Identification of cis sequences required for lytic DNA replication and packaging of murine gammaherpesvirus 68

Human gammaherpesviruses are associated with lymphomas and other malignancies. Murine gammaherpesvirus 68 (MHV-68) infection of mice has emerged as a model for understanding gammaherpesvirus pathogenesis in vivo. In contrast to human gammaherpesviruses, MHV-68 replicates in permissive cell lines in a robust manner, presenting an efficient model to study the basic mechanisms for DNA replication and recombination processes. In addition, MHV-68 also infects a broad range of cells of different tissue types and from different host species, and the viral genome persists as an episome in infected cells. These features make MHV-68 an attractive system on which to build gene delivery vectors. We have therefore undertaken a study to identify the cis elements required for MHV-68 genome replication and packaging. Here we report that an 8.4-kb MHV-68 genomic fragment between ORF66 and ORF73 conferred on the plasmid the ability to replicate; replication required the presence of either de novo viral infection or viral reactivation from latency. We further mapped the origin of lytic replication (oriLyt) to a 1.25-kb region. Moreover, we demonstrated that the terminal repeat of the viral genome is sufficient for packaging of the replicated oriLyt plasmid into mature viral particles. Functional identification of the MHV-68 oriLyt and packaging signal has laid a foundation for investigating the mechanisms controlling gammaherpesvirus DNA replication during the viral lytic phase and will also serve as a base on which to design gene delivery vectors.     

Analysis of a novel strain of murine gammaherpesvirus reveals a genomic locus important for acute pathogenesis

Infection of mice by murine gammaherpesvirus 68 (MHV-68) is an excellent small-animal model of gammaherpesvirus pathogenesis in a natural host. We have carried out comparative studies of another herpesvirus, murine herpesvirus 76 (MHV-76), which was isolated at the same time as MHV-68 but from a different murid host, the yellow-necked mouse (Apodemus flavicollis). Molecular analyses revealed that the MHV-76 genome is essentially identical to that of MHV-68, except for deletion of 9,538 bp at the left end of the unique region. MHV-76 is therefore a deletion mutant that lacks four genes unique to MHV-68 (M1, M2, M3, and M4) as well as the eight viral tRNA-like genes. Replication of MHV-76 in cell culture was identical to that of MHV-68. However, following infection of mice, MHV-76 was cleared more rapidly from the lungs. In line with this, there was an increased inflammatory response in lungs with MHV-76. Splenomegaly was also significantly reduced following MHV-76 infection, and much less latent MHV-76 was detected in the spleen. Nevertheless, MHV-76 maintained long-term latency in the lungs and spleen. We utilized a cosmid containing the left end of the MHV-68 genome to reinsert the deleted sequence into MHV-76 by recombination in infected cells, and we isolated a rescuant virus designated MHV-76(cA8+)4 which was ostensibly genetically identical to MHV-68. The growth properties of the rescuant in infected mice were identical to those of MHV-68. These results demonstrate that genetic elements at the left end of the unique region of the MHV-68 genome play vital roles in host evasion and are critical to the development of splenic pathology.     

The M10 Locus of Murine Gammaherpesvirus 68 Contributes to both the Lytic and the Latent Phases of Infection

