Extended Data Fig. 3: Caspase-6 facilitates MERS-CoV replication.
From: Coronaviruses exploit a host cysteine-aspartic protease for replication
a MERS-CoV-infected BEAS2B cells were incubated with z-VEID-fmk in a time of addition assay. MERS-CoV N gene copy in the supernatant was determined with RT-qPCR at 24hpi (n = 4). b, c Caspase-6 gene (n = 3) (b) and protein expression (c) from caspase-6 stable knockdown A549 and BEAS2B cells. d-e Caspase-6 stable knockdown A549 and BEAS2B cells were infected with MERS-CoV at 0.1MOI. Cell lysates were harvested at 1 hpi to quantify virus entry with RT-qPCR against the MERS-CoV N gene (n = 4) (d). Virus replication at 24 hpi were quantified with RT-qPCR against the MERS-CoV N gene (n = 4) (e). f Caspase-6 stable knockdown or scrambled knockdown A549 and BEAS2B cells were infected with MERS-CoV at 0.1MOI followed by treating the cells with z-VEID-fmk at the indicated concentrations. Cell lysate and supernatant samples were harvested at 24 hpi to quantify virus replication with RT-qPCR against the MERS-CoV N gene (n = 3). g Caspase-6 protein expression from caspase-6 siRNA transfected MDMs. h MDMs were treated with caspase-6 or nontargeting siRNA and infected with MERS-CoV. MERS-CoV N gene copy was quantified at 24 hpi (n = 4). i Caspase-6 protein detection from caspase-6 KO or scrambled KO Huh7 cells. j, k Caspase-6- or caspase-3-overexpressed 293T cells were infected with MERS-CoV at 1MOI. MERS-CoV replication was quantified at 1 and 24 hpi with RT-qPCR against the MERS-CoV N gene (n = 4). The cleaved and uncleaved bands in (j) were obtained from the same experiment using different exposure times. The experiments in (c, g, and i) were repeated three times independently with similar results. Data represented mean and standard deviations from the indicated number of biological repeats. Statistical significance between groups was determined with one way-ANOVA (a), two-way ANOVA (b, d, f, and k), or two-sided Student’s t-test (e and h).
