HIV-1 Intasome Assembly and Function

HIV-1胞内组装和功能 项目总结/摘要 新的HIV-1逆转录病毒感染继续推动全球流行。联合药物疗法有助于 以减弱HIV-1感染者的临床结果。然而，抗药性病毒突变 对这些治疗方案的挑战依然存在，因此确定新的药物靶点至关重要。 HIV-1整合到人类基因组中对于生产性感染是必不可少的。整合是由 逆转录病毒编码的整合酶（IN），其与逆转录病毒的长末端重复序列（LTR）末端形成多聚体复合物。 逆转录的病毒cDNA（称为整合体和/或整合前复合物）。结构 比较显示所有七种逆转录病毒属都保持保守的整合体核心（CIC）构型， 其精确定位LTR末端，用于在整合过程中催化链转移到基因组靶位点。 不同的逆转录病毒家族成员通过附加额外的IN亚基来扩大CIC的大小。为 例如，原型泡沫病毒（PFV）整合体组装成简单的四聚体，而小鼠乳腺病毒（PFV）整合体组装成简单的四聚体。 肿瘤病毒（MMTV）通过将IN二聚体连接到CIC的任一侧而形成八聚体。IN八聚体，十聚体， 已经报道了HIV-1的十二聚体（12-mer）和十六聚体（16-mer）整合体。的贡献 HIV-1生物学和生物物理化学的IN-多聚体结构是未知的。 最终形成HIV-1整合体的组装过程仍然是个谜。越来越多的证据 这表明，含有HIV-1基因组的病毒衣壳被导入细胞核， 整合体与衣壳的解体同时发生。然后整合到基因组染色质中1 - 2 µ m 从衣壳分解位点。包括LEDGF/p75在内的宿主因子促进了HIV-1整合。我们 和其他人发现LEDGF/p75是体外有效的HIV-1整合体组装所必需的。这些 观察结果支持了几个关键的未回答的问题： 组装HIV-1整合体？LEDGF/p75在整合体组装中的作用是什么？什么是影响IN- 多聚体结构对整合体稳定性和基因组靶位点选择的影响？ 我们建议利用创新的实时单分子成像和分析来了解 具有以下特定目的的HIV-1机制的IN-多聚体结构：1.）确定IN亚基 控制多聚体HIV-I整合体结构的组装进展，2.）确定HIV-1的作用 在体外与确定的染色质靶DNA的动态相互作用上的整合体结构，和3.）确定 HIV-1整合体结构在细胞中靶向宿主染色质特征的作用。 这些研究旨在询问支持HIV-1整合体结构的动画过程 目的是鉴定可用作治疗靶点的其他逆转录病毒进展。

HIV-1 INTASOME ASSEMBLY AND FUNCTIONS PROJECT SUMMARY / ABSTRACT New HIV-1 retrovirus infections continue to drive a worldwide pandemic. Combined drug therapies have helped to blunt the clinical outcomes that afflict HIV-1 infected individuals. However, drug-resistance virus mutations that challenge these treatment regimens persist, making identification of new drug targets of crucial importance. HIV-1 integration into the human genome is essential for a productive infection. Integration is catalyzed by the retrovirus encoded integrase (IN), which forms a multimeric complex with the long terminal repeat (LTR) ends of the reverse-transcribed viral cDNA (termed an intasome and/or pre-integration complex). Structural comparisons show that all seven retrovirus genera maintain a conserved intasome core (CIC) configuration, which precisely positions the LTR-ends for catalytic strand-transfer into a genomic target site during integration. Different retrovirus family members expand the size of the CIC by appending additional IN subunits. For example, the prototype foamy virus (PFV) intasome assembles as a simple tetramer while the mouse mammary tumor virus (MMTV) forms an octamer by attaching IN dimers to either side of the CIC. IN octamer, decamer, dodecamer (12-mer) and hexadecamer (16-mer) intasomes have been reported for HIV-1. The contributions of IN-multimer architecture to HIV-1 biology and biophysical chemistry is unknown. The assembly processes that ultimately result in an HIV-1 intasome remain enigmatic. Accumulating evidence suggests that the viral capsid containing the HIV-1 genome is imported into the nucleus where assembly of the intasome occurs in concert with capsid disassembly. Integration into the genomic chromatin then occurs 1-2 µm from the capsid disassembly site. HIV-1 integration is facilitated by host factors that include LEDGF/p75. We and others have found that LEDGF/p75 is required for efficient HIV-1 intasome assembly in vitro. These observations underpin several key unanswered questions: What are the progressions that result in an assembled HIV-1 intasome? What is the role of LEDGF/p75 in intasome assembly? What is the impact of IN- multimer architecture on intasome stability and genomic target site selection in vitro and in vivo? We propose to utilize innovative real-time single molecule imaging and analysis to understand the contributions of IN-multimer architecture on HIV-1 mechanics with the following Specific Aims: 1.) determine the IN-subunit assembly progressions that control multimeric HIV-1 intasome architecture, 2.) determine the role of HIV-1 intasome architecture on the dynamic interactions with defined chromatin target DNA in vitro, and 3.) determine the role of HIV-1 intasome architecture on targeting host chromatin features in cellulo. These studies are designed to interrogate the animated processes that support HIV-1 intasome architecture with the goal of identifying additional retroviral progressions that may be exploited as therapeutic targets.