Study On The Initiation Mechanism Of The Conformational Change Of MHV A59 S Protein

Coronaviruses(CoVs)are large,enveloped,and positive-stranded RNA viruses that infect many mammalian and avian species,and cause respiratory,enteric,gastrointestinal,and neurological diseases.They can be divided into four genera:??,?,and ?.coV genome encodes four structure proteins,spike(S)glycoprotein,envelope protein(E),membrane protein(M),and nucleoprotein(N).Among them,S protein mediates virus entry and membrane fusion.Mouse hepatitis virus(MHV)is a lineage A ?-CoV and is commonly used as a model to study CoV entry,replication,and pathogenesis.MHV utilizes murine carcinoembryonic antigen-related cell adhesion molecule 1(mCEACAM1)as a receptor.The entry of CoV is mediated by the interaction between the receptor and the viral S protein.The S protein is composed of two subunits,S1 and S2.Like most other class 1 viral fusion proteins,S1 contains the receptor binding domain(RBD),while S2 possesses the fusion machinery.S1 can be further divided into two subdomains:the N-terminal domain(NTD)and the C-terminal domain(CTD).MHV uses its NTD to bind mCEACAM1a.There are 14 residues on the NTD making direct contacts with mCEACAM1a in the NTD/mCEACAM1a structure.However,the contribution of each individual contacting residue in the NTD to receptor binding and virus entry has not been fully analyzed.In this study,we systematically investigated the role of most contacting residues of NTDs in receptor binding and infection and found that R20,122,N26,and N172 are critical for receptor binding and virus entry.Surprisingly,we found that G29 was critical for conformational changes of the S protein,although it had no influence on receptor binding.The hydrophobic interaction between Y15,L89,and L160 in NTD and 141 in the receptor was also important but with redundancy.Moreover,we found that G29 was critical for the conformational changes of the S protein triggered by either receptor binding or high pH.Replacement of G29 with A,D,F,K,M,and T,to different extents,caused spontaneous dissociation of S1 from the S protein,resulting in an enhancement of high-pH-triggered receptor-independent syncytium(RIS)formation in HEK293T cells,compared to the wild type(???).In contrast,replacement of G29 with P,a turn-prone residue with a strict conformation,hindered virus entry and conformational changes of the S protein triggered by either receptor binding or pH 8.0,suggesting that the structural turn around G29 and its flexibility are critical.Finally,stabilization of the NTD by G29P had almost no effect on pH-independent RIS induced by the Y320A mutation in the C-terminal domain(CTD)of the S1 subunit,indicating that there might be an absence of cross talk between the NTD and CTD during conformational changes of the S protein.