The ACE-2 receptor accelerates but is not biochemically required for SARS-CoV-2 membrane fusion.

The SARS-CoV-2 coronavirus infects human cells via the ACE-2 receptor. Circumstantial evidence suggests that ACE-2 may not just serve as an attachment factor but also help activate the SARS-CoV-2 spike protein for membrane fusion. Here, we test that hypothesis directly, using DNA-lipid tethering as a synthetic attachment factor in the place of ACE-2. We find that SARS-CoV-2 pseudovirus and viruslike particles are both capable of membrane fusion if attached in the absence of ACE-2 and activated with an appropriate protease. However, addition of soluble ACE-2 speeds the fusion reaction. This is observed for both the Wuhan strain and the B.1.1.529 Omicron variant. Kinetic analysis suggests that there are at least two rate-limiting steps for SARS-CoV-2 membrane fusion, one of which is ACE-2 dependent and one of which is not. These data establish that, in the presence of an alternative attachment factor, ACE-2 is not biochemically required for SARS-CoV-2 membrane fusion. Since ACE-2 serves as the high-affinity attachment factor on human cells, the possibility to replace it with other factors has implications for the evolvability of SARS-CoV-2 and the fitness landscape for future related coronaviruses.