SARS-CoV-2 variants with mutations at the S1/S2 cleavage site are generated in vitro during propagation in TMPRSS2-deficient cells

• Published in: PLoS pathogens Vol. 17; no. 1; p. e1009233
• Main Authors: Sasaki, Michihito, Uemura, Kentaro, Sato, Akihiko, Toba, Shinsuke, Sanaki, Takao, Maenaka, Katsumi, Hall, William W., Orba, Yasuko, Sawa, Hirofumi
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 21.01.2021; Public Library of Science (PLoS)
• Subjects: ACE2; Amino Acid Sequence; Amino acids; Analysis; Angiotensin; Angiotensin-converting enzyme 2; Animals; Binding sites; Biology and life sciences; Caco-2 Cells; Cell Line; Chlorocebus aethiops; Cleavage; Cloning; Coronaviruses; COVID-19; Dilution; Gene mutations; Genetic aspects; Genomes; HEK293 Cells; Humans; Infections; Medicine and health sciences; Mutation; Nucleotides; Pandemics; Peptidyl-dipeptidase A; Progeny; Propagation; Proteins; Receptors; Research and Analysis Methods; S gene; SARS-CoV-2 - classification; SARS-CoV-2 - genetics; SARS-CoV-2 - growth & development; SARS-CoV-2 - physiology; Sequence Alignment; Serial Passage; Serine Endopeptidases - deficiency; Severe acute respiratory syndrome; Severe acute respiratory syndrome coronavirus 2; Spike Glycoprotein, Coronavirus - genetics; Vero Cells; Viral diseases; Viral Tropism; Virions; Viruses; Japan
• ISSN: 1553-7374; 1553-7366; 1553-7374
• DOI: 10.1371/journal.ppat.1009233

The spike (S) protein of Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) binds to a host cell receptor which facilitates viral entry. A polybasic motif detected at the cleavage site of the S protein has been shown to broaden the cell tropism and transmissibility of the virus. Here we examine the properties of SARS-CoV-2 variants with mutations at the S protein cleavage site that undergo inefficient proteolytic cleavage. Virus variants with S gene mutations generated smaller plaques and exhibited a more limited range of cell tropism compared to the wild-type strain. These alterations were shown to result from their inability to utilize the entry pathway involving direct fusion mediated by the host type II transmembrane serine protease, TMPRSS2. Notably, viruses with S gene mutations emerged rapidly and became the dominant SARS-CoV-2 variants in TMPRSS2-deficient cells including Vero cells. Our study demonstrated that the S protein polybasic cleavage motif is a critical factor underlying SARS-CoV-2 entry and cell tropism. As such, researchers should be alert to the possibility of de novo S gene mutations emerging in tissue-culture propagated virus strains.