Determining the role of microtubules and motor proteins during early HIV-1 replication

确定微管和马达蛋白在HIV-1早期复制中的作用 摘要 在感染HIV-1期间，病毒膜与宿主细胞膜融合，将HIV-1病毒传递给宿主 细胞质。一旦进入细胞，HIV-1必须移位到细胞核，使逆转录的DNA 被整合到宿主基因组中。尽管致力于HIV-1感染的研究已有40多年，但令人惊讶的是 关于HIV-1是如何利用宿主细胞的细胞骨架来促进转运到细胞核和 加速感染。这项提案的目标是建立和验证一种检测HIV-1病毒的方法 微管运动蛋白和动力蛋白在体外的微管转运。最近的工作导致了 鉴定两个宿主因子-FEZ1和BicD2-作为通过Kinesin-1运输的货物适配器 胞浆动力蛋白-1。为了研究FEZ1和BicD2在HIV-1运输中的作用，我们将 利用单分子全内反射荧光显微镜监测药物的转运 重组微管上荧光标记的病毒-马达复合体(目标1)。我们将用一本小说 从胶囊中提取的合成二十面体支架用于验证该分析，我们将使用该分析来测量 通过货物转接器与病毒货物结合的运动蛋白质组。然后我们将确定HIV-1是如何 通过测量FEZ1的联合影响来确定微管上的方向性(动蛋白与动力蛋白)， BicD2、激动素-1和动力蛋白(目标2)。通过确定微管马达能够运输 这一建议将为HIV-1的研究提供一条新的途径，并提供新的靶点来阻断 HIV-1感染。

Determining the role of microtubules and motor proteins during early HIV-1 replication SUMMARY During HIV-1 infection, viral membrane fuses with the host cell membrane to deliver the HIV-1 virus to the host cell cytoplasm. Once within the cell, HIV-1 must translocate to the nucleus for the reverse transcribed DNA to be incorporated into the host genome. Despite 40+ years of research devoted to HIV-1 infection, surprisingly little is known about how HIV-1 exploits the host cell cytoskeleton to facilitate transport to the nucleus and accelerate infection. The goal of this proposal is to establish and validate an assay to measure HIV-1 microtubule trafficking in vitro via kinesin and dynein microtubule motor proteins. Recent work has led to the identification of two host factors - FEZ1 and BicD2 - that serve as cargo adaptors for transport via kinesin-1 and cytoplasmic dynein-1, respectively. To study the role of FEZ1 and BicD2 in the transport of HIV-1, we will utilize single-molecule total internal reflection fluorescence (TIRF) microscopy to monitor the transport of fluorescently-labeled virus-motor complexes on reconstituted microtubules (Aim 1). We will use a novel synthetic icosahedral scaffold derived from encapsulins to validate the assay, which we will use to measure the motility of motor protein teams bound to viral cargo via cargo adaptors. We will then determine how HIV-1 determines directionality on microtubules (kinesin vs. dynein) by measuring the combined influences of FEZ1, BicD2, kinesin-1, and dynein (Aim 2). By establishing that microtubule motors are capable of transporting HIV-1 in vitro, this proposal will provide a new avenue in the study of HIV-1 and provide new targets to block HIV-1 infection.