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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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Identification of a novel RTA response element in the M1 promoter.(A) To identify the Rta response element serial truncations of the M1 promoter were tested for RTA transactivation in the P3X63Ag8 plasmacytoma line which constitutively expresses IRF4. The blue box indicates the putative RTA response element; the yellow box denotes the location of the IRF4 binding site. (B) The nucleotide sequence of the putative Rta response element found in the M1 promoter is shown, along with nucleotide sequences sharing the core sequence with the M1 Rta response element (RRE) (denoted by the open blue rectangle) that are located upstream of a number of other MHV68 genes. In addition, the mutation (M1pRREm) introduced into the M1 Rta response element is shown. (C and D) Activity of the M1 promoter-driven reporter construct containing a mutation (indicated in red) in the putative Rta response element (RREm) was assessed in the P3X63Ag8 plasmacytoma cell line for Rta transactivation (panel C) and in 293T cells for IRF4+Rta synergy (panel D). Transient transfection reporter gene assays were conducted in triplicate and three independent assays were performed. Standard error of the mean is shown.
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ppat-1004302-g009: Identification of a novel RTA response element in the M1 promoter.(A) To identify the Rta response element serial truncations of the M1 promoter were tested for RTA transactivation in the P3X63Ag8 plasmacytoma line which constitutively expresses IRF4. The blue box indicates the putative RTA response element; the yellow box denotes the location of the IRF4 binding site. (B) The nucleotide sequence of the putative Rta response element found in the M1 promoter is shown, along with nucleotide sequences sharing the core sequence with the M1 Rta response element (RRE) (denoted by the open blue rectangle) that are located upstream of a number of other MHV68 genes. In addition, the mutation (M1pRREm) introduced into the M1 Rta response element is shown. (C and D) Activity of the M1 promoter-driven reporter construct containing a mutation (indicated in red) in the putative Rta response element (RREm) was assessed in the P3X63Ag8 plasmacytoma cell line for Rta transactivation (panel C) and in 293T cells for IRF4+Rta synergy (panel D). Transient transfection reporter gene assays were conducted in triplicate and three independent assays were performed. Standard error of the mean is shown.

Mentions: Several investigators have identified Rta responsive elements in viral promoters for both KSHV and MHV68 [40]–[44]. To date, the known Rta responsive genes are either regulated through: (i) direct interaction of Rta and DNA through a core Rta binding sequence; or (ii) Rta DNA binding is facilitated through protein-protein interactions – in the case of KSHV Rta, through interaction with the cellular transcription factor RBPJκ (reviewed in [45]). In MHV68 gene 57 promoter, it appears that both types of Rta response elements may be utilized – although a role for RBPJκ in MHV68 Rta activation has not been formally demonstrated [40], [41]. Interestingly, neither of the binding sites identified in the MHV68 gene 57 promoter are present in the M1 promoter, suggesting a novel Rta interaction motif. To identify the Rta response element(s) in the M1 promoter, a series of promoter truncations were generated and tested in the P3X63Ag8 plasmacytoma cell line. A candidate Rta response element was identified by evaluating promoter constructs which lost the ability to be transactivated by Rta. Using this approach we identified a putative Rta response element between −82 and −72 bp in the M1 promoter (Figure 9A). This 12 bp sequence (5′-GGTCAGAAGGCT-3′) failed to show homology to any known Rta response element identified in the gammaherpesvirus family. However, a screen of the MHV68 genome identified a number of candidate sites upstream of other MHV68 replication-associated genes which share significant homology with the core 5′-TCAGAAG-3′ sequence in the putative M1 promoter Rta response element (Figure 9B). Mutations of the three most central residues of the predicted Rta response element (see M1pRREm in Figure 9B) resulted in an ca. 10-fold reduction in transactivation in the plasma cell line (Figure 9C), as well as an ca. 6-fold reduction in Rta and IRF4 synergistic transactivation of the M1 promoter in 293T cells (Figure 9D).


