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

描述（由申请人提供）：HIV-1进入细胞质后，必须通过细胞质、逆转录、脱壳、进行病毒DNA末端的3'加工、穿过核孔、通过核环境、将加工的3'末端整合到染色体中并完成缺口修复。沿着，病毒必须避开或中和许多宿主细胞的防御，其中许多可能是未知的。病毒生命周期中的这一宽泛间隔在许多方面仍然是一个黑匣子。在融入社会之前，向核心人口贩运和通过核心人口贩运是艾滋病毒/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，这些因子要么被慢病毒利用（已确定的因子包括LEDGF，Transportin-3/TNP 03，CPSF 6，Nup 358和其他几种核孔蛋白，亲环蛋白A），要么必须被规避（限制因子，其他先天免疫系统）。这些因素如何组合成一个连续的机械路径目前还不清楚。角色的塑造无疑是不完整的。有趣的是，有人认为不同的慢病毒以不同的、可能灵活的方式谈判核输入。一些宿主细胞因子，最明显的是LEDGF，似乎也会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。在该资助的前一个周期中，我们关注慢病毒整合酶相互作用因子LEDGF的辅因子作用， 以及LEDGF整合酶结合结构域（IBD）介导的显性干扰对核进入前后的影响。在研究这一关键的HIV-1依赖性因素时，我们推进了参与进入后HIV-1复制步骤的其他宿主依赖性和限制因素，最终导致整合。我们还在艾滋病易感物种中建立了一种新的种系转基因技术。目前的更新申请是基于这一广泛的初步数据。我们提出了生化和培养细胞实验，以了解我们所做的观察慢病毒辅因子和显性干扰功能的LEDGF。这包括最近LEDGF和一种不太了解的HIV-1辅助蛋白Vpr之间的相互作用。重要的是，正如我们修改后的本周期标题所示，我们还将包括参与HIV- 1整合前步骤的其他特定宿主依赖性因素。我们将使用生物化学、病毒学、整合位点定位和转录激活因子样效应核酸酶（TALEN）的位点特异性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，这种更新也将使我们的LEDGF研究在体内进行。与小鼠可提供易感模型的许多病毒性疾病不同，慢病毒依赖性因子的体内发病机制作用从未通过易感物种的前瞻性、受控遗传操作（种系基因添加、敲除、敲入）来接近。我们将通过靶向LEDGF建立并分析艾滋病易感物种中HIV-1依赖因子的首次敲除，LEDGF的使用在所有慢病毒中绝对保守。

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.