Identification of HIV-1 Restriction Factors through Functional Genomic Screens

描述（由申请人提供）：HIV需要宿主机器进行复制。反过来，宿主细胞利用抗病毒因子保护自己。对HIV复制过程中的宿主因素的全面了解将为我们提供HIV病毒学和细胞生物学的宝贵知识。它还将有助于开发新的抗艾滋病毒疗法。使用全基因组siRNA文库的功能筛选已经涉及数百种宿主蛋白作为HIV复制的调节剂。在这个拟议的项目中，我们将使用多种RNAi资源来识别限制HIV-1复制的先天宿主蛋白。本项目的其余目标是集中于进一步验证 和功能研究的限制因素，出来的屏幕。目的1是通过全基因组siRNA筛选鉴定HIV-1限制性因子并进行功能分析。我们最近使用多种RNAi资源（三个正交siRNA文库）完成了siRNA筛选。筛选确定了212个潜在的宿主限制因素。为了实现这一目标，我们计划集中精力缩短潜在的抗HIV宿主因子的列表，并进行进一步的验证和功能研究，以确认它们的作用。该计划包括确认 siRNA敲低效率、RNAi效应拯救、在人类原代细胞中的验证以及一些有希望的HIV-1限制因子的机制研究。此外，我们计划测试已确认的限制因子对其他病毒的抑制作用。目的2是通过全基因组siRNA筛选来鉴定介导干扰素诱导的HIV-1限制的因子。人们早就知道，I型干扰素（IFN）可以诱导大量干扰素刺激基因的表达，并建立有效的抗病毒反应。HIV-1复制的早期和晚期步骤都可以被IFN-1阻断。在这个目标中，我们将进行全基因组siRNA筛选，以确定HIV-1的限制性因子，其活性所需的IFN。候选限制因子将通过进行综合生物信息学分析进一步筛选。一组潜在的IFN诱导的HIV-1限制因子将被选择用于进一步的验证和功能研究。我们相信，我们对HIV-1限制因子的研究将为病毒感染的障碍提供新的见解，并可能对识别新的抗病毒靶点产生重要影响。

DESCRIPTION (provided by applicant): HIV requires host machinery to replicate. In turn, the host cell protects itself using antiviral factors. A full understanding of the host factors involve in HIV replication will provide us with valuable knowledge of HIV virology and cell biology. It will also help to develop new anti-HIV therapy. Functional screens using genome-wide siRNA libraries have implicated hundreds of host proteins as modulators of HIV replication. In this proposed project, we will use multiple RNAi resources to identify the innate host proteins that restrict HIV-1 replication. The remaining goals for this project are to focus on further validation and functional study of the restriction factors that come out of screens. Aim 1 is the validation and functional analysis of HIV-1 restriction factors identified through genome-wide siRNA screens. We recently completed siRNA screens using multiple RNAi resources (three orthologous siRNA libraries). The screens identified 212 potential host restriction factors. For this aim, we plan to focus on a shortened list of potential anti-HIV host factors, and perform further validation and functional studies to confirm their roles. The plan includes confirmation of siRNA knockdown efficacy, RNAi effect rescue, validation in human primary cells, and mechanistic studies of a few of promising HIV-1 restriction factors. In addition, we plan to test the inhibitory efforts of confirmed restriction factors on other viruses. Aim 2 is the identificatin of factors that mediate interferon- induced restriction of HIV-1 through genome-wide siRNA screens. It has been long known that Type I interferon (IFN) can induce the expression of a large set of interferon-stimulated genes and establish a potent antiviral response. Both early and late steps of HIV-1 replication can be blocked by IFN-1. In this aim, we will perform genome-wide siRNA screens to identify HIV-1 restriction factors whose activity is required by IFN. The candidate restriction factors will be further filtered by performing comprehensive bioinformatic analysis. A set of potential IFN-induced HIV-1 restriction factors will be selected for further validation and functional studies. We believe that our study of HIV-1 restriction factors will provide new insights into the barriers to viral infection and may have important ramifications for the identification of new antiviral targets.