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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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Low levels of M1 promoter activity are detected in germinal center B cells.C57Bl/6 mice were intranasally infected with 5×105 pfu of the indicated virus and spleens were harvested at 14 days post infection. B cells were defined by CD19+CD3− population. (A) Representative plots show YFP marking (colored) overlayed on total B cell population (gray) and are gated for germinal center B cells defined by GL7HiCD95Hi. (B) Complied results from 3 experiments, with 3–5 mice per group, show the frequency of YFP+ cells with a germinal center B cell phenotype.
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ppat-1004302-g003: Low levels of M1 promoter activity are detected in germinal center B cells.C57Bl/6 mice were intranasally infected with 5×105 pfu of the indicated virus and spleens were harvested at 14 days post infection. B cells were defined by CD19+CD3− population. (A) Representative plots show YFP marking (colored) overlayed on total B cell population (gray) and are gated for germinal center B cells defined by GL7HiCD95Hi. (B) Complied results from 3 experiments, with 3–5 mice per group, show the frequency of YFP+ cells with a germinal center B cell phenotype.

Mentions: We have previously shown that the majority (ca. 70–90%) of virally infected B cells, as indicated by YFP expression, exhibit a germinal center phenotype [23], [27]. Individual mice were assessed for YFP marking and, consistent with previous observations, we found a similar frequency of virus infected (YFP+) B cells with a germinal center phenotype for mice infected with either the MHV68.YFP or MHV68-M1st.YFP viruses, both showing an average of ca. 70% (Figure 3). These results further substantiate that a functional M1 gene is dispensable for establishment of MHV68 latency in B cells. In contrast, few infected germinal center B cells were marked by the M1pYFP virus (an average of ca. 20% of YFP+ cells) – indicating that the majority of M1 expressing cells do not have a germinal center B cell phenotype. Based on the ca. 10-fold lower frequency of splenocytes marked by the M1pYFP virus (see Figure 2), we estimate that M1 promoter activity is only detectable in ca. 5% of infected germinal center B cells. Based on these results it is clear that M1 is predominantly expressed in some other MHV68 infected cellular reservoir.


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)

Low levels of M1 promoter activity are detected in germinal center B cells.C57Bl/6 mice were intranasally infected with 5×105 pfu of the indicated virus and spleens were harvested at 14 days post infection. B cells were defined by CD19+CD3− population. (A) Representative plots show YFP marking (colored) overlayed on total B cell population (gray) and are gated for germinal center B cells defined by GL7HiCD95Hi. (B) Complied results from 3 experiments, with 3–5 mice per group, show the frequency of YFP+ cells with a germinal center B cell phenotype.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004302-g003: Low levels of M1 promoter activity are detected in germinal center B cells.C57Bl/6 mice were intranasally infected with 5×105 pfu of the indicated virus and spleens were harvested at 14 days post infection. B cells were defined by CD19+CD3− population. (A) Representative plots show YFP marking (colored) overlayed on total B cell population (gray) and are gated for germinal center B cells defined by GL7HiCD95Hi. (B) Complied results from 3 experiments, with 3–5 mice per group, show the frequency of YFP+ cells with a germinal center B cell phenotype.
Mentions: We have previously shown that the majority (ca. 70–90%) of virally infected B cells, as indicated by YFP expression, exhibit a germinal center phenotype [23], [27]. Individual mice were assessed for YFP marking and, consistent with previous observations, we found a similar frequency of virus infected (YFP+) B cells with a germinal center phenotype for mice infected with either the MHV68.YFP or MHV68-M1st.YFP viruses, both showing an average of ca. 70% (Figure 3). These results further substantiate that a functional M1 gene is dispensable for establishment of MHV68 latency in B cells. In contrast, few infected germinal center B cells were marked by the M1pYFP virus (an average of ca. 20% of YFP+ cells) – indicating that the majority of M1 expressing cells do not have a germinal center B cell phenotype. Based on the ca. 10-fold lower frequency of splenocytes marked by the M1pYFP virus (see Figure 2), we estimate that M1 promoter activity is only detectable in ca. 5% of infected germinal center B cells. Based on these results it is clear that M1 is predominantly expressed in some other MHV68 infected cellular reservoir.

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