Identification of H209 as Essential for pH 8-Triggered Receptor-Independent Syncytium Formation by S Protein of Mouse Hepatitis Virus A59

Abstract

The spike glycoprotein (S) of murine coronavirus mouse hepatitis virus (MHV) strain A59 uses murine carcinoembryonic antigen-related cell adhesion molecule 1a as its receptor for cell entry, but S protein can also be triggered in the absence of receptor by pH 8.0 alone at 37°C. The mechanism by which conformational changes of this S glycoprotein can be triggered by pH 8.0 has not yet been determined. Here, we show that MHV-A59 S protein is triggered by pH 8.0 at 37°C to induce receptor-independent syncytium (RIS) formation on 293T cells, and that the conformational changes in S proteins triggered by pH 8.0 are very similar to those triggered by receptor binding. We systemically mutated each of 15 histidine residues in S protein and found that H209 is essential for pH 8.0-triggered RIS formation, while H179, H441, H643, and H759 also play important roles in this process. Replacement of H209 with Ala had no effect on receptor binding, but in murine 17Cl.1 cells mutant H209A MHV-A59 showed delayed growth kinetics and was readily outcompeted by wild-type virus when mixed together, indicating that the H209A mutation caused a defect in virus fitness. Finally, the H209A mutation significantly increased the thermostability of S protein in its prefusion conformation, which may raise the energy barrier for conformational change of S protein required for membrane fusion and lead to a decrease in virus fitness in cell culture. Thus, MHV-A59 may have evolved to lower the stability of its S protein in order to increase virus fitness. Enveloped viruses enter cells through fusion of viral and cellular membranes, and the process is mediated by interactions between viral envelope proteins and their host receptors. In the prefusion conformation, viral envelope proteins are metastable, and activation to the fusion conformation is tightly regulated, since premature activation would lead to loss of viral infectivity. The stability of viral envelope proteins greatly influences their activation and virus fitness. Here, we report that, similar to the A82V mutation in Ebola glycoprotein, in the S glycoprotein of murine coronavirus MHV-A59, the histidine residue at position of 209 significantly affects the thermal stability of the S protein, determines whether S protein can be activated at 37°C by either pH 8.0 alone or by receptor binding, and affects viral fitness in cell culture. Thus, the spike glycoprotein of MHV-A59 has evolved to retain histidine at position 209 to optimize virus fitness.

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

“…As shown in Fig. 4A, while treatment of HEK293T cells transiently expressing the WT S protein at pH 6.5 induced levels only slightly above the background level of RIS, large RIS were formed when WT S-expressing cells were incubated at pH 8.0, in agreement with our previous report (27). Surprisingly, G29D showed extensive formation of RIS even at pH 6.5 ( Fig.…”

“…Purified pseudovirions with MHV S proteins were incubated with the soluble receptor at 37°C and treated with limited trypsin digestion on ice. Consistent with our previous report (27), upon limited trypsin digestion, several new bands were generated in WT S proteins triggered by receptor binding at 37°C, including a new ϳ60-kDa band without boiling and dithiothreitol (DTT) treatment ( Fig. 7C, left upon receptor binding and limited trypsin digestion, compared to the controls, indicating that there was a lack of major conformational changes in G29P mutant S proteins when triggered by receptor binding.…”

“…Production of MHV S-pseudotyped lentivirus and transduction of pseudovirions. MHV S protein-pseudotyped viruses were produced as described previously (27), with minor modifications. Briefly, a plasmid encoding either the wild-type or mutant S protein was cotransfected into HEK293T cells with pLenti-Luc-GFP (a gift from Fang Li, Duke University) and psPAX2 (Addgene, Cambridge, MA) at a molar ratio of 1:1:1 using PEI.…”

“…High-pH-triggered and receptor-independent cell-cell fusion assays. High-pH-triggered and receptor-independent cell-cell fusion assays were described previously (27). Briefly, human HEK293T cells were cotransfected with plasmids encoding the WT or mutant MHV-A59 S glycoprotein and green fluorescent protein (GFP) using PEI.…”

“…We previously showed that the S protein of MHV-A59 mediated the formation of receptor-independent syncytia (RIS) in HEK293T cells upon high-pH, such as pH 8.0 but not pH 6.5, triggering (27)(28)(29). As shown in Fig.…”