Spike protein-independent attenuation of SARS-CoV-2 Omicron variant in laboratory mice

Abstract

Despite being more transmissible, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant only causes milder diseases in laboratory animals, often accompanied by a lower viral load compared with previous variants of concern. In this study, we report the structural basis for a robust interaction between the receptor-binding domain of the Omicron spike protein and mouse ACE2. We show that pseudovirus bearing the Omicron spike protein efficiently utilizes mouse ACE2 for entry. By comparing viral load and disease severity among laboratory mice infected by a natural Omicron variant or recombinant ancestral viruses bearing either the entire Omicron spike or only the N501Y/Q493R mutations in its spike, we find that mutations outside the spike protein in the Omicron variant may be responsible for the observed lower viral load. Together, our results imply that a post-entry block to the Omicron variant exists in laboratory mice.

Keywords: Balb/c mice; CP: Microbiology; K18-hACE2; Omicron variant; SARS-CoV-2; attenuation.

Graphical abstract

Figure 1

Pseudovirus bearing the Omicron spike protein efficiently infects cells expressing mACE2 (A) 293T cells were transfected with ACE2 expression plasmids of 10 species and then infected by lentiviral pseudoparticles bearing the spike protein of either the Wuhan SARS-CoV-2 variant or the Omicron variant. Infected cells would be GFP positive (green). Blue dye-stained nuclei. Images shown here are representatives of two independent experiments. (B) A similar experiment was performed as in (A), except pseudoviruses expressing firefly luciferase were employed. (C) Infection of hACE2-and mACE2 stable 293T cells by pseudoviruses bearing the spike protein of either the Wuhan SARS-CoV-2 variant or the Omicron variant. Results in each bar graph contain at least three technical replicates.

Figure 2

The spike protein of Omicron variant carries mutations that confer utility of mouse ACE2 for entry (A) Illustration of all changes found in the Omicron isolate used in this study (hCoV-19/USA/MD-HP20874/2021) compared with USA-WA1/2020. Coincidentally, the Omicron spike protein in pseudoviruses carries an identical number of changes. (B–E) Various coordinations of charged residues at the ACE2-RBD interface: (B) hACE2-wtRBD (buried charge: 0), (C) mACE2-wtRBD (buried charge: −2 e), (D) hACE2-oRBD (buried charge: +2 e), and (E) mACE2-oRBD (buried charge: 0). (F and G) Detailed interfacial coordinations between mACE2 and oRBD. (F) The interfacial binding near the salt bridge R498-D38. (G) The interfacial binding near the salt bridge R493-E35. (H and I) Pseudoviruses carrying WA1 spike with indicated combinations of mutations infected 293T-hACE2 or 293-mACE2 cell lines. (J) Cell-cell fusion is mediated by WA1 spike or Omicron spike or the WA1-Q493R/N501Y spike protein. Results in each bar graph contain at least three technical replicates. Data shown here are representatives of two independent experiments.

Figure 3

Spike-independent attenuation of the Omicron variant in Balb/c mice (A) Overall study design. (B) Weight loss profile. (C) Clinical scores of all three groups of mice overall the period of study. (D–G) Subgenomic RNA (sgmRNA) and total viral RNA levels in nasal turbinates (D and E) and lungs (F and G). (H) Induction of ISG15 at 2 DPI in mouse lungs. (I) Summed pathology scores of all three groups of mice overall the period of study. (J–M) Representative H&E images of the natural Omicron-infected mice. (K) is a closeup image from (J), and (M) is a closeup from (L). (N) An RNAscope image of the natural Omicron variant-infected mouse. (O) A closeup from (N). Blue, nuclei; red, viral RNA. (P) A representative H&E image of the WA1-Omicron S-infected mouse. (Q) A closeup from (P). (R) A representative RNAscope image of the WA1-Omicron S-infected mouse. (S) A closeup from (R). (T) A representative H&E image of the WA1-Q493R/N501Y-infected mouse. (U) A closeup from (T). (V) A presentative RNAscope image of the WA1-Q493R/N501Y-infected mouse. (W) A closeup from (V). Results shown in this figure were derived from one experiment (n = 10–15 for each group). Each solid shape represents one animal in (D)–(H).

Figure 4

WA1-Omicron-S is lethal in K18-hACE2 mice (A) Overall study design. n = 10 per group. (B) Weight loss profile. Note, after 9 DPI there was only one WA1-infected mouse alive. All WA-Omicron-S-infected mice succumbed to infection by day 8. (C) Survival curves. (D) Viral RNA (D) and sgmRNA (E) levels in nasal turbinates (NBs) and the lungs at 6 DPI. (F) Infectious viral titers from lung homogenates at 6 DPI. ^∗p < 0.05. Results shown in this figure were derived from one experiment. Each solid shape represents one animal in (D)–(F).

Figure 5

Omicron bearing WA1 spike is attenuated in K18-hACE2 mice (A) Body weight loss of K18 mice after infection with 10⁴ PFUs of Omicron-WA1 S, WA1-Omicron 5′ UTR-Nsp12, or Omicron or 10³ PFUs WA1. (B) Survival curves after infection. Numbers of mice in each group are indicated in the legend. (C and D) sgmRNA (C) and viral RNA (D) levels in NBs at 2 DPI. (E and F) sgmRNA (E) and viral RNA (F) levels from brains at 6 DPI. (G) Infectious virus titers from lungs at 6 DPI. ^∗p < 0.05. Results shown in this figure were derived from one experiment. Each solid shape represents one animal in (C)–(G).