Dynamic Ca²⁺ sensitivity stimulates the evolved SARS-CoV-2 spike strain-mediated membrane fusion for enhanced entry

Mutations in the spike protein generated a highly infectious and transmissible D614G variant, which is present in newly evolved fast-spreading variants. The D614G, Alpha, Beta, and Delta spike variants of SARS-CoV-2 appear to expedite membrane fusion process for entry, but the mechanism of spike-mediated fusion is unknown. Here, we reconstituted an in vitro pseudovirus-liposome fusion reaction and report that SARS-CoV-2 wild-type spike is a dynamic Ca²⁺ sensor, and D614G mutation enhances dynamic calcium sensitivity of spike protein for facilitating membrane fusion. This dynamic calcium sensitivity for fusion is found to be higher in Alpha and Beta variants and highest in Delta spike variant. We find that efficient fusion is dependent on Ca²⁺ concentration at low pH, and the fusion activity of spike dropped as the Ca²⁺ level rose beyond physiological levels. Thus, evolved spike variants may control the high fusion probability for entry by increasing Ca²⁺ sensing ability.