Murine gammaherpesvirus 68 (MHV-68) is closely related to Epstein-Barr virus and Kaposi''s sarcoma-associated herpesvirus (KSHV) and provides a small-animal model to study the pathogenesis of gammaherpesvirus (γHV) infections. According to the colinear organization of the γHV genomes, the M10 locus is situated at a position equivalent to the K12 locus of KSHV, which codes for proteins of the kaposin family. The M10 locus of MHV-68 has been predicted to code for three overlapping open reading frames (M10a, M10b, and M10c [M10a-c]) with unknown function. In addition, the M10 locus contains a lytic origin of replication (oriLyt). To elucidate the function of the M10 locus during lytic and latent infections, we investigated, both in vitro and in vivo, the following four recombinant viruses which were generated using MHV-68 cloned as a bacterial artificial chromosome: (i) a mutant virus with a deletion which affects both the coding region for M10a-c and the oriLyt; (ii) a revertant virus in which both the M10a-c coding region and the oriLyt were reverted to those of the wild type; (iii) a virus with an ectopic insertion of the oriLyt, which restores the function of the oriLyt but not the M10a-c coding region; and (iv) a mutant virus with a deletion in the oriLyt only. While the mutants were slightly attenuated with regard to lytic replication in cell culture, they showed severe growth defects in vivo. Both lytic replication and latency amplification were strongly reduced. In contrast, both the revertant virus and the virus with the ectopic oriLyt insertion grew very similarly to the parental wild-type virus both in vitro and in vivo. Thus, we provide genetic evidence that mutation of the oriLyt, and not of putative protein coding sequences within the M10a-c region, is responsible for the observed phenotype. We conclude that the oriLyt in the M10 locus plays an important role during infection of mice with MHV-68.Diseases caused by gammaherpesviruses continue to be a challenge for human health. The prototypic gamma-1 herpesvirus Epstein-Barr virus (EBV) is associated with lymphomas and nasopharyngeal carcinoma (22). Human herpesvirus 8 (also called Kaposi''s sarcoma-associated herpesvirus [KSHV]), a gamma-2 herpesvirus, is associated with lymphoproliferative disorders and Kaposi''s sarcoma (24). In vivo studies of gammaherpesvirus pathogenesis have been limited to clinical investigation of the infection because of the restricted host range of these viruses. The murine gammaherpesvirus 68 (MHV-68) is also a member of the gammaherpesvirus subfamily and is closely related to KSHV and EBV. Since there exist no good animal models for KSHV and EBV, MHV-68 serves as a small-animal model to investigate gammaherpesvirus pathogenesis (6, 9, 10, 13, 21, 25, 26, 30). MHV-68 is a natural pathogen of wild rodents (7) and is capable of infecting laboratory mice. The nucleotide sequence of MHV-68 is similar to that of EBV and even more closely related to that of KSHV (29). MHV-68 contains genes which are homologous to cellular genes or to genes of other gammaherpesviruses. In addition, it contains virus-specific genes. Many of the latency- and transformation-associated proteins of the gammaherpesviruses, for example, EBNA and LMP of EBV, appear to be encoded by virus-specific genes, yet it has been suggested that pathogenesis-associated genes of gammaherpesviruses may be contained in similarly positioned genome regions (29). The virus-specific genes of MHV-68 were originally designated M1 to M14 (29). The M10 locus has been predicted to code for three overlapping open reading frames (M10a, M10b, and M10c [M10a-c]) (29). While several MHV-68-specific genes have been shown to code for proteins with important functions, the function of M10 is still unknown. A more recent report even considered M10a-c rather unlikely to code for proteins (21). Importantly, the M10 locus also contains a lytic origin of replication (oriLyt) (3, 8). According to the colinear organization of the gammaherpesvirus genomes, the M10 locus is situated at a position equivalent to that of the K12 locus of KSHV. K12 encodes proteins of the kaposin family. Kaposin proteins are involved in cellular transformation and in stabilization of cytokine mRNAs (16-18,20). Of note, the K12 locus also contains an oriLyt (5).Here, we investigated the function of the M10 locus during lytic and latent infections by studying mutant viruses with deletions in the M10 loci, either affecting both the coding region for M10a-c and the oriLyt or the oriLyt only. While the mutants were slightly attenuated with regard to lytic replication in cell culture, they showed severe growth defects in vivo. Both lytic replication and latency amplification were strongly reduced in mice infected with the mutant viruses. In contrast, a revertant virus in which both the M10a-c coding region and the oriLyt were reverted to those of the wild type and a virus with an ectopic insertion of the oriLyt which restores the function of the oriLyt but not the M10a-c coding region grew very similarly to the parental wild-type virus both in vitro and in vivo. Thus, we provide genetic evidence that mutation of the oriLyt, and not of putative protein coding sequences within the M10a-c region, is responsible for the observed phenotype.     

Multiple Lytic Origins of Replication Are Required for Optimal Gammaherpesvirus Fitness In Vitro and In Vivo

An unresolved question in herpesvirus biology is why some herpesviruses contain more than one lytic origin of replication (oriLyt). Using murine gammaherpesvirus 68 (MHV-68) as model virus containing two oriLyts, we demonstrate that loss of either of the two oriLyts was well tolerated in some situations but not in others both in vitro and in vivo. This was related to the cell type, the organ or the route of inoculation. Depending on the cell type, different cellular proteins, for example Hexim1 and Rbbp4, were found to be associated with oriLyt DNA. Overexpression or downregulation of these proteins differentially affected the growth of mutants lacking either the left or the right oriLyt. Thus, multiple oriLyts are required to ensure optimal fitness in different cell types and tissues.     

