Elucidation of HIV Env acquisition strategies

描述（由申请人提供）：HIV-1感染已造成全球性大流行，夺去了2000多万人的生命，并感染了4000多万人。我们的长期目标是了解HIV-1是如何组装成病毒颗粒的，以及在这个过程中利用了什么样的细胞机制。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。尽管Env和结构蛋白（Gag）之间的直接相互作用被认为有助于这种募集，但长期以来人们已经认识到许多外源病毒糖蛋白也有效地掺入病毒颗粒中。由于这些外来病毒糖蛋白和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，以促进这种共组装。我们推测HIV-1招募外源病毒糖蛋白的机制也被用于招募其天然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，可以对细胞表面上的Env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。使用该测定，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。可以用天然以及一些外源Env蛋白观察到富集。截短Env的胞质尾区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，即使当被认为与Env相互作用的Gag结构域（mativity）被非逆转录病毒膜结合结构域取代时，也可以发生Env在出芽位点的富集。这种新的SEM Env分布测定以及其他测定将用于研究HIV-1如何获得Env。1)我们将首先确定不同的病毒糖蛋白，保留被招募到HIV-1出芽位点的能力。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)这些糖蛋白将被解剖，以确定特定的结构域所需的促进招募到出芽位点。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的哪些组分有助于Env识别。叙述性HIV-1感染已导致全球大流行，夺去了2000多万人的生命，并感染了4000多万人。长期以来，人们已经认识到，包括HIV在内的病毒具有将所有病毒组分募集到细胞内正确的病毒组装位点的独特机制，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.