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

病毒蛋白和宿主限制因子在早期 HIV-1 复制事件中的新作用

• 批准号: 10603583
• 负责人: MARK C WILLIAMS
• 金额: $ 60万
• 依托单位: NORTHEASTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-12 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10603583
• 关键词: Address; Binding; Biochemical; Biological; Biophysics; Capsid; Cations; Cell Nucleus; Cells; Characteristics; Chromatin; Chromatin Structure; Complex; DNA; DNA Binding; DNA Sequence; DNA biosynthesis; Deaminase; Drug Targeting; Event; Evolution; Gene Expression; Genes; Genetic Transcription; HIV; HIV Genome; HIV-1; Human; Infection; Knowledge; Lead; Length; Literature; Measurement; Measures; Minority; Molecular; Monitor; Mutation; Nuclear; Nuclear Pore; Nucleocapsid; Nucleosomes; Physical condensation; Positioning Attribute; Process; Proteins; Proviruses; Resistance; Reverse Transcription; Reverse Transcription Inhibition; Role; Swelling; Testing; Therapeutic; Therapeutic Intervention; Transcriptional Regulation; Viral; Viral Genome; Viral Proteins; Viral Reverse Transcription; Virus; Virus Latency; Virus Replication; ds-DNA; enzyme activity; enzyme mechanism; improved; in vivo; inhibitor; insight; mutant; novel; pressure; prevent; rational design; targeted treatment; transcription factor; viral DNA; vpr Gene Products

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.

项目总结/摘要 在HIV复制周期的早期阶段抑制HIV在治疗上是有利的，这是由于在HIV复制周期的早期阶段， 整合前病毒的持久性和体内病毒潜伏期的持久性。来合理设计 早期复制步骤的新抑制剂，需要详细的分子理解。我们提出 综合的生物化学、生物物理学和细胞方法，以探测早期病毒感染的关键步骤。 复制的最近的研究发现，只有少数衣壳是完整地通过核运输的。 细胞核中的孔和未被膜成功整合。HIV衣壳是一个有希望的治疗靶点， 干预由于其溶解的关键时间，但它仍然是未知的，如何在病毒感染后 DNA合成开始，什么样的分子相互作用控制它的时间。成功的衣壳 在细胞核内发生的脱壳解释了为什么人类宿主APOBEC 3（A3）蛋白必须是 在生产细胞中包装到病毒中以成功抑制逆转录。因为很少有A3 蛋白质被包装，这些蛋白质必须是非常有效的抑制剂。改良型A3超级限流器 但它们如何抑制逆转录病毒复制仍然是一个关键的知识空白。病毒 成功整合后的基因组转录受许多蛋白质调节，包括HIV-1 Vpr。 尽管Vpr改变了细胞和病毒基因组转录，但增强艾滋病毒转录的相互作用 和改变细胞基因表达的方法是未知的。为解决这些问题，提出了三个目标： (1)确定HIV-1核衣壳、IN和Vpr蛋白在调节HIV-1表达时间中的作用。 病毒逆转录过程中衣壳脱壳。为了验证NC压缩双- 为了调节衣壳脱壳，我们将测量野生型的DNA凝聚活性， 和突变的HIV-1 NC。我们将相关的结果与活细胞研究的衣壳脱壳和逆转 在相同的NC突变存在下，转录以及总体病毒复制。(2)确定 APOBEC 3（A3）超级限制因子如何优化HIV-1逆转录抑制。测试 假设不同DNA结合形式的A3超限制因子具有不同的酶活性， HIV-1复制抑制的机制，我们将测量有效的DNA结合特性， 超级限制器。我们将比较我们的结果与测量的A3包装，逆转录 抑制，脱氨酶依赖性和脱氨酶非依赖性复制抑制，以及抗性 HIV-1 Vif对抗措施的超级限制因子。(3)确定HIV-1 Vpr如何调节 核小体可及性和转录。为了验证Vpr结合DNA并改变 核小体促进HIV-1和细胞基因的转录，我们将测量Vpr及其 突变体根据DNA序列改变核小体稳定性。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。