Biochemical Analysis of Coronavirus Spike Glycoprotein Conformational Intermediates during Membrane Fusion

Abstract

A fusion protein expressed on the surface of enveloped viruses mediates fusion of the viral and cellular membranes to facilitate virus infection. Pre-and postfusion structures of viral fusion proteins have been characterized, but conformational changes between them remain poorly understood. Here, we examined the intermediate conformation of the murine coronavirus fusion protein, called the spike protein, which must be cleaved by a cellular protease following receptor binding. Western blot analysis of protease digestion products revealed that two subunits (67 and 69 kDa) are produced from a single spike protein (180 kDa). These two subunits were considered to be by-products derived from conformational changes and were useful for probing the intermediate conformation of the spike protein. Interaction with a heptad repeat (HR) peptide revealed that these subunits adopt packed and unpacked conformations, respectively, and two-dimensional electrophoresis revealed a trimeric assembly. Based on biochemical observations, we propose an asymmetric trimer model for the intermediate structure of the spike protein. Receptor binding induces the membrane-binding potential of the trimer, in which at least one HR motif forms a packed-hairpin structure, while membrane fusion subunits are covered by the receptor-binding subunit, thereby preventing the spike protein from forming the typical homotrimeric prehairpin structure predicted by the current model of class I viral fusion protein. Subsequent proteolysis induces simultaneous packing of the remaining unpacked HRs upon assembly of three HRs at the central axis to generate a six-helix bundle. Our model proposes a key mechanism for membrane fusion of enveloped viruses. IMPORTANCE Recent studies using single-particle cryo-electron microscopy (cryoEM) revealed the mechanism underlying activation of viral fusion protein at the priming stage. However, characterizing the subsequent triggering stage underpinning transition from pre-to postfusion structures is difficult because single-particle cryoEM excludes unstable structures that appear as heterogeneous shapes. Therefore, population-based biochemical analysis is needed to capture features of unstable proteins. Here, we analyzed protease digestion products of a coronavirus fusion protein during activation; their sizes appear to be affected directly by the conformational state. We propose a model for the viral fusion protein in the intermediate state, which involves a compact structure and conformational changes that overcome steric hindrance within the three fusion protein subunits.

Structural and functional properties of SARS-CoV-2 spike protein: potential antivirus drug development for COVID-19

“…The formation of 6-HB is essential for viral fusion. The FP in the N-terminus of SARS-CoV-2 and the two HR domains on S2 is essential for viral fusion [50]. After cleavage of the S protein, the FP of SARS-CoV-2 is exposed and triggers viral fusion.…”

Viral Membrane Fusion and the Transmembrane Domain

“…This tryptophan-rich region and its location with respect to the highly hydrophobic coil of the S protein TMD is critical for S-mediated membrane fusion. This study also suggests there is flexibility in the region adjacent to the membrane region [133], with flexibility in the region between the MPER and the TMD critical for large-scale conformational changes of the overall protein. Flexibility between the TMD and the upstream region is characteristic of many Class I fusion proteins, as it has been demonstrated for several different families (Flexible linker IAV, MPER-TMD HIV), though data from paramyxovirus F proteins do not support flexibility for that system [134].…”

Cryo-EM analysis of the HCoV-229E spike glycoprotein reveals dynamic prefusion conformational changes

“…3). Previous work, including MHV 43,44 and SARS-CoV 34 showed that receptor binding to the S protein can induce its conformational change, exposing the cleavage site for the proteases. This can explain why the C2 presents a small opening conformational change compared with the C1 due to the stabilizing effect of S1 on S2 and lack of hAPN binding.…”

“…9), the conformational transition from pre-to postfusion is difficult. Receptor binding and proteolytic activation can remove the restriction and promote conformational transformation 34,43,44 . Hence, we propose a feasible membrane fusion process for HCoV-229E as shown below (Fig.…”