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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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Generation of YFP reporter viruses.C57Bl/6 mice were intranasally infected with 5×105 pfu of the indicated virus and spleens were harvested at 14 days post infection. (A) To assess M1 promoter activity, a YFP cassette was cloned in place of the M1 open reading frame, allowing marking of infected cells where M1 promoter was active. (B) To determine MHV68 infection in the absence of M1 expression, a translational stop codon was introduced into the M1 open reading frame (ORF) of the MHV68-YFP BAC [23] by allelic exchange as previously described [2].
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ppat-1004302-g001: Generation of YFP reporter viruses.C57Bl/6 mice were intranasally infected with 5×105 pfu of the indicated virus and spleens were harvested at 14 days post infection. (A) To assess M1 promoter activity, a YFP cassette was cloned in place of the M1 open reading frame, allowing marking of infected cells where M1 promoter was active. (B) To determine MHV68 infection in the absence of M1 expression, a translational stop codon was introduced into the M1 open reading frame (ORF) of the MHV68-YFP BAC [23] by allelic exchange as previously described [2].

Mentions: To identify cellular reservoirs in which the M1 gene is expressed in vivo, we generated a series of recombinant viruses that express yellow fluorescent protein (YFP) to mark infected cells. For detection of M1 promoter activity, the M1 coding sequence was replaced with that encoding YFP, creating a M1 promoter-driven YFP mutant (Figure 1A). This strategy allows detection of the cellular reservoirs in which M1 is expressed during infection. Additionally, two important controls were used: MHV68-YFP, in which the YFP transgene under the control of the HCMV IE promoter was cloned into a neutral locus in the viral genome (efficiently marking MHV68 infected B cells and plasma cells) [23]; and (ii) MHV68-M1st.YFP, which contains the M1 translational stop mutation (M1- virus) in the context of the YFP transgene cloned into the neutral locus (Figure 1B). As M1 has previously been identified as a non-essential for both virus replication and for the establishment of latency in vivo[24], we did not anticipate that the M1pYFP recombinant would change the cellular reservoirs infected by MHV68. However, to formally address this issue, we have included analyses of the MHV68-M1st.YFP virus – which like the M1pYFP lacks a functional M1 gene.


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)

Generation of YFP reporter viruses.C57Bl/6 mice were intranasally infected with 5×105 pfu of the indicated virus and spleens were harvested at 14 days post infection. (A) To assess M1 promoter activity, a YFP cassette was cloned in place of the M1 open reading frame, allowing marking of infected cells where M1 promoter was active. (B) To determine MHV68 infection in the absence of M1 expression, a translational stop codon was introduced into the M1 open reading frame (ORF) of the MHV68-YFP BAC [23] by allelic exchange as previously described [2].
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004302-g001: Generation of YFP reporter viruses.C57Bl/6 mice were intranasally infected with 5×105 pfu of the indicated virus and spleens were harvested at 14 days post infection. (A) To assess M1 promoter activity, a YFP cassette was cloned in place of the M1 open reading frame, allowing marking of infected cells where M1 promoter was active. (B) To determine MHV68 infection in the absence of M1 expression, a translational stop codon was introduced into the M1 open reading frame (ORF) of the MHV68-YFP BAC [23] by allelic exchange as previously described [2].
Mentions: To identify cellular reservoirs in which the M1 gene is expressed in vivo, we generated a series of recombinant viruses that express yellow fluorescent protein (YFP) to mark infected cells. For detection of M1 promoter activity, the M1 coding sequence was replaced with that encoding YFP, creating a M1 promoter-driven YFP mutant (Figure 1A). This strategy allows detection of the cellular reservoirs in which M1 is expressed during infection. Additionally, two important controls were used: MHV68-YFP, in which the YFP transgene under the control of the HCMV IE promoter was cloned into a neutral locus in the viral genome (efficiently marking MHV68 infected B cells and plasma cells) [23]; and (ii) MHV68-M1st.YFP, which contains the M1 translational stop mutation (M1- virus) in the context of the YFP transgene cloned into the neutral locus (Figure 1B). As M1 has previously been identified as a non-essential for both virus replication and for the establishment of latency in vivo[24], we did not anticipate that the M1pYFP recombinant would change the cellular reservoirs infected by MHV68. However, to formally address this issue, we have included analyses of the MHV68-M1st.YFP virus – which like the M1pYFP lacks a functional M1 gene.

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