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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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Novel RRE involved in Rta activation of the gene 50 proximal promoter.(A) Analysis of Rta transactivation of MHV68 promoters that contain the core sequence present in the novel RRE identified in the M1 promoter. The indicated promoter-driven luciferase reporter constructs were transfected into 293T cells in the presence and absence of an Rta expression plasmid. (B) Mutation introduced into the putative RRE present in the gene 50 proximal promoter (ORF50pp described in [46]). (C) Analysis of Rta transactivation of the wt and RRE mutant gene 50 proximal promoter transfected into 293T cells.
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ppat-1004302-g010: Novel RRE involved in Rta activation of the gene 50 proximal promoter.(A) Analysis of Rta transactivation of MHV68 promoters that contain the core sequence present in the novel RRE identified in the M1 promoter. The indicated promoter-driven luciferase reporter constructs were transfected into 293T cells in the presence and absence of an Rta expression plasmid. (B) Mutation introduced into the putative RRE present in the gene 50 proximal promoter (ORF50pp described in [46]). (C) Analysis of Rta transactivation of the wt and RRE mutant gene 50 proximal promoter transfected into 293T cells.

Mentions: With the identification of a novel Rta response element, we next wanted to evaluate whether this element was functional in other viral promoters that appear to contain this RRE (see Fig. 9C). Reporter constructs for the putative promoter regions of the M2 gene (encoding an adaptor protein involved in B cell signaling), ORF8 (encoding glycoprotein B), ORF22 (encoding glycoprotein H), ORF63 (encoding a tegument protein), and ORF73 (encoding the MHV68 Latency Associated Nuclear Antigen (LANA) homolog) were generated. In addition, the gene 50 proximal, distal, and N4/N5 promoter constructs previously described in Wakeman et al.[46] were evaluated for response to Rta expression. We observed varying levels of promoter response, with the strongest responses from ORF50pp, ORF8p, ORF22p, ORF63p, intermediate responses from the M1p, ORF50dp and ORF50 N4/N5p, and weak responses from M2p and ORF73p (Figure 10A). To further investigate the role of the Rta response element in the observed transactivation, we engineered the same three nucleotide mutation used in the M1p (Figure 9B) into the proximal ORF50 promoter (Figure 10B). Notably, mutation of this sequence resulted in a 38-fold reduction in Rta transactivation (Figure 10C). Notably, with the exception of the M1 promoter, for all the other reporter construct we failed to observe any synergistic activation by the co-expression of Rta and IRF4 (data not shown).


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)

Novel RRE involved in Rta activation of the gene 50 proximal promoter.(A) Analysis of Rta transactivation of MHV68 promoters that contain the core sequence present in the novel RRE identified in the M1 promoter. The indicated promoter-driven luciferase reporter constructs were transfected into 293T cells in the presence and absence of an Rta expression plasmid. (B) Mutation introduced into the putative RRE present in the gene 50 proximal promoter (ORF50pp described in [46]). (C) Analysis of Rta transactivation of the wt and RRE mutant gene 50 proximal promoter transfected into 293T cells.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004302-g010: Novel RRE involved in Rta activation of the gene 50 proximal promoter.(A) Analysis of Rta transactivation of MHV68 promoters that contain the core sequence present in the novel RRE identified in the M1 promoter. The indicated promoter-driven luciferase reporter constructs were transfected into 293T cells in the presence and absence of an Rta expression plasmid. (B) Mutation introduced into the putative RRE present in the gene 50 proximal promoter (ORF50pp described in [46]). (C) Analysis of Rta transactivation of the wt and RRE mutant gene 50 proximal promoter transfected into 293T cells.
Mentions: With the identification of a novel Rta response element, we next wanted to evaluate whether this element was functional in other viral promoters that appear to contain this RRE (see Fig. 9C). Reporter constructs for the putative promoter regions of the M2 gene (encoding an adaptor protein involved in B cell signaling), ORF8 (encoding glycoprotein B), ORF22 (encoding glycoprotein H), ORF63 (encoding a tegument protein), and ORF73 (encoding the MHV68 Latency Associated Nuclear Antigen (LANA) homolog) were generated. In addition, the gene 50 proximal, distal, and N4/N5 promoter constructs previously described in Wakeman et al.[46] were evaluated for response to Rta expression. We observed varying levels of promoter response, with the strongest responses from ORF50pp, ORF8p, ORF22p, ORF63p, intermediate responses from the M1p, ORF50dp and ORF50 N4/N5p, and weak responses from M2p and ORF73p (Figure 10A). To further investigate the role of the Rta response element in the observed transactivation, we engineered the same three nucleotide mutation used in the M1p (Figure 9B) into the proximal ORF50 promoter (Figure 10B). Notably, mutation of this sequence resulted in a 38-fold reduction in Rta transactivation (Figure 10C). Notably, with the exception of the M1 promoter, for all the other reporter construct we failed to observe any synergistic activation by the co-expression of Rta and IRF4 (data not shown).

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