Analysis of human cytomegalovirus oriLyt sequence requirements in the context of the viral genome

During the lytic phase of infection, replication of herpesvirus genomes initiates at the lytic origin of replication, oriLyt. Many herpesviruses harbor more than one lytic origin, but so far, only one oriLyt has been identified for human cytomegalovirus (HCMV). Evidence for the existence of additional lytic origins of HCMV has remained elusive. On the basis of transient replication assays with cloned viral fragments, HCMV oriLyt was described as a core region of 1.5 kbp (minimal oriLyt) flanked by auxiliary sequences required for maximal replication activity (complete oriLyt). It remained unclear whether minimal oriLyt alone can drive the replication of HCMV in the absence of its accessory regions. To investigate the sequence requirements of oriLyt in the context of the viral genome, mutant genomes were constructed lacking either minimal or complete oriLyt. These genomes were not infectious, suggesting that HCMV contains only one lytic origin of replication. Either minimal or complete oriLyt was then ectopically reinserted into the oriLyt-depleted genomes. Only the mutant genomes carrying complete oriLyt led to infectious progeny. Remarkably, inversion of the 1.5-kbp core origin relative to its flanking regions resulted in a replication-defective genome. Mutant genomes carrying minimal oriLyt plus the left flanking region gave rise to minifoci, but genomes harboring minimal oriLyt together with the right flanking region were noninfectious. We conclude that the previously defined minimal lytic origin is not sufficient to drive replication of the HCMV genome. Rather, our results underline the importance of the accessory regions and their correct arrangement for the function of HCMV oriLyt.     

Unconventional Sequence Requirement for Viral Late Gene Core Promoters of Murine Gammaherpesvirus 68

Infection with the human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi''s sarcoma-associated herpesvirus (KSHV), is associated with several cancers. During lytic replication of herpesviruses, viral genes are expressed in an ordered cascade. However, the mechanism by which late gene expression is regulated has not been well characterized in gammaherpesviruses. In this study, we have investigated the cis element that mediates late gene expression during de novo lytic infection with murine gammaherpesvirus 68 (MHV-68). A reporter system was established and used to assess the activity of viral late gene promoters upon infection with MHV-68. It was found that the viral origin of lytic replication, orilyt, must be on the reporter plasmid to support activation of the late gene promoter. Furthermore, the DNA sequence required for the activation of late gene promoters was mapped to a core element containing a distinct TATT box and its neighboring sequences. The critical nucleotides of the TATT box region were determined by systematic mutagenesis in the reporter system, and the significance of these nucleotides was confirmed in the context of the viral genome. In addition, EBV and KSHV late gene core promoters could be activated by MHV-68 lytic replication, indicating that the mechanisms controlling late gene expression are conserved among gammaherpesviruses. Therefore, our results on MHV-68 establish a solid foundation for mechanistic studies of late gene regulation.     

Glycoprotein M is an essential lytic replication protein of the murine gammaherpesvirus 68

All herpesviruses encode a homolog of glycoprotein M (gM), which appears to function in virion morphogenesis. Despite its conservation, gM is inessential for the lytic replication of alphaherpesviruses. In order to address the importance of gM in gammaherpesviruses, we disrupted it in the murine gammaherpesvirus 68 (MHV-68). The mutant virus completely failed to propagate in normally permissive fibroblasts. The defective genome was rescued by either homologous recombination to restore the wild-type gM in situ or the insertion of an ectopic, intergenic expression cassette encoding gM into the viral genome. Thus, gM was essential for the lytic replication of MHV-68.     

