Among them, we selected HEK293 cells to purify recombinant full-length M protein (see Methods). To prepare the SARS-CoV-2 M protein for structural determination, we examined the expression of M protein using Escherichia coli, baculovirus, and HEK293 expression systems. SARS-CoV-2 M protein forms dimer and higher-order oligomers Here, we report the cryo-EM structures of M protein in two distinct conformational states and M/N/RNA interaction analysis that provide a clue to understanding the mechanism underlying an essential step in virus assembly. These data suggest that the SARS-CoV-2 M protein also functions as a viroporin.ĭespite its critical importance for virus assembly, the precise role of the M protein in this process remains largely unknown. The recently reported cryo-EM structures of SARS-CoV-2 ORF3a revealed that ORF3a mainly forms dimers and functions as a nonselective cation-permeable viroporin 19. Phylogenetic analysis revealed that the Betacoronavirus M protein family is evolutionarily related to ORF3a in Sarbecovirus, ORF5 in Middle East Respiratory Syndrome (MERS) CoV, and M proteins from more distant toroviruses 17, 18. M protein contains three transmembrane helices and a C-terminal intravirion domain (Fig. These data in SARS-CoV-2 and related viruses suggest the versatile and vital role of M protein in coronavirus assembly. In addition, the interactions between M protein and N protein of SARS-CoV-2 15 and between M protein and genome RNA containing a packaging signal of MHV 16 have been reported. The species-specific pairing of M and N proteins is essential for the correct formation of virus particles in SARS-CoV and murine coronavirus mouse hepatitis virus (MHV) 14. This indicates the indispensable role of M protein in virus assembly. Co-expression of SARS-CoV-2 structural proteins in mammalian cells generates virus-like particles (VLPs), where a minimal combination of M + N is required for VLP formation 11, 12, 13. M protein synthesized in the host cells is localized in the endoplasmic reticulum–Golgi intermediate compartment (ERGIC) and provides a platform for recruiting other viral structural proteins 10. Our data shed light on the M protein-driven virus assembly mechanism and provide a structural basis for therapeutic intervention targeting M protein. Moreover, the interaction analyses of M protein with nucleocapsid protein (N) and RNA suggest that the M protein mediates the concerted recruitment of these components through the positively charged intravirion domain. The M protein dimer is unexpectedly similar to SARS-CoV-2 ORF3a, a viral ion channel. A highly conserved hinge region is key for conformational changes. M protein further assembles into higher-order oligomers. M protein forms a mushroom-shaped dimer, composed of two transmembrane domain-swapped three-helix bundles and two intravirion domains. Here, we report the cryo-electron microscopy structure of the SARS-CoV-2 M protein in two different conformations. However, the process of M protein-driven virus assembly are largely unknown. The coronavirus membrane protein (M) is the most abundant viral structural protein and plays a central role in virus assembly and morphogenesis.
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