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生命周期中的早期事件，如融合后病毒核心穿过细胞质的运输， 核孔复合物（NPC）和进入细胞核，仍然知之甚少，由于有限的信息 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，具有多种宿主细胞蛋白质 艾滋病感染（依赖因素）对这一过程至关重要。然而，它们的完整身份和分子 作用机制在很大程度上仍然未知。我们的初步研究结果如下两个 主要发现。1)我们已经将SEC 24 C确定为一种新的、关键的HIV-1宿主依赖性因子。 SEC 24 C是一种主要的细胞质蛋白，其采用苯丙氨酸-甘氨酸（FG）基序， 特异性地与六聚体CA晶格在由两个相邻的 亚单位。这些新的发现，加上其他CA结合FG基序的已知作用， 细胞因子NUP 153和CPSF 6在HIV-1的核输入和整合位点选择中的作用表明， 这些蛋白质共同为HIV-1在不同地区的持续旅程提供了可靠的平台， 在病毒侵入期间，细胞隔室以确保生产性感染。2)我们发现了朊病毒- SEC 24 C、NUP 153和CPSF 6与六聚体的亲和结合的PrLD介导的机制 CA格我们初步的cryo-EM和HDX-MS研究提供了新的结构信息， 除了已知的CPSF 6 FG肽与同源CA疏水口袋结合外， 相互作用使得CPSF 6分子能够沿着相邻CA的延伸晶格沿着进行多价组装 六聚体我们的病毒学实验进一步支持CPSF 6 PrLD在功能性病毒中的重要作用- 感染细胞中的宿主相互作用。为了扩展这些令人兴奋的、范式转变的初步研究，我们 提出了以下三个具体目标：目标1将确定SEC 24 C在HIV-1感染中的作用;目标2将 阐明SEC 24 C和其他CA结合宿主因子之间的相互作用;目的3将确定结构 含FG基序和PrLD的细胞因子对六聚体HIV-1 CA晶格的亲和识别基础 SEC 24 C、NUP 153和CPSF 6。为了实现这些目标，我们组建了一个高度合作的团队 在病毒学、蛋白质组学、生物化学和X射线晶体学方面具有互补的专业知识 （Kvaratskhelia）、冷冻-EM（Asturias）、活细胞显微术（Melikian）和HDX-MS（Griffin）。这些研究 有望揭示HIV-1感染的关键病毒-宿主相互作用机制。此外，委员会认为， 阐明SEC 24 C、NUP 153和CPSF 6的功能相关相互作用的结构决定因素 六聚体CA晶格将为HIV-1结构生物学提供一个新的前沿，并改善我们的研究。 理解这些病毒-宿主相互作用作为重要的治疗靶点。