Murine gammaherpesvirus-68 expands, but does not activate, CD11b+ gr-1+ splenocytes in vivo

ABSTRACT Promyelocytic Leukemia nuclear body (PML NB) proteins mediate an intrinsic cellular host defense response against virus infections. Herpesviruses express proteins that modulate PML or PML-associated proteins by a variety of strategies, including degradation of PML or relocalization of PML NB proteins. The consequences of PML-herpesvirus interactions during infection in vivo have yet to be investigated in detail, largely because of the species-specific tropism of many human herpesviruses. Murine gammaherpesvirus 68 (γHV68) is emerging as a suitable model to study basic biological questions of virus-host interactions because it naturally infects mice. Therefore, we sought to determine whether γHV68 targets PML NBs as part of its natural life cycle. We found that γHV68 induces PML degradation through a proteasome-dependent mechanism and that loss of PML results in more robust virus replication in mouse fibroblasts. Surprisingly, γHV68-mediated PML degradation was mediated by the virion tegument protein ORF75c, which shares homology with the cellular formylglycinamide ribotide amidotransferase enzyme. In addition, we show that ORF75c is essential for production of infectious virus. ORF75 homologs are conserved in all rhadinoviruses but so far have no assigned functions. Our studies shed light on a potential role for this unusual protein in rhadinovirus biology and suggest that γHV68 will be a useful model for investigation of PML-herpesvirus interactions in vivo.

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Characterization of a Spontaneous 9.5-Kilobase-Deletion Mutant of Murine Gammaherpesvirus 68 Reveals Tissue-Specific Genetic Requirements for Latency

Journal of Virology ◽

10.1128/jvi.76.13.6532-6544.2002 ◽

2002 ◽

Vol 76 (13) ◽

pp. 6532-6544 ◽

Cited By ~ 22

Author(s):

Eric T. Clambey ◽

Herbert W. Virgin ◽

Samuel H. Speck

Keyword(s):

Deletion Mutant ◽

Latent Infection ◽

Loss Of Function ◽

Murine Gammaherpesvirus ◽

Peritoneal Cells ◽

Gammaherpesvirus 68 ◽

Murine Gammaherpesvirus 68

ABSTRACT Murine gammaherpesvirus 68 (γHV68 [also known as MHV-68]) establishes a latent infection in mice, providing a small-animal model with which to identify host and viral factors that regulate gammaherpesvirus latency. While γHV68 establishes a latent infection in multiple tissues, including splenocytes and peritoneal cells, the requirements for latent infection within these tissues are poorly defined. Here we report the characterization of a spontaneous 9.5-kb-deletion mutant of γHV68 that lacks the M1, M2, M3, and M4 genes and eight viral tRNA-like genes. Previously, this locus has been shown to contain the latency-associated M2, M3, and viral tRNA-like genes. Through characterization of this mutant, we found that the M1, M2, M3, M4 genes and the viral tRNA-like genes are dispensable for (i) in vitro replication and (ii) the establishment and maintenance of latency in vivo and reactivation from latency following intraperitoneal infection. In contrast, following intranasal infection with this mutant, there was a defect in splenic latency at both early and late times, a phenotype not observed in peritoneal cells. These results indicate (i) that there are different genetic requirements for the establishment of latency in different latent reservoirs and (ii) that the genetic requirements for latency depend on the route of infection. While some of these phenotypes have been observed with specific mutations in the M1 and M2 genes, other phenotypes have never been observed with the available γHV68 mutants. These studies highlight the importance of loss-of-function mutations in defining the genetic requirements for the establishment and maintenance of herpesvirus latency.

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Disruption of CCL21-Induced Chemotaxis In Vitro and In Vivo by M3, a Chemokine-Binding Protein Encoded by Murine Gammaherpesvirus 68

Journal of Virology ◽

10.1128/jvi.77.1.624-630.2003 ◽

2003 ◽

Vol 77 (1) ◽

pp. 624-630 ◽

Cited By ~ 46

Author(s):

Kristian K. Jensen ◽

Shu-Cheng Chen ◽

R. William Hipkin ◽

Maria T. Wiekowski ◽

Martin A. Schwarz ◽

...

