Pro108Ser mutant of SARS-CoV-2 3CL pro Reduces the Enzymatic Activity and Ameliorates COVID-19 Severity in Japan

Abe, Kodai Kabe Yasuaki Uchiyama Susumu W.Iwasaki Yuka Ishizu Hirotsugu Uwamino Yoshifumi Takenouchi Toshiki Uno Shunsuke Ishii Makoto Maruno Takahiro Noda Masanori Murata Mitsuru Hasegawa Naoki Saya Hideyuki Kitagawa Yuko Fukunaga Koichi Amagai Masayuki Siomi Haruhiko Suematsu Makoto Kosaki Kenjiro Project Keio Donner

Background: It is well known that SARS-CoV-2 genome accumulates point mutations constantly. However, whether non-synonymous mutations affect COVID-19 severity through altering viral protein function remains unknown.Methods: We performed SARS-CoV-2 genome sequencing in 90 patients with COVID-19 admitted at Keio University Hospital in Tokyo Metropolitan area between March and August 2020. Viral haplotypes were examined by counting the number of non-synonymous mutations to analyse phylogenic trees and comparative amino acid sequence. Statistically relevant non-synonymous mutations were functionally evaluated with structural analyses.Findings: The number of non-synonymous mutations correlated inversely with the COVID-19 severity. Phylogenic tree analyses identified two predominant groups which were differentiated by a set of six point mutations (four non-synonymous amino acid mutations). Among them, Pro108Ser in 3 chymotrypsin-like protease (3CLpro) and Pro151Leu in nucleocapsid protein occurred at conserved locations among β-coronaviruses. Patients with these mutations indicated significantly lower odds ratio for developing hypoxia which required supplemental oxygen (adjusted odds ratio 0·24 [95% CI 0·07-0·88, p-value = 0·032]) after adjustments for age and sex, versus those lacking this haplotype in the canonical Clade 20B spread in Japan. The Pro108Ser 3CLpro enzyme decreases in the activity by 58%, and the hydrogen/deuterium exchange mass spectrometry reveals that mechanisms for decline-of-function involve structural perturbation at the substrate-binding region which is positioned behind and distant from the 108th amino acid residue of the enzyme.Interpretation: Viral genome sequencing in Tokyo showed that the specific mutant strain containing Pro108Ser mutation in 3CLpro, ameliorates the COVID-19 severity. This Pro108Ser mutant in 3CLpro reduces the catalytic activity of the protein by 50%. The mutant strain rapidly outcompeted pre-existing variants to become the dominant one in Japan. Our results may benefit the efforts under way to design small molecular compounds or antibodies targeting 3CLpro.Funding: Keio Gijuku Academic Development Funds and AMED (Grant Number JP20he0622043)Declaration of Interests: Authors have no conflicts of interests.Ethics Approval Statement: The study protocol was approved by the Ethics Committee of Keio University School of Medicine (approval number: 20200062).