HIV-1 Intasome Assembly and Function

项目摘要/摘要 新的HIV-1感染继续推动全球大流行。联合抗逆转录病毒疗法(CART) 有助于削弱HIV-1感染者的临床结果。然而，耐药突变 继续挑战CART方案，强调确定新的病毒药物靶点的重要性。 HIV-1整合到人类基因组中对生产性感染至关重要。一体化是由 逆转录病毒编码的整合酶(IN)，与病毒的长末端重复序列(LTR)末端形成复合体 通过逆转录HIV-1基因组RNA而产生的c DNA。由此产生的有趣的东西精确地定位在 LTR末端用于催化链转移到基因组靶点。结构比较显示，所有七个 逆转录病毒属维持一个中央保守的内含体核心(CIC)，其中含有IN的四聚体。一些人 逆转录病毒家族成员通过附加额外的IN亚基来扩展CIC周围的结构。为 例如，原型泡沫病毒(PFV)内含体组装成一个简单的四聚体，而小鼠 乳腺肿瘤病毒(MMTV)通过将IN-二聚体附着在CIC的两侧形成IN-八聚体。在八聚体中， 已有关于HIV-1的十聚体、十二聚体(12-聚体)和十六聚体(16-聚体)的报道。 值得注意的是，IN-多聚体结构对HIV-1生物学的贡献在很大程度上是未知的。 导致HIV-1完全形成的IN-组装进展同样未知。在.期间 感染、反转录和内切小体组装发生在核膜或核膜附近。东道主 辅因子LEDGF/p75似乎促进了HIV-1内含体的染色质定位，并缺失了 LEDGF/p75将HIV-1的整合减少了至少10倍。我们发现非保守肽很好地连接在一起- 已知保守IN结构域控制HIV-1 IN-多聚体结构，且LEDGF/p75是 高效的HIV-1端粒酶体外组装。这些观察支撑了几个关键的悬而未决的问题： 引导多聚体进展导致HIV-1完全组装的因素是什么？什么 LEDGF/p75在HIV-1整合体组装和/或染色质相互作用中的作用？HIV-1是如何进入- 多聚体结构影响细胞内基因组靶点的选择？ 我们建议利用创新的实时单分子成像和分析来了解这些贡献 关于HIV-1机制的IN-多聚体结构，具体目标如下：1.确定IN组件 控制HIV-1侵袭体结构的进展，2.确定HIV-1连接体结构在 体外与特定靶DNA和染色质的动态相互作用。确定HIV-1的作用 细胞内靶向宿主染色质特征的融合结构。 这些研究旨在询问支持HIV-1 Intasome结构的动画过程 目的是确定可能被用作治疗靶点的更多逆转录病毒进展。

PROJECT SUMMARY / ABSTRACT New HIV-1 infections continue to drive a worldwide pandemic. Combinatorial anti-retroviral therapies (cART) have helped to blunt the clinical outcomes of HIV-1 infected individuals. However, drug-resistance mutations continue to challenge cART regimens underscoring the importance of identifying new viral drug targets. HIV-1 integration into the human genome is essential for a productive infection. Integration is catalyzed by the retrovirus encoded integrase (IN) that forms a complex with the long terminal repeat (LTR) ends of the viral cDNA, produced by reverse transcription of the HIV-1 genomic RNA. The resulting intasome precisely positions the LTR-ends for catalytic strand-transfer into a genomic target site. Structural comparisons show that all seven retrovirus genera maintain a central Conserved Intasome Core (CIC) containing a tetramer of IN. Some retrovirus family members expand the structure surrounding the CIC by appending additional IN subunits. For example, the prototype foamy virus (PFV) intasome assembles into a simple IN-tetramer while the mouse mammary tumor virus (MMTV) forms an IN-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. Remarkably, the contributions of IN-multimer architecture to HIV-1 biology is largely unknown. The IN-assembly progressions that result in a fully formed HIV-1 intasome are similarly unknown. During infection, reverse transcription and intasome assembly occurs at or near the nuclear membrane. The host cofactor LEDGF/p75 appears to facilitate chromatin localization of the HIV-1 intasomes, and deletion of LEDGF/p75 reduces HIV-1 integration at least 10-fold. We have found the non-conserved peptides linking well- known conserved IN domains control HIV-1 IN-multimer architecture, and that LEDGF/p75 is necessary for efficient HIV-1 intasome assembly in vitro. These observations underpin several key unanswered questions: What are the factors that guide IN multimer progressions resulting in a fully assembled HIV-1 intasome? What is the function of LEDGF/p75 in HIV-1 intasome assembly and/or chromatin interactions? How does HIV-1 IN- multimer architecture impact genomic target site selection in cellulo? 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 assembly progressions that control HIV-1 intasome architecture, 2.) determine the role of HIV-1 intasome architecture on the dynamic interactions with defined target DNA and chromatin 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 might be exploited as therapeutic targets.