Characterisation of protease activity duringSARS-CoV-2 infection identifies novel viralcleavage sites and cellular targets withtherapeutic potential

<p><strong>Virus infections in siRNA-based cellular protein knockdowns - imaging dataset for cell viability</strong><br> Host proteins were knocked-down in A549-Ace2 cells using specific dsiRNAs from IDT. Briefly, A549-Ace2 cells seeded at 1x10$^{4}$ cells/well in 96-well plates. After 24 hours, each well was transfected with 5 pmol of individual dsiRNAs using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the manufacturer&rsquo;s instructions. 24 hours post transfection, the cell culture supernatant was removed and replaced with virus inoculum (MOI of 0.1 PFU/cell). Following a 1 hour adsorption at 37&deg;C, the virus inoculum was removed and replaced with fresh 2\% FBS/DMEM media. Cells were incubated at 37&deg;C for 3 days before supernatants were harvested. Samples were either heat-inactivated at 80&deg;C for 20 min and viral RNA was quantified by RT-qPCR, using previously published SARS-CoV-2 specific primers targeting the N gene \cite{ChuClinChem}. RT-qPCR was performed using the Luna Universal One-Step RT-qPCR Kit (NEB) in an Applied Biosystems QuantStudio 7 thermocycler, using the following cycling conditions: 55 &deg;C for 10 min, 95 &deg;C for 1 min, and 40 cycles of 95 &deg;C for 10 sec, followed by 60 &deg;C for 1 min. The quantity of viral genomes is expressed as PFU equivalents, and was calculated by performing a standard curve with RNA derived from a viral stock with a known viral titer. Alternatively, infectious virus titers were quantified using plaque assays as described above.</p> <p>To quantify siRNA-based cellular protein knockdowns, A549-Ace2 cells were seeded and transfected with individual dsiRNAs as described above. After 24 hours incubation at 37 &deg;C cells were lysed and RNA was extracted using Trizol (ThermoFisher Scientific) followed by purification using the Direct-zol-96 RNA extraction kit (Zymo) following the manufacturer&rsquo;s instructions. RNA levels of target proteins were subsequently quantified by using RT-with the Luna Universal One-Step RT-qPCR Kit (NEB) in an Applied Biosystems QuantStudio 7 thermocycler using gene-specific primers. Expression levels were compared to scrambled dsiRNA-transfected cells und normalized to expression of human beta-actin. Knockdown efficiencies were calculated using &Delta;&Delta;Ct in Matlab.&nbsp;</p> <p>To assess cell viability after siRNA knockdowns, cells were seeded and transfected as described above. 24 hours after transfection cell viability was measured using alamarBlue reagent (ThermoFisher Scientific), &nbsp;media was removed and replaced with alamarBlue and incubated for 1h at 37 &deg;C and fluorescence measured in a Tecan Infinite M200 Pro plate reader. Percentage viability was calculated relative to untreated cells (100\% viability) and cells lysed with 20\% ethanol (0\% viability), included in each plate.</p> <p>For cell counting to determine cell numbers, cells were fixed in formalin to deactivate virus. The fixed cells were stained with 5&micro;g/ml of Hoechst 33258 (Sigma). The assay plates were imaged on an IX-83 automated inverted microscope (Olympus) using a 10x objective. The DAPI settings (Ex UV 377/50, Em 415&ndash;480) were used to image Hoechst 33258. The acquisition setup was configured to image 4 sites per well. The nuclei were identified using the object detection module in the ScanR analysis software.</p> <p>&nbsp;</p> <p>Data provided in two zip files. Sample layout/key within the plates is provided within the zip files.</p>