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)

Identification of a novel RTA response element in the M1 promoter.(A) To identify the Rta response element serial truncations of the M1 promoter were tested for RTA transactivation in the P3X63Ag8 plasmacytoma line which constitutively expresses IRF4. The blue box indicates the putative RTA response element; the yellow box denotes the location of the IRF4 binding site. (B) The nucleotide sequence of the putative Rta response element found in the M1 promoter is shown, along with nucleotide sequences sharing the core sequence with the M1 Rta response element (RRE) (denoted by the open blue rectangle) that are located upstream of a number of other MHV68 genes. In addition, the mutation (M1pRREm) introduced into the M1 Rta response element is shown. (C and D) Activity of the M1 promoter-driven reporter construct containing a mutation (indicated in red) in the putative Rta response element (RREm) was assessed in the P3X63Ag8 plasmacytoma cell line for Rta transactivation (panel C) and in 293T cells for IRF4+Rta synergy (panel D). Transient transfection reporter gene assays were conducted in triplicate and three independent assays were performed. Standard error of the mean is shown.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004302-g009: Identification of a novel RTA response element in the M1 promoter.(A) To identify the Rta response element serial truncations of the M1 promoter were tested for RTA transactivation in the P3X63Ag8 plasmacytoma line which constitutively expresses IRF4. The blue box indicates the putative RTA response element; the yellow box denotes the location of the IRF4 binding site. (B) The nucleotide sequence of the putative Rta response element found in the M1 promoter is shown, along with nucleotide sequences sharing the core sequence with the M1 Rta response element (RRE) (denoted by the open blue rectangle) that are located upstream of a number of other MHV68 genes. In addition, the mutation (M1pRREm) introduced into the M1 Rta response element is shown. (C and D) Activity of the M1 promoter-driven reporter construct containing a mutation (indicated in red) in the putative Rta response element (RREm) was assessed in the P3X63Ag8 plasmacytoma cell line for Rta transactivation (panel C) and in 293T cells for IRF4+Rta synergy (panel D). Transient transfection reporter gene assays were conducted in triplicate and three independent assays were performed. Standard error of the mean is shown.
Mentions: Several investigators have identified Rta responsive elements in viral promoters for both KSHV and MHV68 [40]–[44]. To date, the known Rta responsive genes are either regulated through: (i) direct interaction of Rta and DNA through a core Rta binding sequence; or (ii) Rta DNA binding is facilitated through protein-protein interactions – in the case of KSHV Rta, through interaction with the cellular transcription factor RBPJκ (reviewed in [45]). In MHV68 gene 57 promoter, it appears that both types of Rta response elements may be utilized – although a role for RBPJκ in MHV68 Rta activation has not been formally demonstrated [40], [41]. Interestingly, neither of the binding sites identified in the MHV68 gene 57 promoter are present in the M1 promoter, suggesting a novel Rta interaction motif. To identify the Rta response element(s) in the M1 promoter, a series of promoter truncations were generated and tested in the P3X63Ag8 plasmacytoma cell line. A candidate Rta response element was identified by evaluating promoter constructs which lost the ability to be transactivated by Rta. Using this approach we identified a putative Rta response element between −82 and −72 bp in the M1 promoter (Figure 9A). This 12 bp sequence (5′-GGTCAGAAGGCT-3′) failed to show homology to any known Rta response element identified in the gammaherpesvirus family. However, a screen of the MHV68 genome identified a number of candidate sites upstream of other MHV68 replication-associated genes which share significant homology with the core 5′-TCAGAAG-3′ sequence in the putative M1 promoter Rta response element (Figure 9B). Mutations of the three most central residues of the predicted Rta response element (see M1pRREm in Figure 9B) resulted in an ca. 10-fold reduction in transactivation in the plasma cell line (Figure 9C), as well as an ca. 6-fold reduction in Rta and IRF4 synergistic transactivation of the M1 promoter in 293T cells (Figure 9D).

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