Structure-function studies of the membrane-interacting domains of HIV-1 Env spike

项目摘要 HIV-1等包膜病毒进入宿主细胞的第一个关键步骤是病毒膜融合。 病毒融合蛋白是一种迷人的蛋白质折叠机器，能够采用完全不同的 融合过程中的构象；它们也是重要的疫苗和治疗靶点。上一首 研究揭示了许多病毒融合的可溶性片段在融合前和融合后的构象。 蛋白质，但对它们的融合肽、跨膜和膜近端的结构知之甚少 在脂质双层的背景下的区域。有强有力的证据表明膜的功能作用-- 然而，关于它们如何发挥其功能的机械性研究仍然很少。我们 假设与HIV-1囊膜相关的其他融合蛋白的膜相互作用区 蛋白质(Env)采用特定的寡聚结构，这些结构对稳定性、功能和 膜全长蛋白的抗原性。在我们之前完成的研究中 在资助期，我们已经确定了TM的结构，膜近端外区，以及 利用最新的核磁共振技术，HIV-1env的细胞质尾巴在双胞体中模拟脂质双层。我们发现 这些区域都形成了有序的三聚体团簇，并以构象耦合的形式存在，这破坏了它们 会减少融合，改变整个包膜的抗原结构。在本应用程序中，我们建议应用 我们的核磁共振/双细胞技术研究了SIV Env和最近出现的SARS的膜区- CoV-2 Spike(S)，并用冷冻电子显微镜确定全长蛋白质的结构。 在脂质纳米盘中重组。我们将定义这些关键结构元件在膜融合中的作用 通过深度诱变和功能分析。我们将追求以下具体目标：1)我们将 研究SIV Env的膜相互作用组分；2)我们将研究膜相互作用 3)我们将确定全长SIV环境的结构和 膜环境中的SARS-CoV-2 S；4)我们将阐明SARS-CoV-2膜相互作用结构域的作用 HIV/SIV Env和SARS-CoV-2 S对其稳定性、功能和抗原性的研究

Project Summary The first critical step for enveloped viruses, such as HIV-1, to enter host cells is viral membrane fusion. Viral fusion proteins are fascinating protein folding machineries capable of adopting completely different conformations during the fusion process; they are also important vaccine and therapeutic targets. Previous studies have revealed both pre- and post-fusion conformations of the soluble fragments of many viral fusion proteins, but less is known for structures of their fusion peptide, transmembrane and membrane-proximal regions in the context of lipid bilayers. There is strong evidence for functional roles of the membrane- interacting regions in fusion, and yet mechanistic studies on how they exert their functions remain scarce. We hypothesize that membrane-interacting regions of other fusion proteins related to HIV-1 envelope protein (Env) adopt defined oligomeric structures that are critical for the stability, function and antigenicity of the full-length proteins in membrane. In the studies that we completed during the previous funding period, we have determined the structures of the TM, membrane proximal external region, and cytoplasmic tail of HIV-1 Env in bicelles that mimic lipid bilayers using the latest NMR technology. We find that these regions all form well-ordered trimeric clusters and are conformationally coupled, and that disrupting them can reduce fusion and alter the antigenic structure of the entire Env. In this application, we propose to apply our NMR/bicelle technology to investigate the membrane regions of SIV Env and the recently emerged SARS- CoV-2 spike (S), and to use cryo-electron microscopy to determine structures of the full-length proteins reconstituted in lipid nanodiscs. We will define roles in membrane fusion of critical structural elements of these regions by deep mutagenesis and functional assays. We will purse the following specific aims: 1) we will investigate the membrane-interacting components of SIV Env; 2) we will investigate the membrane-interacting components in the postfusion arrangement; 3) we will determine structures of the full-length SIV Env and SARS-CoV-2 S in the context of membrane; 4) we will elucidate roles of the membrane-interacting domains of HIV/SIV Env and SARS-CoV-2 S in their stability, function and antigenicity.