Distinct pathways for evolution of enhanced receptor binding and cell entry in SARS-like bat coronaviruses

“…WIV1-CoV circulates in the same colony of bats in Yunnan Province, China from which the SHC014-CoV genome was also discovered and sequenced; however, the latter virus was not recovered from these samples [ 14 ]. We found that, unlike the WT SHC014-CoV spike, the WT WIV1-CoV spike could readily bind human ACE2 and mediate viral entry, as observed previously [ 14 – 16 ] and consistent with recent structural observations that the WIV1-CoV spike has a more open conformation than its SHC014-CoV counterpart [ 12 ]. Herein, we show that a single amino acid residue difference in the S1 subunit can fully account for the divergent properties of the WIV1-CoV and SHC014-CoV spikes without altering their propensity to undergo proteolytic cleavage at the S1-S2 boundary.…”

“…We propose that these changes propagate upward to CTD1 and laterally to the FPPR, as described by Cai and Calvaresi [ 3 , 7 ], enhancing RBD availability and priming S2 to undergo its fusion-related conformational changes. Interestingly, the amino acid substitutions in the NTD-RBD linker and the FPPR that we previously identified in the VSV-adapted SHC014-CoV spike [ 12 ] and the FPPR substitutions we identified in the VSV-adapted Rs3367-CoV spike herein ( S9 Fig ) bracket the CTD2 and 630 loop substitutions (V584I and Y623H, respectively), providing further support for the SARS-CoV-2 allosteric model [ 3 , 7 ].…”

“…Here we found that, as expected, the WT WIV1-CoV spike phenocopies the VSV-adapted SHC014-CoV spike in its enhanced capacity to recognize ACE2 and its increased susceptibility to antibody neutralization. Concordantly, recent structural work indicates that the WIV1-CoV spike can adopt the partially open ‘one-RBD-up’ state, whereas the SHC014-CoV spike cannot [ 11 , 12 ]. These findings pointed to one or more amino acid sequence differences between SHC014-CoV and WIV1-CoV as responsible for the correlated phenotypic differences between the spikes of these rhinolophid bat beta-CoVs.…”

“…Because the RBDs of Rs3367-CoV and WIV1-CoV do not differ in amino acid sequence and the latter has been shown to recognize Hs ACE2 with nanomolar affinity [ 13 ], we reasoned that Y623H likely enhances spike:ACE2 binding by increasing the propensity of the WIV1-CoV spike to sample the RBD-up conformation (‘RBD availability’), as we also recently observed for SHC014-CoV spikes bearing growth-adaptive mutations [ 12 ]. To test this, we first compared the sensitivities of scVSVs bearing Rs3367-CoV, WIV1-CoV, and their chimeras to neutralization by Adagio-2 (ADG-2), a monoclonal antibody (mAb) that specifically recognizes the “up” conformer of the RBD [ 19 , 20 ].…”