Characterization of Gammaherpesvirus 68 Gene 50 Transcription

Liu, Shaofan; Pavlova, Iglika V.; Virgin, Herbert W.; Speck, Samuel H.

doi:10.1128/jvi.74.4.2029-2037.2000

Cited by 78 publications

(93 citation statements)

References 46 publications

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“…A 32 Plabeled ORF50 sequence-specific probe detected a distinct ϳ2-kb band of equivalent intensity in all samples, with the exception of the mock. This result is consistent with previous observations of ORF50 transcription in vitro (12). Ethidium bromide staining of the gel prior to blotting indicated that all lanes contained equivalent amounts of rRNA (data not shown).…”

Section: Targeted Insertion Of the M4 Genesupporting

confidence: 82%

The M4 Gene of Murine Gammaherpesvirus Modulates Productive and Latent Infection In Vivo

Townsley

Dutia

Nash

2004

J Virol

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Murine gammaherpesvirus 68 (MHV-68Murine gammaherpesvirus 68 (MHV-68) is a well-established small-animal model for the study of gammaherpesvirus pathogenesis. MHV-68 infects inbred and outbred mice and, after intranasal administration, undergoes productive infection within lung epithelia, followed by lifelong latent infection. B lymphocytes within the spleen comprise the major reservoir of latent virus after intranasal infection (9, 27) although, like Kaposi's sarcoma-associated herpesvirus, latent virus is detectable within several other cell types, specifically dendritic cells, macrophages/monocytes, and lung epithelia (8,21,35). After the initial detection of latently infected cells, the splenic viral latent load continues to increase and then begins to decrease after it reaches a peak at approximately 14 days postinfection (p.i.). We refer to this stage of infection as acute-phase latency. During acute-phase latency, overt splenomegaly is evident, a process dependent on host (CD4 ϩ T cells) and viral factors (13, 25). A related and previously characterized murid herpesvirus, MHV-76, replicates equivalently to MHV-68 in vitro. However, MHV-76 is phenotypically distinguishable from MHV-68 after infection of immunocompetent mice. Lytic replication in the lung differs significantly from MHV-68, since MHV-76 displays accelerated clearance and an increased inflammatory infiltrate. Acute-phase splenic latency is also affected, with peak latency reduced ϳ50-fold with respect to MHV-68. Significantly, splenomegaly is profoundly reduced in MHV-76-infected mice. Macrae et al. (13) have confirmed this altered phenotype of MHV-76 is the result of a 9,538-bp deletion at the left terminus of MHV-68. In MHV-68, this 9,538-bp encompasses four open reading frames (ORFs; M1 to M4) and eight viral tRNA-like sequences. This locus represents a cluster of MHV-68-specific genes (possibly with latency-associated or immunoregulatory functions), a genomic structure akin to other characterized gammaherpesviruses, where virus-specific genes are interspersed between gene blocks conserved among gammaherpesviruses (31). Aside from this deletion, the left termini of the MHV-76 and MHV-68 genomes are effectively identical, but for one amino acid substitution and a trivial discrepancy between terminal repeats. Reinsertion of the deleted 9,538 bp into MHV-76 restored the in vivo phenotype to that of . Furthermore, an independently isolated MHV-68 mutant, with a similar deletion to that of MHV-76, displays a similar defect in splenic latency (4), underscoring the contribution this locus at the left terminus of MHV-68 makes to in vivo pathogenesis.Data concerning the involvement of these MHV-68-specific elements during in vivo infection remains limited. Thus far, only one gene remains functionally characterized: M3 encodes a broad-spectrum chemokine-binding protein that is expressed during lytic replication and persistence (16,30). Recombinant MHV-68 mutants containing targeted loss-of-function mutations provide data on the contribution of viru...

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Section: Targeted Insertion Of the M4 Genesupporting

confidence: 82%

The M4 Gene of Murine Gammaherpesvirus Modulates Productive and Latent Infection In Vivo

Townsley

Dutia

Nash

2004

J Virol

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“…The next step was to identify ␥HV-68 genes responsible for inhibiting antigen presentation. Viral genomic library clones in pUC119 (9) were transfected into L929-H-2K b cells, together with the major immediate-early viral transactivator (ORF 50) (27) and an M-TSINFVKI-ASNENMETM-SIINFEKL polytope, each in pCDNA3. After 48 h, the presentation of H-2K b-SIINFEKL was assayed using B3Z.…”

