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

Abstract

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.

The SARS-CoV-2 and other human coronavirus spike proteins are fine-tuned towards temperature and proteases of the human airways

“…4B, top right panel). This explains why SARS-CoV-2 passaging in Vero E6 cells regularly leads to emergence of viruses bearing substitutions or deletions in the S1/S2 loop (30)(31)(32)(33)(34)(35)(36). For the SARS-1-S and MERS-S mutants, the data concurred with other reports.…”

“…When unprimed, SARS-2-S pseudoviruses are strongly boosted towards the cathepsin B/L route. This likely explains the replication advantage of loop-deletion SARS-CoV-2 mutants in cathepsin L-rich Vero E6 cells (30)(31)(32)(33)(34)(35)(36). Whereas the S1/S2 primed state predisposes to S2ʹ activation by TTSPs, the non-covalently linked S1/S2 form is less stable and a plausible disadvantage for endosomal entry.…”

“…For SARS-2-S, the determinants governing cleavage of its extended S1/S2 loop are still far from clear. In addition, SARS-CoV-2 passaging in Vero cells commonly leads to substitutions or deletions in the S1/S2 cleavage loop (30)(31)(32)(33)(34)(35)(36). In animal models, these viruses exhibit reduced transmission (37) or virulence (38).…”

The furin cleavage site of SARS-CoV-2 spike protein is a key determinant for transmission due to enhanced replication in airway cells

“…The three separate deletions reported in Supplementary Table S1 have not previously been reported but are similar to those seen upon passage in Vero E6 cells. OS5 deletes 4 amino acids after the CS similar to a deletion reported in a recent preprint (Sasaki et al, 2020); OS19-1 overlaps with most of ΔFlank and OS19-2 completely removes the S1/S2 site, similar to ΔCS. We have also observed identical deletions to OS19-2 upon passaging the clonal WT virus in Vero E6 cells.…”

“…Interestingly, SARS-CoV-2 has been shown in multiple independent studies to rapidly lose this polybasic CS upon passage in Vero E6 cells, which has been a popular cell line for isolating and propagating the virus (Davidson et al, 2020; Klimstra et al, 2020; Z. Liu et al, 2020; Ogando et al, 2020; Sasaki et al, 2020; Wong et al, 2020; Y. Zhu et al, 2020).…”