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The Critical Role of Genome Maintenance Proteins in Immune Evasion During Gammaherpesvirus Latency.

ABSTRACT: Gammaherpesviruses are important pathogens that establish latent infection in their natural host for lifelong persistence. During latency, the viral genome persists in the nucleus of infected cells as a circular episomal element while the viral gene expression program is restricted to non-coding RNAs and a few latency proteins. Among these, the genome maintenance protein (GMP) is part of the small subset of genes expressed in latently infected cells. Despite sharing little peptidic sequence similarity, gammaherpesvirus GMPs have conserved functions playing essential roles in latent infection. Among these functions, GMPs have acquired an intriguing capacity to evade the cytotoxic T cell response through self-limitation of MHC class I-restricted antigen presentation, further ensuring virus persistence in the infected host. In this review, we provide an updated overview of the main functions of gammaherpesvirus GMPs during latency with an emphasis on their immune evasion properties.

SUBMITTER: Sorel O

PROVIDER: S-EPMC6333680 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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The Critical Role of Genome Maintenance Proteins in Immune Evasion During Gammaherpesvirus Latency.

Sorel Océane O Dewals Benjamin G BG

Frontiers in microbiology 20190109

Gammaherpesviruses are important pathogens that establish latent infection in their natural host for lifelong persistence. During latency, the viral genome persists in the nucleus of infected cells as a circular episomal element while the viral gene expression program is restricted to non-coding RNAs and a few latency proteins. Among these, the genome maintenance protein (GMP) is part of the small subset of genes expressed in latently infected cells. Despite sharing little peptidic sequence simi ...[more]

PMID: 30687291

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Project description:The rhadinoviruses Kaposi's Sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus (MHV-68) persist in infected hosts in a latent state that is characterized by the absence of virus production and by restricted viral gene expression. Their major latency protein, the latency-associated nuclear antigen (kLANA for KSHV and mLANA for MHV-68), is essential for viral genome maintenance and replication and involved in transcriptional regulation. Both kLANA and mLANA interact with cellular chromatin-associated proteins, among them the Bromodomain and Extra Terminal domain (Brd/BET) proteins, which recruit cellular and viral proteins to acetylated histones through their bromodomains and modulate cellular gene expression. Brd/BET proteins also play a role in the tethering, replication, segregation or integration of a diverse group of viral DNA genomes. In this study we explored if Brd/BET proteins influence the localization of the LANAs to preferential regions in the host chromatin and thereby contribute to kLANA- or mLANA-mediated transcriptional regulation. Using ChIP-Seq, we revealed a genome-wide co-enrichment of kLANA with Brd2/4 near cellular and viral transcriptional start sites (TSS). Treatment with I-BET151, an inhibitor of Brd/BET, displaced kLANA and Brd2/4 from TSS in the viral and host chromatin, but did not affect the direct binding of kLANA to kLANA-binding sites (LBS) in the KSHV latent origin of replication. Similarly, mLANA, but not a mLANA mutant deficient for binding to Brd2/4, also associated with cellular TSS. We compared the transcriptome of KSHV-infected with uninfected and kLANA-expressing human B cell lines, as well as a murine B cell line expressing mLANA or a Brd2/4-binding deficient mLANA mutant. We found that only a minority of cellular genes, whose TSS are occupied by kLANA or mLANA, is transcriptionally regulated by these latency proteins. Our findings extend previous reports on a preferential deposition of kLANA on cellular TSS and show that this characteristic chromatin association pattern is at least partially determined by the interaction of these viral latency proteins with members of the Brd/BET family of chromatin modulators.

