The Interaction of the Gammaherpesvirus 68 orf73 Protein with Cellular BET Proteins Affects the Activation of Cell Cycle Promoters (original) (raw)
Related papers
2006
The Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen 1 (LANA-1) is required for the replication of episomal viral genomes. Regions in its N-terminal and C-terminal domains mediate the interaction with host cell chromatin. Several cellular nuclear proteins, e.g., BRD2/RING3, histones H2A and H2B, MeCP2, DEK, and HP1␣, have been suggested to mediate this interaction. In this work, we identify the double-bromodomain proteins BRD4 and BRD3/ORFX as additional LANA-1 interaction partners. The carboxy-terminal region of the short variant of BRD4 (BRD4 S) containing the highly conserved extraterminal domain directly interacts with an element in the LANA-1 carboxy-terminal domain. We show that ectopically expressed BRD4 S and BRD2/RING3 delay progression into the S phase of the cell cycle in epithelial and B-cell lines and increase cyclin E promoter activity. LANA-1 partly releases epithelial and B cells from a BRD4 Sand BRD2/RING3-induced G 1 cell cycle arrest and also promotes S-phase entry in the presence of BRD4 S and BRD2/RING3. This is accompanied by a reduction in BRD4 S-mediated cyclin E promoter activity. Our data are in keeping with the notion that the direct interaction of KSHV LANA-1 with BRD4 and other BRD proteins could play a role in the G 1 /S phase-promoting functions of KSHV LANA-1. Further, our data support a model in which the LANA-1 C terminus contributes to a functional attachment to acetylated histones H3 and H4 via BRD4 and BRD2, in addition to the recently demonstrated direct interaction (A.
Interaction of Gamma-Herpesvirus Genome Maintenance Proteins with Cellular Chromatin
PLoS ONE, 2013
The capacity of gamma-herpesviruses to establish lifelong infections is dependent on the expression of genome maintenance proteins (GMPs) that tether the viral episomes to cellular chromatin and allow their persistence in latently infected proliferating cells. Here we have characterized the chromatin interaction of GMPs encoded by viruses belonging to the genera Lymphocryptovirus (LCV) and Rhadinovirus (RHV). We found that, in addition to a similar diffuse nuclear localization and comparable detergent resistant interaction with chromatin in transfected cells, all GMPs shared the capacity to promote the decondensation of heterochromatin in the A03-1 reporter cell line. They differed, however, in their mobility measured by fluorescence recovery after photobleaching (FRAP), and in the capacity to recruit accessory molecules required for the chromatin remodeling function. While the AT-hook containing GMPs of LCVs were highly mobile, a great variability was observed among GMPs encoded by RHV, ranging from virtually immobile to significantly reduced mobility compared to LCV GMPs. Only the RHV GMPs recruited the bromo-and extra terminal domain (BET) proteins BRD2 and BRD4 to the site of chromatin remodeling. These findings suggest that differences in the mode of interaction with cellular chromatin may underlie different strategies adopted by these viruses for reprogramming of the host cells during latency.
PLoS Pathogens, 2013
Kaposi sarcoma-associated herpesvirus (KSHV) establishes a lifelong latent infection and causes several malignancies in humans. Murine herpesvirus 68 (MHV-68) is a related c 2-herpesvirus frequently used as a model to study the biology of cherpesviruses in vivo. The KSHV latency-associated nuclear antigen (kLANA) and the MHV68 mLANA (orf73) protein are required for latent viral replication and persistence. Latent episomal KSHV genomes and kLANA form nuclear microdomains, termed 'LANA speckles', which also contain cellular chromatin proteins, including BRD2 and BRD4, members of the BRD/BET family of chromatin modulators. We solved the X-ray crystal structure of the C-terminal DNA binding domains (CTD) of kLANA and MHV-68 mLANA. While these structures share the overall fold with the EBNA1 protein of Epstein-Barr virus, they differ substantially in their surface characteristics. Opposite to the DNA binding site, both kLANA and mLANA CTD contain a characteristic lysine-rich positively charged surface patch, which appears to be a unique feature of c 2-herpesviral LANA proteins. Importantly, kLANA and mLANA CTD dimers undergo higher order oligomerization. Using NMR spectroscopy we identified a specific binding site for the ET domains of BRD2/4 on kLANA. Functional studies employing multiple kLANA mutants indicate that the oligomerization of native kLANA CTD dimers, the characteristic basic patch and the ET binding site on the kLANA surface are required for the formation of kLANA 'nuclear speckles' and latent replication. Similarly, the basic patch on mLANA contributes to the establishment of MHV-68 latency in spleen cells in vivo. In summary, our data provide a structural basis for the formation of higher order LANA oligomers, which is required for nuclear speckle formation, latent replication and viral persistence.
