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

<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’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°C, the virus inoculum was removed and replaced with fresh 2\% FBS/DMEM media. Cells were incubated at 37°C for 3 days before supernatants were harvested. Samples were either heat-inactivated at 80°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 °C for 10 min, 95 °C for 1 min, and 40 cycles of 95 °C for 10 sec, followed by 60 °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. 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 °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’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 ΔΔCt in Matlab. 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), media was removed and replaced with alamarBlue and incubated for 1h at 37 °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. For cell counting to determine cell numbers, cells were fixed in formalin to deactivate virus. The fixed cells were stained with 5µg/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 415–480) 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. Data provided in two zip files. Sample layout/key within the plates is provided within the zip files.基于小干扰RNA（small interfering RNA, siRNA）的细胞蛋白敲降中的病毒感染——细胞活力成像数据集。研究采用IDT公司的特异性双链小干扰RNA（double-stranded siRNA, dsiRNAs）对A549-Ace2细胞内的宿主蛋白进行敲降。简言之，将A549-Ace2细胞以1×10⁴个/孔的密度接种于96孔板中。24小时后，按照制造商说明书，使用Lipofectamine RNAiMAX转染试剂（Thermo Fisher Scientific）将5 pmol的单种dsiRNAs转染至每孔细胞内。转染24小时后，移除细胞培养上清液，替换为病毒接种液（感染复数（multiplicity of infection, MOI）为0.1空斑形成单位（plaque-forming unit, PFU）/细胞）。于37℃吸附1小时后，移除病毒接种液，更换为含2%胎牛血清（FBS）的DMEM培养基。将细胞置于37℃孵育3天，随后收集上清液。部分样品经80℃热灭活20分钟，采用此前发表的针对SARS-CoV-2核衣壳（N）基因的特异性引物，通过逆转录实时定量PCR（RT-qPCR）定量病毒RNA[引用文献：ChuClinChem]。RT-qPCR实验使用Luna Universal One-Step RT-qPCR试剂盒（NEB），在Applied Biosystems QuantStudio 7型热循环仪中完成，循环程序设置为：55℃孵育10分钟，95℃孵育1分钟，随后40个循环的95℃变性10秒、60℃退火延伸1分钟。病毒基因组数量以PFU当量表示，通过使用已知病毒滴度的病毒原液提取的RNA制作标准曲线计算得到。此外，亦可采用前述空斑实验定量感染性病毒滴度。为定量基于dsiRNAs的细胞蛋白敲降效率，按照上述方法接种并转染A549-Ace2细胞。于37℃孵育24小时后裂解细胞，使用Trizol试剂（ThermoFisher Scientific）提取RNA，随后按照制造商说明书使用Direct-zol-96 RNA提取试剂盒（Zymo）完成纯化。后续通过RT-qPCR，使用基因特异性引物定量靶蛋白的RNA水平。以乱序dsiRNAs（scrambled dsiRNAs）转染的细胞作为对照，将靶基因表达水平以人β-肌动蛋白（beta-actin）的表达量作为内参进行归一化处理。敲降效率通过Matlab软件中的ΔΔCt法计算得到。为评估siRNA敲降后的细胞活力，按照上述方法接种并转染细胞。转染24小时后，使用alamarBlue试剂（ThermoFisher Scientific）检测细胞活力：移除培养基，替换为含alamarBlue的培养基，于37℃孵育1小时后，使用Tecan Infinite M200 Pro型多功能酶标仪检测荧光信号。细胞活力百分比以未处理细胞（设为100%活力）和用20%乙醇裂解的细胞（设为0%活力）作为参照进行计算，每个检测板均设置该对照。为通过细胞计数确定细胞数量，将细胞用福尔马林固定以灭活病毒。固定后的细胞用5µg/ml的Hoechst 33258（Sigma）进行染色。使用Olympus IX-83型自动化倒置显微镜，搭配10倍物镜对检测板进行成像。采用DAPI通道（激发波长UV 377/50 nm，发射波长415–480 nm）采集Hoechst 33258的荧光信号，成像设置为每孔采集4个视野。使用ScanR分析软件中的目标检测模块识别细胞核。本数据集包含两个压缩包文件，压缩包内提供了板内样品的布局信息与关键注释。