Murine gammaherpesvirus 68 contains two functional lytic origins of replication

A 1.25-kbp DNA fragment from the right side of the genome containing the lytic origin of replication (oriLyt) of murine gammaherpesvirus 68 (MHV-68) has been identified by a plasmid replication assay. Here we show that a mutant MHV-68 with a deletion of an essential part of this oriLyt, generated by using an MHV-68 bacterial artificial chromosome, was only slightly attenuated and still able to replicate but that a mutant containing an additional deletion on the left side of the genome was replication deficient. The newly identified region was sufficient to support plasmid replication, thus providing evidence for a second 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.     

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.     

Murine gammaherpesvirus 68 open reading frame 31 is required for viral replication

Murine gammaherpesvirus 68 (MHV-68) is genetically related to the human gammaherpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) and Epstein-Barr virus (EBV). It has been proposed as a model for gammaherpesvirus infection and pathogenesis. Open reading frame 31 (ORF31) is conserved among the Beta- and Gammaherpesvirinae subfamily, and there is no known mammalian homologue of this protein. The function of MHV-68 ORF31 and its viral homologues has not yet been determined. We described here a primary characterization of this protein and its requirement for lytic replication. The native MHV-68 ORF31 was detected at peak levels by 24 h postinfection, and the FLAG-tagged and green fluorescent protein fusion ORF31 were localized in the cytoplasm and nucleus in a diffuse pattern. Two independent experimental approaches were then utilized to demonstrate that ORF31 was required for lytic replication. First, small interfering RNA generated against ORF31 expression blocked protein expression and virus production in transfected cells. Then, two-independent bacterial artificial chromosome-derived ORF31-null MHV-68 mutants (31STOP) were generated and found to be defective in virus production in fibroblast cells. This defect can be rescued in trans by MHV-68 ORF31 and importantly by its KSHV homologue. A repair virus of 31STOP was also generated by homologous recombination in fibroblast cells. Finally, we showed that the defect in ORF31 blocked late lytic protein expression. Our results demonstrate that MHV-68 ORF31 is required for viral lytic replication, and its function is conserved in its KSHV homologue.     

Murine gammaherpesvirus 68 lacking thymidine kinase shows severe attenuation of lytic cycle replication in vivo but still establishes latency

The lytic cycle functions of gammaherpesviruses have received relatively little attention to date, at least in part due to the lack of a convenient experimental model. The murine gammaherpesvirus 68 (MHV-68) now provides such a model and allows the roles of individual lytic cycle gammaherpesvirus proteins to be evaluated in vivo. We have used MHV-68 to determine the contribution of a gammaherpesvirus thymidine kinase (TK) to viral lytic replication and latency establishment. MHV-68 mutants with a disrupted TK gene grew normally in vitro but showed a severe attenuation of replication in the lungs after intranasal inoculation, with lytic titers at least 1,000-fold lower than those of wild-type and revertant viruses. Nevertheless, the establishment of latency by the TK-deficient mutants, while delayed, was not prevented by their lytic replication deficit. The viral TK clearly plays a crucial role in the capacity of MHV-68 to replicate efficiently in its natural host but does not seem to be essential to establish a persistent infection. The potential of TK-deficient mutants as gammaherpesvirus vaccines is discussed.     

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.     

Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 RTA reactivates murine gammaherpesvirus 68 from latency

Murine gammaherpesvirus 68 (MHV-68), Kaposi's sarcoma-associated herpesvirus (HHV-8), and Epstein-Barr virus (EBV) are all members of the gammaherpesvirus family, characterized by their ability to establish latency in lymphocytes. The RTA protein, conserved in all gammaherpesviruses, is known to play a critical role in reactivation from latency. Here we report that HHV-8 RTA, not EBV RTA, was able to induce MHV-68 lytic viral proteins and DNA replication and processing and produce viable MHV-68 virions from latently infected cells at levels similar to those for MHV-68 RTA. HHV-8 RTA was also able to activate two MHV-68 lytic promoters, whereas EBV RTA was not. In order to define the domains of RTA responsible for their functional differences in viral promoter activation and initiation of the MHV-68 lytic cycle, chimeric RTA proteins were constructed by exchanging the N-terminal and C-terminal domains of the RTA proteins. Our data suggest that the species specificity of MHV-68 RTA resides in the N-terminal DNA binding domain.     

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.     

