+TIPs as novel host capsid-binding co-factors in early HIV-1 infection

TIP 作为早期 HIV-1 感染中新型宿主衣壳结合辅助因子

• 批准号: 10709142
• 负责人: Mojgan Hosseini Naghavi
• 金额: $ 74.91万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-09 至 2028-02-29
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10709142
• 关键词: Adaptor Signaling Protein; Amino Acids; Area; Automobile Driving; Binding; Binding Sites; Biochemical; Biochemistry; CLIP-170 gene; Capsid; Capsid Proteins; Cell Nucleus; Charge; Coiled-Coil Domain; Complement; Complex; Cone; Conflict (Psychology); Cryoelectron Microscopy; Cyclophilin A; Cytoplasmic Protein; Cytosol; Data; Dynein ATPase; Elongation Factor; Goals; HIV-1; Homologous Gene; Image; In Vitro; Infection; Integration Host Factors; Intracellular Transport; Kinesin; Left; Mediating; Microtubules; Modeling; Motor; Movement; Process; Proteins; Research; Shapes; Structure; Tertiary Protein Structure; Testing; Tubular formation; Viral; Viral Genome; Viral Reverse Transcription; Work; cofactor; dynactin; dynactin 1; genetic regulatory protein; insight; mutant; novel; particle; recruit

PROJECT SUMMARY/ABSTRACT Several aspects of early HIV-1 infection are both unusual and still poorly understood. In particular, HIV-1's cone-shaped viral core consists of pentamers and hexamers of capsid (CA) protein and is known to be metastable, undergoing restructuring and CA loss that is driven by and facilitates reverse transcription of the viral genome. Yet only a relatively small number of host proteins have been both structurally and functionally well-characterized in terms of how they bind to and influence core stability, and to date their binding strategies all center around recognition of hexamers. This includes microtubule motor adaptor proteins, which HIV-1 exploits to indirectly engage motor proteins to regulate both capsid stability and transport to the nucleus. Our recent work was at the forefront in identifying the Kinesin-1 adaptor for HIV-1 as Fasiculation and Elongation Factor Zeta-1 (FEZ1) and determining that negatively charged amino acids in one of FEZ1's coiled-coil domains mediate binding to the positively charged central pore of CA hexamers. While the structural basis of its interactions have yet to be determined, others subsequently found that another coiled-coil domain protein, Bicaudal D Homolog 2 (BICD2) acts as HIV-1's Dynein adaptor. Independently, we discovered that the specialized microtubule regulatory protein, Cytoplasmic Linker Protein 170 (CLIP170) binds to HIV-1 cores and in vitro assembled CA structures in a unique manner that is distinct from currently known co-factors. Specifically, unlike hexamer-binding co-factors, CLIP170 binds to the extreme ends of wildtype CA assemblies and also has a unique ability to bind and stabilize CA-R18L mutant assemblies, which form pentamer-rich rather than hexamer-rich structures. Moreover, CLIP170 binds to the Major Homology Region (MHR) of CA, which is structurally oriented inward and predicted to be inaccessible to cytosolic co-factors. However, cryoEM imaging reveals that native HIV-1 cores contain breaks while we reveal unusual pentamer organizations in R18-derived CA assemblies that create a pore which makes the MHR domain accessible from outside the capsid. From this, we hypothesize that CLIP170 recognizes the MHR upon exposure by natural breaks in the CA lattice of native cores or WT CA assemblies, or through previously unrecognized pores that form in R18- derived assemblies, and functions to then control the HIV-1 metastable state. Furthermore, our data shows that Dynactin 1 (DCTN1), a key component of the primary Dynein adaptor complex, Dynactin, also functions independently to negatively regulate the pro-viral functions of CLIP170. We hypothesize that this makes DCTN1 incompatible with HIV-1's goal of separately engaging motors through its hexamers while using CLIP170 to regulate core metastability, and that this was an evolutionary driver for HIV-1 to instead use BICD2 to engage Dynein. In this proposal, we employ cutting-edge cryoEM, biochemical and functional approaches to test these hypotheses and anticipate that our findings will provide new insights into host co-factors that control capsid metastability along with a better understanding of why HIV-1 exploits less conventional motor adaptors.

项目摘要/摘要 早期HIV-1感染的几个方面都是不寻常的，而且仍然很少了解。特别是HIV-1 锥形病毒核心由五角体和六聚体（CA）蛋白质组成，已知为 可稳定的，进行重组和CA损失，并由 病毒基因组。然而，在结构和功能上，只有相对较少的宿主蛋白既是 在其与核心稳定性的结合和影响核心稳定性方面的特征良好 所有这些都围绕着对六聚体的认可。这包括微管电机适配器蛋白，HIV-1 利用间接接触运动蛋白以调节衣壳稳定性和转运到细胞核。我们的 最近的工作在识别HIV-1的驱动蛋白-1适配器作为效率和伸长率方面处于最前沿 因子Zeta-1（FEZ1），并确定FEZ1盘绕螺旋的一种负电荷的氨基酸 域介导与CA六聚体的正负中心孔的结合。而结构性基础 它的相互作用尚未确定，其他人随后发现另一种盘绕螺旋域蛋白， Bicaudal D同源2（BICD2）充当HIV-1的Dynein适配器。独立地，我们发现 专业的微管调节蛋白，细胞质接头蛋白170（CLIP170）与HIV-1核结合，并结合 体外组装的Ca结构的独特方式与当前已知的共同因素不同。 具体而言，与六聚体结合的联合因子不同，夹子170与野生型Ca组件的极端结合 并且具有绑定和稳定CA-R18L突变体组件的独特能力，形成了五角星富含五聚体的能力 而不是丰富的结构。此外，CLIP170与CA的主要同源区（MHR）结合， 它是在结构上向内的，并且预测是胞质辅助因子无法访问的。但是，冷冻 成像揭示了本地HIV-1核心包含休息时间，而我们在 R18衍生的CA组件创建孔，使MHR域可从外部访问 衣壳。由此，我们假设Clip170在自然断裂暴露时识别MHR 天然核心或WT CA组件的Ca晶格，或通过以前未识别的孔在R18-中形成的毛孔 派生的组件和功能可以控制HIV-1亚稳态。此外，我们的数据显示 Dynactin 1（DCTN1），主要动力蛋白适配器复合物的关键成分，dynactin也起作用 独立地调节CLIP170的促病毒功能。我们假设这使得 DCTN1与HIV-1的目标不兼容，即在使用时通过其六聚体分别吸引电动机 CLIP170调节核心的亚稳定性，这是HIV-1的进化驱动器，而是使用BICD2 参与Dynein。在此提案中，我们采用了尖端的冷冻，生化和功能性的方法 检验这些假设，并预测我们的发现将为控制托管副因素提供新的见解 衣壳的亚稳定性以及更好地理解HIV-1为何利用较少传统的运动适配器。