Glycine 29 Is Critical for Conformational Changes of the Spike Glycoprotein of Mouse Hepatitis Virus A59 Triggered by either Receptor Binding or High pH

Abstract

Mouse hepatitis virus (MHV) uses its N-terminal domain (NTD) of the viral spike (S) protein to bind the host receptor mouse carcinoembryonic antigenrelated cell adhesion molecule 1a (mCEACAM1a) and mediate virus entry. Our previous crystal structure study of the MHV NTD/mCEACAM1a complex (G. Peng, D. Sun, K. R. Rajashankar, Z. Qian, et al., Proc Natl Acad Sci U S A 108:10696 -10701, 2011, https://doi.org/10.1073/pnas.1104306108) reveals that there are 14 residues in the 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 as being essential for receptor binding and virus entry. Unexpectedly, 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 (WT). 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 pHindependent 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. Our study will aid in better understanding the mechanism of how conformational changes of the S protein are triggered. IMPORTANCE Binding of the MHV S protein to the receptor mCEACAM1a triggers conformational changes of S proteins, leading to the formation of a six-helix bundle and viral and cellular membrane fusion. However, the mechanism by which the conformational change of the 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 the receptor binding interface and the center of the structural turn after the ␤1-sheet of the S protein, with D or T triggered spontaneous conformational changes of the S protein and pH-independent RIS, the G29P mutation significantly impeded the conformational changes of S proteins triggered by either receptor binding or pH 8.0. We reason that this structural turn might be critical for conformational changes of the S protein and that altering this structural turn could initiate conformational changes of the S protein, leading to membrane fusion. KEYWORDS mouse hepatitis virus, S protein conformational change, coronavirus spike glycoprotein, initiation of conformational change, receptor-independent syncytium formation C oronaviruses (CoVs) are a large family of enveloped plus-stranded R...

Evolution-guided mutagenesis of the cytoplasmic incompatibility proteins: Identifying CifA’s complex functional repertoire and new essential regions in CifB

“…Global folding abnormalities may block the enzymatic function of trans-acting domains or prevent binding elsewhere in the protein. Local or non-local changes to the structure can inhibit processes close to the mutated site in tertiary structure [44,45]. These hypotheses are applicable to all mutant variants.…”

Fe–Fe Double-Atom Catalysts for Murine Coronavirus Disinfection: Nonradical Activation of Peroxides and Mechanisms of Virus Inactivation

“…The S protein of MHV-A59 is posttranslationally cleaved into an S1 receptor binding unit and an S2 membrane fusion unit with distinct functions (Table S11). The N-terminal domain (NTD) within the S1 unit recognizes the cell receptor of murine carcinoembryonic antigen-related cell adhesion molecule 1a (mCEACAM1a), and this interaction is critical to determine viral infectivity. , Particularly, several amino acid residues in NTD of MHV-A59 (highlighted in red in Table S11) are important because they are responsible for the direct contact between the virus and its cell receptor. Interestingly, the reactivity between these amino acids and PMS is low because PMS only reacts with sulfur-containing cysteine and methionine (Met) rapidly, and the reaction rate constant of Met–PMS is 3–6 orders of magnitude higher than those of other amino acids (Table S12).…”

Research Advances on Swine Acute Diarrhea Syndrome Coronavirus

“…The structural similarities between the NTDs of HKU2 and SADS-CoV, and those of HCoV-NL63 and PEDV, which are classified as subtype I, suggest a possible evolutionary relationship between these coronaviruses. The NTDs of β-coronaviruses, including MERS-CoV [56,57], SARS-CoV [58], and MHV [59,60], share similarities with subtype I in terms of topology, the distribution of disulfide bonds, and structural features, which may influence their immune evasion and receptor-binding abilities [61][62][63]. The α-coronavirus CTD structure can be categorized into two groups, single-layer and double-layer β-folded cores, based on the number of twisted β-folded cores.…”