Modulation of retroviral uncoating by cellular factors

尽管公共和私人机构做了大量的努力，我们仍然没有预防性疫苗 针对HIV-1或根除方案来治疗感染患者。最近，全世界的实验室都在 报告说，对一种或多种成分具有耐药性的HIV-1病毒株的传播增加了。 抗逆转录病毒（ART）鸡尾酒。为了找到新的治疗靶点，对HIV-1复制的基本了解 是迫切需要的。人类免疫缺陷病毒（HIV-1）复制的早期步骤包括： 病毒核心进入宿主细胞的细胞质。病毒核心是由~1500个 HIV-1衣壳蛋白的单体，其容纳病毒RNA基因组。我们研究了HIV-1的脱壳， 逆转录和核输入，通过利用不同的细胞因子的能力， HIV-1的复制。“脱壳”是单体衣壳从HIV-1核心解离。蛋白 破坏HIV-1核心的稳定性并加速细胞质中外壳的脱落的药物会阻止逆转录。 与逆转录与脱壳有关的概念一致，我们已经表明，基因或 逆转录的药理学抑制可防止脱包被。在上一个财政年度，我们 发现稳定HIV-1核心的细胞蛋白允许逆转录，但不允许核输入。 这些结果支持了一种模型，其中HIV-1核心是一个“容器”，容纳了 逆转录打开“容器”会破坏逆转录。保持“容器”关闭 允许完成逆转录，但阻止核输入。这些结果表明， 在感染过程中受到严格调节。亲环素A（CypA）占细胞蛋白质的0.4%， 与HIV-1衣壳和核心相互作用。然而，CypA在HIV-1感染中的作用尚不清楚。 我们的初步数据表明，CypA消耗抑制HIV-1感染的原代CD 4 + T细胞的100倍， 支持使用该模型来了解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，作为HIV-1感染的中心调节剂发挥作用 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、鲁班博士和科瓦尔拉马尼博士，以下具体目标将检验这一假设。目标1将 确定CypA调节人类原代CD 4 + T细胞中HIV-1感染的机制。目的 2将确定CypA对细胞核中HIV-1衣壳的影响。目标3将研究其他组织的作用 HIV-1复制中的亲环素。目的4将确定Nup 358亲环蛋白结构域（Cyp 358）对细胞凋亡的影响。 HIV-1核输入。该提案将解决HIV-1领域的两个重要问题： 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 生产性感染

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.