Keyword(s):

Lymphoid Tissues ◽

Transgenic Expression ◽

Murine Gammaherpesvirus ◽

Chemokine Signaling ◽

Specific Subset ◽

Gammaherpesvirus 68 ◽

Murine Gammaherpesvirus 68 ◽

Human Chemokines

ABSTRACT Chemokine-binding proteins represent a novel class of antichemokine agents encoded by poxviruses and herpesviruses. One such protein is encoded by the M3 gene present in the murine gammaherpesvirus 68 (MHV-68) genome. The M3 gene encodes a secreted 44-kDa protein that binds with high affinity to certain murine and human chemokines and has been shown to block chemokine signaling in vitro. However, there has been no direct evidence that M3 blocks chemokine activity in vivo, nor has the nature of M3-chemokine interaction been defined. To better understand the ability of M3 to block chemokine activity in vivo, we examined its interaction with a specific subset of chemokines expressed in lymphoid tissues, areas where gammaherpesviruses characteristically establish latency. Here we show that M3 blocks in vitro chemotaxis induced by CCL19 and CCL21, chemokines expressed constitutively in secondary lymphoid tissues. Moreover, we provide evidence that chemokine M3 binding exhibits positive cooperativity. In vivo, the expression of M3 in the pancreas of transgenic mice inhibits recruitment of lymphocytes induced by transgenic expression of CCL21 in this organ. The ability of M3 to block the biological activity of chemokines may represent an important strategy used by MHV-68 to evade immune detection and favor viral replication in the infected host.

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Evidence for a Multiprotein Gamma-2 Herpesvirus Entry Complex

Journal of Virology ◽

10.1128/jvi.01141-07 ◽

2007 ◽

Vol 81 (23) ◽

pp. 13082-13091 ◽

Cited By ~ 21

Author(s):

Laurent Gillet ◽

Philip G. Stevenson

Keyword(s):

B Cells ◽

Specific Antibody ◽

Cell Binding ◽

Murine Gammaherpesvirus ◽

Single Entry ◽

Single Complex ◽

Gammaherpesvirus 68 ◽

Murine Gammaherpesvirus 68 ◽

Better Than

ABSTRACT 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.

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Murine Gammaherpesvirus 68 ORF48 Is an RTA-Responsive Gene Product and Functions in both Viral Lytic Replication and Latency duringIn VivoInfection

Journal of Virology ◽

10.1128/jvi.00406-15 ◽

2015 ◽

Vol 89 (11) ◽

pp. 5788-5800 ◽

Cited By ~ 8

Author(s):

Jing Qi ◽

Chuanhui Han ◽

Danyang Gong ◽

Ping Liu ◽

Sheng Zhou ◽

...

Keyword(s):

Lytic Replication ◽

Laboratory Mice ◽

Transcription Activator ◽

Murine Gammaherpesvirus ◽

Responsive Gene ◽

Gammaherpesvirus 68 ◽

Murine Gammaherpesvirus 68 ◽

Lytic Genes

ABSTRACTReplication and transcription activator (RTA) of gammaherpesvirus is an immediate early gene product and regulates the expression of many downstream viral lytic genes. ORF48 is also conserved among gammaherpesviruses; however, its expression regulation and function remained largely unknown. In this study, we characterized the transcription unit ofORF48from murine gammaherpesvirus 68 (MHV-68) and analyzed its transcriptional regulation. We showed that RTA activates theORF48promoter via an RTA-responsive element (48pRRE). RTA binds to 48pRRE directlyin vitroand also associates with ORF48 promoterin vivo. Mutagenesis of 48pRRE in the context of the viral genome demonstrated that the expression of ORF48 is activated by RTA through 48pRRE duringde novoinfection. Through site-specific mutagenesis, we generated an ORF48-null virus and examined the function of ORF48in vitroandin vivo. The ORF48-null mutation remarkably reduced the viral replication efficiency in cell culture. Moreover, through intranasal or intraperitoneal infection of laboratory mice, we showed that ORF48 is important for viral lytic replication in the lung and establishment of latency in the spleen, as well as viral reactivation from latency. Collectively, our study identifiedORF48as an RTA-responsive gene and showed that ORF48 is important for MHV-68 replication bothin vitroandin vivo.IMPORTANCEThe replication and transcription activator (RTA), conserved among gammaherpesviruses, serves as a molecular switch for the virus life cycle. It works as a transcriptional regulator to activate the expression of many viral lytic genes. However, only a limited number of such downstream genes have been uncovered for MHV-68. In this study, we identifiedORF48as an RTA-responsive gene of MHV-68 and mapped theciselement involved. By constructing a mutant virus that is deficient in ORF48 expression and through infection of laboratory mice, we showed that ORF48 plays important roles in different stages of viral infectionin vivo. Our study provides insights into the transcriptional regulation and protein function of MHV-68, a desired model for studying gammaherpesviruses.

