The role of FEZ1 in early HIV-1 infection

虽然广泛使用的联合抗逆转录病毒疗法（cART）有效地延长了患者的寿命， 尽管有许多感染者，但HIV-1仍然是发达国家和贫困国家的一个主要公共卫生问题 资源设置。因此，了解其复制周期的基本机制有助于 开发治疗感染的新方法。HIV-1利用不寻常的，错综复杂的早期感染 涉及逆转录、衣壳核心的解体（也称为"脱壳"）和 运输到核。尽管其确切的时间和地点仍有争议，但越来越多的证据表明， 表明在转运到细胞核的过程中，细胞质中至少发生了部分脱膜。的确， 进入的HIV-1颗粒表现出基于微管（MT）的双向运动性，提示它们的关联 与内向（动力蛋白）和外向（驱动蛋白）MT马达，最近的研究表明，相反的 由这些马达产生的力促进了去涂层。尽管如此，HIV-1似乎并不与马达结合， 直接使用电机适配器，直到最近几年，其身份仍然是个谜。我们的工作 在上一个周期资助的一项研究中，将HIV-1驱动蛋白-1适配器鉴定为成束和延伸因子Zeta 1（FEZ 1）。我们进一步确定了FEZ1在进入的病毒颗粒的运输和脱壳中的核心作用 在天然靶细胞中，其通过控制驱动蛋白-1活性的FEZ1磷酸化来调节。 此外，我们发现HIV-1核心与微管相关调节激酶2（MARK2）结合， 控制病毒颗粒上的FEZ1磷酸化。我们进一步表明，HIV-1颗粒也高度结合 专门的MT调节蛋白，以诱导形成稳定的MT网络，MT丝的子集 受到驱动蛋白马达的青睐。利用创新的结构和功能研究与熊 在耶鲁大学的实验室，我们的初步数据揭示了HIV-1使用的一种常见的高亲和力结合策略， 接合FEZ1用于由衣壳六聚体和FEZ1中的四个卷曲螺旋结构域之一介导的转运。 数据还表明，FEZ1和MARK2以控制FEZ1的程度的方式竞争结合。 HIV-1衣壳上的磷酸化。此外，我们确定了一个新的宿主因子，我们的数据表明， FEZ1中不同的卷曲螺旋区域，并被传入的病毒颗粒利用，以增强MT的稳定性， 细胞外围。累积起来，我们的数据表明FEZ 1中不同的卷曲螺旋结构域介导了衣壳蛋白的表达。 结合，运动募集和MT稳定，以协调早期HIV-1转运的几个方面， 去涂层在这项提案中，我们的目标是确定FEZ 1和MARK 2如何在HIV-1衣壳上发挥作用， 促进早期感染，并扩展我们的新发现，FEZ1在早期感染中起多功能作用。 通过招募MT稳定性的马达和调节剂来进入HIV-1颗粒来感染。的结果 我们的研究将为FEZ 1的多功能性提供重要的机理见解，并扩大我们的研究范围。 更广泛地了解HIV-1如何控制天然靶细胞早期感染的几个重要步骤。

Although widespread use of combination antiretroviral therapy (cART) has effectively increased the life span of many infected individuals, HIV-1 continues to be a major public health issue in both developed and poor resource settings. As such, understanding the basic mechanisms of its replication cycle is instrumental to the development of new approaches to treat infection. HIV-1 employs unusual, intricately intertwined early infection strategies involving reverse transcription, disassembly of capsid core (also known as “uncoating”) and transport to the nucleus. Although its precise timing and location remain contentious, growing evidence suggests that at least partial uncoating occurs in the cytoplasm during transport to the nucleus. Indeed, incoming HIV-1 particles exhibit microtubule (MT) based bi-directional motility suggestive of their association with both inward (dynein) and outward (kinesin) MT motors, and recent studies suggest that the opposing forces generated by these motors facilitate uncoating. Despite this, HIV-1 does not appear to bind motors directly but instead, uses motor adaptors whose identity remained enigmatic until recent years. Our work funded in the previous cycle identified the HIV-1 kinesin-1 adaptor as Fasiculation and Elongation Factor Zeta 1 (FEZ1). We further established FEZ1’s central role in the transport and uncoating of incoming viral particles in natural target cells, which is regulated through FEZ1 phosphorylation that controls kinesin-1 activity. Moreover, we found that HIV-1 cores bind microtubule associated regulatory kinase 2 (MARK2) to locally control FEZ1 phosphorylation on viral particles. We further showed that HIV-1 particles also bind highly specialized MT regulatory proteins to induce the formation of stable MT networks, a subset of MT filaments favored by kinesin motors. Using innovative structural and functional studies in collaboration with the Xiong Lab at Yale University, our preliminary data reveals an usual and high affinity binding strategy used by HIV-1 to engage FEZ1 for transport that is mediated by capsid hexamers and one of four coiled-coil domains in FEZ1. Data also suggests that FEZ1 and MARK2 compete for binding in a manner that controls the extent of FEZ1 phosphorylation on HIV-1 capsids. In addition, we identify a new host factor that our data suggests binds distinct coiled-coil regions in FEZ1 and is exploited by incoming viral particles to enhance MT stabilization at the cell periphery. Cumulatively, our data suggests that distinct coiled-coil domains in FEZ1 mediate capsid binding, motor recruitment and MT stabilization to coordinate several aspects of early HIV-1 transport and uncoating. In this proposal, we aim to determine how FEZ1 and MARK2 function on the HIV-1 capsid to promote early infection and expand upon our new findings that FEZ1 plays a multifunctional role in early infection by recruiting both motors and regulators of MT stability to incoming HIV-1 particles. The outcome of our studies will provide important mechanistic insights into the multifunctionality of FEZ1 and expand our broader understanding of how HIV-1 controls several important steps in early infection of natural target cells.