Figure 5.

Proposed viral membrane fusion mechanism. (A) Two major pathways for cell entry. Direct entry is the primary pathway; however, endocytosis is also utilized. After the receptor binding, spikes may have more opportunities to facilitate the fusion steps by interacting with other attachment factors. (B) Typical receptors of SARS-CoV-2 for the membrane fusion. ACE2 is a primary receptor; however, the C-terminal of S1 subunit binds to neuropilin 1 (NRP1). The NRP1 pathway is considered to be utilized when the viral load is high. Note that the CendR motif binding to NRP1 is a unique insertion at the S1/S2 site in the SARS-CoV-2 spike. (C) The membrane fusion mechanism proposed in this study. Step 0 (PRE state): After binding a primary receptor such as ACE2, some spikes approach the surface of the host membrane. The RBDs are in thermal equilibrium, taking Up and Down forms. Soluble proteases such as furin can cleave the S1/S2 site in the spike during or before circulation (Table S8). Step 1 (PRE state RBD Up forms): A spike trimer approaches the host membrane surface and interacts with any attachment factors (Table S7) on the membrane surface, leading to the equilibrium shift toward the RBD Up form. Step 2 (INT state): The intermediate (INT) state between PRE and POST states before the S2′ cleavage. A structural transition to INT is triggered by the conditions (RBD Up forms, S1/S2 cleavages). When the two conditions are satisfied, subsequently the unwinding of the stalk occurs, which unlocks the structural transition (untwisting activation mechanism). As a result of the structural transition from PRE to INT, the stalk in S2′ unfolds toward the host membrane and wraps around the S1 trimer. Concurrently, the stem integrates into the viral membrane and N-terms of S1/S2-S2′ expand onto the inner surface of the viral membrane to stabilize the stem. The S1 trimer is facing against the host membrane surface, and eventually the S1 trimer integrates into the host membrane. Step 3 (POST state): The S2′ cleavage event activates two pieces of fusion peptides (uFP and dFP) located at the S2′ cleavage site and triggers membrane fusion. In concert with the N-term of S1/S2-S2′, uFP and dFP moderate and facilitate the curvature of the membrane. Step 4: Viral and host membranes are fused. S1 subunits dissociate and are shed into the host cell. The rest of the proteolytically processed spikes, namely the S1/S2-S2′ and S2′ subunits, are possibly disassembled and delivered into the host cell as well (PRE, INT, and POST structures are colored by subunits).