Roles of HIV-1 capsid-binding FG-motif containing cellular cofactors in infection

含有细胞辅助因子的 HIV-1 衣壳结合 FG 基序在感染中的作用

• 批准号: 10569058
• 负责人: Francisco J Asturias
• 金额: $ 78.29万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-08 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10569058
• 关键词: Affinity; Binding; Binding Proteins; Binding Sites; Biochemistry; Biological Assay; Biology; Capsid; Capsid Proteins; Cell Nucleus; Cells; Complement; Complex; Cone; Coupled; Cryoelectron Microscopy; Cytoplasm; Cytoplasmic Protein; Dependence; Drug Industry; Ensure; Event; Foundations; Future; Genetic Code; Glycine; Goals; HIV-1; Highly Active Antiretroviral Therapy; Hydrophobicity; In Vitro; Infection; Integration Host Factors; Knowledge; Label; Life Cycle Stages; Mediating; Molecular; Molecular Mechanisms of Action; Monitor; Nuclear Import; Nuclear Pore Complex; Peptides; Phase III Clinical Trials; Phenylalanine; Productivity; Proteins; Proteomics; Recombinants; Reporting; Role; Shapes; Surface; Testing; Time; Tube; Tubular formation; Viral; Virus; Work; X-Ray Crystallography; activation-induced cytidine deaminase; base; cofactor; experimental study; frontier; improved; inhibitor; insight; integration site; live cell microscopy; next generation; novel; novel virus; prion-like; recruit; retrograde transport; spatiotemporal; structural biology; structural determinants; synergism; therapeutic target; trafficking; virology; virus host interaction

Abstract Early events in HIV-1 lifecycle, such as post-fusion trafficking of viral cores across the cytoplasm, through the nuclear pore complex (NPC) and into the nucleus, remain poorly understood due to limited information about virus-host interactions. Interactions of the core’s surface, which is composed of the capsid protein (CA) arranged into large hexameric lattices and exactly 12 pentamers, with a variety of host cell proteins that aid infection (dependency factors) are crucial for this journey. However, their full identity and molecular mechanisms of action remain largely unknown. Our preliminary studies resulted in the following two principal discoveries. 1) We have identified SEC24C as a new, crucial HIV-1 host dependency factor. SEC24C is a predominantly cytoplasmic protein that employs a phenylalanine-glycine (FG)-motif to specifically interact with the hexameric CA lattice at the hydrophobic pocket comprised of two adjoining subunits. These novel findings, coupled with the known roles of other CA-binding FG-motif containing cellular factors NUP153 and CPSF6 in nuclear import and integration site selection of HIV-1, suggest that these proteins collectively provide a dependable platform for continuous HIV-1 journey throughout different cellular compartments during the virus ingress to ensure productive infection. 2) We discovered a prion- like domain (PrLD)-mediated mechanism for avid binding of SEC24C, NUP153 and CPSF6 to hexameric CA lattices. Our preliminary cryo-EM and HDX-MS studies provide novel structural information indicating that, in addition to known CPSF6 FG peptide binding to the cognate CA hydrophobic pocket, PrLD-PrLD interactions enable polyvalent assembly of CPSF6 molecules along the extended lattices of adjoining CA hexamers. Our virology experiments further support an essential role of CPSF6 PrLD in functional virus- host interactions in infected cells. To extend these exciting, paradigm-shifting preliminary studies we propose the following three specific aims: Aim 1 will define a role of SEC24C in HIV-1 infection; Aim 2 will elucidate interplay between SEC24C and other CA-binding host factors; Aim 3 will determine the structural basis for avid recognition of hexameric HIV-1 CA lattices by FG-motif and PrLD containing cellular factors SEC24C, NUP153 and CPSF6. To accomplish these aims we have assembled a highly collaborative team with complementary expertise in virology, proteomics, biochemistry and X-ray crystallography (Kvaratskhelia), cryo-EM (Asturias), live cell microscopy (Melikian) and HDX-MS (Griffin). These studies are expected to uncover novel virus-host interaction mechanisms crucial for HIV-1 infection. Furthermore, elucidating structural determinants for functionally relevant interactions of SEC24C, NUP153 and CPSF6 to hexameric CA lattices will provide a new frontier in HIV-1 structural biology and improve our understanding of these virus-host interactions as an important therapeutic target.

摘要 HIV-1生命周期的早期事件，如融合后病毒核心跨细胞质运输，通过 由于信息有限，人们对核孔复合体(NPC)和进入细胞核的分子仍知之甚少 关于病毒与宿主的相互作用。由衣壳蛋白组成的核心表面的相互作用 (CA)排列成大的六聚体晶格和正好12个五聚体，带有各种宿主细胞蛋白质 这种辅助感染(依赖因素)对这一旅程至关重要。然而，它们的完整身份和分子 行动机制在很大程度上仍然不为人知。我们的初步研究产生了以下两个结果 主要发现。1)我们已经确定SEC24C是一种新的、关键的HIV-1宿主依赖因子。 SEC24C是一种主要的细胞质蛋白，它利用苯丙氨酸-甘氨酸(FG)-基序 在疏水口袋处与六聚体CA晶格特别地相互作用 亚单位。这些新的发现，再加上其他CA结合FG基序的已知作用，包含 细胞因子NUP153和CPSF6在HIV-1核进口和整合位点选择中的作用 这些蛋白质共同为HIV-1在不同时期的持续旅程提供了可靠的平台 在病毒进入过程中的细胞隔间，以确保有效感染。2)我们发现了一个普里恩- 类结构域(PrLD)介导的SEC24C、NUP153和CPSF6与六聚体亲和结合机制 CA晶格。我们的冷冻-EM和HDX-MS初步研究提供了新的结构信息 除了已知的CPSF6 FG肽与同源CA疏水口袋结合外，PrLD-PrLD 相互作用使CPSF6分子能够沿着相邻CA的扩展晶格进行多价组装 六角人。我们的病毒学实验进一步支持CPSF6 PrLD在功能病毒中的重要作用- 感染细胞中的宿主相互作用。为了扩展这些令人兴奋的、改变范式的初步研究，我们 提出以下三个具体目标：目标1将确定SEC24C在艾滋病毒-1感染中的作用；目标2将 阐明SEC24C和其他CA结合宿主因子之间的相互作用；目标3将决定结构 含细胞因子的FG-Motif和PrLD对HIV-1六聚体CA晶格的亲和识别基础 SEC24C、NUP153和CPSF6。为了实现这些目标，我们组建了一个高度协作的团队 在病毒学、蛋白质组学、生物化学和X射线结晶学方面具有互补的专业知识 (Kvaratskhelia)、冷冻-EM(Asturias)、活细胞显微镜(Melikian)和HDX-MS(Griffin)。这些研究 有望发现对HIV-1感染至关重要的新的病毒-宿主相互作用机制。此外， SEC24C、NUP153和CPSF6功能相关相互作用的结构决定因素 六面体CA晶格将为HIV-1结构生物学提供一个新的前沿，并改善我们的 了解这些病毒与宿主的相互作用是一个重要的治疗目标。