Elucidation of HIV Env acquisition strategies

描述(申请人提供)：艾滋病毒-1感染已引起一场全球大流行，夺走了2000多万人的生命，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发生病毒萌发的准确位置。尽管Env和结构蛋白(Gag)之间的直接相互作用被认为是导致这种募集的原因，但长期以来人们一直认为许多外源病毒糖蛋白也有效地整合到病毒颗粒中。由于这些外来病毒糖蛋白与HIV-1Env之间没有序列相似性，因此必须存在其他机制，可能涉及宿主细胞机制，以促进这种联合组装。我们推测，HIV-1用来招募外来病毒糖蛋白的机制也被用来招募其本土的Env蛋白。对这些机制的理解将有助于了解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)的分析方法，可以定性和定量地成像细胞表面的Env分布，同时还可以成像单个HIV-1萌芽部位的位置。使用这项检测，我们可以清楚地表明，在病毒萌发部位，病毒Env被浓缩了50倍以上。可以观察到天然的和一些外来的Env蛋白都可以浓缩。截断Env的细胞质尾部可以消除萌发部位的浓缩，但不能消除被动的Env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(Matrx)被非逆转录病毒膜结合域取代时，也可以在萌芽位置发生Env的浓缩。这种新的扫描电子显微镜环境分布分析，以及其他测试，将被用于研究HIV-1是如何获得环境病毒的。1)我们将首先确定不同的病毒糖蛋白，它们保留了被招募到HIV-1萌芽部位的能力。因为这些外来蛋白质不太可能直接与HIV-1 Gag结合，所以对这些蛋白质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，以确定促进萌发部位招募所需的特定结构域。3)HIV-1 Gag的各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的哪些组成部分有助于环境识别。非典型HIV-1感染已导致全球大流行，已夺走2000多万人的生命，并感染4000多万人。人们早就认识到，包括艾滋病毒在内的病毒具有不同的机制，可以将所有病毒成分招募到细胞内正确的病毒组装位置，但这些机制仍然知之甚少。对这些机制的理解将有助于阐明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.