and then assayed for early gene expression by real-time RT-PCR using primers specific for ORF6. Early transcripts increased 5,000- to 9,000-fold in cells infected with wt MHV-68(BAC) or the null-revertant, but only 6-fold in cells infected with the ORF45-null mutant (Fig. 6C). We concluded that viral gene transcription was blocked during infection with ORF45-deficient viruses.

Next, we assessed the amounts of extracellular and intracellular viruses produced in cultures infected with the wt or 45STOP mutant virus. BHK-21 cells were infected with wt, 45STOP, or 45STOP.R at an MOI of 50 genome copies per cell, and DNAs were isolated from the supernatants or cell pellets at 0 or 48 h p.i. Viral genome copies were analyzed by a real-time PCR using primers specific for the viral genome (ORF65). The results revealed that the numbers of viral genome copies increased approximately 1,000-fold for extracellular and intracellular viruses produced from infection with the wt or 45STOP.R virus. In contrast, the numbers of viral genome copies decreased about 10-fold in cells infected with the 45STOP virus (Fig. 6D). These results coincided with the appearance of a cytopathic effect in cell monolayers infected with the wt or null-revertant virus, but not with the ORF45-null mutant. Consistently, late viral protein expression was completely blocked for the ORF45-null mutant (Fig. 4B and 7A). Thus, our results indicated that the ORF45-null mutant is deficient in viral DNA replication and viral late protein expression.



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  		FIG. 7. Analysis of functional domain(s) of ORF45 in viral replication. (A) BHK-21 cells were mock transfected or cotransfected with the wt, ORF45-null (45STOP), or null-revertant (45STOP.R) BAC DNA plus an expression plasmid containing GFP or GFP fused to full-length ORF45, ORF45{Delta}NLS, or ORF45{Delta}C23, as indicated at the top. At 5 days p.t., the cells were lysed and analyzed by Western blotting for capsid protein (ORF26) (upper) and ORF45 (middle panel) expression. A cellular protein recognized by the polyclonal antibody against ORF26 is indicated by an asterisk (upper panel). Actin was also probed to provide a loading control (lower panel). Note that GFP-ORF45 fusion proteins from expression plasmids were below the detection level by Western blotting (middle panel). (B) Viral DNAs were isolated from the supernatants of transfected cultures as described for panel A and then analyzed by a real-time PCR assay using primers to ORF65. Copy numbers were normalized and expressed per 10 µl of supernatant. The data shown are fold increases compared to cotransfection with 45STOP plus GFP (which was arbitrarily defined as 1.0). Data were compiled from two independent experiments. Error bars represent standard deviations.

Rescue of ORF45-null mutant for viral replication by the NLS deletion mutant, but not the C-terminal 23-amino-acid deletion mutant, of the GFP-ORF45 fusion protein. The MHV-68 ORF45 protein shares a conserved C-terminal region with KSHV ORF45 and EBV BKRF4 and contains a putative NLS (Fig. 2A). We chose to use a transcomplementation assay to define the functional domain of ORF45. An expression vector containing GFP or GFP fused to full-length ORF45 or an ORF45 mutant with a deletion of the putative NLS or the C-terminal 23 amino acids was cotransfected into BHK-21 cells with wt or 45STOP BAC DNA. At 5 days p.t., the cells were lysed and analyzed by Western blotting for late viral protein expression with an antibody to a capsid protein (ORF26). ORF26 was detected in cells that were cotransfected with wt MHV-68(BAC) and GFP but not in cells that were cotransfected with 45STOP and GFP (Fig. 7A, top panel, lanes 1 and 2). Cotransfection with GFP-ORF45 rescued late viral protein expression for the ORF45-null mutant (lane 3). Notably, GFP-ORF45{Delta}NLS showed a similar function as the full-length ORF45 product (lane 4). Surprisingly, the deletion of the C-terminal 23 amino acids failed to complement the function of ORF45 in viral protein expression (lane 5), indicating its essential role in the function of ORF45. Viral late protein expression was also rescued in the null-revertant virus (lane 6). We next examined the abundance of the ORF45 protein in each transcomplementation assay. ORF45 was expressed in cells that were cotransfected with wt or 45STOP.R plus the GFP expression plasmid, but not in cells that were cotransfected with 45STOP plus GFP or a GFP-ORF45 fusion protein expression plasmid. The expression level of GFP-fused ORF45 was below the detection level by Western blotting, indicating the instability of the GFP fusion proteins (Fig. 7A, middle panel). These results also suggested that a small amount of ORF45 might be sufficient to initiate viral replication. We then quantitated the viral genome copies in the extracellular virions (Fig. 7B). Compared with 45STOP/GFP transfection, the number of extracellular viral genome copies in cells transfected with 45STOP/GFP-ORF45 increased >100-fold (columns 2 and 3). The deletion of the NLS decreased virion production twofold compared to that of the full-length GFP-ORF45 fusion protein (column 4). Notably, the deletion of the C-terminal 23 amino acids dramatically decreased the production of viruses, which was >100-fold lower than that after complementation with the full-length GFP-ORF45 fusion protein (column 5). The replication defect for the ORF45-null mutant was completely rescued in the null-revertant (45STOP.R), which produced extracellular virions similar to those of wt MHV-68(BAC) (columns 1 and 6). From these results, we concluded that the conserved C-terminal region of ORF45 is required for the function of ORF45 in viral replication.


