Mrc 5 cells

All cells were maintained in humidified 37°C incubators with 5% CO₂ and 20% O₂. Vero E6 (ATCC), HEK293T cells (ATCC), HeLa Tet-Off cells (Clontech) and HeLa Flp-In TREx GFP-Dcp1a cells (a generous gift from Dr. Anne-Claude Gingras) were cultured in DMEM (Thermo Fisher) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine (Thermo Fisher) and 10% heat-inactivated fetal bovine serum (FBS, Thermo Fisher). Calu3 (ATCC) and MRC-5 cells (ATCC, a generous gift from Dr. David Proud) were cultured in EMEM (ATCC) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine and 10% FBS (Thermo Fisher). HUVECs (Lonza) were cultured in endothelial cell growth medium (EGM-2) (Lonza). HUVECs were seeded onto tissue culture plates or glass coverslips coated with 0.1% (w/v) porcine gelatin (Sigma) in 1x PBS. For sodium arsenite treatment, HUVECs were treated with 0.25 mM sodium arsenite (Sigma) or a vehicle control for 30 min.

All cell lines and PBMC were cultured with 5% CO₂ at 37°C. VeroE6 cells expressing ACE2 and TMPRSS2 (VAT) and A549 cells expressing ACE2 (AA) were a gift from the University of Glasgow Centre for Virus Research (Glasgow, United Kingdom) (69 (link)). Human lung fibroblast MRC-5 cells were obtained from ATCC (CCL-171), and Lenti-X 293T cells were obtained from Takara. All were propagated in complete DMEM (Sigma-Aldrich) containing 10% FBS (HyClone) and 200 IU/mL penicillin/streptomycin (Invitrogen). Ancestral SARS-CoV-2 was recovered from a BAC containing the complete genome of a strain that matches the original Wuhan-Hu-1 (69 (link)), and the Omicron (BA.1) variant was a gift from Arvind Patel (University of Glasgow Centre for Virus Research). Both were propagated in VAT cells. Virions were concentrated and purified by pelleting through a 30% sucrose cushion and titrated by plaque assay in AA cells, as previously described (11 (link)). All virus seed stocks were verified by whole-genome sequencing on the Illumina platform.

Human A549 lung epithelial cells were cultured in complete Dulbecco modified Eagle medium (DMEM; Gibco, USA) containing nonessential amino acids (NEAA), sodium pyruvate, and l‐glutamine, and supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, ON, Canada) following the protocol described previously (10 (link)). Human fetal lung MRC-5 cells were purchased from ATCC (catalog no. CCL-171) and maintained in ATCC Eagle minimum essential medium (EMEM; catalog no. 30-2003) supplemented with 10% FBS. All cells were incubated at 37°C in 5% CO₂ and passaged three times each week to maintain them as monolayers. Human influenza virus strains A/Mexico/INDRE4487/2009 (H1N1; pdm09) and A/WSN/1933 (H1N1; WSN) and mouse-adapted strain A/PR/8/34 (H1N1; PR8) were used in this study. MDCK cells were infected at an MOI of 0.01 (PFU/cell), and viruses were harvested from the supernatant after 48 h. The virus stocks were concentrated by centrifugation at 64,000 × g for 2 h at 4°C and resuspended in phosphate-buffered saline (PBS) supplemented with 10% glycerol, and aliquots were frozen at −80°C until used.

MRC-5 cells were purchased from ATCC (#CCL-171, USA). HAdV-7 was separated and cultured at the Center Laboratory (Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, Guangzhou, China). RAD7EGFP was donated by the Guangzhou Institute of Respiratory Disease (Guangzhou, China). Fetal bovine serum (FBS, 10099141C), Dulbecco’s Modified Eagle’s Medium containing 4.5 g/L glucose (DMEM, C11995500BT), Iscove’s Modified Dulbecco’s Medium GlutaMAX (IMDM, 31980030), and PBS (10010049) were bought from Thermo Fisher Scientific (Waltham, MA, USA). Leukocyte Activation Cocktail (550583) and BFA (347688) from BD Biosciences were used in this study.

The A549 cell-line (ECACC) and derivatives (Appendix Table A.1) were maintained in DMEM with 10% fetal bovine serum (FBS) (DMEM-10). A549 reporter cell-line derivatives expressing HCV NS3-4A, RSV NS1/NS2 or PIV5 V were generated using a self-inactivating lentiviral constitutive expression system (Demaison et al., 2002 (link)). A549 reporter cell-line derivatives expressing CMV IE1 and corresponding negative controls (luciferase (Luc) and IE1dl410-420 (Harwardt et al., 2016 (link))) were generated using the Lenti-X Tet-One inducible expression system (Clontech). Lentiviruses were generated in 293T cells (ECACC), used to transduce the appropriate reporter cell-line and maintained under antibiotic selection. A Hep2 cell-line derivative constitutively expressing PIV2 V (Precious et al., 2005 (link)) or BVDV Npro (Hilton et al., 2006 (link)), MRC-5 cells (ATCC), Vero cells (ECACC) and STAT2-deficient primary skin fibroblasts (Hambleton et al., 2013 (link)) were maintained in DMEM-10.