Cloning and mutagenesis of the murine gammaherpesvirus 68 genome as an infectious bacterial artificial chromosome

Gammaherpesviruses cause important infections of humans, in particular in immunocompromised patients. Recently, murine gammaherpesvirus 68 (MHV-68) infection of mice has been developed as a small animal model of gammaherpesvirus pathogenesis. Efficient generation of mutants of MHV-68 would significantly contribute to the understanding of viral gene functions in virus-host interaction, thereby further enhancing the potential of this model. To this end, we cloned the MHV-68 genome as a bacterial artificial chromosome (BAC) in Escherichia coli. During propagation in E. coli, spontaneous recombination events within the internal and terminal repeats of the cloned MHV-68 genome, affecting the copy number of the repeats, were occasionally observed. The gene for the green fluorescent protein was incorporated into the cloned BAC for identification of infected cells. BAC vector sequences were flanked by loxP sites to allow the excision of these sequences using recombinase Cre and to allow the generation of recombinant viruses with wild-type genome properties. Infectious virus was reconstituted from the BAC-cloned MHV-68. Growth of the BAC-derived virus in cell culture was indistinguishable from that of wild-type MHV-68. To assess the feasibility of mutagenesis of the cloned MHV-68 genome, a mutant virus with a deletion of open reading frame 4 was generated. Genetically modified MHV-68 can now be analyzed in functionally modified mouse strains to assess the role of gammaherpesvirus genes in virus-host interaction and pathogenesis.     

Identification of the Rhesus Macaque Rhadinovirus Lytic Origin of DNA Replication

We have identified a lytic origin of DNA replication (oriLyt) for rhesus macaque rhadinovirus (RRV), the rhesus macaque homolog of human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus. RRV oriLyt maps to the region of the genome between open reading frame 69 (ORF69) and ORF71 (vFLIP) and is composed of an upstream A+T-rich region followed by a short (300-bp) downstream G+C-rich DNA sequence. A set of overlapping cosmids corresponding to the entire genome of RRV was capable of complementing oriLyt-dependent DNA replication only when additional ORF50 was supplied as an expression plasmid in the transfection mixture, suggesting that the level of ORF50 protein originating from input cosmid DNA was insufficient. The requirement of RRV ORF50 in the cotransfection replication assay may also suggest a direct role for this protein in DNA replication. RRV oriLyt shares a high degree of nucleotide sequence and G+C base distribution with the corresponding loci in HHV-8.     

The Epstein-Barr virus replication protein BBLF2/3 provides an origin-tethering function through interaction with the zinc finger DNA binding protein ZBRK1 and the KAP-1 corepressor

Herpesviruses encode a set of core proteins essential for lytic replication of their genomes. Three of these proteins form a tripartite helix-primase complex that, in the case of Epstein-Barr virus (EBV), consists of the helicase BBLF4, the primase BSLF1, and the linker protein BBLF2/3. BBLF2/3 and its homologs in the other herpesviruses remain relatively poorly characterized. To better understand the contribution to replication made by BBLF2/3, a yeast two-hybrid screen was performed with BBLF2/3 as the bait protein. This screen identified as interactors a number of cell replication-related proteins such as DNA polymerase beta and subunits of DNA polymerase delta along with the EBV-encoded DNase BGLF5. The screen also identified the DNA binding zinc finger protein ZBRK1 and the ZBRK1 corepressor KAP-1 as BBLF2/3 interactors. Interaction between BBLF2/3 and ZBRK1 and KAP-1 was confirmed in coimmunoprecipitation assays. A binding site for ZBRK1 in the EBV oriLyt enhancer was identified by electrophoretic mobility shift assay. ZBRK1, KAP-1, and the ZBRK1 binding protein BRCA1 were shown by indirect immunofluorescence to be present in replication compartments in lytically induced D98-HR1 cells, and additionally, chromatin immunoprecipitation assays determined that these proteins associated with oriLyt DNA. Replication of an oriLyt plasmid and a variant oriLyt (DeltaZBRK1) plasmid was examined in lytically induced D98-HR1 cells. Exogenous ZBRK1, KAP-1, or BRCA1 increased the efficiency of oriLyt replication, while deletion of the ZBRK1 binding site impaired replication. These experiments identify ZBRK1 as another cell protein that, through BBLF2/3, provides a tethering point on oriLyt for the EBV replication complex. The data also suggest that BBLF2/3 may serve as a contact interface for cell proteins involved in replication of EBV oriLyt.     

Murine gammaherpesvirus 68 open reading frame 45 plays an essential role during the immediate-early phase of viral replication

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.     

