Dynamics of HIV Core Interactions

摘要，项目1 拟议研究的总体目标是：1)阐明动态的、多方面的相互作用 HIV-1和调节感染的宿主细胞以及ii)将病毒与宿主的相互作用定义为治疗靶点。 HIV-1融合后穿过细胞质并通过核孔复合体(NPC)的旅程受 锥形病毒衣壳与多种宿主蛋白的动态相互作用 (依赖因素)或抑制(限制因素)感染。然而，完整的身份和定义 HIV-1衣壳的细胞结合伙伴的作用机制在很大程度上仍不清楚。此外， 目前尚不清楚HIV-1是如何进化的，以至于它的衣壳可以选择性地与不同的和表面上看起来 决定感染结果的无关的调节宿主蛋白。在目标1中，我们将发现和 描述HIV-1衣壳和宿主细胞在病毒入侵过程中的新的、动态的相互作用。 具体地说，我们的努力将集中在揭开以前未被识别的、动态的蜂窝网络 调节HIV-1的蛋白质‘S穿过细胞质，通过鼻咽癌。此外，我们还将 阐明这些蛋白与HIV-1衣壳选择性和亲和性结合的结构决定因素。这个 与多种HIV-1宿主依赖因子结合的疏水性衣壳口袋已经成功 以小分子抑制剂为靶点。然而，相对较低的抵抗电流衣壳的障碍 以抗病毒药物为目标是一个严重的问题。在目标2中，我们将描述现有的和新的小型 HIV-1衣壳上具有不同位点的分子抑制剂。具体地说，我们将定义已知的HIV-1衣壳 抑制剂影响锥形HIV-1衣壳的动态结构。此外，我们将发现新奇的 小分子结合在HIV-1衣壳上的不同位置，并保持对耐药病毒变体的活性 到目前的抑制剂。为了实现上述两个目标，我们组建了一个高度协作的B-HIVE团队 在冷冻-ET、冷冻-EM、X射线结晶学、HDX-MS、Native方面拥有互补的多学科专业知识 MS、分子建模、活细胞显微镜、病毒学、分子生物学、生物化学和医学 化学反应。我们的研究将对调节HIV的病毒-宿主相互作用产生前所未有的洞察力- 1‘S在病毒入侵期间的旅程，并将这些相互作用确定为主要的治疗靶点。 此外，我们对多个不同的小分子结合位点的结构和机理表征 将为制药行业正在进行的努力提供强有力的手段 合理开发新一代长效衣壳蛋白抑制剂 改变对HIV-1感染者的护理。

ABSTRACT, PROJECT 1 The overarching goals of the proposed studies are to i) elucidate dynamic, multifaceted interplay between HIV-1 and host cell that regulates infection and ii) define the virus-host interactions as therapeutic targets. The post-fusion journey of HIV-1 across the cytoplasm and via nuclear pore complex (NPC) is regulated by dynamic interactions of the conically shaped virus capsid shell with variety host proteins that either aid (dependency factors) or inhibit (restriction factors) infection. However, full identity and definitions of mechanisms of action of the cellular binding partners of HIV-1 capsid remain largely unknown. Furthermore, it is not clear how HIV-1 has evolved so that its capsid shell can bind selectively to diverse and seemingly unrelated regulatory host proteins that dictate the outcome of infection. In Aim 1, we will discover and characterize novel, dynamic interactions between HIV-1 capsid and host cell during virus ingress. Specifically, our efforts will focus on uncovering previously unrecognized, dynamic networks of cellular proteins that regulate HIV-1’s journey across the cytoplasm and through the NPC. Furthermore, we will elucidate structural determinants for selective and avid binding of these proteins to HIV-1 capsid. The hydrophobic capsid pocket, which engages several HIV-1 host dependency factors, has been successfully targeted by small molecule inhibitors. However, a relatively low barrier to resistance to current capsid targeting antivirals is a serious concern. In Aim 2, we will characterize interactions of existing and novel small molecule inhibitors with distinct sites on HIV-1 capsid. Specifically, we will define how known HIV-1 capsid inhibitors influence a dynamic structure of the conical HIV-1 capsid shell. Moreover, we will discover novel small molecules that bind at distinct sites on HIV-1 capsid and retain activity against viral variants resistant to current inhibitors. To accomplish above two aims we have assembled a highly collaborative B-HIVE team with complementary, multidisciplinary expertise in cryo-ET, cryo-EM, X-ray crystallography, HDX-MS, native MS, molecular modeling, live cell microscopy, virology, molecular biology, biochemistry, and medicinal chemistry. Our research will generate unprecedented insight into virus-host interactions that regulate HIV- 1’s journey during the virus ingress and define these interactions as the principal therapeutic target. Moreover, our structural and mechanistic characterization of multiple, distinct small molecule binding sites on the conical capsid surface will provide powerful means for ongoing efforts in pharmaceutical industry to rationally develop next generation of long-acting capsid inhibitors with enhanced barrier to resistance to transform care of the people living with HIV-1.