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DISCUSSION
 
ORF45 is conserved among the members of the Gammaherpesvirinae subfamily and has been suggested to be a virion tegument protein in both KSHV and MHV-68 (3, 61). The repression of ORF45 expression by RNA interference inhibits MHV-68 viral replication. However, the product of MHV-68 ORF45 and its function have not yet been characterized. In this report, we showed that the MHV-68 ORF45 protein is a phosphorylated nuclear protein. Through functional analysis of an ORF45-null MHV-68 mutant (45STOP), we demonstrated that the ORF45 protein is essential for viral replication and that its truncation causes a repression of viral gene expression, inhibition of viral DNA replication, and depletion of late viral protein synthesis. These defects in viral replication are completely rescued by providing ORF45 in trans or in an ORF45-null revertant virus. Using a transcomplementation assay, we showed that the function of ORF45 in viral replication is conserved with its KSHV homologue. Finally, we found that the last 23 amino acids at the C terminus are critical for the function of ORF45 in viral replication.

The highly conserved C-terminal region is required for the function of ORF45 in viral replication, which strongly suggests the significance of sequence conservation among gammaherpesviruses. The gene products of ORF45 and its homologues in gammaherpesviruses are predicted to be approximately 200 amino acids long, with the exception of KSHV ORF45, which has 407 amino acids. An alignment of the predicted sequences of KSHV ORF45 and its homologues in other gammaherpesviruses revealed an extra 200 amino acids in the KSHV ORF45 N terminus. However, the C terminus of KSHV ORF45 is well conserved with its homologues in other gammaherpesviruses, including MHV-68 ORF45. It has been suggested that the KSHV ORF45 gene product interacts with interferon regulatory factor 7, but the C terminus is not essential for its binding activity (60). The extra 200 amino acids in the N terminus of KSHV ORF45 may be uniquely acquired and may play an additional function in KSHV infection. We found that the last 23 amino acids at the C terminus of MHV-68 ORF45 are critical for rescuing virus replication. These 23 amino acids are well conserved among the ORF45 homologues in the gammaherpesviruses. Therefore, the function of ORF45 in viral replication is conserved with that of KSHV ORF45 and possibly with other ORF45 homologues among the gammaherpesvirus subfamily.

A disruption of ORF45 blocked viral immediate-early and early gene expression, suggesting that ORF45 plays an essential role in viral infection immediately after the viral genome enters the nucleus. It seemed that the mutant virus failed to establish latency. Studies of alpha- and betaherpesvirus tegument proteins indicated that some tegument proteins can specifically modify the intracellular environment to the advantage of incoming virions during the establishment of infection in a naïve cell. In this sense, they function during the immediate-early phase of viral replication. Several tegument proteins with transactivation functions have been found to be essential for virus replication. These include the herpes simplex virus type I (HSV-1) UL48 (VP16) immediate-early transactivators and the human cytomegalovirus pUL69, pUL82 (pp71), UL47, and pUL26 proteins, which have important roles in the transactivation of immediate-early viral genes (1, 5, 6, 18, 58). Human cytomegalovirus pp71 is important for the accumulation of the immediate-early proteins IE72, which modulates the cell cycle, and IE86, which is an immediate-early transactivator (6). Tegument proteins have also been suggested to be involved in an immediate regulation of host cells by the packaging and transport of viral mRNA transcripts in the incoming virions (4, 41). The HSV-1 protein UL49 (VP22), an abundant component of the tegument, may bind and carry these transcripts into naïve cells. Another means of transcriptional regulation of host cells is provided by the HSV-1 virion host shutoff (Vhs) tegument protein encoded by the UL41 gene (43). Vhs globally increases the rate of host cell mRNA degradation in the cytoplasm, allowing tight control of the transcriptional and translational machinery by the incoming virus. However, gammaherpesviruses do not encode homologues of the tegument proteins described above and therefore may use different proteins to facilitate the infection process. MHV-68 ORF45 may play a role similar to that of the alpha- and betaherpesvirus tegument proteins, as viral immediate-early and early gene expression was severely restricted during infection by the ORF45-null mutant virus (Fig. 6). The mechanism by which the ORF45 protein affects the immediate-early phase of viral replication needs to be further addressed.


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ACKNOWLEDGMENTS
 
We thank the members of Ren Sun's laboratory for helpful discussions.

This work was supported by NIH grants CA91791, DE14153, and DE15752 and by the Stop Cancer Foundation (R.S.).


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Molecular and Medical Pharmacology, University of California at Los Angeles, Los Angeles, CA 90095. Phone: (310) 794-5557. Fax: (310) 794-5123. E-mail: rsun{at}mednet.ucla.edu. Back


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Journal of Virology, April 2005, p. 5129-5141, Vol. 79, No. 8
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.8.5129-5141.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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