Integration of Retroviral DNA: Accessing Host Target DNA

描述（由申请人提供）：这一竞争性续期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，这种相互作用决定了人类细胞中逆转录病毒基因表达的成功或失败，由表观遗传沉默调节。早在几十年前，人们就已经确定，原型禽肉瘤病毒（ASV） DNA前病毒在哺乳动物细胞中会发生表观遗传沉默，这一系统将继续作为一种模型进行研究。申请人已经发现ASV的表观遗传沉默可以通过抗病毒宿主接头蛋白Daxx的快速参与来介导，Daxx招募细胞表观遗传沉默机制。此外，通过开发和实施一个创新的，强大的，全面的基于sirna的筛选，许多新的和预期的在这种反应中起作用的人类宿主细胞因子被确定。这些发现开辟了令人兴奋的新研究领域，以追求这一竞争更新。在Aim 1中，将确定控制Daxx作为抗病毒因子功能的分子机制。这些研究结果将为了解宿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筛选鉴定的几个新因子的功能机制细节，重点关注那些可能参与病毒DNA序列的因子。假设特定的病毒DNA结合因子参与沉默因子的成核或作为转录屏障，将被测试。目的3将探索新的全面和系统的方法来区分在感染（起始）和长期传代（维持）后不久建立的沉默前病毒的物理和功能表观遗传特征。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、分化成人组织和人类疾病（包括癌症）中对基因表达的调控中发挥着重要作用。因此，拟议研究的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.