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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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Related in: MedlinePlus

Efficient RTA transactivation of M1 promoter is dependent on a functional IRF4 binding site.(A) To evaluate the ability of Rta to transactivate the M1 promoter, a dosage response using increasing amounts of Rta expression vector war performed in P3X63Ag8 cells. (B) Mutations to the IRF4 binding motif were then generated and tested for the ability to respond to Rta in P3X63Ag8 cells. (C) Synergistic effects of RTA and IRF4 on WT and mutated M1 promoter were evaluated by expressing Rta or IRF4 alone or in combination (normalized with pCDNA empty vector) in 293T cells. (D) Lysates from 293T cells transfected with Rta or IRF4 alone or in combination were used for co-immunoprecipitation with IRF4 or Flag antibody. Membranes were probed with the anti-Flag or anti-IRF4 to for detection. The data shown for the reporter gene assays (panels A–C) were done with triplicate samples, and were repeated in 2–3 independent experiments. Standard error of the mean is shown.
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ppat-1004302-g008: Efficient RTA transactivation of M1 promoter is dependent on a functional IRF4 binding site.(A) To evaluate the ability of Rta to transactivate the M1 promoter, a dosage response using increasing amounts of Rta expression vector war performed in P3X63Ag8 cells. (B) Mutations to the IRF4 binding motif were then generated and tested for the ability to respond to Rta in P3X63Ag8 cells. (C) Synergistic effects of RTA and IRF4 on WT and mutated M1 promoter were evaluated by expressing Rta or IRF4 alone or in combination (normalized with pCDNA empty vector) in 293T cells. (D) Lysates from 293T cells transfected with Rta or IRF4 alone or in combination were used for co-immunoprecipitation with IRF4 or Flag antibody. Membranes were probed with the anti-Flag or anti-IRF4 to for detection. The data shown for the reporter gene assays (panels A–C) were done with triplicate samples, and were repeated in 2–3 independent experiments. Standard error of the mean is shown.

Mentions: Several studies have established a link between gammaherpesvirus reactivation from latency and plasma cell differentiation [27], [34]–[39]. Given that our data shows: (i) M1 promoter expression is detected from plasma cells during in vivo infection; (ii) basal M1 promoter activity requires a functional IRF4 site; and (iii) viral reactivation is linked with plasma cell differentiation, we set out to evaluate whether the M1 promoter is responsive to the MHV68 viral lytic transactivator Rta. Expressing increasing amounts of Rta with an M1 promoter-driven reporter construct in the P3X63Ag8 plasmacytoma cell line resulted in a dosage dependent increase in M1 promoter activity (Figure 8A). Moreover, the ability of Rta to efficiently transactivate the M1 promoter in the P3X63Ag8 cell line was dependent on the presence of an intact IRF4 binding site (Figure 8B). To further assess whether Rta functionally synergizes with IRF4 to activate the M1 promoter, we chose a cell line (293T cells) which lacks expression of Rta and IRF4. In 293T cells we observed that either factor alone led to very modest increase in M1 promoter activity (ca. 5–10 fold) (Figure 8C). However, when the two factors were co-expressed there was a significant increase in promoter activity (ca. 250-fold) (Figure 8C). Importantly, disruption of the IRF4 binding site dramatically impaired the ability of IRF4 and Rta to synergistically activate the M1 promoter (Figure 8C).


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)

Efficient RTA transactivation of M1 promoter is dependent on a functional IRF4 binding site.(A) To evaluate the ability of Rta to transactivate the M1 promoter, a dosage response using increasing amounts of Rta expression vector war performed in P3X63Ag8 cells. (B) Mutations to the IRF4 binding motif were then generated and tested for the ability to respond to Rta in P3X63Ag8 cells. (C) Synergistic effects of RTA and IRF4 on WT and mutated M1 promoter were evaluated by expressing Rta or IRF4 alone or in combination (normalized with pCDNA empty vector) in 293T cells. (D) Lysates from 293T cells transfected with Rta or IRF4 alone or in combination were used for co-immunoprecipitation with IRF4 or Flag antibody. Membranes were probed with the anti-Flag or anti-IRF4 to for detection. The data shown for the reporter gene assays (panels A–C) were done with triplicate samples, and were repeated in 2–3 independent experiments. Standard error of the mean is shown.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004302-g008: Efficient RTA transactivation of M1 promoter is dependent on a functional IRF4 binding site.(A) To evaluate the ability of Rta to transactivate the M1 promoter, a dosage response using increasing amounts of Rta expression vector war performed in P3X63Ag8 cells. (B) Mutations to the IRF4 binding motif were then generated and tested for the ability to respond to Rta in P3X63Ag8 cells. (C) Synergistic effects of RTA and IRF4 on WT and mutated M1 promoter were evaluated by expressing Rta or IRF4 alone or in combination (normalized with pCDNA empty vector) in 293T cells. (D) Lysates from 293T cells transfected with Rta or IRF4 alone or in combination were used for co-immunoprecipitation with IRF4 or Flag antibody. Membranes were probed with the anti-Flag or anti-IRF4 to for detection. The data shown for the reporter gene assays (panels A–C) were done with triplicate samples, and were repeated in 2–3 independent experiments. Standard error of the mean is shown.
Mentions: Several studies have established a link between gammaherpesvirus reactivation from latency and plasma cell differentiation [27], [34]–[39]. Given that our data shows: (i) M1 promoter expression is detected from plasma cells during in vivo infection; (ii) basal M1 promoter activity requires a functional IRF4 site; and (iii) viral reactivation is linked with plasma cell differentiation, we set out to evaluate whether the M1 promoter is responsive to the MHV68 viral lytic transactivator Rta. Expressing increasing amounts of Rta with an M1 promoter-driven reporter construct in the P3X63Ag8 plasmacytoma cell line resulted in a dosage dependent increase in M1 promoter activity (Figure 8A). Moreover, the ability of Rta to efficiently transactivate the M1 promoter in the P3X63Ag8 cell line was dependent on the presence of an intact IRF4 binding site (Figure 8B). To further assess whether Rta functionally synergizes with IRF4 to activate the M1 promoter, we chose a cell line (293T cells) which lacks expression of Rta and IRF4. In 293T cells we observed that either factor alone led to very modest increase in M1 promoter activity (ca. 5–10 fold) (Figure 8C). However, when the two factors were co-expressed there was a significant increase in promoter activity (ca. 250-fold) (Figure 8C). Importantly, disruption of the IRF4 binding site dramatically impaired the ability of IRF4 and Rta to synergistically activate the M1 promoter (Figure 8C).

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