Abstract
Virus propagation methods generally use transformed cell lines to grow viruses from clinical specimens, which may force viruses to rapidly adapt to cell culture conditions, a process facilitated by high viral mutation rates. Upon propagation in VeroE6 cells, SARS-CoV-2 may mutate or delete the multibasic cleavage site (MBCS) in the spike protein. Previously, we showed that the MBCS facilitates serine protease-mediated entry into human airway cells (Mykytyn et al., 2021). Here, we report that propagating SARS-CoV-2 on the human airway cell line Calu-3 - that expresses serine proteases - prevents cell culture adaptations in the MBCS and directly adjacent to the MBCS (S686G). Similar results were obtained using a human airway organoid-based culture system for SARS-CoV-2 propagation. Thus, in-depth knowledge on the biology of a virus can be used to establish methods to prevent cell culture adaptation.
Highlights
41 42 Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease (COVID-19) pandemic
98 99 In this study, we investigated whether mutations in the SARS-CoV-2 spike multibasic cleavage site (MBCS) could be prevented in a human airway cell line (Calu-3) and 2D air-liquid interface (ALI) airway organoids in which SARS-CoV-2 enters using serine proteases. 103 Results 104 105 SARS-CoV-2 isolates that are cultured in the lab rapidly lose their spike MBCS (Davidson et al., 2020; Klimstra et al, 2020; Lau et al, 2020; Liu et al, 2020; Ogando et al, 2020).To investigate the extent of cell culture adaptation in our SARS-CoV-2 stocks, we deep108 sequenced passage 2, 3 and 4 stocks (P2, P3 and P4) of the BavPat-1 or Munich-1 strain 109 propagated on VeroE6 cells
The stable expression of TMPRSS2 in VeroE6 cells leads to entry of wild type (WT) pseudoviruses via this protease instead of cathepsin-mediated entry, but SARS-CoV-2 MBCS mutants and to a lesser extent the S686G mutant retained partial cathepsin mediated entry (Figure 3F-I). In addition, a green fluorescent protein (GFP)-complementation fusion assay, in which cell-cell fusion occurs at the plasma membrane, showed that MBCS mutations and to a lesser extent the S686G mutation abrogated fusion in VeroE6, VeroE6-TMPRSS2 and Calu-3 cells (Figure 4A-C)
Summary
41 42 Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease (COVID-19) pandemic. The most widely used cell line in virology is the Vero cell line, which is derived from the kidney of an African green monkey This cell line and its derivatives (e.g. VeroE6, Vero118, etc) contain genomic deletions of genes involved in the antiviral interferon response (Osada et al, 2014). Such mutations are common in transformed cell lines and allow unbridled virus replication, facilitating the production of high titer virus stocks and allowing research on a wide range of viruses. These isolated viruses are often adapted to their cell culture systems due to their high mutation rates. Proteolytic cleavage at the S1/S2 site and the more C-te i al S ’ site is required for coronavirus infectivity as this turns on the fusogenic activity of the S2
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