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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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The majority of M1 promoter activity is detected in splenic plasma cells.C57Bl/6 mice were infected with 5×105 pfu/IN of the indicated virus and spleens were harvested at 14 days post infection. Cells were gated on CD3− population for analysis. (A) Representative plots show YFP marking (colored) overlayed on total CD3− population (gray) and are gated for plasma cells defined by CD138+B220Lo. (B). Compiled results from 3 experiments, with 3–5 mice per group, show the frequency of YFP+ cells with a plasma cell phenotype.
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ppat-1004302-g004: The majority of M1 promoter activity is detected in splenic plasma cells.C57Bl/6 mice were infected with 5×105 pfu/IN of the indicated virus and spleens were harvested at 14 days post infection. Cells were gated on CD3− population for analysis. (A) Representative plots show YFP marking (colored) overlayed on total CD3− population (gray) and are gated for plasma cells defined by CD138+B220Lo. (B). Compiled results from 3 experiments, with 3–5 mice per group, show the frequency of YFP+ cells with a plasma cell phenotype.

Mentions: The other major cell population in the spleen that is infected by MHV68 are plasma cells (CD138hi, B220low) [23], [27]. During infection, virus infection (YFP marking) of splenic plasma cells reaches peak levels at day 14 post-infection (ca. 10–20% of virus infected splenocytes) and begins to wane by day 18 post-infection (ca. 5–10% of virus infected splenocytes) [23]. We observed marking of splenic plasma cells for both MHV68-YFP and MHV68-M1st.YFP infected mice at day 14 post-infection consistent with previous observations, with ca. 10% YFP+ cells exhibiting a plasma cell phenotype (no significant difference between these 2 groups) (Figure 4). Strikingly, when assessing YFP marking of the splenic plasma cell population by the M1pYFP virus, the vast majority of YFP+ cells exhibited a plasma cell phenotype (on average >75% of YFP+ cells) (Figure 4). Thus, this strongly argues that M1 gene expression is largely limited to the infected plasma cell population. Notably, MHV68 reactivation from latently infected splenocytes is tightly linked to plasma cell differentiation [27], which suggests that M1 expression is coupled to virus reactivation from B cells. Finally, when considering the frequency of M1pYFP marked cells with the frequency of MHV68-YFP and MHV68-M1st.YFP marked splenic plasma cells, it appears that the majority of virus infected plasma cells express M1.


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)

The majority of M1 promoter activity is detected in splenic plasma cells.C57Bl/6 mice were infected with 5×105 pfu/IN of the indicated virus and spleens were harvested at 14 days post infection. Cells were gated on CD3− population for analysis. (A) Representative plots show YFP marking (colored) overlayed on total CD3− population (gray) and are gated for plasma cells defined by CD138+B220Lo. (B). Compiled results from 3 experiments, with 3–5 mice per group, show the frequency of YFP+ cells with a plasma cell phenotype.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004302-g004: The majority of M1 promoter activity is detected in splenic plasma cells.C57Bl/6 mice were infected with 5×105 pfu/IN of the indicated virus and spleens were harvested at 14 days post infection. Cells were gated on CD3− population for analysis. (A) Representative plots show YFP marking (colored) overlayed on total CD3− population (gray) and are gated for plasma cells defined by CD138+B220Lo. (B). Compiled results from 3 experiments, with 3–5 mice per group, show the frequency of YFP+ cells with a plasma cell phenotype.
Mentions: The other major cell population in the spleen that is infected by MHV68 are plasma cells (CD138hi, B220low) [23], [27]. During infection, virus infection (YFP marking) of splenic plasma cells reaches peak levels at day 14 post-infection (ca. 10–20% of virus infected splenocytes) and begins to wane by day 18 post-infection (ca. 5–10% of virus infected splenocytes) [23]. We observed marking of splenic plasma cells for both MHV68-YFP and MHV68-M1st.YFP infected mice at day 14 post-infection consistent with previous observations, with ca. 10% YFP+ cells exhibiting a plasma cell phenotype (no significant difference between these 2 groups) (Figure 4). Strikingly, when assessing YFP marking of the splenic plasma cell population by the M1pYFP virus, the vast majority of YFP+ cells exhibited a plasma cell phenotype (on average >75% of YFP+ cells) (Figure 4). Thus, this strongly argues that M1 gene expression is largely limited to the infected plasma cell population. Notably, MHV68 reactivation from latently infected splenocytes is tightly linked to plasma cell differentiation [27], which suggests that M1 expression is coupled to virus reactivation from B cells. Finally, when considering the frequency of M1pYFP marked cells with the frequency of MHV68-YFP and MHV68-M1st.YFP marked splenic plasma cells, it appears that the majority of virus infected plasma cells express M1.

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