Structure of the full-length spike protein of SARS-CoV-2 in the context of membrane

膜背景下 SARS-CoV-2 全长刺突蛋白的结构

• 批准号: 10117733
• 负责人: Bing Chen
• 金额: $ 53.1万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-08 至 2020-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10117733
• 关键词: 2019-nCoV; Adopted; Angiotensin Receptor; Antibody Therapy; Back; Binding; Camels; Cells; Cessation of life; China; Chiroptera; Cities; Complex; Coronavirus; Coronavirus spike protein; Cryoelectron Microscopy; Cytoplasmic Tail; Diagnostic; Disease Outbreaks; Dissociation; Epidemic; Eye; Future; Goals; HIV; Human; Infection; Influenza Hemagglutinin; Integration Host Factors; International; Iran; Italy; Knowledge; Lead; Length; Lipid Bilayers; Lipids; Membrane; Membrane Fusion; Membrane Proteins; Middle East Respiratory Syndrome; Middle East Respiratory Syndrome Coronavirus; Modernization; Molecular Conformation; Names; Peptide Hydrolases; Peptidyl-Dipeptidase A; Play; Property; Proteins; Protocols documentation; Province; Public Health; Quarantine; RNA Viruses; Receptor Cell; Reporting; Resolution; Role; SARS coronavirus; Series; Severe Acute Respiratory Syndrome; Sister; Site; South Korea; Structure; Surface Antigens; Taxonomy; Therapeutic; Transmembrane Domain; Vaccines; Viral; Viral Fusion Proteins; Viral Physiology; Virus; base; digital media; nanodisc technology; nanodisk; neutralizing antibody; pandemic disease; particle; protein function; protein structure; protein structure function; receptor binding; reconstitution; response; social; structural biology; therapeutic development; vaccine development; virus envelope

Coronaviruses (CoVs) are enveloped positive-stranded RNA viruses that caused the outbreaks of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS). To meet the urgent needs for diagnostics, therapeutics and vaccines to contain the current crisis, we need to gain deep understanding of structure-function of the viral proteins and the relevant host factors. Viral membrane fusion is the first key step for enveloped viruses, including CoVs, to enter host cells and establish infection. The spike (S) protein of CoV catalyzes membrane fusion by a spring-loaded mechanism, similar to many other class I viral fusion proteins (e.g., HIV envelope spike (Env) and influenza hemagglutinin (HA)), and it is also the major surface antigen inducing neutralizing antibodies. The protein is first produced as a single-chain precursor that trimerizes and may undergo cleavage by a host protease into two noncovalently associated fragments: the receptor-binding fragment S1 and the fusion fragment S2, at the S1/S2 cleavage site. Binding to a host cell receptor (angiotensin converting enzyme 2 (ACE2) for both SARS-CoV and SARS-CoV-2) and further proteolytic cleavage at a second site in S2 (S2’ site) are believed to trigger possible dissociation of S1 and irreversible refolding of S2. The large conformational changes in the S protein bring the two membranes close together and ultimately lead to membrane fusion. There have been extensive structural studies of the soluble fragments of the CoV S proteins, including those reported in the last few weeks on SARS-CoV-2, but the structure of the full-length S protein, in particular, in the context of membrane, remains unknown, and yet the regions near the membrane are known to play important structural and functional roles. In a series of recent studies, we have determined the structures of the transmembrane domain (TMD), membrane proximal external region (MPER) and the cytoplasmic tail (CT) of HIV Env in lipid bilayers. We find that these regions all form well-ordered trimeric structures in the presence of a lipid bilayer and that disruption of any of them reduces membrane fusion efficiency and alters the antigenic structure of the entire Env. Based on these results, we hypothesize that the transmembrane and membrane-proximal regions of the CoV S protein also adopt defined oligomeric structures that are critical for the stability, function and antigenicity of the full-length protein in membrane. We will capitalize on the recent advances in cryoEM and lipid nanodisc technology and plan to determine structures of the intact S protein from SARS-CoV-2 reconstituted in lipid bilayers and its complex with human ACE2 or neutralizing antibodies. Our goal is to gain a full understanding of the S protein structure-function and to facilitate vaccine and therapeutic development.

冠状病毒(CoV)是一种包膜正链RNA病毒，可引起严重急性呼吸综合征(SARS)和中东呼吸综合征(MERS)的暴发。为了满足对诊断、治疗和疫苗的迫切需求，以遏制当前的危机，我们需要深入了解病毒蛋白的结构和功能以及相关的宿主因素。病毒膜融合是包括冠状病毒在内的包膜病毒进入宿主细胞并建立感染的第一个关键步骤。冠状病毒的Spike(S)蛋白类似于许多其他I类病毒融合蛋白(如HIV包膜刺蛋白(Env)和流感血凝素(HA))，通过弹簧加载的机制催化膜融合，也是诱导中和抗体的主要表面抗原。该蛋白首先以单链前体的形式产生，然后在S1/S2裂解位点被宿主蛋白酶切割成两个非共价结合的片段：受体结合片段S1和融合片段S2。与宿主细胞受体(SARS-CoV和SARS-CoV-2的血管紧张素转换酶2(ACE2))的结合以及S2第二个位点(S2‘位点)的进一步蛋白水解性切割被认为可能引发S1的解离和S2的不可逆重折叠。S蛋白的大的构象变化使两个膜紧密地结合在一起，最终导致膜融合。对冠状病毒S蛋白的可溶性片段进行了广泛的结构研究，包括最近几周在SARS-CoV-2上报道的结构，但S蛋白的全长结构，特别是膜背景下的结构尚不清楚，但膜附近的区域已知在结构和功能上发挥着重要作用。在最近的一系列研究中，我们确定了HIV env跨膜区(TMD)、膜近端外区(MPER)和细胞质尾部(CT)在脂双层中的结构。我们发现，在脂质双层存在的情况下，这些区域都形成了有序的三聚体结构，其中任何一个区域的破坏都会降低膜融合效率，并改变整个Env的抗原结构。基于这些结果，我们推测，冠状病毒S蛋白的跨膜区和膜近端也具有特定的寡聚结构，这些结构对膜上全长蛋白的稳定性、功能和抗原性至关重要。我们将利用低温电子显微镜和脂质纳米盘技术的最新进展，计划确定在脂质双层中重组的SARS-CoV-2完整S蛋白的结构及其与人血管紧张素转换酶2或中和抗体的复合物。我们的目标是充分了解S蛋白的结构和功能，为疫苗和治疗的开发提供便利。