The Interplay of Furin Cleavage and D614G in Modulating SARS-CoV-2 Spike Protein Dynamics.

We report a detailed analysis of the full-length SARS-CoV-2 spike dynamics within a native-like membrane environment and variants inaccessible to studies on soluble constructs by conducting hydrogen-deuterium exchange mass spectrometry (HDX-MS) on enveloped virus-like particles (eVLPs) displaying various spike constructs. We find that the previously identified open-interface trimer conformation is sampled in all eVLP-displayed spike variants studied including sequences from engineered vaccine constructs and native viral sequences. The D614G mutation, which arose early in the pandemic, favors the canonical 'closed-interface' prefusion conformation, potentially mitigating premature S1 shedding in the presence of a cleaved furin site and providing an evolutionary advantage to the virus. Remarkably, furin cleavage at the S1/S2 boundary allosterically increases the flexibility of the S2' site, which may facilitate increased TMPRSS2 processing, enhancing viral infectivity. The use of eVLPs in HDX-MS studies provides a powerful platform for studying viral and membrane proteins in near-native environments.