Deubiquitinating activity of SARS-CoV-2 papain-like protease does not influence virus replication or innate immune responses in vivo
Fig 3
SARS-CoV-2 DUB mutant viruses display unaltered growth kinetics in human lung cells.
(A) SARS-CoV-2 stock titers were determined by plaque assay on VeroE6 cells. Representative examples of the plaque morphology of wild-type and PLpro mutant SARS-CoV-2 are shown. (B) Calu-3 cells were infected at MOI 0.1 with wild-type or DUB mutant SARS-CoV-2 to determine the replication kinetics. Virus titers in the supernatant were determined by plaque assay with a limit of detection of 40 plaque-forming units (pfu)/ml (indicated by the dotted line). At 2 hpi, inoculum was removed and cells were washed three times. The last wash was collected and taken along in the plaque assay. No plaques were detected, meaning that the amount of residual virus was below 40 pfu/ml. (n = 5). Data represented as mean ± s.e.m. (C-D) Intracellular (C) and extracellular (D) viral RNA levels were determined by RT-qPCR using primers targeting the E and RdRp coding regions (n = 3). Representative examples are shown. FEH(M) = F69S/E70K/H73G(/M208S). Data was analyzed by one-way ANOVA with Dunnett’s multiple comparisons test, comparing each group to wild-type SARS-CoV-2. No significant differences were found.