Latent antigen vaccination in a model gammaherpesvirus infection

Vaccines that can reduce the load of latent gammaherpesvirus infections are eagerly sought. One attractive strategy is vaccination against latency-associated proteins, which may increase the efficiency with which T cells recognize and eliminate latently infected cells. However, due to the lack of tractable animal model systems, the effect of latent-antigen vaccination on gammaherpesvirus latency is not known. Here we use the murine gammaherpesvirus model to investigate the impact of vaccination with the latency-associated M2 antigen. As expected, vaccination had no effect on the acute lung infection. However, there was a significant reduction in the load of latently infected cells in the initial stages of the latent infection, when M2 is expressed. These data show for the first time that latent-antigen vaccination can reduce the level of latency in vivo and suggest that vaccination strategies involving other latent antigens may ultimately be successfully used to reduce the long-term latent infection.     

Identification and sequencing of a novel rodent gammaherpesvirus that establishes acute and latent infection in laboratory mice

Gammaherpesviruses encode numerous immunomodulatory molecules that contribute to their ability to evade the host immune response and establish persistent, lifelong infections. As the human gammaherpesviruses are strictly species specific, small animal models of gammaherpesvirus infection, such as murine gammaherpesvirus 68 (γHV68) infection, are important for studying the roles of gammaherpesvirus immune evasion genes in in vivo infection and pathogenesis. We report here the genome sequence and characterization of a novel rodent gammaherpesvirus, designated rodent herpesvirus Peru (RHVP), that shares conserved genes and genome organization with γHV68 and the primate gammaherpesviruses but is phylogenetically distinct from γHV68. RHVP establishes acute and latent infection in laboratory mice. Additionally, RHVP contains multiple open reading frames (ORFs) not present in γHV68 that have sequence similarity to primate gammaherpesvirus immunomodulatory genes or cellular genes. These include ORFs with similarity to major histocompatibility complex class I (MHC-I), C-type lectins, and the mouse mammary tumor virus and herpesvirus saimiri superantigens. As these ORFs may function as immunomodulatory or virulence factors, RHVP presents new opportunities for the study of mechanisms of immune evasion by gammaherpesviruses.     

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.     

Characterization of Omental Immune Aggregates during Establishment of a Latent Gammaherpesvirus Infection

Herpesviruses are characterized by their ability to establish lifelong latent infection. The gammaherpesvirus subfamily is distinguished by lymphotropism, establishing and maintaining latent infection predominantly in B lymphocytes. Consequently, gammaherpesvirus pathogenesis is closely linked to normal B cell physiology. Murine gammaherpesvirus 68 (MHV68) pathogenesis in laboratory mice has been extensively studied as a model system to gain insights into the nature of gammaherpesvirus infection in B cells and their associated lymphoid compartments. In addition to B cells, MHV68 infection of macrophages contributes significantly to the frequency of viral genome-positive cells in the peritoneal cavity throughout latency. The omentum, a sheet of richly-vascularized adipose tissue, resides in the peritoneal cavity and contains clusters of immune cell aggregates termed milky spots. Although the value of the omentum in surgical wound-healing has long been appreciated, the unique properties of this tissue and its contribution to both innate and adaptive immunity have only recently been recognized. To determine whether the omentum plays a role in gammaherpesvirus pathogenesis we examined this site during early MHV68 infection and long-term latency. Following intraperitoneal infection, immune aggregates within the omentum expanded in size and number and contained virus-infected cells. Notably, a germinal-center B cell population appeared in the omentum of infected animals with earlier kinetics and greater magnitude than that observed in the spleen. Furthermore, the omentum harbored a stable frequency of viral genome-positive cells through early and into long-term latency, while removal of the omentum prior to infection resulted in a slight decrease in the establishment of splenic latency following intraperitoneal infection. These data provide the first evidence that the omentum is a site of chronic MHV68 infection that may contribute to the maintenance of chronic infection.     

Granzymes and caspase 3 play important roles in control of gammaherpesvirus latency

Gammaherpesviruses can establish lifelong latent infections in lymphoid cells of their hosts despite active antiviral immunity. Identification of the immune mechanisms which regulate gammaherpesvirus latent infection is therefore essential for understanding how gammaherpesviruses persist for the lifetime of their host. Recently, an individual with chronic active Epstein-Barr virus infection was found to have mutations in perforin, and studies using murine gammaherpesvirus 68 (gammaHV68) as a small-animal model for gammaherpesvirus infection have similarly revealed a critical role for perforin in regulating latent infection. These results suggest involvement of the perforin/granzyme granule exocytosis pathway in immune regulation of gammaherpesvirus latent infection. In this study, we examined gammaHV68 infection of knockout mice to identify specific molecules within the perforin/granzyme pathway which are essential for regulating gammaherpesvirus latent infection. We show that granzymes A and B and the granzyme B substrate, caspase 3, are important for regulating gammaHV68 latent infection. Interestingly, we show for the first time that orphan granzymes encoded in the granzyme B gene cluster are also critical for regulating viral infection. The requirement for specific granzymes differs for early versus late forms of latent infection. These data indicate that different granzymes play important and distinct roles in regulating latent gammaherpesvirus infection.     

