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

细胞因素在逆转录病毒脱壳和病毒 DNA 合成中的作用

• 批准号: 7930231
• 负责人: Felipe Diaz-Griffero
• 金额: $ 41.5万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-15 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7930231
• 关键词: AIDS prevention; Address; Affect; Buffers; Capsid; Capsid Proteins; Cell Nucleus; Cells; Complex; Cytoplasm; DNA; DNA biosynthesis; Defect; Equilibrium; Event; Exhibits; Fibroblast Growth Factor; Gene Expression; Genome; Goals; HIV; HIV-1; Infection; Integration Host Factors; Isotonic Exercise; Knowledge; Lead; Learning; Light; Molecular; Mutagenesis; Mutation; Nuclear Import; Phase; Process; Production; Proteasome Inhibitor; Proteins; RNA Splicing; RNA-Directed DNA Polymerase; Reverse Transcription; Ribonucleoproteins; Role; Structure; TRIM Motif; Testing; Therapeutic; Time; Variant; Viral; Viral Genes; Virus; blocking factor; monomer; multicatalytic endopeptidase complex; mutant; particle; public health relevance; research study; viral DNA; viral RNA

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.

描述（由申请人提供）：人类免疫缺陷病毒（HIV-1）复制的早期步骤涉及将病毒核递送到宿主细胞的细胞质中。当病毒核心到达细胞质时，发生了一种称为“脱衣”的过程。脱衣是单体衣壳从逆转录病毒核心脱落的过程。同时，病毒RNA基因组转化为病毒DNA（VDNA），随后将其转移到细胞核中并整合到细胞DNA中，从而使新病毒颗粒的表达和产生。尽管尚不清楚衣壳脱落与病毒DNA（VDNA）合成之间的关系，但细胞质中病毒核的稳定性对于生产性感染似乎很重要。逆转录病毒核心的稳定性的变化似乎会影响VDNA合成：1）HIV-1的衣壳蛋白中的突变，从而降低（或增加）HIV-1核心的稳定性干扰VDNA合成； 2）TRIM51蛋白质通过改变逆转录病毒核的稳定性和破坏VDNA合成的稳定性来阻断HIV-1复制； 3）在没有辅助蛋白（例如VIF，VPR或NEF）的情况下，从产生的颗粒中分离出的HIV-1核表现低稳定性，并且由于VDNA合成缺陷而导致感染性较差。 4）在HIV-1感染期间使用蛋白酶体抑制剂会增加核心的稳定性，同时增加了VDNA的合成； 5）分离的HIV-1核需要细胞提取物以进行VDNA合成，表明宿主因子的要求； 6）分离的逆转录病毒核在胞质提取物中的持续时间比等渗缓冲液中的时间更长，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合成涉及紧密交织的事件的微妙平衡。在此提案中，我们将检验以下假设：逆转录病毒核心和细胞因子的最佳稳定性调节细胞质中不涂层的速率和程度，并且发生完全病毒DNA合成所必需的这种速率和脱覆的速率和程度。我们将通过执行以下实验来检验这一假设：1）我们将研究使逆转录病毒核心对病毒DNA合成的因素的影响，2）我们将研究蛋白酶体对逆转录病毒脱膜的贡献，3）我们将通过逆转型ribon的相互作用研究VDNA合成的调节。这些实验的结果将在HIV-1的脱膜中产生基本知识。脱衣是在HIV-1治疗药物上探索不当的一步。但是，我们已经从诸如TRIM5之类的限制因素中学到了脱涂层，这可能是HIV-1治疗剂的非常有效的靶标。 公共卫生相关性：该提案的主要目的是了解HIV-1的不涂层过程，这是一个研究不足的过程。脱涂层的巨大治疗潜力是通过阻止HIV-1复制（例如TRIM5）的自然因素的分离揭示的。由于像TRIM5靶标HIV-1这样的蛋白质在复制方面实现了非常强大的障碍，因此我们认为，彻底了解逆转录病毒脱涂层将产生新的机会，以开发有效的HIV-1控制和艾滋病预防。