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CD4 T Cells Mediate Killing during Persistent Gammaherpesvirus 68 Infection

Journal of Virology ◽

10.1128/jvi.02240-08 ◽

2009 ◽

Vol 83 (9) ◽

pp. 4700-4703 ◽

Cited By ~ 29

Author(s):

Kathleen A. Stuller ◽

Emilio Flaño

Keyword(s):

T Cells ◽

Cd4 T Cells ◽

Persistently Infected ◽

Murine Gammaherpesvirus ◽

Cytotoxicity Assays ◽

Gammaherpesvirus 68 ◽

In Vivo Cytotoxicity ◽

Murine Gammaherpesvirus 68

ABSTRACT CD4 T cells are critical for the control of gammaherpesvirus persistence, but their direct effector mechanisms of virus control in vivo are still poorly understood. In this study, we use murine gammaherpesvirus 68 (γHV68) in in vitro and in vivo cytotoxicity assays to show CD4-dependent killing of γHV68-loaded cells in mice persistently infected with γHV68. Our results underscore the cytotoxic capacity of CD4 T cells during γHV68 persistence.

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

Journal of Virology ◽

10.1128/jvi.01717-08 ◽

2008 ◽

Vol 83 (3) ◽

pp. 1474-1482 ◽

Cited By ~ 35

Author(s):

Lisa M. Gargano ◽

J. Craig Forrest ◽

Samuel H. Speck

Keyword(s):

B Cell ◽

Cell Activation ◽

B Cell Activation ◽

Toll Like Receptors ◽

Murine Gammaherpesvirus ◽

Latently Infected ◽

Gammaherpesvirus 68 ◽

Murine Gammaherpesvirus 68 ◽

Stimulation Of

ABSTRACT 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.

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Murine Gammaherpesvirus 68 Expressing Kaposi Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen (LANA) Reveals both Functional Conservation and Divergence in LANA Homologs

Journal of Virology ◽

10.1128/jvi.00992-17 ◽

2017 ◽

Vol 91 (19) ◽

Cited By ~ 7

Author(s):

Arundhati Gupta ◽

Darby G. Oldenburg ◽

Eduardo Salinas ◽

Douglas W. White ◽

J. Craig Forrest

Keyword(s):

Chronic Infection ◽

Ex Vivo ◽

Kaposi Sarcoma ◽

Lytic Replication ◽

Nuclear Antigen ◽

Content Type ◽

Murine Gammaherpesvirus ◽

Gammaherpesvirus 68 ◽

Murine Gammaherpesvirus 68

ABSTRACT Latency-associated nuclear antigen (LANA) is a multifunctional protein encoded by members of the Rhadinovirus genus of gammaherpesviruses. Studies using murine gammaherpesvirus 68 (MHV68) demonstrated that LANA is important for acute replication, latency establishment, and reactivation in vivo. Despite structural similarities in their DNA-binding domains (DBDs), LANA homologs from Kaposi sarcoma-associated herpesvirus (KSHV) and MHV68 exhibit considerable sequence divergence. We sought to determine if KSHV and MHV68 LANA homologs are functionally interchangeable. We generated an MHV68 virus that encodes KSHV LANA (kLANA) in place of MHV68 LANA (mLANA) and evaluated the virus's capacity to replicate, establish and maintain latency, and reactivate. kLANA knock-in (KLKI) MHV68 was replication competent in vitro and in vivo but exhibited slower growth kinetics and lower titers than wild-type (WT) MHV68. Following inoculation of mice, KLKI MHV68 established and maintained latency in splenocytes and peritoneal cells but did not reactivate efficiently ex vivo. kLANA repressed the MHV68 promoter for ORF50, the gene that encodes the major lytic transactivator protein RTA, while mLANA did not, suggesting a likely mechanism for the KLKI MHV68 phenotypes. Bypassing this repression by providing MHV68 RTA in trans rescued KLKI MHV68 replication in tissue culture and enabled detection of KLKI MHV68 reactivation ex vivo. These data demonstrate that kLANA and mLANA are functionally interchangeable for establishment and maintenance of latency and suggest that repression of lytic replication by kLANA, as previously shown with KSHV, is a kLANA-specific function that is transferable to MHV68. IMPORTANCE Kaposi sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68) are members of the Rhadinovirus genus of gammaherpesviruses. These viruses establish lifelong infections that place their respective human and murine hosts at risk for cancer. Latency-associated nuclear antigen (LANA) is a conserved Rhadinovirus protein that is necessary for long-term chronic infection by these viruses. To better understand the conserved functions performed by LANA homologs, we generated a recombinant MHV68 virus that encodes the KSHV LANA protein in place of the MHV68 LANA homolog. We determined that the KSHV LANA protein is capable of supporting MHV68 latency in a mouse model of chronic infection but also functions to repress viral replication. This work describes an in vivo model system for defining evolutionarily conserved and divergent functions of LANA homologs in Rhadinovirus infection and disease.

