Delineating HIV-1 nuclear import mechanisms through capsid interaction with MxB and the nuclear pore complex

摘要 人类免疫缺陷病毒1型(HIV-1)会导致终身感染，这需要服用抗病毒药物来 控制感染。目前还没有针对这种病毒的疫苗。HIV-1锥形衣壳核心，由 衣壳蛋白(CA)，保护HIV-1基因组，在HIV-1感染中起主要作用，并与几个 宿主因子参与病毒复合体的核输入和整合到宿主基因组中。粘病毒 抗性蛋白B(MXB)被认为通过抑制脱壳和阻止核进入来限制HIV-1感染， 后者是通过核内的2个长末端重复(2-LTR)圆来测量的。然而，MXB并非如此 即使在感染被抑制的情况下，也能抑制感染细胞的细胞核内CA的出现。另外， 作为HIV-1核进口的标志，测量细胞核中的2-LTR环与 生殖性感染。目前，人们对HIV-1脱壳的动力学和部位以及MXB的作用知之甚少 在调控这一过程中。HIV-1核进口也没有完全被理解。几种核孔素(NUP)， 形成核孔复合体(NPC)，与CA相互作用促进HIV-1核输入。全国人大是 以前被认为是内径约40纳米的静态结构，太小了，不能允许核进口 60纳米宽的HIV-1衣壳核心，导致假设HIV-1在进入细胞核之前必须脱去外壳。 然而，最近的证据表明，鼻咽癌是一个动态的结构，可以扩张，而艾滋病毒-1锥体- 成形的核心可以进入原子核。关于NPC和MXB是如何与HIV-1 CA相互作用的知之甚少 分别促进或抑制HIV-1感染。因此，我假设鼻咽癌相关的MXB阻断 禁止去涂层的核进口，以及艾滋病毒-1核心的核进口需要扩大 核孔复合体。在这项提议的两个具体目标中，我将调查艾滋病毒-1核进口 和CA核心在MXB存在下的解体(目标1)，并描绘了核孔结构的变化 由艾滋病毒-1引起的(目标2)使用先进的显微镜技术。在目标1中，我将确定艾滋病毒-1核进入 荧光标记整合酶-超折叠绿色荧光蛋白(INsfGFP)对MXB的抑制作用 在原子核内部。我还将通过可视化丢失荧光标记的亲环素来检查HIV-1未包被的情况 一种与CA结合的衍生物，亲和力很高。在目标2中，我将研究核孔大小的变化 用超分辨显微镜观察HIV-1感染情况。我将用HIV-1 CA突变体感染NUP标记的细胞，它 不能转运到核质中，并与鼻咽癌相关。然后我会想象全国人大 受激发射耗尽(STED)显微镜和随机光学重建显微镜 (暴风雨)。这个项目将为我提供不同的病毒学、细胞生物学和高级显微镜方面的培训 技术，并将促进我们对艾滋病毒-1核进口机制的理解。从长远来看，我的工作 可支持开发可减轻HIV-1感染的新型抗病毒药物。

ABSTRACT Human Immunodeficiency Virus 1 (HIV-1) causes life-long infection, which requires taking antiviral drugs to control the infection. No vaccines are available against this virus. The HIV-1 cone-shaped capsid core, made up of capsid protein (CA), protects the HIV-1 genome, plays a major role in HIV-1 infection and interacts with several host factors involved in nuclear import of viral complexes and integration into the host genome. The myxovirus resistance protein B (MxB) is thought to restrict HIV-1 infection by inhibiting uncoating and blocking nuclear entry, the latter measure by 2-Long Terminal Repeats (2-LTR) circles inside the nucleus. However, MxB does not inhibit the appearance of CA inside the nucleus of infected cells, even when infection is inhibited. Additionally, measuring 2-LTR circles in the nucleus, which is a marker for HIV-1 nuclear import, does not correlate with productive infection. Currently, little is known about the kinetics and sites of HIV-1 uncoating and the role of MxB in regulating this process. HIV-1 nuclear import is also not completely understood. Several nucleoporins (NUPs), which form the nuclear pore complex (NPC), interact with CA to promote HIV-1 nuclear import. The NPC was previously thought to be a static structure with an inner diameter of ~40nm, too small to allow nuclear import of the 60nm-wide HIV-1 capsid core, leading to an assumption that HIV-1 has to uncoat before entering the nucleus. However, recent evidence suggests that NPC is a dynamic structure that may expand and that HIV-1 cone- shaped cores can enter the nucleus. Little is known about how the NPC and MxB interactions with HIV-1 CA respectively promote or inhibit HIV-1 infection. Therefore, I hypothesize that the NPC-associated MxB blocks nuclear import by inhibiting uncoating and that the nuclear import of HIV-1 core requires expansion of the nuclear pore complex. In the two Specific Aims of this proposal, I will investigate the HIV-1 nuclear import and CA core disassembly in the presence of MxB (Aim 1) and delineate the changes in nuclear pore architecture caused by HIV-1 (Aim 2) using advance microscopy techniques. In Aim 1, I will determine HIV-1 nuclear entry inhibition by MxB through visualizing the presence of fluorescently labeled integrase-superfolderGFP (INsfGFP) inside the nucleus. I will also examine HIV-1 uncoating by visualizing loss of a fluorescently labeled cyclophilin A derivative, which binds to CA with high avidity. In Aim 2, I will examine changes in the nuclear pore size upon HIV-1 infection using super-resolution microscopy. I will infect NUP-labeled cells with HIV-1 CA mutant, which fails to transport to the nucleoplasm and remains associated with NPC. I will then visualize the NPC with Stimulated Emission Depletion (STED) microscopy and Stochastic Optical Reconstruction Microscopy (STORM). This project will provide me with diverse training in virology, cell biology and advanced microscopy techniques and will advance our understanding of HIV-1 nuclear import mechanisms. In the long-term, my work may support development of novel antiviral drugs that can mitigate HIV-1 infection.