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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 exhibits basal activity in a plasmablast cell line.Serial truncations of the M1 promoter region were cloned into a luciferase reporter vector and tested for luciferase activity in the P3X63Ag8 cell line from ATCC. P3X63Ag8 cells were nucleofected and 48(pGL4.10). Experiments were done with triplicate samples and repeated in two independent experiments.
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ppat-1004302-g006: M1 promoter exhibits basal activity in a plasmablast cell line.Serial truncations of the M1 promoter region were cloned into a luciferase reporter vector and tested for luciferase activity in the P3X63Ag8 cell line from ATCC. P3X63Ag8 cells were nucleofected and 48(pGL4.10). Experiments were done with triplicate samples and repeated in two independent experiments.

Mentions: To identify the regulatory elements controlling M1 gene expression we next set out to characterize the M1 promoter. Serial truncations of the putative M1 promoter region were cloned into a luciferase reporter vector and tested for promoter activity in a variety of cell lines. Notably, minimal activity was detected in the murine B cell lines A20, WEHI, NSO, and BCL1-3B3 (data not shown) – perhaps consistent with the failure to observe significant M1 promoter-driven YFP activity in most splenic B cell populations with the MHV68-M1pYFP virus in mice. In addition, we failed to detect significant activity from these reporter constructs in the murine macrophage cell line RAW264.7 (data not shown). However, when these reporter constructs were transfected into the P3X68Ag8 murine plasmacytoma cell line significant basal promoter activity was observed (Figure 6). Similar levels of M1 promoter-driven luciferase activity were observed for the longer M1 promoter constructs (M1p/−1025 bp, M1p/−525 bp, and M1p/−245 bp), while truncation of sequences upstream of −100 bp significantly decreased activity (Figure 6). Activity was further decreased to near background levels when sequences upstream of −50 bp were deleted (Figure 6).


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 exhibits basal activity in a plasmablast cell line.Serial truncations of the M1 promoter region were cloned into a luciferase reporter vector and tested for luciferase activity in the P3X63Ag8 cell line from ATCC. P3X63Ag8 cells were nucleofected and 48(pGL4.10). Experiments were done with triplicate samples and repeated in two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004302-g006: M1 promoter exhibits basal activity in a plasmablast cell line.Serial truncations of the M1 promoter region were cloned into a luciferase reporter vector and tested for luciferase activity in the P3X63Ag8 cell line from ATCC. P3X63Ag8 cells were nucleofected and 48(pGL4.10). Experiments were done with triplicate samples and repeated in two independent experiments.
Mentions: To identify the regulatory elements controlling M1 gene expression we next set out to characterize the M1 promoter. Serial truncations of the putative M1 promoter region were cloned into a luciferase reporter vector and tested for promoter activity in a variety of cell lines. Notably, minimal activity was detected in the murine B cell lines A20, WEHI, NSO, and BCL1-3B3 (data not shown) – perhaps consistent with the failure to observe significant M1 promoter-driven YFP activity in most splenic B cell populations with the MHV68-M1pYFP virus in mice. In addition, we failed to detect significant activity from these reporter constructs in the murine macrophage cell line RAW264.7 (data not shown). However, when these reporter constructs were transfected into the P3X68Ag8 murine plasmacytoma cell line significant basal promoter activity was observed (Figure 6). Similar levels of M1 promoter-driven luciferase activity were observed for the longer M1 promoter constructs (M1p/−1025 bp, M1p/−525 bp, and M1p/−245 bp), while truncation of sequences upstream of −100 bp significantly decreased activity (Figure 6). Activity was further decreased to near background levels when sequences upstream of −50 bp were deleted (Figure 6).

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