Biophysics of Protein-Mediated Membrane Fusion

蛋白质介导的膜融合的生物物理学

• 批准号: 10684697
• 负责人: Gregory B Melikian
• 金额: $ 50.67万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-08-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10684697
• 关键词: Actins; Architecture; Attenuated; Binding; Biological Assay; Biophysics; CCR5 gene; CXCR4 gene; Cell fusion; Cell membrane; Cell surface; Cells; Complement; Cryoelectron Microscopy; Data; Fluorescence; Foundations; Glycoproteins; Goals; HIV Envelope Protein gp120; HIV resistance; HIV-1; Imaging Techniques; In Vitro; Infection; Integration Host Factors; Interferons; Lateral; Lipids; Maps; Mediating; Membrane; Membrane Fusion; Membrane Lipids; Membrane Proteins; Modeling; Molecular; Mutation; Process; Property; Protein Family; Proteins; Receptor Signaling; Reporting; Resistance; Role; Serine; Signal Transduction; Site; Specificity; Structure; Techniques; Tertiary Protein Structure; Testing; Transmembrane Domain; V3 Loop; Viral; Viral Physiology; Viral Proteins; Virion; Virus; Visualization; comparison control; env Glycoproteins; imaging approach; insight; mutant; mutation screening; particle; receptor; response; single molecule; superresolution imaging; superresolution microscopy

HIV-1 initiates infection by fusing with the target cell membrane through process that is triggered by Env glycoprotein following the engagement of CD4 and coreceptors (CCR5 or CXCR4). At least two host factors, SERINC5 (serine incorporator 5) and IFITM (interferon-induced transmembrane) protein families, are known to incorporate into HIV-1 particles and reduce their infectivity by inhibiting the virus-cell fusion. The mechanisms by which these restriction factors inhibit viral fusion are poorly understood. The main difference in the antiviral activity of these two proteins is the range of restricted viruses. Whereas IFITMs inhibit fusion of many unrelated viruses, SERINC5 is only reported to interfere with retroviral fusion. However, SERINC5 and IFITMs also share striking similarities, including the ability to reduce HIV-1 fusion when expressed in target cells and the varied resistance of HIV-1 strains to these factors that maps to the gp120 variable loop 3. The most accepted model of IFITM-mediated inhibition of fusion, which is supported by our pilot results, is through increasing the membrane stiffness and curvature. Based on the above similarities, the lack of strong evidence for Env-SERINC5 binding and our super-resolution imaging results showing poor colocalization of these molecules, we hypothesize that SERINC5 and IFITMs inhibit HIV-1 fusion by a similar indirect mechanism that involves altering the properties of viral membrane. To test this hypothesis, we will imlpement several cutting-edge techniques to assess the membrane protein and lipid distributions and dynamics on single HIV-1 particles. Specifically, in Aim 1, we will identify SERINC5-resistant Env mutants by deep mutational scanning and characterize these mutants using flow virometry, super-resolution microscopy and correlative cryo-EM. Aim 2 will focus on delineating the effects of SERINC5 on the lipid order and mobility in the viral membrane. In Aim 3, we will investigate whether, like SERINC5, IFITMs alter the architecture and/or dynamics of the HIV-1 membrane. Finally, Aim 4 will test the hypothesis that plasma membrane tension increases, which are mediated by Env-receptor/coreceptor signaling, drive the late stages of HIV-1 fusion at the cell surface and that SERINC5 may inhibit HIV-1 fusion by attenuating these cellular responses. Completion of the proposed Specific Aims will elucidate the mechanism(s) of HIV-1 restriction by unrelated host factors. If these factors inhibit viral fusion through a conserved indirect mechanism involving modulation of membrane properties, and not through direct interactions with viral proteins or cellular receptors, this would be a paradigm-shifting discovery. The results of this project will also delineate the role of cellular signaling and plasma membrane tension in promoting HIV-1 fusion.

HIV-1通过Env触发的过程与靶细胞膜融合来启动感染 在CD 4和辅助受体（CCR 5或CXCR 4）接合后，糖蛋白的表达。至少有两个宿主因子， 已知SERINC 5（丝氨酸蛋白酶5）和IFITM（干扰素诱导的跨膜）蛋白家族， 整合到HIV-1颗粒中并通过抑制病毒与细胞融合来降低其感染性。的机制 这些限制性因子抑制病毒融合的机制尚不清楚。抗病毒药物的主要区别在于 这两种蛋白的活性范围是病毒的限制。而IFITMs抑制许多不相关的融合， 病毒，SERINC 5仅报道干扰逆转录病毒融合。然而，SERINC 5和IFITM也共享 惊人的相似之处，包括在靶细胞中表达时减少HIV-1融合的能力，以及不同的 HIV-1菌株对这些因子的抗性，这些因子映射到gp 120可变环3。最受欢迎的模型 IFITM介导的融合抑制，这是由我们的试点结果支持，是通过增加膜 硬度和曲率。基于上述相似性，缺乏Env-SERINC 5结合的有力证据， 我们的超分辨率成像结果显示这些分子的共定位性很差，我们假设， SERINC 5和IFITM通过类似的间接机制抑制HIV-1融合，该机制涉及改变 病毒膜为了验证这一假设，我们将实施几项尖端技术来评估 单个HIV-1颗粒的膜蛋白和脂质分布和动力学。具体而言，在目标1中，我们将 通过深度突变扫描鉴定SERINC 5抗性Env突变体并使用流式细胞术表征这些突变体 超分辨率显微镜和相关的冷冻电镜。目标2将侧重于描述 SERINC 5对病毒膜中的脂质顺序和流动性的影响。在目标3中，我们将研究是否，如 SERINC 5、IFITM改变HIV-1膜的结构和/或动力学。最后，目标4将测试 假设质膜张力增加，这是由Env受体/辅助受体信号传导介导的， 驱动HIV-1在细胞表面融合的晚期阶段，并且SERINC 5可以通过减弱HIV-1在细胞表面的表达来抑制HIV-1融合。 这些细胞反应。完成拟议的具体目标将阐明HIV-1的机制 受无关宿主因素的限制。如果这些因子通过保守的间接机制抑制病毒融合 涉及膜特性的调节，而不是通过与病毒蛋白或细胞 受体，这将是一个范式转变的发现。该项目的结果还将说明 细胞信号传导和质膜张力促进HIV-1融合。