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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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Basal activity of the M1 promoter is dependent on IRF4 binding.(A) To identify DNA-protein interaction an electrophoretic mobility shift assay was performed using radiolabeled probe with P3X nuclear extracts. A competition assay to determine the specificity of DNA-protein complexes used 2 and 20 fold molar excess of unlabeled competitor DNA containing either the WT or mutant IRF4 binding sequence. An antibody supershift assay was performed by preincubating samples with anti-IRF4, or an isotype control antibody (anti-pSTAT1) prior to electrophoresis. The asterisk denotes the anti-IRF4 supershifted complex. (B) Point mutations in the M1 promoter were made in the IRF4 binding site and assessed in the P3X63Ag8 plasmacytoma cell line.
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ppat-1004302-g007: Basal activity of the M1 promoter is dependent on IRF4 binding.(A) To identify DNA-protein interaction an electrophoretic mobility shift assay was performed using radiolabeled probe with P3X nuclear extracts. A competition assay to determine the specificity of DNA-protein complexes used 2 and 20 fold molar excess of unlabeled competitor DNA containing either the WT or mutant IRF4 binding sequence. An antibody supershift assay was performed by preincubating samples with anti-IRF4, or an isotype control antibody (anti-pSTAT1) prior to electrophoresis. The asterisk denotes the anti-IRF4 supershifted complex. (B) Point mutations in the M1 promoter were made in the IRF4 binding site and assessed in the P3X63Ag8 plasmacytoma cell line.

Mentions: To characterize IRF4 binding to the candidate IRF site in the M1 promoter, an electrophoretic mobility shift assay (EMSA) was carried out (Figure 7A). EMSA was performed using nuclear extracts from P3X63Ag8 cells grown under normal conditions, along with a [32P]-labeled oligonucleotide probe containing the candidate M1 promoter IRF4 binding site. As expected we observed shifted complexes, which could be competed away using unlabeled double stranded DNA probes containing the M1p IRF4 binding site, but not with a competitor containing an IRF binding site mutation which has previously been shown to disrupt IRF8 binding with DNA [33] (Figure 7A). Furthermore, binding of IRF4 was confirmed by supershift analysis using an antibody against IRF4 (Figure 7A). This analysis was extended by generating M1 promoter-driven luciferase reporter constructs in which mutations were introduced into the IRF binding site. Two mutations in the core interferon response sequence, which have previously been shown to ablate IRF8 DNA:protein interaction [33], were introduced into the M1 promoter. Notably, either mutation led to a significant loss in basal M1 promoter activity (ca. 8-fold decrease in promoter activity) (Figure 7B).


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)

Basal activity of the M1 promoter is dependent on IRF4 binding.(A) To identify DNA-protein interaction an electrophoretic mobility shift assay was performed using radiolabeled probe with P3X nuclear extracts. A competition assay to determine the specificity of DNA-protein complexes used 2 and 20 fold molar excess of unlabeled competitor DNA containing either the WT or mutant IRF4 binding sequence. An antibody supershift assay was performed by preincubating samples with anti-IRF4, or an isotype control antibody (anti-pSTAT1) prior to electrophoresis. The asterisk denotes the anti-IRF4 supershifted complex. (B) Point mutations in the M1 promoter were made in the IRF4 binding site and assessed in the P3X63Ag8 plasmacytoma cell line.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004302-g007: Basal activity of the M1 promoter is dependent on IRF4 binding.(A) To identify DNA-protein interaction an electrophoretic mobility shift assay was performed using radiolabeled probe with P3X nuclear extracts. A competition assay to determine the specificity of DNA-protein complexes used 2 and 20 fold molar excess of unlabeled competitor DNA containing either the WT or mutant IRF4 binding sequence. An antibody supershift assay was performed by preincubating samples with anti-IRF4, or an isotype control antibody (anti-pSTAT1) prior to electrophoresis. The asterisk denotes the anti-IRF4 supershifted complex. (B) Point mutations in the M1 promoter were made in the IRF4 binding site and assessed in the P3X63Ag8 plasmacytoma cell line.
Mentions: To characterize IRF4 binding to the candidate IRF site in the M1 promoter, an electrophoretic mobility shift assay (EMSA) was carried out (Figure 7A). EMSA was performed using nuclear extracts from P3X63Ag8 cells grown under normal conditions, along with a [32P]-labeled oligonucleotide probe containing the candidate M1 promoter IRF4 binding site. As expected we observed shifted complexes, which could be competed away using unlabeled double stranded DNA probes containing the M1p IRF4 binding site, but not with a competitor containing an IRF binding site mutation which has previously been shown to disrupt IRF8 binding with DNA [33] (Figure 7A). Furthermore, binding of IRF4 was confirmed by supershift analysis using an antibody against IRF4 (Figure 7A). This analysis was extended by generating M1 promoter-driven luciferase reporter constructs in which mutations were introduced into the IRF binding site. Two mutations in the core interferon response sequence, which have previously been shown to ablate IRF8 DNA:protein interaction [33], were introduced into the M1 promoter. Notably, either mutation led to a significant loss in basal M1 promoter activity (ca. 8-fold decrease in promoter activity) (Figure 7B).

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