Role of Cellular Factors in Retroviral Uncoating and Synthesis of Viral DNA

描述(申请人提供)：人类免疫缺陷病毒(HIV-1)复制的早期步骤包括将病毒核心送入宿主细胞的细胞质。当病毒核心到达细胞质时，就会发生一种称为“脱壳”的过程。脱壳是单体衣壳从逆转录病毒核心脱落的过程。同时，病毒RNA基因组被转化为病毒DNA(VDNA)，病毒DNA随后被转移到细胞核并整合到细胞DNA中，从而允许表达和生产新的病毒颗粒。虽然衣壳脱落和病毒DNA(VDNA)合成之间的关系尚不清楚，但病毒核心在细胞质中的稳定性似乎对产生性感染很重要。逆转录病毒核心稳定性的变化似乎影响VDNA的合成：1)HIV-1衣壳蛋白的突变降低(或增加)HIV-1核心的稳定性干扰VDNA的合成；2)TRIM51蛋白通过改变逆转录病毒核心的稳定性而阻止HIV-1的复制并干扰VDNA的合成；3)从没有辅助蛋白如vif、vpr或nef的颗粒中分离出的HIV-1核心显示出低稳定性，并且由于VDNA合成的缺陷而感染性差；4)在HIV-1感染期间使用蛋白酶体抑制剂增加了核心的稳定性，同时增加了VDNA的合成；5)分离的HIV-1核心需要细胞提取液才能进行VDNA合成，这表明宿主因素的需要；以及6)分离的逆转录病毒核心在胞浆提取液中比在等渗缓冲液中持续时间更长，这表明存在对VDNA合成至关重要的核心稳定因子。总之，这些证据表明，病毒核心的稳定性和成功的VDNA合成涉及紧密交织的事件的微妙平衡。在这项提案中，我们将检验这样一个假设，即逆转录病毒核心和细胞因素的最佳稳定性调节细胞质中脱壳的速度和程度，并且这种脱壳速度和程度是发生完整的病毒DNA合成所必需的。我们将通过以下实验来验证这一假设：1)我们将研究不稳定逆转录病毒核心的因素对病毒DNA合成的影响，2)我们将研究蛋白酶体对逆转录病毒脱壳的贡献，3)我们将研究逆转录酶与逆转录病毒核糖核蛋白复合体相互作用对VDNA合成的调控。这些实验的结果将产生揭开HIV-1外衣的基本知识。去涂层是HIV-1疗法中探索得很少的一步；然而，我们已经从限制因素，如TRIM5，了解到去涂层可能是HIV-1疗法非常有效的靶点。 与公共卫生相关：这项提案的主要目标是了解HIV-1的脱壳过程，这是一个研究较少的过程。通过发现阻止HIV-1复制的自然因素，如TRIM5，揭示了去涂层的巨大治疗潜力。由于像TRIM5这样的蛋白质针对的是HIV-1去涂层，从而在复制中实现了非常强大的阻断，我们相信彻底了解逆转录病毒去涂层将为开发有效的HIV-1控制和艾滋病预防创造新的机会。

DESCRIPTION (provided by applicant): Early steps in human immunodeficiency virus (HIV-1) replication involve delivery of the viral core into the cytoplasm of the host cell. When the viral core has reached the cytoplasm a process known as "uncoating" takes place. Uncoating is the process by which monomeric capsid sheds from the retroviral core. Simultaneously, the viral RNA genome is converted into viral DNA (vDNA), which is subsequently translocated to the nucleus and integrated into the cellular DNA, allowing expression and production of new viral particles. Although the relationship between capsid shedding and synthesis of viral DNA (vDNA) is not understood, the stability of the viral core in the cytoplasm seems to be important for productive infection. Changes in stability of the retroviral core seem to affect vDNA synthesis: 1) mutations in the capsid protein of HIV-1 that diminish (or increase) the stability of the HIV-1 core interfere with vDNA synthesis; 2) TRIM51 proteins block HIV-1 replication by altering the stability of the retroviral core and disrupt vDNA synthesis; 3) HIV-1 cores isolated from particles produced in the absence of accessory proteins such as vif, vpr or nef exhibit low stability and are poorly infectious due to a defect in vDNA synthesis; 4) the use of proteasome inhibitors during HIV-1 infection increases the stability of the core, and at the same time augments the synthesis of vDNA; 5) Isolated HIV-1 cores require cellular extracts in order to undergo vDNA synthesis, indicating the requirement of host factors; and 6) isolated retroviral cores last longer in cytosolic extracts than in isotonic buffer, suggesting the existence of core-stabilizing factors that are essential for vDNA synthesis. Altogether, this evidence indicates that viral core stability and successful vDNA synthesis involve a delicate balance of tightly interwoven events. In this proposal we will test the hypothesis that optimal stability of the retroviral core and cellular factors modulate the rate and extent of uncoating in the cytoplasm, and that this rate and extent of uncoating is required for the occurrence of complete viral DNA synthesis. We will test this hypothesis by performing the following experiments: 1) we will study the effect of factors that destabilize the retroviral core on viral DNA synthesis, 2) we will study the contribution of the proteasome to retroviral uncoating, and 3) we will study the modulation of vDNA synthesis by the interaction of reverse transcriptase with the retroviral ribonucleoprotein complex. The results from these experiments will generate basic knowledge in the uncoating of HIV-1. Uncoating is a poorly explored step on HIV-1 therapeutics; however, we have learned from restriction factors, such as TRIM5, that uncoating is potentially a very effective target for HIV-1 therapeutics. PUBLIC HEALTH RELEVANCE: The main goal of this proposal is to gain understanding on the uncoating process of HIV-1, a poorly studied process. The great therapeutic potential of uncoating was revealed by the discov- ery of natural factors that block HIV-1 replication, such as TRIM5. Because proteins like TRIM5 target HIV-1 uncoating achieving a very strong block in replication, we believe that a thorough un- derstanding of retroviral uncoating will generate new opportunities to develop effective HIV-1 con- trol and AIDS prevention.