Viral and host strategies for regulation of KSHV infection

卡波西肉瘤相关疱疹病毒（KSHV）是一种致癌的伽玛疱疹病毒，它在人类中建立终身感染。KSHV可导致卡波西肉瘤、原发性积液性淋巴瘤（PEL）和免疫功能低下患者侵袭性多中心Castleman病的发展。KSHV的生命周期有潜伏期和裂解复制两个阶段。虽然潜伏感染传统上与KSHV肿瘤发生有关，但越来越多的证据表明，KSHV的裂解复制不仅对病毒的传播很重要，而且对KSHV相关的肿瘤发生也很重要。KSHV的裂解周期是由KSHV编码的复制转录激活子（RTA）的表达启动的。RTA是诱导潜伏感染细胞的KSHV裂解再激活的必要条件和充分条件。尽管RTA发挥了重要作用，但人们对RTA如何在高度染色质化的病毒和宿主基因组上快速诱导多种靶基因仍知之甚少，而这些基因是KSHV裂解复制所必需的。为了揭示RTA在kshv感染细胞中介导的基因调控机制，我们一直在研究RTA在再激活的PEL细胞中的转录功能。我们进行了ChIP-seq分析，以确定潜伏期和裂解再激活期间病毒和宿主基因组上RTA的结合和激活组蛋白修饰的富集。RNA-seq还用于分析PEL细胞潜伏和裂解再激活期间病毒和宿主基因的差异基因表达。这些分析揭示了RTA的直接基因靶点以及与RTA结合相关的染色质变化。对一个RTA突变体的功能研究表明，RTA可以调控RNA聚合酶II的伸长。与此一致的是，使用再激活的PEL细胞纯化RTA复合物表明，RTA与几种与DNA复制和转录相关的病毒因子、宿主转录延伸因子、染色质修饰酶和核小体重塑因子相互作用。基于我们的初步结果，我们假设RTA利用染色质调控和转录延伸宿主因子与病毒辅助因子一起诱导其病毒和宿主靶基因，以促进潜伏期的KSHV再激活并诱导病毒复制。总的来说，本研究的目标是确定RTA对病毒和宿主基因组转录伸长调控的全基因组效应（Aim 1），并解剖RTA如何利用染色质修饰和重塑宿主因子、转录伸长宿主因子和病毒辅助因子来促进KSHV裂解再激活过程中RTA靶基因上RNA聚合酶II的进展的机制（Aim 2）。这是一个高度创新的建议，将基因组学与生化分析相结合，有助于更好地了解潜伏感染B细胞的裂解再激活的分子机制，这在KSHV发病机制中具有生物学意义。

Kaposi's sarcoma-associated herpesvirus (KSHV) is a cancer-causing gamma herpesvirus, which establishes life-long infection in humans. KSHV accounts for the development of Kaposi's sarcoma, primary effusion lymphoma (PEL), and aggressive forms of multicentric Castleman's disease in immunocompromised patients. Life cycle of KSHV has two distinct phases: latency and lytic replication. While latent infection has been traditionally linked to KSHV tumorigenesis, accumulating evidence suggests that lytic replication of KSHV is important for not only the spreading of the virus but also KSHV-associated oncogenesis. The lytic cycle of KSHV is initiated by the expression of the replication and transcription activator (RTA) encoded by KSHV. RTA is required and sufficient for inducing lytic reactivation of KSHV from latently infected cells. Despite the essential role of RTA, it is still poorly understood how RTA can manage to rapidly induce a large variety of its target genes on the highly chromatinized viral and host genomes that are required for lytic replication of KSHV. To uncover the mechanisms of RTA-mediated gene regulation in KSHV-infected cells, we have been studying the transcriptional function of RTA in PEL cells undergoing reactivation. We performed ChIP-seq analyses to determine the binding of RTA and the enrichment of activating histone modifications on both viral and host genomes during latency and lytic reactivation. RNA-seq was also used to analyze the differential gene expression of viral and host genes between latency and lytic reactivation in PEL cells. These analyses revealed the direct gene targets of RTA and the chromatin changes associated with RTA-binding. The functional study of an RTA mutant showed that RTA can play a role in the regulation of RNA polymerase II elongation. In agreement with this, RTA complex purification using reactivated PEL cells showed that RTA interacts with several viral factors associated with DNA replication and transcription, host transcription elongation factors, chromatin modifying enzymes, and nucleosome remodeling factors. Based on our preliminary results, we hypothesize that RTA utilizes the chromatin regulatory and transcription elongation host factors in concert with viral co-factors to induce its viral and host target genes for promoting KSHV reactivation from latency and induce viral replication. Collectively, the goal of this proposal is to determine the genome-wide effect of RTA on the regulation of transcription elongation on both the viral and host genomes (Aim 1), and dissect the mechanisms of how RTA utilizes the chromatin modifying and remodeling host factors, the transcription elongation host factors, and viral co-factors to facilitate the progression of RNA polymerase II at RTA target genes during lytic reactivation of KSHV (Aim 2). This is a highly innovative proposal, which combines genomics with biochemical assays to help better understand the molecular mechanisms of lytic reactivation from latently infected B cells, which has biological importance in KSHV pathogenesis.