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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 promoter activity is detected in a subset of MHV68 infected splenocytes.C57Bl/6 mice were intranasally infected with 5×105 pfu of the indicated virus and spleens were harvested at 14 days post infection. Splenocytes were gated on the CD3-negative fraction to eliminate auto-fluorescing cells, as previously described [23]. (A) Representative plots show YFP marking of infected splenocytes. YFP-positive gates were based on mice infected with wild type MHV68 lacking a YFP expression cassette. (B) Complied results from 3 experiments, with 3–5 mice per group, show the frequency of YFP+ cells in spleens of infected mice.
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ppat-1004302-g002: M1 promoter activity is detected in a subset of MHV68 infected splenocytes.C57Bl/6 mice were intranasally infected with 5×105 pfu of the indicated virus and spleens were harvested at 14 days post infection. Splenocytes were gated on the CD3-negative fraction to eliminate auto-fluorescing cells, as previously described [23]. (A) Representative plots show YFP marking of infected splenocytes. YFP-positive gates were based on mice infected with wild type MHV68 lacking a YFP expression cassette. (B) Complied results from 3 experiments, with 3–5 mice per group, show the frequency of YFP+ cells in spleens of infected mice.

Mentions: Analysis of MHV68 infection of splenocytes at day 14 post-infection revealed robust marking of splenocytes by both the MHV68-YFP and MHV68-M1stYFP viruses (Figure 2). We have previously noted that there is significant mouse to mouse variation in the frequency of infected splenocytes for a given virus [25], and have recently determined that this directly correlates with the frequency of the CD4+ T follicular helper (TFH) response [26]. For these analyses we observed on average ca. 0.5% and 1.0% of splenocytes were YFP+ for the MHV68-YFP and MHV68-M1stYFP viruses, respectively (Figure 2). The latter result confirms that M1 function is dispensable for the establishment of latency in splenocytes. In contrast, only ca. 0.04% of splenocytes were YFP+ with the M1pYFP virus, indicating that the M1 promoter is active in only ca. 5–10% of infected splenocytes.


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 promoter activity is detected in a subset of MHV68 infected splenocytes.C57Bl/6 mice were intranasally infected with 5×105 pfu of the indicated virus and spleens were harvested at 14 days post infection. Splenocytes were gated on the CD3-negative fraction to eliminate auto-fluorescing cells, as previously described [23]. (A) Representative plots show YFP marking of infected splenocytes. YFP-positive gates were based on mice infected with wild type MHV68 lacking a YFP expression cassette. (B) Complied results from 3 experiments, with 3–5 mice per group, show the frequency of YFP+ cells in spleens of infected mice.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004302-g002: M1 promoter activity is detected in a subset of MHV68 infected splenocytes.C57Bl/6 mice were intranasally infected with 5×105 pfu of the indicated virus and spleens were harvested at 14 days post infection. Splenocytes were gated on the CD3-negative fraction to eliminate auto-fluorescing cells, as previously described [23]. (A) Representative plots show YFP marking of infected splenocytes. YFP-positive gates were based on mice infected with wild type MHV68 lacking a YFP expression cassette. (B) Complied results from 3 experiments, with 3–5 mice per group, show the frequency of YFP+ cells in spleens of infected mice.
Mentions: Analysis of MHV68 infection of splenocytes at day 14 post-infection revealed robust marking of splenocytes by both the MHV68-YFP and MHV68-M1stYFP viruses (Figure 2). We have previously noted that there is significant mouse to mouse variation in the frequency of infected splenocytes for a given virus [25], and have recently determined that this directly correlates with the frequency of the CD4+ T follicular helper (TFH) response [26]. For these analyses we observed on average ca. 0.5% and 1.0% of splenocytes were YFP+ for the MHV68-YFP and MHV68-M1stYFP viruses, respectively (Figure 2). The latter result confirms that M1 function is dispensable for the establishment of latency in splenocytes. In contrast, only ca. 0.04% of splenocytes were YFP+ with the M1pYFP virus, indicating that the M1 promoter is active in only ca. 5–10% of infected splenocytes.

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