Elucidation of HIV Env acquisition strategies

说明（由申请人提供）：艾滋病毒-1感染已在世界范围内造成流行病，夺去了2 000多万人的生命，另有4 000多万人受到感染。我们的长期目标是了解HIV-1如何组装成病毒颗粒，以及在这个过程中利用了什么细胞机制。在HIV-1组装过程中，跨膜表面糖蛋白（Env）必须运输到宿主细胞内病毒出芽发生的精确位置。虽然人们认为Env和结构蛋白（Gag）之间的直接相互作用有助于这种招募，但人们早就认识到许多外来病毒糖蛋白也有效地结合到病毒颗粒中。由于这些外来病毒糖蛋白与HIV-1 Env之间没有序列相似性，因此必须有其他机制（可能涉及宿主细胞机制）促进这种共组装。我们假设HIV-1募集外源病毒糖蛋白的机制也被用于募集其原生的Env蛋白。对这些机制的理解将揭示HIV-1在组装过程中如何与宿主细胞相互作用，并可能导致抗病毒治疗的新靶点。我们开发了一种基于扫描电子显微镜（SEM）的检测方法，可以对Env在细胞表面的分布进行定性和定量成像，同时对单个HIV-1出芽位点的位置进行成像。利用这个实验，我们可以清楚地表明，病毒Env在病毒出芽位点富集超过50倍。可以观察到天然和一些外源Env蛋白的富集。截断Env的细胞质尾部可以消除出芽位点的富集，但不能消除Env的被动掺入。令人惊讶的是，即使被认为与Env（基质）相互作用的Gag结构域被非逆转录病毒膜结合结构域取代，出芽位点的Env富集也会发生。这种新型的SEM Env分布分析以及其他分析将用于研究HIV-1如何获得Env。1)我们将首先确定保留被招募到HIV-1出芽位点能力的不同病毒糖蛋白。由于这些外源蛋白不太可能直接与HIV-1 Gag结合，因此使用这些蛋白的研究不太可能受到多种相互作用模式的阻碍。2)将对这些糖蛋白进行解剖，以确定促进向出芽位点招募所需的特定结构域。3) HIV-1 Gag的单个结构域将与互补的逆转录病毒和非逆转录病毒结构域交换，以确定HIV-1 Gag的哪些成分有助于Env识别。艾滋病毒-1感染已造成世界范围的大流行病，夺去了2 000多万人的生命，另有4 000多万人受到感染。人们早就认识到，包括HIV在内的病毒具有独特的机制来招募所有病毒成分到细胞内正确的病毒组装位点，但这些机制仍然知之甚少。对这些机制的理解将揭示HIV-1如何与宿主细胞相互作用，并可能导致抗病毒治疗的新靶点。

DESCRIPTION (provided by applicant): HIV-1 infections have caused a worldwide pandemic that has claimed the lives of over 20 million people and infected over 40 million more. Our long term goal is to understand how HIV-1 assembles into a viral particle and what cellular machinery is utilized in this process. During HIV-1 assembly, the transmembrane surface glycoprotein (Env) has to traffic to the precise location within the host cell where viral budding occurs. Although a direct interaction between Env and the structural protein (Gag) is believed to contribute to this recruitment, it has long been recognized that many foreign viral glycoproteins are also efficiently incorporated into viral particles. Since there is no sequence similarity between these foreign viral glycoproteins and HIV-1 Env, there must be additional mechanisms, likely involving host cell machinery, that facilitate this co-assembly. We hypothesize that the mechanisms utilized by HIV-1 to recruit foreign viral glycoproteins are also utilized to recruit its native Env protein. An understanding of these mechanisms will shed light on how HIV-1 interacts with the host cell during the assembly process and could lead to new targets for antiviral therapies. We have developed a scanning electron microscopy (SEM)-based assay that allows qualitative and quantitative imaging of the distribution of Env on the cell surface while simultaneously imaging the location of individual HIV-1 budding sites. Using this assay, we can clearly show that viral Env is enriched over 50-fold at viral budding sites. Enrichment can be observed with native as well as some foreign Env proteins. Truncation of the cytoplasmic tail of Env can eliminate enrichment at budding sites, but it does not eliminate passive Env incorporation. Surprisingly, enrichment of Env at budding sites can occur even when the domain of Gag believed to interact with Env (matirx) is replaced with a non-retroviral membrane-binding domain. This novel SEM Env distribution assay, as well as other assays, will be used to study how Env acquisition by HIV-1 occurs. 1) We will first identify divergent viral glycoproteins that retain the ability to be recruited to HIV-1 budding sites. Because such foreign proteins are unlikely to directly bind to HIV-1 Gag, studies with these proteins are less likely to be hampered by multiple modes of interaction. 2) These glycoproteins will be dissected to determine the specific domains required to facilitate recruitment to budding sites. 3) The individual domains of HIV-1 Gag will be exchanged with complementing retroviral and non-retroviral domains to determine what components of HIV-1 Gag contribute to Env recognition.NARRATIVE HIV-1 infections have caused a worldwide pandemic that has claimed the lives of over 20 million people and infected over 40 million more. It has long been recognized that viruses, including HIV, have distinct mechanisms for recruiting all the viral components to the correct viral assembly site within the cell, but these mechanisms remain poorly understood. An understanding of these mechanisms will shed light on how HIV-1 interacts with the host cell and could lead to new targets for antiviral therapies.