Biphasic Euchromatin-to-Heterochromatin Transition on the KSHV Genome Following De Novo Infection

et al. (2013) Biphasic Euchromatin-to-Heterochromatin Transition on the KSHV Genome Following De Novo Infection. PLoS Pathog 9(12): e1003813. doi:10.1371/journal.ppat.1003813 Biphasic Euchromatin-to-Heterochromatin Transition on the KSHV Genome Following De Novo Infection Zsolt Toth 0 Kevin Brulois 0 Hye-Ra Lee 0 Yoshihiro Izumiya 0 Clifford Tepper 0 Hsing-Jien Kung 0 Jae U. Jung 0 Dirk P. Dittmer, University of North Carolina at Chapel Hill, United States of America 0 1 Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, California, United States of America, 2 Department of Dermatology, University of California Davis School of Medicine, University of California Davis Comprehensive Cancer Center, Sacramento, California, United States of America, 3 Department of Biological Chemistry and Molecular Medicine, University of California Davis School of Medicine, University of California Davis Comprehensive Cancer Center , Sacramento, California , United States of America The establishment of latency is an essential step for the life-long persistent infection and pathogenesis of Kaposi's sarcomaassociated herpesvirus (KSHV). While the KSHV genome is chromatin-free in the virions, the viral DNA in latently infected cells has a chromatin structure with activating and repressive histone modifications that promote latent gene expression but suppress lytic gene expression. Here, we report a comprehensive epigenetic study of the recruitment of chromatin regulatory factors onto the KSHV genome during the pre-latency phase of KSHV infection. This demonstrates that the KSHV genome undergoes a biphasic chromatinization following de novo infection. Initially, a transcriptionally active chromatin (euchromatin), characterized by high levels of the H3K4me3 and acetylated H3K27 (H3K27ac) activating histone marks, was deposited on the viral episome and accompanied by the transient induction of a limited number of lytic genes. Interestingly, temporary expression of the RTA protein facilitated the increase of H3K4me3 and H3K27ac occupancy on the KSHV episome during de novo infection. Between 24-72 hours post-infection, as the levels of these activating histone marks declined on the KSHV genome, the levels of the repressive H3K27me3 and H2AK119ub histone marks increased concomitantly with the decline of lytic gene expression. Importantly, this transition to heterochromatin was dependent on both Polycomb Repressive Complex 1 and 2. In contrast, upon infection of human gingiva-derived epithelial cells, the KSHV genome underwent a transcription-active euchromatinization, resulting in efficient lytic gene expression. Our data demonstrate that the KSHV genome undergoes a temporally-ordered biphasic euchromatin-to-heterochromatin transition in endothelial cells, leading to latent infection, whereas KSHV preferentially adopts a transcriptionally active euchromatin in oral epithelial cells, resulting in lytic gene expression. Our results suggest that the differential epigenetic modification of the KSHV genome in distinct cell types is a potential determining factor for latent infection versus lytic replication of KSHV. - Funding: This work was partly supported by DE023926, CA082057, CA31363, CA115284, CA180779, AI105809, AI073099, Hastings Foundation, and Fletcher Jones Foundation (JUJ) and CA14779 (YI). The UC Davis Comprehensive Cancer Center Genomics Shared Resource is supported by Cancer Center Support Grant P30 CA093373 (R.W. dV.W.) from the NCI. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Kaposis sarcoma-associated herpesvirus (KSHV, Human herpesvirus 8 or HHV-8) is one of the seven currently known human tumor viruses and is associated with the pathogenesis of the multifocal, angiogenic and inflammatory cancer called Kaposis sarcoma (KS) and certain B cell-originated neoplasias, including primary effusion lymphoma (PEL) and multicentric Castlemans disease (MCD) [1,2]. KSHV results in persistent infection in immunocompetent humans by establishing latency in CD19+ B cells [3]. The establishment of latency is the most fundamental immune evasion strategy of KSHV, as the severely limited viral gene expression characteristic of latently infected cells allows the virus to escape detection by the host immune system. However, immune suppression along with other environmental and physiological factors can trigger the reactivation of KSHV from latency, leading to the temporally ordered expression of viral genes and release of infectious virus [4,5]. In KSHV-associated tumors, the majority of tumor cells harbor KSHV in the latent phase and virus production is restricted to a small population, indicating that it is the latently infected cells that play a critical role in the development of KSHV-associated cancers [6]. Indeed, the latent proteins of KSHV have several important roles, such as the promotion of malignant transformation by facilitating the proliferation and survival of infected cells, as well as the maintenance of the KSHV genome in dividing cells [7]. During latency, the KSHV genome exists as a circular episome in the nucleus and adopts a nucleosome structure similar to the bulk chromatinized cellular genome [8,9]. In this latent phase, the latent genes of KSHV are continuously expressed, while the lytic genes are repressed. Since chromatinization limits the access of transcription factors to the promoter regions of viral genes, Although the KSHV genome is linear and chromatin-free in the virions, it circularizes and adopts a repressive chromatin structure in latently infected cells, inhibiting the majority of viral gene expression. In this study, we investigate the epigenetic regulatory mechanism of the pre-latency phase of KSHV infection. We found that upon de novo infection, the KSHV genome undergoes distinct chromatin states in a temporally ordered manner prior to the establishment of latency. Initially, the KSHV genome carried a transcriptionally permissive chromatin structure to allow expression of a subset of viral lytic genes. Subsequently, cellular Polycomb Repressive Complex 1 (PRC1) and PRC2 were recruited to the KSHV genome, resulting in the deposition of repressive histone marks onto the viral chromatin and the accumulation of heterochromatin structures, both of which were critical for the establishment of viral latency. In contrast to the biphasic chromatinization and genome-wide inhibition of lytic genes observed in de novo-infected SLK and TIME cells, KSHV adopts a transcriptionally permissive chromatin form in human gingiva-derived epithelial cells, resulting in prolonged and robust lytic gene expression. Thus, our results suggest that the differential epigenetic modification of the KSHV genome in distinct cell types is a potential det (...truncated)