Pro108Ser mutation of SARS-CoV-2 3CLpro reduces the enzyme activity and ameliorates the clinical severity of COVID-19.

Recently, an international randomized controlled clinical trial showed that patients with SARS-CoV-2 infection treated orally with the 3-chymotrypsin-like protease (3CLpro) inhibitor PF-07321332 within three days of symptom onset showed an 89% lower risk of COVID-19-related hospital admission/ death from any cause as compared with the patients who received placebo. Lending support to this critically important result of the aforementioned trial, we demonstrated in our study that patients infected with a SARS-Cov-2 sub-lineage (B.1.1.284) carrying the Pro108Ser mutation in 3CLpro tended to have a comparatively milder clinical course (i.e., a smaller proportion of patients required oxygen supplementation during the clinical course) than patients infected with the same sub-lineage of virus not carrying the mutation. Characterization of the mutant 3CLpro revealed that the Kcat/Km of the 3CLpro enzyme containing Ser108 was 58% lower than that of Pro108 3CLpro. Hydrogen/deuterium-exchange mass spectrometry (HDX-MS) revealed that the reduced activity was associated with structural perturbation surrounding the substrate-binding region of the enzyme, which is positioned behind and distant from the 108th amino acid residue. Our findings of the attenuated clinical course of COVID-19 in patients infected with SARS-CoV-2 strains with reduced 3CLpro enzymatic activity greatly endorses the promising result of the aforementioned clinical trial of the 3CLpro inhibitor.

Severity of COVID-19 is inversely correlated with increased number counts of non-synonymous mutations in Tokyo (preprint)

Background: SARS-CoV-2 genome accumulates point mutations in a constant manner. Whether the accumulation of point mutations is correlated with milder manifestations of COVID-19 remains unknown. Methods: We performed SARS-CoV-2 genome sequencing in 90 patients with COVID-19 infection treated at a tertiary medical center in Tokyo between March and August 2020. The possible association between disease severity and viral haplotype was then assessed by counting the number of mutations in addition to performing phylogenic tree analysis, comparative amino acid sequence analysis among {beta}-coronaviruses, and mathematical prediction of the functional relevance of amino acid substitutions. Results: The number of non-synonymous mutations was inversely correlated with COVID-19 severity, as defined by requiring oxygen supplementation. Phylogenic tree analysis identified two predominant groups which were separated by a set of 6 single nucleotide substitutions, including four leading to non-synonymous amino acid substitutions. Among those four, Pro108Ser in 3 chymotrypsin-like protease (3CLpro) and Pro151Leu in nucleocapsid protein occurred at conserved locations and were predicted to be deleterious. Patients with Pro108Ser in 3CLpro and Pro151Leu in nucleocapsid protein had a lower odds ratio for developing hypoxia requiring supplemental oxygen (odds ratio of 0.24 [95% confidence interval of 0.07-0.88, P-value = 0.032]) after adjustments for age and sex, compared with patients lacking this haplotype in Clade 20B. Conclusion: Viral genome sequencing in 90 patients treated in the Tokyo Metropolitan area showed that the accumulation of point mutations, including Pro108Ser in 3CLpro and Pro151Leu in nucleocapsid protein, was inversely correlated with COVID-19 severity. Further in vitro research is awaited.