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The murine gammaherpesvirus immediate-early Rta synergizes with IRF4, targeting expression of the viral M1 superantigen to plasma cells.

O'Flaherty BM, Soni T, Wakeman BS, Speck SH - PLoS Pathog. (2014)

Bottom Line: In addition, we show that M1 gene transcription is regulated by both the essential viral immediate-early transcriptional activator Rta and cellular interferon regulatory factor 4 (IRF4), which together potently synergize to drive M1 gene expression.Finally, we show that IRF4, a cellular transcription factor essential for plasma cell differentiation, can directly interact with Rta.The latter observation raises the possibility that the interaction of Rta and IRF4 may be involved in regulating a number of viral and cellular genes during MHV68 reactivation linked to plasma cell differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia, United States of America; Emory Vaccine Center, Emory University School of Medicine, Atlanta, Georgia, United States of America.

ABSTRACT
MHV68 is a murine gammaherpesvirus that infects laboratory mice and thus provides a tractable small animal model for characterizing critical aspects of gammaherpesvirus pathogenesis. Having evolved with their natural host, herpesviruses encode numerous gene products that are involved in modulating host immune responses to facilitate the establishment and maintenance of lifelong chronic infection. One such protein, MHV68 M1, is a secreted protein that has no known homologs, but has been shown to play a critical role in controlling virus reactivation from latently infected macrophages. We have previous demonstrated that M1 drives the activation and expansion of Vβ4+ CD8+ T cells, which are thought to be involved in controlling MHV68 reactivation through the secretion of interferon gamma. The mechanism of action and regulation of M1 expression are poorly understood. To gain insights into the function of M1, we set out to evaluate the site of expression and transcriptional regulation of the M1 gene. Here, using a recombinant virus expressing a fluorescent protein driven by the M1 gene promoter, we identify plasma cells as the major cell type expressing M1 at the peak of infection in the spleen. In addition, we show that M1 gene transcription is regulated by both the essential viral immediate-early transcriptional activator Rta and cellular interferon regulatory factor 4 (IRF4), which together potently synergize to drive M1 gene expression. Finally, we show that IRF4, a cellular transcription factor essential for plasma cell differentiation, can directly interact with Rta. The latter observation raises the possibility that the interaction of Rta and IRF4 may be involved in regulating a number of viral and cellular genes during MHV68 reactivation linked to plasma cell differentiation.

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M1 transcript mapping identifies initiation and termination sites which result in a 1.3 and 1.5(A) 5′ and 3′ rapid amplification of cDNA ends (RACE) was performed using RNA isolated from infected NIH3T12 fibroblasts and stimulated MHV68 infected A20-HE2 cells. The number of RACE clones that were identified for transcript initiation and termination sites are summarized. (B) A diagram of the transcript initiation and termination sites is shown with the genomic coordinates.
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ppat-1004302-g005: M1 transcript mapping identifies initiation and termination sites which result in a 1.3 and 1.5(A) 5′ and 3′ rapid amplification of cDNA ends (RACE) was performed using RNA isolated from infected NIH3T12 fibroblasts and stimulated MHV68 infected A20-HE2 cells. The number of RACE clones that were identified for transcript initiation and termination sites are summarized. (B) A diagram of the transcript initiation and termination sites is shown with the genomic coordinates.

Mentions: Having identified the reservoir where M1 is expressed in vivo, we sought to characterize the structure of the M1 transcript and to identify the M1 promoter. Rapid amplification of cDNA ends (RACE) was done to identify the transcript initiation and termination sites in two cell lines: (i) infected NIH3T12 fibroblasts; and (ii) reactivated A20-HE2 cells. A20-HE2 cells are a stable lymphoblast B cell line which carry the MHV68 genome where viral reactivation can be induced by tetradecanoylphorbol acetate (TPA) [28]. RNA and protein were collected from both cell lines and lytic gene expression was confirmed prior to analysis (data not shown). Transcript analysis revealed four initiation sites and a single termination site from an unspliced transcript (Figure 5A, 5B). Though all transcript initiation sites were found in infected 3T12 cells, only transcripts starting at bp 2003 and bp 2013 were detected from reactivated A20-HE2 cells. The sizes of the predicted unspliced M1 transcripts were confirmed by northern analyses of RNA prepared from: (i) TPA stimulated A20-HE2 cells (a MHV68 latently infected B cells); and (ii) MHV68 infected NIH 3T12 fibroblasts (data not shown).


