Distinct Roles for Sialoside and Protein Receptors in Coronavirus Infection

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

An analogous MERS-CoV spike mutation similarly increases hDPP4-independent cell binding and membrane fusion. We next determined whether analogous changes in S1A of the related MERS-CoV also modified viral attachment. The S1A interaction with sialic acids has biological significance, as antibodies against S1A protect mice from lethal MERS-CoV infections (55) , and S1A acquires adaptive mutations in humans (56) and in mouse models of human MERS-CoV lung infection (57, 58) . In mice, this selectivity was at Asn222 (57, 58), a known glycan addition site (17, 59, 60) , and a documented hypervariable residue, evident in a group of MERS-CoV-related HKU4 bat viruses but absent in the HKU5 bat virus groups (Fig. 6A) . Remarkably, in the context of CoV S1A structure, the Asn222 position overlaps closely with B-CoV residue Glu170 and MHV-CoV residue Gly176 (Fig. 6B) . We constructed MERS VLPs to determine whether this Asn222 change impacted cell binding. In initial tests, we constructed MERS-CoV VLPs and evaluated their sialate binding properties. Human red blood cells (RBCs) were agglutinated by MERS VLPs, while neuraminidase-treated RBCs were not (Fig. S4) , indicating that VLPs do bind sialosides. We then constructed wild-type and ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) N222D VLPs containing internal Rluc and compared their Rluc and S-protein levels relative to VLPs lacking S proteins (Fig. 6C ). VLPs were applied to human lungderived Calu3 cells, which express the human DPP4 receptor for MERS-CoV, and to mouse lung-derived LET-1 and mouse brain-derived DBT cells, neither of which express human DPP4 and thus do not bind MERS-CoV via a protein receptor. VLP binding was assessed by quantifying cell-associated Rluc. Asn222 change did not change VLP binding to human Calu3 cells but clearly enhanced VLP binding to murine DBT and LET-1 cells (Fig. 6D) . We next considered whether this adaptation for increased cell binding had consequences in MERS S-mediated cell-cell membrane fusion, similar to the JHM-CoV G176E mutant. We found that the N222D mutant S proteins did indeed exhibit enhanced cell fusion, relative to wild-type S proteins (Fig. 6E) , but this was only observed by eliminating hDPP4 (Fig. 6E) , which removes the dominant S1B-protein receptor interaction and demands S1A utilization. To further probe the relationships between S1A-cell binding and cell fusion, we determined whether N222D S1A-Fc proteins would suppress cell fusion more effectively than WT S1A-Fc. Indeed, we found that exogenously added N222D S1A-Fc proteins suppressed cell fusions more potently than the corresponding wild-type S1A-Fc did (Fig. 6F and Fig. S5) , further reinforcing the direct relationship between S1A binding to cells and resultant cell-cell fusion. Altogether, these findings support the hypothesis that MERS-CoV and JHM-CoV can acquire increased cell binding through S1A mutation, which consequently allows for more robust cell-cell fusion capability. Both of these CoVs can procure these cell binding and cell fusion properties through similarly localized S1A mutations.