+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.

项目总结/摘要 早期HIV-1感染的几个方面既不寻常，也仍然知之甚少。特别是HIV-1 锥形病毒核心由衣壳（CA）蛋白的五聚体和六聚体组成， 亚稳态，经历重组和CA损失，这是由逆转录驱动，并促进 病毒基因组然而，只有相对少数的宿主蛋白质在结构上和功能上都是如此。 在它们如何结合和影响核心稳定性方面得到了很好的表征，并确定了它们的结合策略 都是围绕六聚体的识别。这包括微管马达衔接蛋白，HIV-1 利用间接接合马达蛋白来调节衣壳稳定性和向细胞核的转运。我们 最近的工作是在确定HIV-1的驱动蛋白-1适配器为成束和延伸方面处于领先地位 因子Zeta-1（FEZ 1）及其卷曲螺旋中带负电荷氨基酸的测定 结构域介导与CA六聚体的带正电荷的中心孔的结合。虽然结构基础 其相互作用尚未确定，其他人随后发现另一种卷曲螺旋结构域蛋白， 双尾D同源物2（BICD 2）充当HIV-1的动力蛋白衔接子。独立地，我们发现 一种专门的微管调节蛋白，细胞质连接蛋白170（CLIP 170）结合HIV-1核心， 以不同于目前已知的辅因子的独特方式体外组装CA结构。 具体而言，与六聚体结合辅因子不同，CLIP 170结合野生型CA组装体的末端 并且还具有结合和稳定CA-R18 L突变体组装体的独特能力，所述CA-R18 L突变体组装体形成富含五聚体的 而不是富含六聚体的结构。此外，CLIP 170结合CA的主要同源区（MHR）， 其在结构上向内取向并且被预测为细胞溶质辅因子不可接近。然而，cryoEM 成像显示，天然HIV-1核心含有断裂，而我们发现， R18衍生的CA组装体，其产生使MHR结构域可从细胞外进入的孔。 衣壳。由此，我们假设CLIP 170在暴露后通过细胞中的自然断裂识别MHR。 天然核心或WT CA组装体的CA晶格，或通过R18中形成的先前未识别的孔隙- 衍生的组装体，然后控制HIV-1亚稳态的功能。此外，我们的数据显示 Dynactin 1（DCTN 1）是主要动力蛋白接头复合物Dynactin的关键成分， 独立地负调节CLIP 170的前病毒功能。我们假设这使得 DCTN 1与HIV-1的目标不相容，即在使用时通过其六聚体单独接合马达。 CLIP 170调节核心亚稳定性，这是HIV-1使用BICD 2的进化驱动力 与Dynein交战在这项提案中，我们采用尖端的cryoEM，生物化学和功能方法， 测试这些假设，并预计我们的研究结果将提供新的见解主机辅因子，控制 衣壳亚稳定性沿着更好地理解为什么HIV-1利用不太传统的马达适配器。