KSHV-Ori-Lyt-Dependent DNA Replication

描述(申请人提供)：卡波西肉瘤相关疱疹病毒(KSHV)，也被称为人类疱疹病毒-8(HHV-8)，是一种人类病原体，可导致几种与艾滋病相关的恶性肿瘤，包括卡波西肉瘤(KS)、原发渗出性淋巴瘤(PEL)和多中心Castleman病(MCD)。越来越多的证据表明，病毒的裂解生命周期在KS和其他KSHV相关疾病的发展中起着关键作用。在KS病变中，持续的裂解复制维持着潜伏感染细胞的数量，否则随着纺锤体细胞的分裂，潜伏的病毒表型分离会迅速失去这些细胞。疱疹病毒的DNA裂解复制需要顺式作用元件、起始点和反式作用因子，包括病毒编码的起始点结合蛋白、核心复制机制蛋白和细胞因子。此外，一些表观遗传效应，如核位置、染色体结构和RNA转录，也会影响DNA的复制。为了了解KSHV裂解DNA复制，我们最近在KSHV基因组中鉴定了两个Ori-Lyt，并鉴定了Ori-Lyts中的几个关键顺式作用元件。此外，还发现两种调控蛋白K8和RTA与起始点结合，并研究了它们在启动DNA复制中的作用。这些发现为进一步研究KSHV裂解DNA复制的机制奠定了基础，这将在这里提出的研究中进行探索。在下一个资助阶段，重点将放在启动KSHV Ori-Lyt相关的DNA复制上。(1)我们将对KSHV DNA复制复合体进行鉴定。K8和RTA在复合体形成或募集到Ori-Lyt上的作用将被研究。(2)已鉴定出多种可能参与KSHV DNA复制的细胞蛋白。可能涉及起源DNA解旋和核基质附着的两种细胞蛋白质将被进一步研究，因为这两种事件被认为是复制分叉形成之前的步骤。(3)研究Ori-Lyt相关转录与DNA复制之间的相互作用。我们将检验这一假设，即转录改变DNA或染色质结构，并促进DNA复制的启动。这一结果将极大地加深我们对KSHV复制调控机制的理解。此外，这些研究将导致新的治疗策略，以阻断KSHV DNA复制和治疗KSHV相关疾病。这一建议代表了我们实验室在了解控制和调控KSHV裂解DNA复制的机制方面的效果。我们将从病毒复制体的形成及其在Ori-Lyt上的募集、细胞因素在病毒DNA复制中的参与以及表观遗传效应对病毒DNA复制的调控三个方面来研究DNA复制的启动。这项研究旨在寻找新的治疗策略来阻断KSHV的复制并治疗与KSHV相关的人类疾病。这项建议代表了我们实验室在了解控制和调控KSHV裂解DNA复制的机制方面的效果。我们将从病毒复制体的形成及其在Ori-Lyt上的募集、细胞因素在病毒DNA复制中的参与以及表观遗传效应对病毒DNA复制的调控三个方面来研究DNA复制的启动。这项研究旨在寻找新的治疗策略来阻断KSHV的复制和治疗KSHV相关的人类疾病。

DESCRIPTION (provided by applicant): Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8 (HHV-8), is a human pathogen responsible for several AIDS associated malignancies including Kaposi's sarcoma (KS), primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). Increasing evidence suggests that the lytic life cycle of the virus plays a critical role in the development of KS and other KSHV-associated diseases. In KS lesions, constant lytic replication sustains the population of latently infected cells that otherwise are quickly lost by segregation of latent viral episomes as spindle cells divide. Lytic DNA replication of herpesviruses requires both cis-acting element, the origin, and trans-acting factors including virally encoded origin-binding proteins, core replication machinery proteins and cellular factors. In addition, some epigenetic effects such as nuclear location, chromosome structure and RNA transcription influence DNA replication. To understand KSHV lytic DNA replication, we recently identified two ori-Lyts in the KSHV genome and characterized several critical cis-acting elements in the ori-Lyts. Furthermore, two regulatory proteins, K8 and RTA, were found to bind to the origins and their roles in initiation of DNA replication were investigated. These discoveries set a foundation for further investigation into the mechanism of KSHV lytic DNA replication that will be explored in the studies proposed here. In next funding period, emphasis will be placed on initiation of KSHV ori-Lyt-associated DNA replication. (1) We will characterize KSHV DNA replication complex. Roles of K8 and RTA in the complex formation or recruitment of the complex onto ori-Lyt will be investigated. (2) Many cellular proteins that might be involved in KSHV DNA replication have been identified. Two cellular proteins that may involve origin DNA unwinding and nuclear matrix attachment will be further studied because these two events are believed to be antecedent steps before replication fork formation. (3) We will study interplay between ori-Lyt-associated transcription and DNA replication. We will test the hypothesis that the transcription alters either DNA or chromatin structure and facilitates initiation of DNA replication. The results will greatly enhance our understanding on mechanisms that regulate initiation of KSHV replication. In addition, the studies will lead to new therapeutic strategies to block KSHV DNA replication and to treat KSHV-related diseases. This proposal represents the effects of our laboratory in understanding mechanisms that control and regulate KSHV lytic DNA replication. We will investigate the initiation of DNA replication in three aspects, i.e. formation of viral replisomes and their recruitment onto ori-Lyt, involvement of cellular factors in viral DNA replication and regulation of viral DNA replication by epigenetic effects. The study aims to new therapeutic strategies to block KSHV replication and treat KSHV-associated human diseases PUBLIC HEALTH RELEVANCE This proposal represents the effects of our laboratory in understanding mechanisms that control and regulate KSHV lytic DNA replication. We will investigate the initiation of DNA replication in three aspects, i.e. formation of viral replisomes and their recruitment onto ori-Lyt, involvement of cellular factors in viral DNA replication and regulation of viral DNA replication by epigenetic effects. The study aims to new therapeutic strategies to block KSHV replication and treat KSHV-associated human diseases.