Specific mutation of a gammaherpesvirus-expressed antigen in response to CD8 T cell selection in vivo

Herpesviruses are thought to be highly genetically stable, and their use as vaccine vectors has been proposed. However, studies of the human gammaherpesvirus, Epstein-Barr virus, have found viral isolates containing mutations in HLA class I-restricted epitopes. Using murine gammaherpesvirus 68 expressing ovalbumin (OVA), we examined the stability of a gammaherpesvirus antigenic locus under strong CD8 T cell selection in vivo. OVA-specific CD8 T cells selected viral isolates containing mutations in the OVA locus but minimal alterations in other genomic regions. Thus, a CD8 T cell response to a gammaherpesvirus-expressed antigen that is not essential for replication or pathogenesis can result in selective mutation of that antigen in vivo. This finding may have relevance for the use of herpesvirus vectors for chronic antigen expression in vivo.     

Ataxia Telangiectasia Mutated Kinase Controls Chronic Gammaherpesvirus Infection

Gammaherpesviruses, such as Epstein-Barr virus (EBV), are ubiquitous cancer-associated pathogens that interact with DNA damage response, a tumor suppressor network. Chronic gammaherpesvirus infection and pathogenesis in a DNA damage response-insufficient host are poorly understood. Ataxia-telangiectasia (A-T) is associated with insufficiency of ataxia-telangiectasia mutated (ATM), a critical DNA damage response kinase. A-T patients display a pattern of anti-EBV antibodies suggestive of poorly controlled EBV replication; however, parameters of chronic EBV infection and pathogenesis in the A-T population remain unclear. Here we demonstrate that chronic gammaherpesvirus infection is poorly controlled in an animal model of A-T. Intriguingly, in spite of a global increase in T cell activation and numbers in wild-type (wt) and ATM-deficient mice in response to mouse gammaherpesvirus 68 (MHV68) infection, the generation of an MHV68-specific immune response was altered in the absence of ATM. Our finding that ATM expression is necessary for an optimal adaptive immune response against gammaherpesvirus unveils an important connection between DNA damage response and immune control of chronic gammaherpesvirus infection, a connection that is likely to impact viral pathogenesis in an ATM-insufficient host.     

TLR9 contributes to antiviral immunity during gammaherpesvirus infection

The human gammaherpesviruses Kaposi's sarcoma-associated herpesvirus and EBV cause important infections. As pathogenetic studies of the human infections are restricted, murine gammaherpesvirus 68 serves as a model to study gammaherpesvirus pathogenesis. TLRs are a conserved family of receptors detecting microbial molecular patterns. Among the TLRs, TLR9 recognizes unmethylated CpG DNA motifs present in bacterial and viral DNA. The aim of this study was to assess the role of TLR9 in gammaherpesvirus pathogenesis. Upon stimulation with murine gammaherpesvirus 68, Flt3L-cultured bone marrow cells (dendritic cells) from TLR9-/- mice secreted reduced levels of IL-12, IFN-alpha, and IL-6, when compared with dendritic cells from wild-type mice. Intranasal infection of TLR9-/- and wild-type mice did not reveal any differences during lytic and latent infection. In contrast, when infected i.p., TLR9-/- mice showed markedly higher viral loads both during lytic and latent infection. Thus, we show for the first time that TLR9 is involved in gammaherpesvirus pathogenesis and contributes to organ-specific immunity.     

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.     

Detection of a novel gammaherpesvirus in koalas (Phascolarctos cinereus)

A novel gammaherpesvirus was detected in wild koalas (Phascolarctos cinereus) captured at different locations during 2010. Sequence analysis of the DNA polymerase gene revealed that the virus was genetically distinct from all known gammaherpesviruses. This is the first herpesvirus to be definitively identified in the Vombatiforme suborder (koalas and wombats).     

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.     

