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

基本信息

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
项目类别：
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

项目摘要

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）确定病毒核进入和整合位点的偏好。通过发展追踪建立感染的单个病毒复合物的新型工具，我们最近发现该点突变在CA（N74D）中，影响病毒整合到周围的靶向而不是核的内部由野生型病毒首选。 CA的核作用仍然不受欢迎，可能是从与核前整合复合物（图片）有关的CA分子的子集。我们假设与PIC相关的CA分子与细胞副因素CPSF6之间的相互作用指导 HIV-1向积极转录基因进行整合的核斑点区域的运输。这该提议的科学前提是表征CA/CPSF6依赖的核HIV-1转运到集成站点。我们将（1）将活细胞成像与光激活技术结合使用以可视化 CPSF6与荧光CA标记的图片及其运输到整合位点的相互作用。（2）确定a 通过确定单个单个的扩散系数，CPSF6/PICS相互作用的作用在存在废除CA/CPSF6相互作用的药物和CA突变体的情况下的颗粒。（3）开发一个活细胞成像分析以可视化集成的VDNA，并将图片消失的位置与集成相关联。（4）确定CA中与病毒RNA或蛋白质相互作用的CA中的氨基酸残基，以及识别VRNP中CA的结合伙伴。这个新的研究方向将描述CA在图片与HIV-1整合位置的相互作用和核内运输，这是病毒寿命的重要一步循环仍然受到良好的欣赏。