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

项目摘要 包膜病毒（如HIV-1）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蛋白质结构。 它们在融合过程中具有构象;它们也是重要的疫苗和治疗靶标。先前 研究已经揭示了许多病毒融合蛋白的可溶性片段的融合前和融合后构象 蛋白质，但较少知道其融合肽，跨膜和膜近端的结构 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- 相互作用的区域融合，但机制的研究，他们如何发挥其功能仍然很少。我们 假设与HIV-1包膜相关其他融合蛋白的膜相互作用区域 蛋白（Env）采用确定的寡聚体结构，这些结构对稳定性、功能和 膜中全长蛋白的抗原性。在我们之前完成的研究中， 资金期间，我们已经确定了TM的结构，膜近端外部区域， 使用最新的NMR技术，在模拟脂质双层的双胞中检测HIV-1 Env的胞质尾区。我们发现 这些区域都形成有序的三聚体簇，并且在构象上偶联， 可以减少融合并改变整个Env的抗原结构。在本申请中，我们建议 我们的NMR/bicelle技术来研究SIV Env和最近出现的SARS的膜区域， CoV-2 spike（S），并使用冷冻电子显微镜确定全长蛋白质的结构 在脂质纳米盘中重构。我们将定义这些关键结构元件在膜融合中的作用， 区域通过深度诱变和功能测定。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SIV Env的膜相互作用组分。 融合后安排中的成分; 3）我们将确定全长SIV Env的结构， SARS-CoV-2S在膜环境中的作用; 4）我们将阐明SARS-CoV-2S的膜相互作用结构域的作用。 HIV/SIV Env和SARS-CoV-2S的稳定性、功能和抗原性。

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.