Journal of Virology, 2013
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Brd4 links chromatin targeting to HPV transcriptional silencing
Genes & Development, 2006
The E2 protein encoded by human papillomaviruses (HPVs) inhibits expression of the viral E6 oncoprotein, which, in turn, regulates p53 target gene transcription. To identify cellular proteins involved in E2-mediated transcriptional repression, we isolated an E2 complex from human cells conditionally expressing HPV-11 E2. Surprisingly, the double bromodomain-containing protein Brd4, which is implicated in cell cycle control and viral genome segregation, was found associated with E2 and conferred on E2 the ability to inhibit AP-1-dependent HPV chromatin transcription in an E2-binding site-specific manner as illustrated by in vitro reconstituted chromatin transcription experiments. Knockdown of Brd4 in human cells alleviates E2-mediated repression of HPV transcription. The E2-interacting domain at the extreme C terminus and the chromatin targeting activity of a bromodomain-containing region are both essential for the corepressor activity of Brd4. Interestingly, E2-Brd4 blocks the recruitment of TFIID and RNA polymerase II to the HPV E6 promoter region without inhibiting acetylation of nucleosomal histones H3 and H4, indicating an acetylation-dependent role of Brd4 in the recruitment of E2 for transcriptional silencing of HPV gene activity. Our finding that Brd4 is a component of the virus-assembled transcriptional silencing complex uncovers a novel function of Brd4 as a cellular cofactor modulating viral gene expression.
Virology, 2011
Association of herpesvirus DNA with histones has important implications for lytic and latent infection; thus herpesviruses arbitrate interactions with histones to productively infect host cells. While regulation of alpha and betaherpesvirus chromatin during lytic infection has been actively investigated, very little is known about interaction of gammaherpesvirus DNA with histones upon de novo lytic infection. Murine gammaherpesvirus-68 (MHV68) is a rodent pathogen that offers a tractable system to study gammaherpesvirus lytic infection in primary cells. In this study we report that MHV68 promoter and orilyt sequences underwent dynamic association with histone H3 during de novo lytic infection of primary macrophages and fibroblasts. Similar to HSV-1, the degree of MHV68 DNA association with histone H3 was dependent on the multiplicity of infection and was further regulated by viral DNA synthesis. Our work sets a precedent for future studies of gammaherpesvirus chromatin during de novo lytic infection.