Persistent gammaherpesvirus replication and dynamic interaction with the host in vivo

Gammaherpesviruses establish life-long persistency inside the host and cause various diseases during their persistent infection. However, the systemic interaction between the virus and host in vivo has not been studied in individual hosts continuously, although such information can be crucial to control the persistent infection of the gammaherpesviruses. For the noninvasive and continuous monitoring of the interaction between gammaherpesvirus and the host, a recombinant murine gammaherpesvirus 68 (MHV-68, a gammaherpesvirus 68) was constructed to express a firefly luciferase gene driven by the viral M3 promoter (M3FL). Real-time monitoring of M3FL infection revealed novel sites of viral replication, such as salivary glands, as well as acute replication in the nose and the lung and progression to the spleen. Continuous monitoring of M3FL infection in individual mice demonstrated the various kinetics of transition to different organs and local clearance, rather than systemically synchronized clearance. Moreover, in vivo spontaneous reactivation of M3FL from latency was detected after the initial clearance of acute infection and can be induced upon treatment with either a proteasome inhibitor Velcade or an immunosuppressant cyclosporine A. Taken together, our results demonstrate that the in vivo replication and reactivation of gammaherpesvirus are dynamically controlled by the locally defined interaction between the virus and the host immune system and that bioluminescence imaging can be successfully used for the real-time monitoring of this dynamic interaction of MHV-68 with its host in vivo.     

Characterization of murine gammaherpesvirus 68 glycoprotein B (gB) homolog: similarity to Epstein-Barr virus gB (gp110).

Murine gammaherpesvirus 68 (MHV-68) is a natural pathogen of murid rodents and displays similar pathobiological characteristics to those of the human gammaherpesvirus Epstein-Barr virus (EBV). However, in contrast to EBV, MHV-68 will replicate in epithelial cells in vitro. It has therefore been proposed that MHV-68 may be of use as a model for the study of gammaherpesviruses, EBV in particular, both in vitro and in vivo. The EBV homolog of herpes simplex virus glycoprotein B (gB), termed gp110, is somewhat unusual compared with those of many other herpesviruses. We therefore decided to characterize the homolog of gB encoded by MHV-68 (termed MHV gB) to observe the properties of a gammaherpesvirus gB produced in epithelial cells and also to test the relatedness of MHV-68 and EBV. The MHV gB-coding sequence was determined from cloned DNA. The predicted amino acid sequence shared closest homology with gammaherpesvirus gB homologs. Biochemical analysis showed that MHV gB was a glycoprotein with a molecular weight of 105,000. However, the glycans were of the N-linked, high-mannose type, indicating retention in the endoplasmic reticulum. In line with this, MHV gB was localized to the cytoplasm and nuclear margins of infected cells but was not detected on the cell surface or in virions. Additionally, anti-MHV gB antisera were nonneutralizing. Thus, the MHV gB was unlike many other herpesvirus gBs but was extremely similar to the EBV gB. This highlights the close relationship between MHV-68 and EBV and underlines the potential of MHV-68 as a model for EBV in epithelial cells both in vitro and in vivo.     

Identification of proteins associated with murine gammaherpesvirus 68 virions

Murine gammaherpesvirus 68 (MHV68 [also known as gammaHV-68]) is distinguished by its ability to replicate to high titers in cultured cells, making it an excellent candidate for studying gammaherpesvirus virion composition. Extracellular MHV68 virions were isolated, and abundant virion-associated proteins were identified by mass spectrometry. Five nucleocapsid protein homologues, the tegument protein homologue encoded by open reading frame (ORF) 75c, and envelope glycoproteins B and H were detected. In addition, gene products from MHV68 ORF20, ORF24, ORF28, ORF45, ORF48, and ORF52 were identified in association with virions, suggesting that these gammaherpesvirus genes are involved in the early phase of infection or virion assembly and egress.     

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: a model for the study of Epstein-Barr virus infections and related diseases

Epstein-Barr virus (EBV) is a ubiquitous human gammaherpesvirus (GHV) that causes acute infection and establishes life-long latency. EBV is associated with the development of B-cell lymphoproliferative disorders, several malignant cancers, the syndrome of infectious mononucleosis, and chronic interstitial lung disease. Although the molecular biology of EBV has been characterized extensively, the associated disease conditions and their pathogenesis are difficult to study in human populations because of variation in human environments and genetics, the well-documented effect of stressors on pathogenesis, and the chronic and latent properties of the virus. GHV are highly species-specific, and suitable animal models for EBV are not available. However, in 1980, a murine gammaherpesvirus (MuGHV, also known as MHV68 and gammaHV68) was identified as a natural pathogen of bank voles and wood mice. Experimental MuGHV infections in laboratory mice share many features of EBV infections in humans, including facets of the clinical human syndrome known as infectious mononucleosis. These features make MuGHV a valuable experimental model for studying the pathophysiology of a GHV in a natural host.     

De novo infection of B cells during murine gammaherpesvirus 68 latency

The mechanisms by which gammaherpesviruses maintain latency are unclear. Here we used a murine gammaherpesvirus model to show that previously uninfected B cells in immunocompetent mice can acquire virus during latency. In vivo depletion of T cells allowed viral reactivation, as measured by increased viral loads, but not enhanced transfer of virus to new cells. In the absence of both immune T cells and antibody following the transfer of latently infected cells into naïve animals, there was robust infection of new B cells. These data confirm that both T cells and antibody contribute to the control of gammaherpesvirus latency, reactivation, and spread.