Glycine 29 is critical for conformational changes of spike glycoprotein of MHV-A59 triggered by either receptor binding or high pH.

Mouse hepatitis virus (MHV) uses its N-terminal domain (NTD) of viral spike (S) protein to bind the host receptor， mouse carcinoembryonic antigen-related cell adhesion molecule 1a (mCEACAM1a)， and mediate virus entry. Our previous crystal structure study of MHV NTD/mCEACAM1a complex (1) reveals that there are 14 residues in NTD interacting with the receptor. However， their contribution to receptor binding and virus entry has not been fully investigated. Here we analyzed 13 out of 14 contact residues by mutagenesis， and identified I22 essential for receptor binding and virus entry. Unexpectedly， we found that G29 was critical for the conformational changes of S protein triggered either by receptor binding or high pH. Substitution of G29 with A， D， F， K， M， and T， to different extents， caused spontaneous dissociation of S1 from S protein， resulting in enhancement of high pH triggered receptor-independent syncytia (RIS) formation in 293T cells， compared to WT. By contrast， replacement of G29 with P， a turn prone residue with strict conformation， hindered virus entry and conformational changes of 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 NTD by G29P almost had no effect on pH-independent RIS induced by Y320A mutation in C-terminal domain (CTD) of S1 subunit， indicating that there might be absence of crosstalk between NTD and CTD during conformational changes of S protein. Our study will aid better understanding the mechanism how conformational changes of S protein is triggered. Binding of MHV S protein to the receptor， mCEACAM1a， triggers the conformational changes of S proteins， leading to formation of six-helix bundle and viral and cellular membrane fusion. However， the mechanism by which the conformational change of S protein is initiated after receptor binding has not been determined. In this study， we showed that， while replacement of G29， a residue at the edge of receptor binding interface and the center of structural turn after β1-sheet of S protein， with D or T triggered spontaneous conformational change of S protein and pH-independent RIS， G29P mutation significantly impeded the conformational changes of S proteins triggered either by receptor binding and pH 8.0. We reason that this structural turn might be critical for conformational change of S protein and altering this structural turn could initiate the conformational changes of S protein， leading to membrane fusion.