Identification and characterization of murine gammaherpesvirus 68 gp150: a virion membrane glycoprotein.

Murine gammaherpesvirus 68 (MHV-68) is a naturally occurring virus of murid rodents which displays pathobiological characteristics similar to those of other gammaherpesviruses, including Epstein-Barr virus (EBV). However, unlike EBV and many other gammaherpesviruses, MHV-68 replicates in epithelial cells in vitro and infects laboratory strains of mice and therefore provides a good model for the study of gammaherpesviruses. Studies of sequences around the center of the MHV-68 genome identified a gene (designated BPRF1 for BamHI P fragment rightward open reading frame 1) whose putative product had motifs reminiscent of a transmembrane glycoprotein. All other gammaherpesviruses have a glycoprotein in this genomic position, but the BPRF1 gene showed sequence homology with only the EBV membrane antigen gp340/220. Biochemical analysis showed that the product of BPRF1 was a glycoprotein present on the surface of infected cells, and immunoelectron microscopy showed that it was present in the virus particle. In addition, antibodies to the BPRF1 product raised by using a bacterial fusion protein neutralized the virus in the absence of complement. The predominant molecular weights of the protein were 150,000 and 130,000. Pulse-chase analysis and endoglycosidase-H digestion showed that the 130,000-molecular-weight form was a precursor of the 150,000-molecular-weight form, and cell surface labelling showed that the 150,000-molecular-weight form alone was on the cell surface. We therefore named the protein gp150. Since gp150 is the first virion-associated glycoprotein and neutralizing determinant of MHV-68 to be characterized, it provides a valuable tool for the future study of virus-host interactions.     

Characterization of murine gammaherpesvirus 68 glycoprotein B

Murine gammaherpesvirus 68 (MHV-68) glycoprotein B (gB) was identified in purified virions by immunoblotting, immunoprecipitation, and immunoelectron microscopy. It was synthesized as a 120-kDa precursor in infected cells and cleaved into 65-kDa and 55-kDa disulfide-linked subunits close to the time of virion release. The N-linked glycans on the cleaved, virion gB remained partially endoglycosidase H sensitive. The processing of MHV-68 gB therefore appears similar to that of Kaposi's sarcoma-associated herpesvirus gB and human cytomegalovirus gB.     

In vivo function of a gammaherpesvirus virion glycoprotein: influence on B-cell infection and mononucleosis

The human gammaherpesviruses Epstein-Barr virus and Kaposi Sarcoma-associated herpesvirus both contain a glycoprotein (gp350/220 and K8.1, respectively) that mediates binding to target cells and has been studied in great detail in vitro. However, there is no direct information on the role that these glycoproteins play in pathogenesis in vivo. Infection of mice by murid herpesvirus 4 strain 68 (MHV-68) is an established animal model for gammaherpesvirus pathogenesis and expresses an analogous glycoprotein, gp150. To elucidate the in vivo function of gp150, a recombinant MHV-68 deficient in gp150 production was generated (vgp150Delta). The productive viral replication in vitro and in vivo was largely unaffected by mutation of gp150, aside from a partial defect in the release of extracellular virus. Likewise, B-cell latency was established. However, the transient mononucleosis and spike in latently infected cells associated with the spread of MHV-68 to the spleen was significantly reduced in vgp150Delta-infected mice. A soluble, recombinant gp150 was found to bind specifically to B cells but not to epithelial cells in culture. In addition, gp150-deficient MHV-68 derived from mouse lungs bound less well to spleen cells than wild-type virus. Thus, gp150 is highly similar in function in vitro to the Epstein-Barr virus gp350/220. These results suggest a role for these analogous proteins in mononucleosis and have implications for their use as vaccine antigens.     

