Integration of Retroviral DNA: Accessing Host Target DNA

描述(由申请人提供)：这项竞争性续签申请继续关注逆转录病毒复制的早期阶段。这项应用的首要目标是揭示关键的细胞-病毒相互作用，这些相互作用决定了逆转录病毒基因在人类细胞中表达的成败，就像表观遗传沉默所调节的那样。禽肉瘤病毒(ASV)DNA前病毒的原型在哺乳动物细胞中受到表观遗传沉默的影响，这一系统将继续作为模型进行研究。申请人发现，ASV的表观遗传沉默可以通过快速参与抗病毒宿主适配器蛋白Daxx的参与来介导，Daxx招募了细胞表观遗传沉默机制。此外，通过开发和实施一种创新的、强大的和全面的基于siRNA的筛选，确定了一些在这种反应中发挥作用的新的和预期的人类宿主细胞因子。这些发现开辟了令人兴奋的新调查领域，将在这一相互竞争的更新中进行追求。在目标1中，将确定Daxx作为抗病毒因子发挥作用的分子机制。这些研究的结果将为了解寄主的一般内在防御机制提供有价值的见解。在AIM 2中，将研究通过siRNA筛选确定的几个新因子的功能的机制细节，重点是那些可能参与病毒DNA序列的因子。特定的病毒DNA结合因子要么参与沉默因子的成核，要么作为转录屏障，这一假说将得到检验。目的3将开发新的全面和系统的方法来区分沉默前病毒的物理和功能表观遗传学特征，这些特征是在感染(开始)后不久和在长期传代(维持)之后建立的。关于抗病毒因子的作用以及前病毒和细胞染色质在沉默中的相互作用的具体假设将被考虑。表观遗传机制在发育过程中基因表达的调节、在分化的成人组织中以及在包括癌症在内的人类疾病中具有重要的作用。因此，拟议的研究结果将与病毒学以及正常细胞和癌细胞的细胞生物学高度相关。

DESCRIPTION (provided by applicant): This competitive renewal application continues to focus on the early stages of retroviral replication. The overarching goal of this application is to uncover critical cell-virus interactions that determine the success or failure of retroviral gene expression in human cells, as modulated by epigenetic silencing. It was established decades ago that the prototype avian sarcoma virus (ASV) DNA provirus is subject to epigenetic silencing in mammalian cells, and this system will continue to be studied as a model. The applicants have discovered that epigenetic silencing of ASV can be mediated by the rapid engagement of an antiviral host adaptor protein, Daxx, which recruits the cellular epigenetic silencing machinery. Furthermore, through development and implementation of an innovative, robust, and comprehensive siRNA-based screen, a number of novel and expected human host cell factors that play a role in this response were identified. These findings have opened up the exciting new areas of investigation to be pursued in this competing renewal. In Aim 1, the molecular mechanisms that govern Daxx function as an antiviral factor will be determined. Results of these studies will provide valuable insight into general intrinsic host defense mechanisms. In Aim 2 mechanistic details for the function of several novel factors identified by siRNA screening will be studied, with a focus on those likely to engage viral DNA sequences. The hypotheses that specific viral DNA binding factors either participate in nucleation of silencing factors or act as a transcriptional barrier, will be tested. Aim 3 will exploit new comprehensive and systematic approaches to distinguish the physical and functional epigenetic features of silent proviruses that are established soon after infection (initiation) and after long-term passage (maintenance). Specific hypotheses concerning the roles of antiviral factors, and the interplay of proviral and cellular chromatin, in silencing will be considered. Epigenetic mechanisms have an essential role in the regulation of gene expression during development, in differentiated adult tissues, and in human disease, including cancer. Consequently, results from the proposed research will be highly relevant to virology, as well as the cellular biology of normal and cancer cells.