scholarly journals Characterization of Murine Gammaherpesvirus 68 Glycoprotein B

2004 ◽  
Vol 78 (23) ◽  
pp. 13370-13375 ◽  
Author(s):  
Filipa B. Lopes ◽  
Susanna Colaco ◽  
Janet S. May ◽  
Philip G. Stevenson
Keyword(s):  
Immunoelectron Microscopy ◽  
Human Cytomegalovirus ◽  
Glycoprotein B ◽  
Murine Gammaherpesvirus ◽  
Virion Release ◽  
Infected Cells ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68 ◽  
Endoglycosidase H

ABSTRACT 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.

scholarly journals Murine Gammaherpesvirus 68 Lacking gp150 Shows Defective Virion Release but Establishes Normal Latency In Vivo

2004 ◽  
Vol 78 (10) ◽  
pp. 5103-5112 ◽  
Author(s):  
Brigitte D. de Lima ◽  
Janet S. May ◽  
Philip G. Stevenson
Keyword(s):  
Plasma Membranes ◽  
Lytic Replication ◽  
Virus Propagation ◽  
Murine Gammaherpesvirus ◽  
Virion Release ◽  
Infected Cells ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68

ABSTRACT 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.

scholarly journals Identification and functional characterization of the left origin of lytic replication of murine gammaherpesvirus 68

Virology ◽  
2009 ◽  
Vol 387 (2) ◽  
pp. 285-295 ◽  
Author(s):  
Danyang Gong ◽  
Jing Qi ◽  
Vaithilingaraja Arumugaswami ◽  
Ren Sun ◽  
Hongyu Deng
Keyword(s):  
Functional Characterization ◽  
Lytic Replication ◽  
Murine Gammaherpesvirus ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68

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

2002 ◽  
Vol 76 (13) ◽  
pp. 6532-6544 ◽  
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.

scholarly journals Selective Uptake of Small RNA Molecules in the Virion of Murine Gammaherpesvirus 68

2008 ◽  
Vol 83 (5) ◽  
pp. 2321-2326 ◽  
Author(s):  
Anna R. Cliffe ◽  
Anthony A. Nash ◽  
Bernadette M. Dutia
Keyword(s):  
Small Rna ◽  
Nuclear Staining ◽  
Rna Molecules ◽  
Murine Gammaherpesvirus ◽  
Barr Virus ◽  
Genes Encoding ◽  
Infected Cells ◽  
Epstein Barr ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68

ABSTRACT Noncoding RNAs are a feature of many herpesvirus genomes. They include microRNAs, whose function is the subject of intense investigation, in addition to longer RNA molecules such as the Epstein-Barr virus-encoded RNAs and herpesvirus saimiri U RNAs, which have been known for some time but whose function is still not well defined. Murine gammaherpesvirus 68 (MHV-68) encodes eight viral tRNA-like molecules (vtRNAs) of unknown function. Investigating the kinetics of expression of the vtRNAs, we observed that they were present directly after infection with the virus. This strongly suggested that vtRNAs were part of the virion structure, which was confirmed by their detection within various purified, RNase-treated preparations. Although both viral and cellular mRNAs were also detected within the MHV-68 virion, the major RNA species present were small RNAs of around 70 nucleotides in length. Interestingly, incorporation of viral mRNA was not related to the relative abundance in infected cells, as M11 mRNA, which is present at low abundance, was found in virions. MHV-76, which lacks the genes encoding the vtRNAs, also incorporated small RNA molecules within the virion, suggesting a requirement for these molecules for virion maturation. In productively infected cells the vtRNAs localized predominantly within the cytoplasm, although they also exhibited a globular pattern of nuclear staining. Their presence in the cytoplasm is consistent with interaction with virion components prior to maturation of virus particles. The significance of these findings for virion architecture and function is discussed.

scholarly journals Murine Gammaherpesvirus 68 Encodes a Functional Regulator of Complement Activation

1999 ◽  
Vol 73 (9) ◽  
pp. 7658-7670 ◽  
Author(s):  
Sharookh B. Kapadia ◽  
Hector Molina ◽  
Victor van Berkel ◽  
Samuel H. Speck ◽  
Herbert W. Virgin
Keyword(s):  
Complement Activation ◽  
Flow Cytometric Analysis ◽  
Fluid Phase ◽  
Northern Blotting ◽  
Protein Isoforms ◽  
Murine Gammaherpesvirus ◽  
Infected Cells ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68 ◽  
Gene 4