Project description:UnlabelledLatency-associated nuclear antigen (LANA) is a conserved, multifunctional protein encoded by members of the rhadinovirus subfamily of gammaherpesviruses, including Kaposi sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68). We previously demonstrated that MHV68 LANA (mLANA) is required for efficient lytic replication. However, mechanisms by which mLANA facilitates viral replication, including interactions with cellular and viral proteins, are not known. Thus, we performed a mass spectrometry-based interaction screen that defined an mLANA protein-protein interaction network for lytic viral replication consisting of 15 viral proteins and 191 cellular proteins, including 19 interactions previously reported in KSHV LANA interaction studies. We also employed a stable-isotope labeling technique to illuminate high-priority mLANA-interacting host proteins. Among the top prioritized mLANA-binding proteins was a cellular chaperone, heat shock cognate protein 70 (Hsc70). We independently validated the mLANA-Hsc70 interaction through coimmunoprecipitation and in vitro glutathione S-transferase (GST) pulldown assays. Immunofluorescence and cellular fractionation analyses comparing wild-type (WT) to mLANA-null MHV68 infections demonstrated mLANA-dependent recruitment of Hsc70 to nuclei of productively infected cells. Pharmacologic inhibition and small hairpin RNA (shRNA)-mediated knockdown of Hsc70 impaired MHV68 lytic replication, which functionally correlated with impaired viral protein expression, reduced viral DNA replication, and failure to form viral replication complexes. Replication of mLANA-null MHV68 was less affected than that of WT virus by Hsc70 inhibition, which strongly suggests that Hsc70 function in MHV68 lytic replication is at least partially mediated by its interaction with mLANA. Together these experiments identify proteins engaged by mLANA during the MHV68 lytic replication cycle and define a previously unknown role for Hsc70 in facilitating MHV68 lytic replication.ImportanceLatency-associated nuclear antigen (LANA) is a conserved gamma-2-herpesvirus protein important for latency maintenance and pathogenesis. For MHV68, this includes regulating lytic replication and reactivation. While previous studies of KSHV LANA defined interactions with host cell proteins that impact latency, interactions that facilitate productive viral replication are not known. Thus, we performed a differential proteomics analysis to identify and prioritize cellular and viral proteins that interact with the MHV68 LANA homolog during lytic infection. Among the proteins identified was heat shock cognate protein 70 (Hsc70), which we determined is recruited to host cell nuclei in an mLANA-dependent process. Moreover, Hsc70 facilitates MHV68 protein expression and DNA replication, thus contributing to efficient MHV68 lytic replication. These experiments expand the known LANA-binding proteins to include MHV68 lytic replication and demonstrate a previously unappreciated role for Hsc70 in regulating viral replication.

Project description:In the process of characterizing the requirements for expression of the essential immediate-early transcriptional activator (RTA) encoded by gene 50 of murine gammaherpesvirus 68 (MHV68), a recombinant virus was generated in which the known gene 50 promoter was deleted (G50pKO). Surprisingly, the G50pKO mutant retained the ability to replicate in permissive murine fibroblasts, albeit with slower kinetics than wild-type MHV68. 5'-rapid amplification of cDNA ends analyses of RNA prepared from G50pKO-infected fibroblasts revealed a novel upstream transcription initiation site, which was also utilized during wild-type MHV68 infection of permissive cells. Furthermore, the region upstream of the distal gene 50/RTA transcription initiation site exhibited promoter activity in both permissive NIH 3T12 fibroblasts as well as in the murine macrophage cell line RAW 264.7. In addition, in RAW 264.7 cells the activity of the distal gene 50/RTA promoter was strongly upregulated (>20-fold) by treatment of the cells with lipopolysaccharide. Reverse transcriptase PCR analyses of RNA prepared from Kaposi's sarcoma-associated herpesvirus- and Epstein-Barr virus-infected B-cell lines, following induction of virus reactivation, also revealed the presence of gene 50/RTA transcripts initiating upstream of the known transcription initiation site. The latter argues that alternative initiation of gene 50/RTA transcription is a strategy conserved among murine and human gammaherpesviruses. Infection of mice with the MHV68 G50pKO demonstrated the ability of this mutant virus to establish latency in the spleen and peritoneal exudate cells (PECs). However, the G50pKO mutant was unable to reactivate from latently infected splenocytes and also exhibited a significant reactivation defect from latently infected PECs, arguing in favor of a model where the proximal gene 50/RTA promoter plays a critical role in virus reactivation from latency, particularly from B cells. Finally, analyses of viral genome methylation in the regions upstream of the proximal and distal gene 50/RTA transcription initiation sites revealed that the distal promoter is partially methylated in vivo and heavily methylated in MHV68 latently infected B-cell lines, suggesting that DNA methylation may serve to silence the activity of this promoter during virus latency.

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The Critical Role of Genome Maintenance Proteins in Immune Evasion During Gammaherpesvirus Latency.

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The Critical Role of Genome Maintenance Proteins in Immune Evasion During Gammaherpesvirus Latency.

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