BET-Inhibitors Disrupt Rad21-Dependent Conformational Control of KSHV Latency
Kaposi's Sarcoma-associated Herpesvirus (KSHV) establishes stable latent infection in B-lymphocytes and pleural effusion lymphomas (PELs). During latency, the viral genome persists as an epigenetically constrained episome with restricted gene expression programs. To identify epigenetic regulators of KSHV latency, we screened a focused small molecule library containing known inhibitors of epigenetic factors. We identified JQ1, a Bromodomain and Extended Terminal (BET) protein inhibitor, as a potent activator of KSHV lytic reactiva-tion from B-cells carrying episomal KSHV. We validated that JQ1 and other BET inhibitors efficiently stimulated reactivation of KSHV from latently infected PEL cells. We found that BET proteins BRD2 and BRD4 localize to several regions of the viral genome, including the LANA binding sites within the terminal repeats (TR), as well as at CTCF-cohesin sites in the latent and lytic control regions. JQ1 did not disrupt the interaction of BRD4 or BRD2 with LANA, but did reduce the binding of LANA with KSHV TR. We have previously demonstrated a cohesin-dependent DNA-loop interaction between the latent and lytic control regions that restrict expression of ORF50/RTA and ORF45 immediate early gene transcripts. JQ1 reduced binding of cohesin subunit Rad21 with the CTCF binding sites in the latency and lytic control regions. JQ1 also reduced DNA-loop interaction between latent and lytic control regions. These findings implicate BET proteins BRD2 and BRD4 in the maintenance of KSHV chromatin architecture during latency and reveal BET inhibitors as potent activators of KSHV reactivation from latency. KSHV is an oncogenic human herpesvirus implicated as the causative agent of KS and cofactor in pleural effusion lymphomas (PELs). The latent virus persists in PELs as an epi-genetically regulated episome. We found that small molecule inhibitors of BET family have potent activity in triggering the lytic switch during latent infection in PELs. The BET family inhibitor JQ1 disrupted the latent virus from maintaining a closed DNA loop con-formation. These findings have implications for treatment of KSHV-associated malignan-cies with epigenetic modulators of the BET inhibitor family.
Journal of Virology, 2005
Latency-associated nuclear antigen 1 (LANA-1) of Kaposi's sarcoma-associated herpesvirus (KSHV) mediates the episomal replication of the KSHV genome, as well as transcriptional regulation, in latently infected cells. Interaction of LANA-1 with cellular chromatin is required for both these functions. An N-terminal heterochromatin-binding site in LANA-1 is essential for the replication and maintenance of latent episomes, as well as transcriptional regulation. We have recently described a C-terminal domain in LANA-1 that modulates the interaction with cellular interphase chromatin or elements of the nuclear matrix. Here, we used a series of LANA-1 deletion mutants to investigate the relationship between the different functions of LANA-1 and its interaction with the host chromatin-binding protein Brd2/RING3. Our findings suggest that the C-terminal chromatin-binding domain in LANA-1 is required for multiple LANA-1 functions, including the ability to bind to and replicate viral episo...
The DR 6 protein from human herpesvirus-6 B induces p 53-independent cell cycle arrest in G 2 / M
2014
HHV-6B infection inhibits cell proliferation in G2/M, but no protein has so far been recognized to exert this function. Here we identify the protein product of direct repeat 6, DR6, as an inhibitor of G2/M cell-cycle progression. Transfection of DR6 reduced the total number of cells compared with mocktransfected cells. Lentiviral transduction of DR6 inhibited host cell DNA synthesis in a p53-independent manner, and this inhibition was DR6 dose-dependent. A deletion of 66 amino acids from the N-terminal part of DR6 prevented efficient nuclear translocation and the ability to inhibit DNA synthesis. DR6induced accumulation of cells in G2/M was accompanied by an enhanced expression of cyclin B1 that accumulated predominantly in the cytoplasm. Pull-down of cyclin B1 brought down pCdk1 with the inactivating phosphorylation at Tyr15. Together, DR6 delays cell cycle with an accumulation of cells in G2/M and thus might be involved in HHV-6B-induced cell-cycle arrest. & 2014 Elsevier Inc. All...
Virus Research, 2015
The E2 protein of the carcinogen human papillomavirus 16 (HPV16) regulates replication and transcription of the viral genome in association with viral and cellular proteins. Our previous work demonstrated that E2 can regulate transcription from the host genome. E2 can activate transcription from adjacent promoters when located upstream using E2 DNA binding sequences and this activation is dependent upon the cellular protein Brd4; this report demonstrates that a Brd4 binding E2 mutant alters host genome expression differently from wild type E2. Of particular note is that highly down regulated genes are mostly not affected by failure to interact with Brd4 suggesting that the E2-Brd4 interaction is more responsible for the transcriptional activation of host genes rather than repression. Therefore failure to interact efficiently with Brd4, or altered levels of Brd4, would alter the ability of E2 to regulate the host genome and could contribute to determining the outcome of infection.