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

冠状病毒是一种包膜正链RNA病毒，曾导致严重急性呼吸综合征（SARS）和中东呼吸综合征（MERS）的爆发。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子。病毒膜融合是包膜病毒（包括冠状病毒）进入宿主细胞并建立感染的第一步。冠状病毒的刺突(S)蛋白通过弹簧负载机制催化膜融合，类似于许多其他I类病毒融合蛋白（如HIV包膜刺突（Env）和流感血凝素（HA）），它也是主要的表面抗原诱导中和抗体。该蛋白首先作为单链前体产生，三聚体化，并可能被宿主蛋白酶切割成两个非共价相关的片段：受体结合片段S1和融合片段S2，在S1/S2切割位点。与宿主细胞受体（SARS-CoV和SARS-CoV-2的血管紧张素转换酶2 (angiotensin converting enzyme 2, ACE2)）结合，并在S2的第二个位点（S2 '位点）进一步进行蛋白水解裂解，被认为可能触发S1的解离和S2的不可逆再折叠。S蛋白的巨大构象变化使两层膜靠近并最终导致膜融合。人们已经对冠状病毒S蛋白的可溶性片段进行了广泛的结构研究，包括最近几周关于SARS-CoV-2的报道，但全长S蛋白的结构，特别是在膜的背景下，仍然未知，但已知膜附近的区域起着重要的结构和功能作用。在最近的一系列研究中，我们确定了HIV Env在脂质双层中的跨膜结构域（TMD）、膜近端外区（MPER）和细胞质尾部（CT）的结构。我们发现这些区域在存在脂质双分子层的情况下都形成有序的三聚体结构，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，我们假设冠状病毒S蛋白的跨膜和膜近端区域也采用确定的寡聚结构，这些结构对膜中全长蛋白的稳定性、功能和抗原性至关重要。我们将利用低温电镜和脂质纳米盘技术的最新进展，并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ACE2或中和抗体的复合物。我们的目标是全面了解S蛋白的结构和功能，促进疫苗和治疗的发展。

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.