Section: Infection With ␥Hv-68 Leads To Diminished Cell-surface Mhc Cmentioning

confidence: 99%

Inhibition of MHC class I-restricted antigen presentation by γ2-herpesviruses

Stevenson

Efstathiou

Doherty

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

201

180

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The ␥-herpesviruses, in contrast to the ␣-and ␤-herpesviruses, are not known to inhibit antigen presentation to CD8 ؉ cytotoxic T lymphocytes (CTLs) during lytic cycle replication. However, murine ␥-herpesvirus 68 causes a chronic lytic infection in CD4 ؉ T celldeficient mice despite the persistence of a substantial CTL response, suggesting that CTL evasion occurs. Here we show that, distinct from host protein synthesis shutoff, ␥-herpesvirus 68 down-regulates surface MHC class I expression on lytically infected fibroblasts and inhibits their recognition by antigen-specific CTLs. The viral K3 gene, encoding a zinc-finger-containing protein, dramatically reduced the half-life of nascent class I molecules and the level of surface MHC class I expression and was by itself sufficient to block antigen presentation. The homologous K3 and K5 genes of the related Kaposi's sarcoma-associated virus also inhibited antigen presentation and decreased cell surface expression of HLA class I antigens. Thus it appears that an immune evasion strategy shared by at least two ␥-herpesviruses allows continued lytic infection in the face of strong CTL immunity.

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“…RNA was isolated from A20, WEHI-231, or NSO cells 24 h after infection at a multiplicity of infection (MOI) of 10 PFU/cell using guanidium isothiocyanate-phenol as previously described (3). Reverse transcription-PCR (RT-PCR) for spliced orf50 or orf73 transcripts was performed as previously described (3,37). RNA was isolated from A20-HE1 or A20-HE2 cells prior to or at 8 h and 24 h after treatment with 20 ng/ml PMA, contaminating DNA was removed by DNase I digestion (Invitrogen), and RNA was reverse transcribed using SuperScript II (Invitrogen) as previously described (21).…”

mentioning

confidence: 99%

Establishment of B-Cell Lines Latently Infected with Reactivation-Competent Murine Gammaherpesvirus 68 Provides Evidence for Viral Alteration of a DNA Damage-Signaling Cascade

Forrest

Speck

2008

J Virol

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Gammaherpesvirus 68 (␥HV68, or MHV68) is a naturally occurring rodent pathogen that replicates to high titer in cell culture and is amenable to in vivo experimental evaluation of viral and host determinants of gammaherpesvirus disease. However, the inability of MHV68 to transform primary murine B cells in culture, the absence of a robust cell culture latency system, and the paucity of MHV68-positive tumor cell lines have limited an understanding of the molecular mechanisms by which MHV68 modulates the host cell during latency and reactivation. To facilitate a more complete understanding of viral and host determinants that regulate MHV68 latency and reactivation in B cells, we generated a recombinant MHV68 virus that encodes a hygromycin resistance protein fused to enhanced green fluorescent protein as a means to select cells in culture that harbor latent virus. We utilized this virus to infect the A20 murine mature B-cell line and evaluate reactivation competence following treatment with diverse stimuli to reveal viral gene expression, DNA replication, and production of progeny virions. Comparative analyses of parental and infected A20 cells indicated a correlation between infection and alterations in DNA damage signaling following etoposide treatment. The data described in this study highlight the potential utility of this new cell culture-based system to dissect molecular mechanisms that regulate MHV68 latency and reactivation, as well as having the potential of illuminating biochemical alterations that contribute to gammaherpesvirus pathogenesis. In addition, such cell lines may be of value in evaluating targeted therapies to gammaherpesvirus-related tumors.Gammaherpesvirus 68 (␥HV68 or MHV68) is a rodent rhadinovirus found naturally in European bank voles and wood mice (6, 7). As a rodent pathogen, MHV68 infection of laboratory mice offers an experimental system to investigate aspects of gammaherpesvirus pathogenesis. Following experimental inoculation of laboratory mice, MHV68 undergoes productive replication at the site of inoculation, which facilitates dissemination to distal organs and is followed by quiescence or latency, a form of infection where viral gene expression is restricted and progeny virions are not produced (41,51,71). In the case of MHV68, latent infection is established in B lymphocytes, as well as epithelial cells, macrophages, and dendritic cells (19,56,58,74). Latency is maintained for the lifetime of the host, and latently infected cells retain the capacity to reinitiate the productive replication cycle, a process termed reactivation (45, 51, 52).The genetic malleability of MHV68 enables the identification of viral genes involved in specific aspects of viral pathogenesis by targeted mutagenesis (1). For example, such approaches have demonstrated the importance of the unique MHV68 M2 gene product in latency establishment, reactivation from latency, and promoting B-cell differentiation (24, 26), as well as defining the roles of conserved genes, such as orf73 (LANA homolog) in latency mai...

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Characterization of Gammaherpesvirus 68 Gene 50 Transcription

Cited by 78 publications

References 46 publications

The M4 Gene of Murine Gammaherpesvirus Modulates Productive and Latent Infection In Vivo

The M4 Gene of Murine Gammaherpesvirus Modulates Productive and Latent Infection In Vivo

Inhibition of MHC class I-restricted antigen presentation by γ2-herpesviruses

Establishment of B-Cell Lines Latently Infected with Reactivation-Competent Murine Gammaherpesvirus 68 Provides Evidence for Viral Alteration of a DNA Damage-Signaling Cascade

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