Critical ACE2 Determinants of SARS-CoV-2 and Group 2B Coronavirus Infection and Replication

Abstract

The angiotensin-converting enzyme 2 (ACE2) receptor is a major severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) host range determinant, and understanding SARS-CoV-2-ACE2 interactions will provide important insights into COVID-19 pathogenesis and animal model development. SARS-CoV-2 cannot infect mice due to incompatibility between its receptor binding domain and the murine ACE2 receptor. Through molecular modeling and empirical in vitro validation, we identified 5 key amino acid differences between murine and human ACE2 that mediate SARS-CoV-2 infection, generating a chimeric humanized murine ACE2. Additionally, we examined the ability of the humanized murine ACE2 receptor to permit infection by an additional preemergent group 2B coronavirus, WIV-1, providing evidence for the potential pan-virus capabilities of this chimeric receptor. Finally, we predicted the ability of these determinants to inform host range identification of preemergent coronaviruses by evaluating hot spot contacts between SARS-CoV-2 and additional potential host receptors. Our results identify residue determinants that mediate coronavirus receptor usage and host range for application in SARS-CoV-2 and emerging coronavirus animal model development.IMPORTANCE SARS-CoV-2 (the causative agent of COVID-19) is a major public health threat and one of two related coronaviruses that have caused epidemics in modern history. A method of screening potential infectible hosts for preemergent and future emergent coronaviruses would aid in mounting rapid response and intervention strategies during future emergence events. Here, we evaluated determinants of SARS-CoV-2 receptor interactions, identifying key changes that enable or prevent infection. The analysis detailed in this study will aid in the development of model systems to screen emergent coronaviruses as well as treatments to counteract infections.

Keywords: COVID-19; SARS-CoV-2; coronavirus; host range; receptors; virus-host interactions.

FIG 1

Engineered mutations in the mACE2 background reestablish contacts with SARS-CoV-2 RBD and support SARS-CoV-2 infection and replication. (a) Residues modified to incrementally increase humanization of mACE2 (blue, mouse; pink, human). (b) Contacts between the SARS-CoV-2 RBD and the three hACE2 interaction hot spots. (c) Lost contacts between mACE2 hot spots and SARS-CoV-2 RBD Q493/G496 (red). (d to g) Key contacts gained and lost through mACE2 humanization of one amino acid change (Q493 and G496 gained, F456 and Y489 lost) (d), three amino acid changes (Q493, F456, and Y489 maintained, G496 lost) (e), five amino acid changes (all regained) (f), and a swap of the entire hACE2 binding interface (all maintained) (g). (h) Western blotting confirming ACE2 receptor expression (anti-6×-His) and SARS-CoV-2 (anti-nucleocapsid) infection. (i) qPCR 24 h after infection by SARS-like virus SHC014. (j) qPCR. (k) Genome equivalents. (l) Luciferase. (m) Titer 24 h following virus infection. n = 3 replicates per sample. Data analyzed by 2-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons. Error bars represent standard error about the mean. *, comparisons to mock where P ≤ 0.0001; x, comparisons to mock where P ≤ 0.001; +, comparisons to mACE2 where P ≤ 0.0001. sgRNA, single guide RNA.

FIG 2

hmACE2 contacts with WIV-1 and P5L are predicted to support replication. (a) Aligned contact residues of SARS-CoV-2, SARS-CoV, WIV-1, and P5L. (b and c) Contacts identified between mACE2 and WIV-1 (b) and those gained between hmACE2 and WIV-1 (c) include Y482 and D481. (d and e) Contacts identified between mACE2 and P5L (d) and gained between hmACE2 and P5L (e) include G496, E493, and F456 (right). (f and g) qPCR (f) and titer (g) 24 h following WIV-1 infection. Data analyzed by 2-way ANOVA followed by Dunnett’s multiple comparisons. Error bars represent standard error about the mean. *, comparisons to mock where P ≤ 0.0001. Plus signs represent comparisons to mACE2 (+, P ≤ 0.05; ++, P ≤ 0.01; +++, P ≤ 0.001; ++++, P ≤ 0.0001).