Dependency Factors in HIV-1 Cytoplasmic-Nuclear Transit and Integration

描述(申请人提供)：HIV-1进入细胞质后，必须穿过细胞质，反转录，脱去外壳，对病毒DNA末端进行3‘处理，穿过核孔，协商核环境，将处理后的3’末端整合到染色体中，并完成缺口修复。在此过程中，病毒必须避免或中和许多宿主细胞防御，其中许多可能是未知的。病毒生命周期中的这一广泛间隔在许多方面仍然是一个黑匣子。融合前贩运进入和通过核贩运是艾滋病毒/艾滋病研究中最重要的问题之一。研究这些早期事件的研究人员最近发现了一些宿主细胞因子，它们要么被慢病毒利用(已确定的因子包括LEDGF、Transportin-3/TNP03、CPSF6、Nup358和其他几种核孔蛋白、亲环素A)，要么必须回避(限制因子，其他先天性免疫系统)。目前还不清楚这些因素是如何结合在一起形成一条连续的机制路径的。毫无疑问，演员阵容是不完整的。有趣的是，有建议说，不同的慢病毒以不同的、可能是灵活的方式谈判核进口。一些宿主细胞因素，最明显的是LEDGF，似乎也影响整合位点模式和转录活性，这对潜伏期领域具有重要意义。在这项资助前一个周期中，我们重点研究了慢病毒整合酶互作因子LEDGF AS的辅助因子作用 以及LEDGF整合酶结合域(IBD)介导的显性干扰在核进入前后的影响。在研究这一关键的HIV-1依赖因素的同时，我们推动了进入后HIV-1复制步骤中涉及的额外宿主依赖和限制因素，最终导致整合。我们还在一种易患艾滋病的物种中建立了一种新的生殖系转基因技术。目前的续期申请就是基于这一广泛的初步数据。我们建议进行生化和培养细胞实验，以了解我们对LEDGF的慢病毒辅助因子和显性干扰功能的观察。这包括最近LEDGF和鲜为人知的HIV-1辅助蛋白之一VPR之间的相互作用。重要的是，正如我们为本周期修订的标题所表明的那样，我们还将包括艾滋病毒-1整合前步骤中涉及的具体其他宿主依赖因素。我们将利用生物化学、病毒学、整合位点定位和转录激活物样效应核酸酶(TALENS)的定点基因靶向来确定病毒核前运输、核输入和整合的机制。重要的是，这一更新也将使我们的LEDGF研究在体内进行。与许多小鼠可以提供易感模型的病毒疾病不同，慢病毒依赖因子在体内的致病作用从未通过对易感物种进行预期的、可控的遗传操作(生殖系基因添加、基因敲除、敲入)来接近。我们将通过靶向LEDGF来建立和分析有史以来第一次在艾滋病易感物种中敲除HIV-1依赖因子，LEDGF的使用是所有慢病毒绝对保守的。

DESCRIPTION (provided by applicant): After entry into the cytoplasm, HIV-1 must transit the cytoplasm, reverse transcribe, uncoat, carry out 3' processing of the viral DNA ends, traverse the nuclear pore, negotiate the nuclear milieu, integrate the processed 3' termini into a chromosome, and complete gap repair. Along the way, the virus must avoid or neutralize numerous host cell defenses, many of which are likely unknown. This broad interval in the viral life cycle remains a black box in many ways. Pre-integration trafficking into and through the nucleus is one of the most significant problems in HIV/AIDS research. Researchers studying these early events have recently implicated a number of host cell factors that are either exploited by lentiviruses (identified ones include LEDGF, Transportin-3/TNP03, CPSF6, Nup358 and several other nucleoporins, Cyclophilin A) or that must be evaded (restriction factors, other innate immunity systems). How these factors fit together into a sequential mechanistic pathway is murky at present. The cast of characters is without doubt incomplete. Intriguingly, there are suggestions that different lentiviruses negotiate nuclear import in variant and possibly flexible ways. Some of the host cell factors, most clearly LEDGF, also appear to impact integration site patterns and transcriptional activity, which has significance for the latency field. In the previou cycle of this grant we focused on the cofactor role of the lentiviral integrase interactor LEDGF as well as pre- and post-nuclear entry impacts of LEDGF integrase binding domain (IBD)-mediated dominant interference. While studying this key HIV-1 dependency factor, we pushed forward to additional host dependency and restriction factors involved in the post-entry HIV-1 replication steps that culminate in integration. We also founded a new germline transgenesis technology in an AIDS-susceptible species. The present renewal application is based on this extensive preliminary data. We propose biochemical and cultured cell experiments to understand observations we have made on the lentiviral cofactor and dominant interference functions of LEDGF. This includes most recently an interplay between LEDGF and one of the less understood HIV-1 accessory proteins, Vpr. Importantly, as our revised title for this cycle indicates, we will also include specific other host dependency factors involved in HIV- 1 pre-integration steps. We will use biochemistry, virology, integration site mapping, and site-specific gene targeting with transcription activator-like effector nucleases (TALENs) to determine mechanisms of viral pre- nuclear trafficking, nuclear import and integration. Importantly, this renewal will take our LEDGF research in vivo as well. Unlike numerous viral diseases for which mice can provide susceptible models, the in vivo pathogenesis roles of lentiviral dependency factors have never been approachable by prospective, controlled genetic manipulation (germline gene addition, knockout, knock-in) of a susceptible species. We will establish and analyze the first-ever knockout of an HIV-1 dependency factor in an AIDS-susceptible species, by targeting LEDGF, use of which is absolutely conserved by all lentiviruses.