Expression in a recombinant murid herpesvirus 4 reveals the in vivo transforming potential of the K1 open reading frame of Kaposi's sarcoma-associated herpesvirus

Murid herpesvirus 4 (commonly called MHV-68) is closely related to Kaposi's sarcoma-associated herpesvirus (KSHV) and provides an excellent model system for investigating gammaherpesvirus-associated pathogenesis. MHV-76 is a naturally occurring deletion mutant of MHV-68 that lacks 9,538 bp of the left end of the unique portion of the genome encoding nonessential pathogenesis-related genes. The KSHV K1 protein has been shown to transform rodent fibroblasts in vitro and common marmoset T lymphocytes in vivo. Using homologous recombination techniques, we successfully generated recombinants of MHV-76 that encode green fluorescent protein (MHV76-GFP) and KSHV K1 (MHV76-K1). The replication of MHV76-GFP and MHV76-K1 in cell culture was identical to that of MHV-76. However, infection of BALB/c mice via the intranasal route revealed that MHV76-K1 replicated to a 10-fold higher titer than MHV76-GFP in the lungs at day 5 postinfection (p.i.). We observed type 2 pneumocyte proliferation in areas of consolidation and interstitial inflammation of mice infected with MHV76-K1 at day 10 p.i. MHV76-K1 established a 2- to 3-fold higher latent viral load than MHV76-GFP in the spleens of infected mice on days 10 and 14 p.i., although this was 10-fold lower than that established by wild-type MHV-76. A salivary gland tumor was present in one of four mice infected with MHV76-K1, as well as an increased inflammatory response in the lungs at day 120 p.i. compared with that of mice infected with MHV-76 and MHV76-GFP.     

Virus reconstituted from infectious bacterial artificial chromosome (BAC)-cloned murine gammaherpesvirus 68 acquires wild-type properties in vivo only after excision of BAC vector sequences

We studied the in vivo biological properties of viruses reconstituted from the genome of murine gammaherpesvirus 68 (MHV-68) cloned as an infectious bacterial artificial chromosome (BAC). Recombinant virus RgammaHV68A98.01, containing BAC vector sequences, is attenuated in vivo as determined by (i) viral titers in the lungs during the acute phase of infection, (ii) the extent of splenomegaly, and (iii) the number of latently infected spleen cells reactivating virus in an ex vivo reactivation assay. Since the BAC vector sequences were flanked by loxP sites, passaging the virus in fibroblasts expressing Cre recombinase resulted in the generation of recombinant virus RgammaHV68A98.02, with biological properties comparable to those of wild-type MHV-68. On the basis of these data we conclude (i) that excision of BAC vector sequences from cloned MHV-68 genomes is critical for reconstitution of the wild-type phenotypic properties of this virus and (ii) that the BAC-cloned MHV-68 genome is suitable for the construction of mutants and mutant libraries whose phenotypes can be reliably assessed in vivo.     

Murine gammaherpesvirus 68 ORF52 encodes a tegument protein required for virion morphogenesis in the cytoplasm

The tegument, a semiordered matrix of proteins overlying the nucleocapsid and underlying the virion envelope, in viruses in the gamma subfamily of Herpesviridae is poorly understood. Murine gammaherpesvirus 68 (MHV-68) is a robust model for studying gammaherpesvirus virion structure, assembly, and composition, as MHV-68 efficiently completes the lytic phase and productively infects cultured cells. We have found that MHV-68 ORF52 encodes an abundant tegument protein conserved among gammaherpesviruses. Detergent sensitivity experiments revealed that the MHV-68 ORF52 protein is more tightly bound to the virion nucleocapsid than the ORF45 tegument protein but could be dissociated from particles that retained the ORF65 small capsomer protein. ORF52, tagged with enhanced green fluorescent protein or FLAG epitope, localized to the cytoplasm. A recombinant MHV-68 bacterial artificial chromosome mutant with a nonsense mutation incorporated into ORF52 exhibited viral DNA replication, expression of late lytic genes, and capsid assembly and packaging at levels near those of the wild type. However, the MHV-68 ORF52-null virus was deficient in the assembly and release of infectious virion particles. Instead, partially tegumented capsids produced by the ORF52-null mutant accumulated in the cytoplasm, containing conserved capsid proteins, the ORF64 and ORF67 tegument proteins, but virtually no ORF45 tegument protein. Thus, ORF52 is essential for the tegumentation and egress of infectious MHV-68 particles in the cytoplasm, suggesting an important conserved function in gammaherpesvirus virion morphogenesis.