Delineating a role for CA in HIV-1 nuclear transport to sites of integration

描述 CA 在 HIV-1 核转运至整合位点中的作用

• 批准号: 10342316
• 负责人: Ashwanth Christopher Francis
• 金额: $ 23.1万
• 依托单位: FLORIDA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-01 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10342316
• 关键词: Active Biological Transport; Address; Amino Acids; Antiviral Agents; Binding; Biological Assay; Biotin; CD4 Positive T Lymphocytes; Capsid Proteins; Cell Nucleus; Cells; Chimeric Proteins; Co-Immunoprecipitations; Complementary DNA; Complex; Cone; DNA; Data; Development; Diffusion; DsRed; Electron Microscopy; Enzymes; Exposure to; Future; Genes; Genome; Genomic Segment; Guide RNA; HIV-1; Human Parainfluenza Virus 2; Image; Infection; Integrase; Label; Lentivirus; Life Cycle Stages; Ligation; Location; Maps; Measures; Mediating; Molecular Biology; Movement; Mutation; NPC1 gene; Nature; Nuclear; Nuclear Envelope; Nuclear Import; Nuclear Pore Complex; Nucleocapsid Proteins; Pathway interactions; Peripheral; Pharmaceutical Preparations; Point Mutation; Polyadenylation; Positioning Attribute; Proteolytic Processing; Public Health; RNA-Directed DNA Polymerase; Research; Residual state; Ribonucleoproteins; Role; Structure; System; Techniques; Testing; Viral; Viral Genome; Viral Proteins; Virion; Virus; Virus Integration; Visualization; base; biochemical tools; cell type; design; drug development; gag Gene Products; improved; innate immune sensing; insight; integration site; live cell imaging; mRNA Cleavage and Polyadenylation Factors; macrophage; monocyte; mutant; novel; novel virus; nucleocytoplasmic transport; particle; passive transport; photoactivation; preference; small molecule inhibitor; tool; trafficking; vector; viral RNA

Project Summary HIV-1 capsid protein (CA) determines the virus nuclear entry and integration site preference. By developing novel tools to track single viral complexes that establish infection, we have recently found that point mutations in CA (N74D) influence the targeting of viral integration to the periphery as opposed to interior of the nucleus preferred by wild-type virus. A nuclear role for CA, which remains poorly appreciated, is potentially derived from the subset of CA molecules that remain associated with nuclear pre-integration complexes (PICs). We hypothesize that interaction between PIC-associated CA molecules and cellular co-factor CPSF6 directs the transport of HIV-1 to nuclear speckle regions that are rich in actively transcribing genes for integration. The scientific premise of this proposal is to characterize the CA/CPSF6 dependent nuclear HIV-1 transport to the sites of integration. We will, (1) apply live-cell imaging in combination with photoactivation techniques to visualize CPSF6 interaction with fluorescent CA-labeled PICs and their transport to sites of integration. (2) Determine a role for CPSF6/PICs interaction in the HIV-1 nuclear transport by determining diffusion coefficients of single particles in the presence of drugs and CA mutants that abrogate CA/CPSF6 interactions. (3) Develop a live-cell imaging assay to visualize the integrated vDNA, and correlate the location of PICs disappearance to integration. (4) Determine the amino acid residues in CA involved in its interaction with viral RNA or proteins in PICs and identify the binding partner of CA in vRNPs. This new direction of research will delineate a role for CA in interactions and intra-nuclear trafficking of PICs to locations of HIV-1 integration, an important step in viral life cycle that remains poorly appreciated.

项目摘要 HIV-1衣壳蛋白（CA）决定了病毒进入核和整合位点的偏好。发展中 新的工具来追踪建立感染的单一病毒复合体，我们最近发现， 在CA（N74 D）中，影响病毒整合到细胞核的外周而不是内部的靶向 优选野生型病毒。CA的核作用仍然没有得到很好的认识，可能来自于 CA分子的子集保持与核前整合复合物（PIC）相关联。我们 假设PIC相关CA分子和细胞辅因子CPSF 6之间的相互作用指导了 将HIV-1转运至富含用于整合的活跃转录基因的核斑点区域。的 该建议的科学前提是表征CA/CPSF 6依赖的核HIV-1转运至 融合的场所。我们将，（1）应用活细胞成像结合光活化技术， CPSF 6与荧光CA标记的PIC的相互作用及其向整合位点的转运。(2)确定 通过确定单个CPSF的扩散系数，CPSF 6/PIC相互作用在HIV-1核转运中的作用 在药物和消除CA/CPSF 6相互作用的CA突变体的存在下，(3)开发一个活细胞 成像测定以使整合的vDNA可视化，并将PIC消失的位置与整合相关联。 (4)确定CA中参与与PIC中病毒RNA或蛋白质相互作用的氨基酸残基， 鉴定vRNP中CA的结合伴侣。这一新的研究方向将描绘CA在以下方面的作用 相互作用和核内运输PIC到HIV-1整合的位置，这是病毒生命的重要一步 这一周期仍然不太受重视。