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

描述（由申请人提供）：人类免疫缺陷病毒（HIV-1）复制的早期步骤包括将病毒核心传递到宿主细胞的细胞质中。当病毒核心到达细胞质时，就会发生一个被称为“脱壳”的过程。脱包衣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)，随后易位到细胞核并整合到细胞DNA中，从而允许表达和产生新的病毒颗粒。尽管衣壳脱落与病毒DNA （vDNA）合成之间的关系尚不清楚，但细胞质中病毒核的稳定性似乎对生产感染很重要。逆转录病毒核心稳定性的变化似乎影响vDNA的合成：1)HIV-1衣壳蛋白的突变会降低（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和破坏vDNA合成来阻断HIV-1复制；3)由于vDNA合成缺陷，从缺乏vif、vpr或nef等辅助蛋白的颗粒中分离出的HIV-1核具有较低的稳定性和较差的传染性；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核细胞需要细胞提取物才能进行vDNA合成，表明宿主因子的要求；6)分离的逆转录病毒核在细胞质提取物中比在等渗缓冲液中持续时间更长，这表明核稳定因子的存在是vDNA合成所必需的。总之，这一证据表明，病毒核心的稳定性和成功的vDNA合成涉及紧密交织事件的微妙平衡。在本提案中，我们将测试逆转录病毒核心和细胞因子的最佳稳定性调节细胞质中脱壳的速度和程度的假设，并且这种脱壳的速度和程度是发生完整病毒DNA合成所必需的。我们将通过以下实验来验证这一假设：1)我们将研究破坏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物相互作用对vDNA合成的调节。这些实验的结果将为揭开HIV-1的外壳提供基础知识。在HIV-1治疗中，剥去涂层是一个探索不足的步骤；然而，我们从限制因子（如TRIM5）中了解到，脱膜是HIV-1治疗的潜在非常有效的靶点。

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.