Novel roles of viral proteins and host restriction factors in early HIV-1 replication events

项目摘要/摘要 在艾滋病毒复制周期的早期阶段抑制艾滋病毒在治疗上是有利的，因为 整合前病毒的持久性和体内病毒潜伏期的持久性。合理设计 新的早期复制步骤的抑制剂，需要详细的分子理解。我们建议 综合生化、生物物理和细胞方法探测早期病毒的关键步骤 复制。最近的研究发现，只有少数衣壳原封不动地通过核运输。 胞核内的毛孔和脱膜成功融合。HIV衣壳是一个很有希望的治疗靶点 由于其溶解的关键时机而进行干预，但仍不清楚病毒感染后如何去涂层 DNA合成是启动的，什么分子相互作用控制着它的时间。那个成功的衣壳 核内脱壳解释了为什么人类宿主APOBEC3(A3)蛋白必须是 被包装到病毒的产生细胞中，成功地抑制了逆转录。因为很少有A3 蛋白质是包装的，这些蛋白质一定是极其有效的抑制剂。改进型A3超级限制器 已经开发出了它们，但它们如何抑制逆转录病毒复制仍然是一个关键的知识缺口。病毒式传播 整合成功后的基因组转录受多种蛋白质的调控，其中包括HIV-1vpr。 尽管VPR改变了细胞和病毒基因组的转录，但增强HIV转录的相互作用 以及改变细胞基因的表达是未知的。为了解决这些问题，我们提出了三个目标： (1)确定HIV-1核衣壳蛋白、IN蛋白和VPR蛋白在调节HIV-1感染时机中的作用 病毒逆转录过程中衣壳被揭开。为了检验NC压实双倍的假设 为了调节衣壳的脱壳，我们将测量野生型的DNA凝集活性 和突变型HIV-1 NC。我们将把结果与衣壳去涂层和反转的活细胞研究相关联。 转录，以及整体病毒复制，在存在相同的NC突变的情况下。(二)确定 APOBEC3(A3)超级限制子如何优化HIV-1逆转录酶的抑制。要测试 假设不同DNA结合形式的A3超限制子具有不同的酶活性和 抑制HIV-1复制的机制，我们将测量有效的DNA结合特性 超级限制器。我们将把我们的结果与A3包装、逆转录的测量结果进行比较 抑制、依赖于脱氨酶和非依赖于脱氨酶的复制抑制及其耐药性 针对HIV-1 VIF对策的超级限制器。(3)确定HIV-1 vpr如何调节 核小体可及性和转录。要检验VPR结合DNA并改变DNA的假设 为了促进HIV-1和细胞基因的转录，我们将测量VPR和它的 突变体改变核小体的稳定性作为DNA序列的函数。结果将直接与 野生型和突变型vpr对HIV-1和细胞基因表达的影响。

Project Summary/Abstract It is therapeutically advantageous to inhibit HIV at early stages of its replication cycle owing to the permanence of the integrated provirus and the persistence of viral latency in vivo. To rationally design new inhibitors of early replication steps, a detailed molecular understanding is required. We propose integrated biochemical, biophysical, and cellular approaches to probe critical steps in early viral replication. Recent studies found that only a minority of capsids are transported intact through the nuclear pore and uncoat in the nucleus successfully integrate. The HIV capsid is a promising target for therapeutic intervention owing to the critical timing of its dissolution, but it remains unknown how uncoating after viral DNA synthesis is initiated and what molecular interactions control its timing. That successful capsid uncoating occurs inside the nucleus explains why human host APOBEC3 (A3) proteins must be packaged into the virus in producer cells to successfully inhibit reverse transcription. Because so few A3 proteins are packaged, these proteins must be extremely efficient inhibitors. Modified A3 super-restrictors have been developed but how they inhibit retroviral replication remains a critical knowledge gap. Viral genome transcription after successful integration is regulated by many proteins, including HIV-1 Vpr. Although Vpr alters cellular and viral genome transcription, the interactions that enhance HIV transcription and alter cellular gene expression are unknown. To address these questions, three aims are proposed: (1) Determine the roles of HIV-1 nucleocapsid, IN and Vpr proteins in regulating the timing of capsid uncoating during viral reverse transcription. To test the hypothesis that NC compacts double- stranded DNA to regulate capsid uncoating, we will measure the DNA condensing activity of wild type and mutant HIV-1 NC. We will correlate the results with live cell studies of capsid uncoating and reverse transcription, as well as overall viral replication, in the presence of the same NC mutations. (2) Determine how APOBEC3 (A3) super-restrictors optimize inhibition of HIV-1 reverse transcription. To test the hypothesis that different DNA-bound forms of A3 super-restrictors have different enzyme activities and mechanisms of HIV-1 replication inhibition, we will measure the DNA binding characteristics of effective super-restrictors. We will compare our results with measurements of A3 packaging, reverse transcription inhibition, deaminase-dependent and deaminase-independent replication inhibition, and the resistance of the super-restrictors to HIV-1 Vif countermeasures. (3) Determine how HIV-1 Vpr regulates nucleosome accessibility and transcription. To test the hypothesis that Vpr binds DNA and alters nucleosomes to facilitate transcription of HIV-1 and cellular genes, we will measure how Vpr and its mutants alter nucleosome stability as a function of DNA sequence. Results will be directly compared with the effects of wild type and mutant Vpr on HIV-1 and cellular gene expression.