SARS-CoV-2 Delta and Omicron variants resist spike cleavage by human airway trypsin-like protease

Abstract

Soluble host factors in the upper respiratory tract can serve as the first line of defense against SARS-CoV-2 infection. In this study, we described the identification and function of a human airway trypsin-like protease (HAT), capable of reducing the infectivity of ancestral SARS-CoV-2. Further, in mouse models, HAT analogue expression was upregulated by SARS-CoV-2 infection. The antiviral activity of HAT functioned through the cleavage of the SARS-CoV-2 spike glycoprotein at R682. This cleavage resulted in inhibition of the attachment of ancestral spike proteins to host cells, which inhibited the cell-cell membrane fusion process. Importantly, exogenous addition of HAT notably reduced the infectivity of ancestral SARS-CoV-2 in vivo. However, HAT was ineffective against the Delta variant and most circulating Omicron variants, including the BQ.1.1 and XBB.1.5 subvariants. We demonstrate that the P681R mutation in Delta and P681H mutation in the Omicron variants, adjacent to the R682 cleavage site, contributed to HAT resistance. Our study reports what we believe to be a novel soluble defense factor against SARS-CoV-2 and resistance of its actions in the Delta and Omicron variants.

Keywords: COVID-19; Molecular biology.

Figure 1. Defense-related component existing in the respiratory tract inhibits infection of ancestral SARS-CoV-2 but not Delta and Omicron variants.

(A–C) Infectivity of WT (A), Delta (B), and Omicron BA.1 (C) pseudoviruses pretreated with nasal wash samples (NW) from young (20–30 years) and old (55–70 years) participants (n = 10). (D) Infectivity of WT pseudovirus with respiratory tract proteases or proteins (n = 3). (E) WT pseudovirus treated with NW, with or without HAT inhibitors aprotinin and soybean trypsin inhibitor (STI) (n = 10). (F) HAT (2 μg/mL) pretreated with spike proteins, incubated with WT pseudovirus, determined infectivity (n = 3). (G and H) Representative images (G) and quantification (H) of CPE in Vero E6 cells infected with live ancestral viruses, with or without HAT (0.5–5 μg/mL) (n = 3). (I and J) Cell lysates collected 48 hours after infection with live viruses for gRNA (I) and sgRNA (J) assays (n = 3). (K) The mRNA levels of tmprss11d in trachea (left) and lung (right) tissues in mice infected with SARS-CoV-2 on day 3 after infection (n = 3 mice each group). (L and M) The representative images displayed conjugates (arrow) formed with SARS-CoV-2 spike protein (green) and released trypsin-like protease (red) in bronchial tissues of infected mice (L) and primates (M). (N) The images indicated SARS-CoV-2 particles (green) trapped by filamentous HAT (red) in the sputum of patients with COVID-19. The inset shows magnified conjugates in merged images. Data are representative (D, F, G–J, and K) of 2 independent experiments with 3 replicates each. Unpaired, 2-tailed Student’s t test was performed in D and K, and 1-way ANOVA analysis followed by Tukey’s multiple comparison post hoc test was conducted in A–C, E, F, and H–J. Scale bars: 200 μm in G; 4 μm in L and M; and 10 μm in N. Data are presented as mean values ± SEM in A–K. *P <0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 2. Delta and Omicron variants resist the HAT-induced antiviral effects.

(A) The viral loads in trachea and bronchus from nonhuman primates challenged with ancestral, Delta, or Omicron BA.1 strains. The data were retrospectively collated from animals in control group that previously used in evaluation of vaccine. All animals were sedated and challenged with 1 × 10⁶ PFU of live viruses via intranasal (0.5 mL) and intratracheal (0.5 mL) routes, and euthanized 7 days after infection for viral loads assay (n = 9 nonhuman primates in ancestral virus group, n = 10 in Delta group, and n = 7 in BA.1 group). (B–J) The antiviral effects of HAT against WT (B), Delta (C), and Omicron subvariants BA.1 (D), BA.2 (E), BA.3 (F), BA.4/5 (G), BA.2.12.1 (H), BQ.1.1 (I), and XBB.1.5 (J) pseudoviruses were determined. (n = 3 each group). (K) The infectivity of WT, Delta, and Omicron BA.1 pseudoviruses on TMPRSS2-positive Calu-3 cells in the presence or absence of the HAT (0.5–5 μg/mL) (n = 3). (L and M) The representative pictures (L) and quantification analysis (M) of cytopathogenic effects (CPE) in Delta and Omicron variant-infected Vero E6 cells (n = 3). Scale bars: 200 μm in L. (N and O) Cell lysates from infected Vero E6 cells were collected 48 hours after infection with live viruses for detection of the levels of gRNA (N) and sgRNA (O) (n = 3). (P) The gRNA levels of viruses in infected Calu-3 cells with or without preincubation of HAT (n = 3). 2-way ANOVA followed by Tukey’s multiple comparison post hoc test was conducted in A. 1-way ANOVA followed by Tukey’s multiple comparison post hoc test was conducted in B–K and M–P. Data are presented as mean values ± SEM A–K and M–P. *P <0.05; **P < 0.01; ****P < 0.0001.

