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，必须穿越细胞质，逆转录，脱衣，对病毒DNA末端进行3′加工，穿过核孔，与核环境协商，将加工后的3′末端整合到染色体中，完成间隙修复。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。在许多方面，病毒生命周期的这个大间隔仍然是一个黑盒子。融入前贩运进入和通过核心是艾滋病毒/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暗示了一些宿主细胞因子，这些因子要么被慢病毒利用（已确定的包括LEDGF， Transportin-3/TNP03, CPSF6， Nup358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）。这些因素是如何结合在一起形成一个有序的机制途径的，目前尚不清楚。毫无疑问，角色阵容是不完整的。有趣的是，有人认为不同的慢病毒以不同的、可能是灵活的方式进行核输入。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，这对潜伏期具有重要意义。在本基金的前一个周期中，我们重点研究了慢病毒整合酶相互作用因子LEDGF as的辅助因子作用

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.