Elucidation of HIV Env acquisition strategies

HIV Env 获取策略的阐明

• 批准号: 7749936
• 负责人: Marc C Johnson
• 金额: $ 29.53万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-01-01 至 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7749936
• 关键词: Affect; Agreement; Antiviral Therapy; Binding; Biological Assay; Cell surface; Cells; Collaborations; Complement; Cytoplasmic Tail; Data; Future; Gagging; Glycoproteins; Goals; Grant; HIV; HIV-1; Image; Individual; Infection; Lead; Life; Light; Location; Maps; Membrane; Membrane Glycoproteins; PH Domain; Process; Proteins; Published Comment; Recruitment Activity; Scanning Electron Microscopy; Signal Transduction; Site; Structural Protein; Tertiary Protein Structure; Trematoda; Viral; Virus; Work; Writing; base; design; env Gene Products; novel; pandemic disease; particle; research study; sound; trafficking

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.

描述（由申请人提供）：HIV-1 感染已引起世界范围内的大流行，已夺去了超过 2000 万人的生命，并感染了超过 4000 万人。我们的长期目标是了解 HIV-1 如何组装成病毒颗粒以及在此过程中使用了哪些细胞机制。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，但人们早已认识到许多外源病毒糖蛋白也能有效地掺入病毒颗粒中。由于这些外源病毒糖蛋白和 HIV-1 Env 之间不存在序列相似性，因此必须存在其他机制（可能涉及宿主细胞机制）来促进这种共组装。我们假设 HIV-1 用于招募外源病毒糖蛋白的机制也用于招募其天然 Env 蛋白。了解这些机制将有助于了解 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) 的检测方法，可以对细胞表面的 Env 分布进行定性和定量成像，同时对各个 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包膜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。 Env 细胞质尾部的截断可以消除​​出芽位点的富集，但不能消除被动 Env 掺入。令人惊讶的是，即使当被认为与 Env (matirx) 相互作用的 Gag 结构域被非逆转录病毒膜结合结构域取代时，Env 在出芽位点的富集也会发生。这种新颖的 SEM Env 分布测定以及其他测定将用于研究 HIV-1 获取 Env 是如何发生的。 1) 我们将首先鉴定出不同的病毒糖蛋白，这些糖蛋白保留了被招募到 HIV-1 出芽位点的能力。由于此类外源蛋白不太可能直接与 HIV-1 Gag 结合，因此对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) 将解剖这些糖蛋白以确定促进招募到出芽位点所需的特定结构域。 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进行交换，以确定 HIV-1 Gag 的哪些成分有助于 Env 识别。 叙述 HIV-1 感染已引起世界范围内的大流行，已夺去了超过 2000 万人的生命，并感染了超过 4000 万人。人们早就认识到，包括艾滋病毒在内的病毒具有独特的机制，可以将所有病毒成分招募到细胞内正确的病毒组装位点，但人们对这些机制仍然知之甚少。了解这些机制将有助于了解 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。