Modulation of retroviral uncoating by cellular factors

尽管公共和私人机构做出了大量努力，但我们还没有预防性疫苗 抗HIV-1或根除方案来治疗受感染的患者。最近，世界各地的实验室都有 报告说，对艾滋病毒的一种或多种成分产生抗药性的艾滋病毒-1毒株的传播有所增加 抗逆转录病毒(ART)鸡尾酒。为了寻找新的治疗靶点，对HIV-1复制的基本了解 是迫切需要的。复制人类免疫缺陷病毒(HIV-1)的早期步骤包括交付 病毒核心进入宿主细胞的细胞质。病毒核心是由~1500组成的圆锥形结构 HIV-1衣壳蛋白的单体，包含病毒RNA基因组。我们研究了HIV-1病毒的脱壳， 通过利用不同细胞因子影响早期的能力来逆转录和核进口 HIV-1复制事件。“脱壳”是指单体衣壳从HIV-1核心解离。蛋白质 而破坏HIV-1核心稳定并加速胞浆脱壳的药物可以阻止逆转录。 与逆转录与去涂层有关的概念一致，我们已经证明了基因或 通过药物抑制逆转录可防止脱壳。在上一个资助期内，我们 研究发现，稳定HIV-1核心的细胞蛋白质允许逆转录，但不允许核进口。 这些结果支持这样一个模型，即HIV-1核心是一个“容器”，它容纳了 逆转录。打开“容器”会扰乱逆转录。把“集装箱”关起来 允许完成反转录，但阻止核进口。这些结果表明，核心稳定性 在感染期间受到严密的调节。亲环素A(CypA)占细胞蛋白的0.4%， 与HIV-1衣壳和核心相互作用。然而，CypA在HIV-1感染中扮演的角色尚不清楚。 我们的初步数据表明，CypA的缺失可以100倍地抑制原代CD4+T细胞中的HIV-1感染， 支持使用这一模型来理解CypA在HIV-1感染中的作用。我们假设CypA 通过控制细胞因子之间的相互作用发挥HIV-1感染的中心调节器的作用 和HIV-1感染早期阶段的HIV-1核心。在Hultquist博士的专家支持下， 坎贝尔、鲁班和凯瓦拉马尼博士表示，以下具体目标将检验这一假说。目标1将 确定CypA调节人类原代CD4+T细胞中HIV-1感染的机制。目标 2将确定CypA对核内HIV-1衣壳的影响。目标3将研究其他人的角色 HIV-1复制中的亲环素。目的4将确定Nup358亲环素结构域(Cyp358)对 HIV-1核进口。这项提议将解决艾滋病毒-1领域的两个重要问题：1) CypA在HIV-1复制中的作用，25年来一直难以捉摸；2)核衣壳在HIV-1中的作用 生殖性感染。

Despite of a great deal of effort by public and private institutions, we don't have a prophylactic vaccine against HIV-1 or an eradication protocol to treat infected patients. Recently, laboratories all over the word have reported an increase in the transmission of HIV-1 strains that are resistant to one or more components of the antiretroviral (ART) cocktail. In order to find novel therapeutic targets, basic understanding of HIV-1 replication is urgently needed. Early steps in the replication of human immunodeficiency virus (HIV-1) involve delivery of the viral core into the cytoplasm of the host cell. The viral core is a conical structure composed of ~1500 monomers of the HIV-1 capsid protein, which houses the viral RNA genome. We studied HIV-1 uncoating, reverse transcription, and nuclear import by exploiting the ability of different cellular factors to affect early events of HIV-1 replication. “Uncoating” is the dissociation of monomeric capsids from the HIV-1 core. Proteins and drugs that destabilize the HIV-1 core and accelerate uncoating in the cytosol prevent reverse transcription. Consistent with the concept that reverse transcription is linked to uncoating, we have shown that genetic or pharmacologic inhibition of reverse transcription prevents uncoating. During the previous funding period, we found that cellular proteins that stabilize the HIV-1 core allow reverse transcription but not nuclear import. These results support a model in which the HIV-1 core is a “container” that holds the elements required for reverse transcription. Opening the “container” disrupts reverse transcription. Keeping the “container” closed allows completion of reverse transcription but prevents nuclear import. These results suggest that core stability is tightly modulated during infection. Cyclophilin A (CypA) accounts for up to 0.4% of cellular protein and interacts with the HIV-1 capsid and core. However, the role that CypA plays in HIV-1 infection is not known. Our preliminary data indicate that CypA depletion inhibits HIV-1 infection in primary CD4+ T cells by 100 fold, supporting the use of this model to understand the role of CypA in HIV-1 infection. We hypothesize that CypA functions as a central modulator of HIV-1 infection by controlling interactions between cellular factors and the HIV-1 core during the early steps of HIV-1 infection. With the expert support of Dr. Hultquist, Dr. Campbell, Dr. Luban and Dr. KewalRamani, the following specific aims will test this hypothesis. Aim 1 will determine the mechanisms through which CypA modulates HIV-1 infection in human primary CD4+ T cells. Aim 2 will determine the effects of CypA on the HIV-1 capsid in the nucleus. Aim 3 will examine the roles of other cyclophilins in HIV-1 replication. Aim 4 will determine the effects of the Nup358 cyclophilin domain (Cyp358) on HIV-1 nuclear import. This proposal will address two important questions on the HIV-1 field: 1) The role of CypA in HIV-1 replication, which has been elusive for 25 years, and 2) the role of nuclear capsid in HIV-1 productive infection.