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

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.