The murine gammaherpesvirus 68 ORF27 gene product contributes to intercellular viral spread

Herpesviruses remain predominantly cell associated within their hosts, implying that they spread between cells by a mechanism distinct from free virion release. We previously identified the efficient release of murine gammaherpesvirus 68 (MHV-68) virions as a function of the viral gp150 protein. Here we show that the MHV-68 ORF27 gene product, gp48, contributes to the direct spread of viruses from lytically infected to uninfected cells. Monoclonal antibodies to gp48 identified it on infected cell surfaces and in virions. gp48-deficient viruses showed no obvious deficit in virion cell binding, single-cycle replication, or virion release but had reduced lytic propagation between cells. After intranasal infection of mice, ORF27-deficient viruses were impaired predominantly in lytic replication in the lungs. There was a small deficit in latency establishment, but long-term latency appeared normal. Since ORF27 has homologs in both Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, it is likely part of a conserved mechanism employed by gammaherpesviruses to disseminate lytically in their hosts.     

Murine gammaherpesvirus targets type I IFN receptor but not type III IFN receptor early in infection

The innate immune response represents a primary line of defense against invading viral pathogens. Since epithelial cells are the primary site of gammaherpesvirus replication during infection in vivo and there are no information on activity of IFN-III signaling against gammaherpesviruses in this cell type, in present study, we evaluated the expression profile and virus-host interactions in mouse mammary epithelial cell (NMuMG) infected with three strains of murine gammaherpesvirus, MHV-68, MHV-72 and MHV-4556. Studying three strains of murine gammaherpesvirus, which differ in nucleotide sequence of some structural and non-structural genes, allowed us to compare the strain-dependent interactions with host organism. Our results clearly demonstrate that: (i) MHV-68, MHV-72 and MHV-4556 differentially interact with intracellular signaling and dysregulate IFN signal transduction; (ii) MHV-68, MHV-72 and MHV-4556 degrade type I IFN receptor in very early stages of infection (2–4 hpi), but not type III IFN receptor; (iii) type III IFN signaling might play a key role in antiviral defense of epithelial cells in early stages of murine gammaherpesvirus replication; (iv) NMuMG cells are an appropriate model for study of not only type I IFN signaling, but also type III IFN signaling pathway. These findings are important for better understanding of individual virus-host interactions in lytic as well as in persistent gammaherpesvirus replication and help us to elucidate IFN-III function in early events of virus infection.     

Murine gammaherpesvirus-68 ORF38 encodes a tegument protein and is packaged into virions during secondary envelopment

Tegument is the unique structure of a herpesvirion which occupies the space between nucleocapsid and envelope. Accumulating data have indicated that interactions among tegument proteins play a key role in virion morphogenesis. Morphogenesis of gammaherpesviruses including Kaposi’s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) is poorly understood due to the lack of efficient de novo lytic replication in cell culture. Murine gammaherpesvirus-68 (MHV-68) is genetically related to these two human herpesviruses and serves as an effective model to study the lytic replication of gammaherpesviruses. We previously showed that ORF33 of MHV-68 encodes a tegument protein and plays an essential role in virion maturation in the cytoplasm. However, the molecular mechanism of how ORF33 participates in virion morphogenesis has not been elucidated. In this study we demonstrated that ORF38 of MHV-68 is also a tegument protein and is localized to cytoplasmic compartments during both transient transfection and viral infection. Immuno-gold labeling assay showed that ORF38 is only present on virions that have entered the cytoplasmic vesicles, indicating that ORF38 is packaged into virions during secondary envelopment. We further showed that ORF38 co-localizes with ORF33 during viral infection; therefore, the interaction between ORF38 and ORF33 is conserved among herpesviruses. Notably, we found that although ORF33 by itself is distributed in both the nucleus and the cytoplasm, in the presence of ORF38, ORF33 is co-localized to trans-Golgi network (TGN), a site where secondary envelopment takes place.     

A replication-defective gammaherpesvirus efficiently establishes long-term latency in macrophages but not in B cells in vivo

Murine gammaherpesvirus 68 (γHV68 or MHV68) is genetically related to the human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), providing a useful system for in vivo studies of the virus-host relationship. To begin to address fundamental questions about the mechanisms of the establishment of gammaherpesvirus latency, we previously generated a replication-defective γHV68 lacking the expression of the single-stranded DNA binding protein encoded by orf6. In work presented here, we demonstrate that this mutant virus established a long-term infection in vivo that was molecularly identical to wild-type virus latency. Thus, despite the absence of an acute phase of lytic replication, the mutant virus established a chronic infection in which the viral genome (i) was maintained as an episome and (ii) expressed latency-associated, but not lytic replication-associated, genes. Macrophages purified from mice infected with the replication-defective virus harbored viral genome at a frequency that was nearly identical to that of wild-type γHV68; however, the frequency of B cells harboring viral genome was greatly reduced in the absence of lytic replication. Thus, this replication-defective gammaherpesvirus efficiently established in vivo infection in macrophages that was molecularly indistinguishable from wild-type virus latency. These data point to a critical role for lytic replication or reactivation in the establishment or maintenance of latent infection in B cells.