SARS-CoV-2 Delta and Omicron variants resist spike cleavage by human airway trypsin-like protease
Wenyan Ren, et al.
Wenyan Ren, et al.
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Research Article

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.

Authors

Wenyan Ren, Weiqi Hong, Jingyun Yang, Jun Zou, Li Chen, Yanan Zhou, Hong Lei, Aqu Alu, Haiying Que, Yanqiu Gong, Zhenfei Bi, Cai He, Minyang Fu, Dandan Peng, Yun Yang, Wenhai Yu, Cong Tang, Qing Huang, Mengli Yang, Bai Li, Jingmei Li, Junbin Wang, Xuelei Ma, Hongbo Hu, Wei Cheng, Haohao Dong, Jian Lei, Lu Chen, Xikun Zhou, Jiong Li, Wei Wang, Guangwen Lu, Guobo Shen, Li Yang, Jinliang Yang, Zhenling Wang, Guowen Jia, Zhaoming Su, Bin Shao, Hanpei Miao, Johnson Yiu-Nam Lau, Yuquan Wei, Kang Zhang, Lunzhi Dai, Shuaiyao Lu, Xiawei Wei

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Figure 5

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

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The mutations P681R in Delta and P681H in Omicron spike proteins result ...
(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.