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递送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），其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。尽管衣壳脱落和病毒DNA（vDNA）合成之间的关系尚不清楚，但细胞质中病毒核心的稳定性似乎对生产性感染很重要。逆转录病毒核心稳定性的变化似乎会影响vDNA的合成：1）HIV-1衣壳蛋白的突变，（或增加）HIV-1核心的稳定性干扰vDNA合成; 2）TRIM 51蛋白通过改变逆转录病毒核心的稳定性阻断HIV-1复制并破坏vDNA合成; 3）从在不存在辅助蛋白如vif、vpr或nef的情况下产生的颗粒中分离的HIV-1核心表现出低稳定性，并且由于vDNA合成缺陷而感染性差; 4）在HIV-1感染过程中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核心需要细胞提取物才能进行vDNA合成，这表明需要宿主因子;和6）分离的逆转录病毒核心在胞质提取物中比在等渗缓冲液中持续更长时间，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，我们将测试的假设，逆转录病毒核心和细胞因子的最佳稳定性调节的速度和程度的脱壳在细胞质中，而这种速度和程度的脱壳是需要的发生完整的病毒DNA合成。我们将通过进行以下实验来验证这一假设：1）我们将研究使逆转录病毒核心不稳定的因素对病毒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HIV-1的脱壳提供基础知识。在HIV-1治疗中，去包被是一个探索不足的步骤;然而，我们从限制因子（如TRIM 5）中了解到，去包被可能是HIV-1治疗的一个非常有效的靶点。

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.