Murine gammaherpesvirus 68 lacking gp150 shows defective virion release but establishes normal latency in vivo

All gammaherpesviruses encode a virion glycoprotein positionally homologous to Epstein-Barr virus gp350. These glycoproteins are thought to be involved in cell binding, but little is known of the roles they might play in the whole viral replication cycle. We have analyzed the contribution of murine gammaherpesvirus 68 (MHV-68) gp150 to viral propagation in vitro and host colonization in vivo. MHV-68 lacking gp150 was viable and showed normal binding to fibroblasts and normal single-cycle lytic replication. Its capacity to infect glycosaminoglycan (GAG)-deficient CHO-K1 cells and NS0 and RAW264.7 cells, which express only low levels of GAGs, was paradoxically increased. However, gp150-deficient MHV-68 spread poorly through fibroblast monolayers, with reduced cell-free infectivity, consistent with a deficit in virus release. Electron microscopy showed gp150-deficient virions clustered on infected-cell plasma membranes. MHV-68-infected cells showed reduced surface GAG expression, suggesting that gp150 prevented virions from rebinding to infected cells after release by making MHV-68 infection GAG dependent. Surprisingly, gp150-deficient viruses showed only a transient lag in lytic replication in vivo and established normal levels of latency. Cell-to-cell virus spread and the proliferation of latently infected cells, for which gp150 was dispensable, therefore appeared to be the major route of virus propagation in an infected host.     

The MHV68 M2 protein drives IL-10 dependent B cell proliferation and differentiation

Murine gammaherpesvirus 68 (MHV68) establishes long-term latency in memory B cells similar to the human gammaherpesvirus Epstein Barr Virus (EBV). EBV encodes an interleukin-10 (IL-10) homolog and modulates cellular IL-10 expression; however, the role of IL-10 in the establishment and/or maintenance of chronic EBV infection remains unclear. Notably, MHV68 does not encode an IL-10 homolog, but virus infection has been shown to result in elevated serum IL-10 levels in wild-type mice, and IL-10 deficiency results in decreased establishment of virus latency. Here we show that a unique MHV68 latency-associated gene product, the M2 protein, is required for the elevated serum IL-10 levels observed at 2 weeks post-infection. Furthermore, M2 protein expression in primary murine B cells drives high level IL-10 expression along with increased secretion of IL-2, IL-6, and MIP-1alpha. M2 expression was also shown to significantly augment LPS driven survival and proliferation of primary murine B cells. The latter was dependent on IL-10 expression as demonstrated by the failure of IL10-/- B cells to proliferate in response to M2 protein expression and rescue of M2-associated proliferation by addition of recombinant murine IL-10. M2 protein expression in primary B cells also led to upregulated surface expression of the high affinity IL-2 receptor (CD25) and the activation marker GL7, along with down-regulated surface expression of B220, MHC II, and sIgD. The cells retained CD19 and sIgG expression, suggesting differentiation to a pre-plasma memory B cell phenotype. These observations are consistent with previous analyses of M2-null MHV68 mutants that have suggested a role for the M2 protein in expansion and differentiation of MHV68 latently infected B cells-perhaps facilitating the establishment of virus latency in memory B cells. Thus, while the M2 protein is unique to MHV68, analysis of M2 function has revealed an important role for IL-10 in MHV68 pathogenesis-identifying a strategy that appears to be conserved between at least EBV and MHV68.     

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 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.     

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.     

Murine gammaherpesvirus 68 open reading frame 11 encodes a nonessential virion component

Open reading frame 11 (ORF11) is a conserved gammaherpesvirus gene that remains undefined. We identified the product of murine gammaherpesvirus 68 (MHV-68) ORF11, p43, as a virion component with a predominantly perinuclear distribution in infected cells. MHV-68 lacking p43 grew normally in vitro but showed reduced lytic replication in vivo and a delay in seeding to the spleen. Subsequent latency amplification was normal. Thus, MHV-68 ORF11 encoded a virion component that was important for in vivo lytic replication but dispensable for the establishment of latency.     