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Host Shutoff Is a Conserved Phenotype of Gammaherpesvirus Infection and Is Orchestrated Exclusively from the Cytoplasm

Journal of Virology ◽

10.1128/jvi.01051-09 ◽

2009 ◽

Vol 83 (18) ◽

pp. 9554-9566 ◽

Cited By ~ 67

Author(s):

Sergio Covarrubias ◽

Justin M. Richner ◽

Karen Clyde ◽

Yeon J. Lee ◽

Britt A. Glaunsinger

Keyword(s):

Murine Gammaherpesvirus ◽

Functional Conservation ◽

Barr Virus ◽

Infected Cells ◽

Host Shutoff ◽

Epstein Barr ◽

Gammaherpesvirus 68 ◽

Murine Gammaherpesvirus 68 ◽

Cellular Transcriptome

ABSTRACT Lytic infection with the two human gammaherpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), leads to significant depletion of the cellular transcriptome. This host shutoff phenotype is driven by the conserved herpesviral alkaline exonuclease, termed SOX in KSHV and BGLF5 in EBV, which in gammaherpesviruses has evolved the genetically separable ability to target cellular mRNA. We now show that host shutoff is also a prominent consequence of murine gammaherpesvirus 68 (MHV68) infection, which is widely used as a model system to study pathogenesis of these viruses in vivo. The effector of MHV68-induced host shutoff is its SOX homolog, here termed muSOX. There is remarkable functional conservation of muSOX host shutoff activities with those of KSHV SOX, including the recently described ability of SOX to induce mRNA hyperadenylation in the nucleus as well as cause nuclear relocalization of the poly(A) binding protein. SOX and muSOX localize to both the nucleus and cytoplasm of infected cells. Using spatially restricted variants of these proteins, we go on to demonstrate that all known host shutoff-related activities of SOX and muSOX are orchestrated exclusively from the cytoplasm. These results have important mechanistic implications for how SOX and muSOX target nascent cellular transcripts in the nucleus. Furthermore, our findings establish MHV68 as a new, genetically tractable model to study host shutoff.

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Glycoprotein L Disruption Reveals Two Functional Forms of the Murine Gammaherpesvirus 68 Glycoprotein H

Journal of Virology ◽

10.1128/jvi.01616-06 ◽

2006 ◽

Vol 81 (1) ◽

pp. 280-291 ◽

Cited By ~ 40

Author(s):

Laurent Gillet ◽

Janet S. May ◽

Susanna Colaco ◽

Philip G. Stevenson

Keyword(s):

Epithelial Cells ◽

Membrane Fusion ◽

Major Function ◽

Murine Gammaherpesvirus ◽

Glycoprotein H ◽

Functional Forms ◽

Infected Cells ◽

Gammaherpesvirus 68 ◽

Murine Gammaherpesvirus 68

ABSTRACT The herpesvirus glycoprotein H (gH) and gL associate to form a heterodimer that plays a central role in virus-driven membrane fusion. When archetypal alpha- or betaherpesviruses lack gL, gH misfolds and progeny virions are noninfectious. In order to define the role that gL plays in gamma-2 herpesvirus infections, we disrupted its coding sequence in murine gammaherpesvirus-68 (MHV-68). MHV-68 lacking gL folded gH into a conformation antigenically distinct from the form that normally predominates on infected cells. gL-deficient virions bound less well than the wild type to epithelial cells and fibroblasts. However, they still incorporated gH and remained infectious. The cell-to-cell spread of gL-deficient viruses was remarkably normal, as was infection, dissemination, and latency establishment in vivo. Viral membrane fusion was therefore gL independent. The major function of gL appeared to be allowing gH to participate in cell binding prior to membrane fusion. This function was most important for the entry of MHV-68 virions into fibroblasts and epithelial cells.

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