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

项目摘要/摘要 早期HIV-1感染的几个方面既不寻常，也仍然知之甚少。尤其是艾滋病毒-1‘S 锥形病毒核心由衣壳蛋白的五聚体和六聚体组成，已知的是 亚稳定的，正在进行重组和CA损失，由逆转录驱动并促进 病毒基因组。然而，只有数量相对较少的宿主蛋白在结构和功能上都是 在它们如何约束和影响核心稳定性以及它们的约束策略方面具有良好的特点 所有这些都围绕着对六人组的认可展开。这包括微管运动适配器蛋白，HIV-1 利用间接结合马达蛋白来调节衣壳的稳定性和向细胞核的运输。我们的 最近的工作是在确定HIV-1的Kinesin-1接头为卷曲和延伸方面走在前列 Zeta-1因子与FEZ1‘S线圈中带负电荷氨基酸的测定 结构域介导与CA六聚体带正电的中心孔的结合。虽然结构的基础是 它的相互作用尚未确定，其他人随后发现另一种螺旋卷曲结构域蛋白， 双尾D同源2(BICD2)作为HIV-1‘S动力蛋白接头。独立地，我们发现 专门的微管调节蛋白，细胞质连接蛋白170(CLIP170)与HIV-1核心结合并 在体外组装的CA结构以一种独特的方式，不同于目前已知的辅助因子。 具体地说，与六聚体结合辅助因子不同，CLIP170结合到野生型CA组件的末端 并且还具有结合和稳定CA-R18L突变组件的独特能力，这些突变组件形成富含五聚体的 而不是富含六聚体的结构。此外，CLIP170与CA的主要同源区域(MHR)结合， 它在结构上是向内的，并预测不能与胞质辅助因子结合。然而，低温电子显微镜 成像显示，天然HIV-1核心含有断裂，而我们揭示了不寻常的五聚体组织 R18衍生的CA组件，它创建一个孔，使MHR结构域可以从外部访问 凯西德。由此，我们假设CLIP170在暴露于MHR时，通过 原生岩心或WT CA组件的CA晶格，或通过R18中形成的以前未识别的孔- 衍生的组件，并具有控制HIV-1亚稳态的功能。此外，我们的数据显示 动力蛋白1(DCTN1)是主要的动力蛋白接头复合体的关键成分，也起作用 独立地负向调节CLIP170的亲病毒功能。我们假设这会使 与HIV-1不相容的DCTN1‘S的目标是在使用时通过其六角体单独接触电机 CLIP170调节核心亚稳性，这是HIV-1转而使用BICD2的进化驱动因素 与Dynein交战。在这项提案中，我们使用了尖端的低温EM、生化和功能方法来 测试这些假设，并期待我们的发现将为控制宿主辅助因素提供新的见解 衣壳亚稳定，以及更好地理解为什么艾滋病毒-1利用不那么传统的运动适配器。

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.