Distinct Roles for Sialoside and Protein Receptors in Coronavirus Infection

Qing, Enya; Hantak, Michael; Perlman, Stanley; Gallagher, Tom

A JHM-CoV spike mutation increases mCEACAM-independent cell binding and membrane fusion. To date, analyses of the multidomain CoV S proteins have revealed that the S1A domains interact with sialic acids (18, 19, 21, 41) and that most of the S1B domains bind to protein receptors (22, 52, 53) . The exceptions are the MHV beta-CoVs, which have S1A domains that bind to protein (CEACAM) receptors (25) . From these findings, we inferred that the JHM-CoV S1A domains contain a novel dual-receptor binding capability, able to bind both sialic acid and CEACAM receptors. Sialic acidbinding sites on beta-CoV S proteins were originally inferred from mutagenesis studies (19) , and most recently, from structural resolution of S proteins in complex with sialosides (18, 54) . Mutations in the inferred site did decrease sialoside binding (19, 41) , even though they are distal from the structurally resolved sialoside binding grooves (18, 54) . Here, we noted a mutation in the original inferred site. Among a stretch of four residues important for sialate-binding activity of bovine CoV (B-CoV) S1A, JHM differed only at residue 176, where there is a Gly on a divergent loop in place of the orthologous B-CoV Glu170 (Fig. 4A ). To evaluate the importance of this divergence to sialic acid binding, we constructed a G176E mutant JHM-CoV S protein. G176E S-protein expression, proteolytic processing, and VLP incorporation were all comparable to the wild type (WT) (Fig. 4B) . Notably, VLPs with G176E spikes bound more tightly to HeLa cells, and G176E spike-containing VLP binding was suppressed by neuraminidase pretreatment of the cells (Fig. 4C ). To correlate this increased cell binding with membrane fusion, we compared the syncytial potency of JHM wild-type and G176E mutant S proteins. Cell-cell fusions were measured by DSP 1-7 ϪDSP 8 -11 complementation (Fig. 4D ), visualized as GFP-positive syncytia (Fig. 4E) , and quantified by measuring Rluc signals (Fig. 4F ). Of note, when mCEACAM receptors were present, synyctia induced by wild type and mutant S proteins developed comparably, but in the absence of mCEACAM, the G176E spikes were significantly more robust in their syncytiuminducing activity (Fig. 4F) . We concluded that a single point mutation in the JHM-CoV S1A domain increases the cell binding and membrane fusion activities of S proteins, independent of the prototype JHM protein receptor.