Figure 3. HAT blocked the binding of spike proteins to hACE2 receptor and inhibited the cell-cell membrane fusion process.

(A–E) Flow cytometry analysis of the percentages of spike (S) proteins from WT (A), Delta (B), and Omicron BA.1 (C), BA.4 (D), or BA.5 (E) strains pretreated with HAT binding to 293T/ACE2 cells (n = 3). (F–I) Effect of HAT on the process of cell-cell fusion. Representative images (F) and quantitative analysis (G–I) of syncytia in the cell–cell fusion. The effector cells expressing spike proteins and EGFP were pretreated with HAT for 2 hours, and the effector cells were then collected and added to target cells that express the hACE2 receptor (n = 3). Unpaired, 2-tailed Student’s t test was conducted in A–E and G–I. Scale bars: 50 μm in F. Data are presented as mean values ± SEM. **P < 0.01; ****P < 0.0001.

Figure 4. The effect of HAT on the infectivity of ancestral SARS-CoV-2, Delta, and Omicron variants in vivo.

(A) 5 × 10⁴ PFU of ancestral SARS-CoV-2, Delta, and Omicron (BA.1) variants were preincubated with PBS or 80 ng HAT in a total volume of 40 μL. After incubation at 37°C for 2 hours, 6–8 week-old female transgenic hACE2 (hACE2-KI/NIFDC) mice were intranasally instilled with the mixtures. The lung tissues were collected on day 3 after infection to determine the histopathological changes and the viral loads (n = 5 mice each group). (B and C) Representative images of histopathological changes (B) and pathological score (C) in the lung tissues in each group. Scale bars represent 100 μm in B. (D) The levels of gRNA in mouse lung tissues on day 3 after infection in each group were detected by RT-qPCR. 1-way ANOVA followed by Tukey’s multiple comparison post hoc test was conducted in C and D. Data are presented as mean values ± SEM in C and D. ****P < 0.0001.

Figure 5. The mutations P681R in Delta and P681H in Omicron spike proteins result in resistance to the HAT antiviral effect.

(A) Coomassie staining analysis of spike protein cleavage by HAT. Spike protein alone was used as control. Numbers represent normalized band intensities. (B and C) Western blot assessed spike protein cleavage by HAT using anti-S1(B) and anti-S2 (C) antibodies. (D) Live viruses produced in Vero E6 cells, preincubated with HAT (2 μg/mL) with or without aprotinin, then assayed by Western blot using S1 (top) and nucleocapsid (bottom) antibodies. (E) The calculated spike cleavage rate in D. (F) A molecular model of HAT interacting with SARS-CoV-2 S proteins S1/S2 cleavage site. Proteins are shown in ribbon format, with HAT in cyan and the cleavage site in yellow. Important residues, including the catalytic triad H227, D272, and S368 and salt bridge R682–D362, are shown in stick form. (G) RMSD time evolution of SARS-CoV-2 S proteins S1/S2 cleavage site. (H) Time evolution of HAT-S1/S2 cleavage site contact area for SARS-CoV-2 S proteins. (I and J) Cleavage products of spike protein for mass spectrometry: in-gel collection, enzymatic digestion, and analysis. HAT cleaves spike protein at R682 site. (K) Diagram of cleavage and surrounding mutation sites in SARS-CoV-2 variants. (L) Infectivity of Mut-1 (R681P in Delta), Mut-2 (K679N in BA.1), and Mut-3 (H681P in BA.1) pseudoviruses preincubated with HAT (2 μg/mL) (n = 3). (M) Infectivity of WT pseudovirus carrying P681R mutation pretreated with or without HAT (0.5–2 μg/mL) (n = 3). (N) Time evolution of the RMSD of the S1/S2 cleavage site for BA.1 (N679K) or (P681H) mutation. (O) Time evolution of contact interface area between HAT and S1/S2 site for BA.1 (N679K) or (P681H) mutation. 2-way ANOVA followed by Šidák’s multiple comparisons test was conducted in L and M. Data are presented as mean values ± SEM. **P < 0.01; ****P < 0.0001.