The murine gammaherpesvirus immediate-early Rta synergizes with IRF4, targeting expression of the viral M1 superantigen to plasma cells.

O'Flaherty BM, Soni T, Wakeman BS, Speck SH - PLoS Pathog. (2014)

M1 transcript mapping identifies initiation and termination sites which result in a 1.3 and 1.5(A) 5′ and 3′ rapid amplification of cDNA ends (RACE) was performed using RNA isolated from infected NIH3T12 fibroblasts and stimulated MHV68 infected A20-HE2 cells. The number of RACE clones that were identified for transcript initiation and termination sites are summarized. (B) A diagram of the transcript initiation and termination sites is shown with the genomic coordinates.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4125235&req=5

ppat-1004302-g005: M1 transcript mapping identifies initiation and termination sites which result in a 1.3 and 1.5(A) 5′ and 3′ rapid amplification of cDNA ends (RACE) was performed using RNA isolated from infected NIH3T12 fibroblasts and stimulated MHV68 infected A20-HE2 cells. The number of RACE clones that were identified for transcript initiation and termination sites are summarized. (B) A diagram of the transcript initiation and termination sites is shown with the genomic coordinates.
Mentions: Having identified the reservoir where M1 is expressed in vivo, we sought to characterize the structure of the M1 transcript and to identify the M1 promoter. Rapid amplification of cDNA ends (RACE) was done to identify the transcript initiation and termination sites in two cell lines: (i) infected NIH3T12 fibroblasts; and (ii) reactivated A20-HE2 cells. A20-HE2 cells are a stable lymphoblast B cell line which carry the MHV68 genome where viral reactivation can be induced by tetradecanoylphorbol acetate (TPA) [28]. RNA and protein were collected from both cell lines and lytic gene expression was confirmed prior to analysis (data not shown). Transcript analysis revealed four initiation sites and a single termination site from an unspliced transcript (Figure 5A, 5B). Though all transcript initiation sites were found in infected 3T12 cells, only transcripts starting at bp 2003 and bp 2013 were detected from reactivated A20-HE2 cells. The sizes of the predicted unspliced M1 transcripts were confirmed by northern analyses of RNA prepared from: (i) TPA stimulated A20-HE2 cells (a MHV68 latently infected B cells); and (ii) MHV68 infected NIH 3T12 fibroblasts (data not shown).

Bottom Line: In addition, we show that M1 gene transcription is regulated by both the essential viral immediate-early transcriptional activator Rta and cellular interferon regulatory factor 4 (IRF4), which together potently synergize to drive M1 gene expression.Finally, we show that IRF4, a cellular transcription factor essential for plasma cell differentiation, can directly interact with Rta.The latter observation raises the possibility that the interaction of Rta and IRF4 may be involved in regulating a number of viral and cellular genes during MHV68 reactivation linked to plasma cell differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia, United States of America; Emory Vaccine Center, Emory University School of Medicine, Atlanta, Georgia, United States of America.

ABSTRACT
MHV68 is a murine gammaherpesvirus that infects laboratory mice and thus provides a tractable small animal model for characterizing critical aspects of gammaherpesvirus pathogenesis. Having evolved with their natural host, herpesviruses encode numerous gene products that are involved in modulating host immune responses to facilitate the establishment and maintenance of lifelong chronic infection. One such protein, MHV68 M1, is a secreted protein that has no known homologs, but has been shown to play a critical role in controlling virus reactivation from latently infected macrophages. We have previous demonstrated that M1 drives the activation and expansion of Vβ4+ CD8+ T cells, which are thought to be involved in controlling MHV68 reactivation through the secretion of interferon gamma. The mechanism of action and regulation of M1 expression are poorly understood. To gain insights into the function of M1, we set out to evaluate the site of expression and transcriptional regulation of the M1 gene. Here, using a recombinant virus expressing a fluorescent protein driven by the M1 gene promoter, we identify plasma cells as the major cell type expressing M1 at the peak of infection in the spleen. In addition, we show that M1 gene transcription is regulated by both the essential viral immediate-early transcriptional activator Rta and cellular interferon regulatory factor 4 (IRF4), which together potently synergize to drive M1 gene expression. Finally, we show that IRF4, a cellular transcription factor essential for plasma cell differentiation, can directly interact with Rta. The latter observation raises the possibility that the interaction of Rta and IRF4 may be involved in regulating a number of viral and cellular genes during MHV68 reactivation linked to plasma cell differentiation.

Show MeSH
Related in: MedlinePlus