ABSTRACT Sequence analysis of the murine gammaherpesvirus 68 (γHV68) genome revealed an open reading frame (gene 4) which is homologous to a family of proteins known as the regulators of complement activation (RCA proteins) (H. W. Virgin, P. Latreille, P. Wamsley, K. Hallsworth, K. E. Weck, A. J. Dal Canto, and S. H. Speck, J. Virol. 71:5894–5904, 1997). The predicted gene 4 product has homology to other virally encoded RCA homologs, as well as to the complement-regulatory proteins decay-accelerating factor and membrane cofactor protein. Analyses by Northern blotting and rapid amplification of cDNA ends revealed that gene 4 is transcribed as a 5.2-kb bicistronic transcript of the late kinetic class. Three γHV68 RCA protein isoforms (60 to 65 kDa, 50 to 55 kDa, and 40 to 45 kDa) were detected by Western blotting of infected murine NIH 3T12 fibroblast cells. A soluble 40- to 45-kDa isoform was detected in the supernatants of virally infected cells. Flow cytometric analysis revealed that the γHV68 RCA protein was expressed on the surfaces of infected cells. Supernatants from virally infected cells contained an activity that inhibited murine complement activation as measured by inhibition of C3 deposition on activated zymosan particles. Recombinant γHV68 RCA protein, containing the four conserved short consensus repeats, inhibited murine C3 deposition on zymosan via both classical and alternative pathways and inhibited deposition of human C3 on activated zymosan particles. Expression of this inhibitor of complement activation, both at the cell surface and in the fluid phase, may be important for γHV68 pathogenesis via the inhibition of innate and adaptive immunity.

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

2009 ◽  
Vol 83 (18) ◽  
pp. 9554-9566 ◽  
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.

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

2006 ◽  
Vol 81 (1) ◽  
pp. 280-291 ◽  
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.

scholarly journals Identification of Infected B-Cell Populations by Using a Recombinant Murine Gammaherpesvirus 68 Expressing a Fluorescent Protein

2009 ◽  
Vol 83 (13) ◽  
pp. 6484-6493 ◽  
Author(s):  
Christopher M. Collins ◽  
Jeremy M. Boss ◽  
Samuel H. Speck
Keyword(s):  
B Cells ◽  
Virus Infection ◽  
Plasma Cells ◽  
Fluorescent Protein ◽  
Viral Latency ◽  
Murine Gammaherpesvirus ◽  
Latently Infected ◽  
Infected Cells ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68

ABSTRACT Infection of inbred mice with murine gammaherpesvirus 68 (MHV68) has proven to be a powerful tool to study gammaherpesvirus pathogenesis. However, one of the limitations of this system has been the inability to directly detect infected cells harvested from infected animals. To address this issue, we generated a transgenic virus that expresses the enhanced yellow fluorescent protein (YFP), driven by the human cytomegalovirus immediate-early promoter and enhancer, from a neutral locus within the viral genome. This virus, MHV68-YFP, replicated and established latency as efficiently as did the wild-type virus. During the early phase of viral latency, MHV68-YFP efficiently marked latently infected cells in the spleen after intranasal inoculation. Staining splenocytes for expression of various surface markers demonstrated the presence of MHV68 in distinct populations of splenic B cells harboring MHV68. Notably, these analyses also revealed that markers used to discriminate between newly formed, follicular and marginal zone B cells may not be reliable for phenotyping B cells harboring MHV68 since virus infection appears to modulate cell surface expression levels of CD21 and CD23. However, as expected, we observed that the overwhelming majority of latently infected B cells at the peak of latency exhibited a germinal center phenotype. These analyses also demonstrated that a significant percentage of MHV68-infected splenocytes at the peak of viral latency are plasma cells (ca. 15% at day 14 and ca. 8% at day 18). Notably, the frequency of virus-infected plasma cells correlated well with the frequency of splenocytes that spontaneously reactivate virus upon explant. Finally, we observed that the efficiency of marking latently infected B cells with the MHV68-YFP recombinant virus declined at later times postinfection, likely due to shut down of transgene expression, and indicating that the utility of this marking strategy is currently limited to the early stages of virus infection.

scholarly journals Analysis of a Novel Strain of Murine Gammaherpesvirus Reveals a Genomic Locus Important for Acute Pathogenesis

2001 ◽  
Vol 75 (11) ◽  
pp. 5315-5327 ◽  
Author(s):  
Alastair I. Macrae ◽  
Bernadette M. Dutia ◽  
Steven Milligan ◽  
David G. Brownstein ◽  
Deborah J. Allen ◽  
...  
Keyword(s):  
Small Animal ◽  
Genomic Locus ◽  
Unique Region ◽  
Murine Gammaherpesvirus ◽  
Growth Properties ◽  
Infected Cells ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68 ◽  
Yellow Necked Mouse

ABSTRACT 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.

Sign in / Sign up

Export Citation Format

Share Document