Evidence for a multiprotein gamma-2 herpesvirus entry complex

Herpesviruses use multiple virion glycoproteins to enter cells. How these work together is not well understood: some may act separately or they may form a single complex. Murine gammaherpesvirus 68 (MHV-68) gB, gH, gL, and gp150 all participate in entry. gB and gL are involved in binding, gB and gH are conserved fusion proteins, and gp150 inhibits cell binding until glycosaminoglycans are engaged. Here we show that a gH-specific antibody coprecipitates gB and thus that gH and gB are associated in the virion membrane. A gH/gL-specific antibody also coprecipitated gB, implying a tripartite complex of gL/gH/gB, although the gH/gB association did not require gL. The association was also independent of gp150, and gp150 was not demonstrably bound to gB or gH. However, gp150 incorporation into virions was partly gL dependent, suggesting that it too contributes to a single entry complex. gp150⁻ and gL⁻ gp150⁻ mutants bound better than the wild type to B cells and readily colonized B cells in vivo. Thus, gp150 and gL appear to be epithelial cell-adapted accessories of a core gB/gH entry complex. The cell binding revealed by gp150 disruption did not require gL and therefore seemed most likely to involve gB.     

Signaling through Toll-Like Receptors Induces Murine Gammaherpesvirus 68 Reactivation In Vivo

Murine gammaherpesvirus 68 (MHV68) establishes a lifelong infection in mice and is used as a model pathogen to study the role of viral and host factors in chronic infection. The maintenance of chronic MHV68 infection, at least in some latency reservoirs, appears to be dependent on the capacity of the virus to reactivate from latency in vivo. However, the signals that lead to MHV68 reactivation in vivo are not well characterized. Toll-like receptors (TLRs), by recognizing the specific patterns of microbial components, play an essential role in the activation of innate immunity. In the present study, we investigated the capacity of TLR ligands to induce MHV68 reactivation, both in vitro and in vivo. The stimulation of latently infected B cell lines with ligands for TLRs 3, 4, 5, and 9 enhanced MHV68 reactivation; the ex vivo stimulation of latently infected primary splenocytes, recovered from infected mice, with poly(I:C), lipopolysaccharide, flagellin, or CpG DNA led to early B-cell activation, B-cell proliferation, and a significant increase in the frequency of latently infected cells reactivating the virus. In vivo TLR stimulation also induced B-cell activation and MHV68 reactivation, resulting in heightened levels of virus replication in the lungs which correlated with an increase in MHV68-specific CD8⁺ T-cell responses. Importantly, TLR stimulation also led to an increase in MHV68 latency, as evidenced by an increase in viral genome-positive cells 2 weeks post-in vivo stimulation by specific TLR ligands. Thus, these data demonstrate that TLR stimulation can drive MHV68 reactivation from latency and suggests that periodic pathogen exposure may contribute to the homeostatic maintenance of chronic gammaherpesvirus infection through stimulating virus reactivation and reseeding latency reservoirs.     

Polar release of pathogenic Old World hantaviruses from renal tubular epithelial cells

Background

Murine gammaherpesvirus 68 (MHV-68) is used as a model to study the function of gammaherpesvirus glycoproteins. gp150 of MHV-68, encoded by open reading frame M7, is a positional homolog of gp350/220 of EBV and of gp35/37 of KSHV. Since it had been proposed that gp350/220 of EBV might be a suitable vaccine antigen to protect from EBV-associated diseases, gp150 has been applied as a model vaccine in the MHV-68 system. When analyzing the function of gp150, previous studies yielded conflicting results on the role of gp150 in latency amplification, and disparities between the mutant viruses which had been analyzed were blamed for the observed differences.

Results

To further develop MHV-68 as model to study the function of gammaherpesvirus glycoproteins in vivo, it is important to know whether gp150 contributes to latency amplification or not. Thus, we re-evaluated this question by testing a number of gp150 mutants side by side. Our results suggest that gp150 is dispensable for latency amplification. Furthermore, we investigated the effect of vaccination with gp150 using gp150-containing exosomes. Vaccination with gp150 induced a strong humoral and cellular immune response, yet it did not affect a subsequent MHV-68 challenge infection.

Conclusions

In this study, we found no evidence for a role of gp150 in latency amplification. The previously observed contradictory results on the role of gp150 in latency amplification were not related to differences between the mutant viruses which had been used.