Hek293t 17

Human lung epithelial cell lines A549 (ATCC CCL-185) and Calu-3 (ATCC HTB-55); HEK 293T cells, a human kidney epithelial cell line (HEK 293T/17, ATCC CRL-11268); and Vero E6 cells (Vero 76, clone E6, Vero E6, ATCC CRL-1586), an African Green Monkey kidney epithelial cell line, were authenticated by ATCC (American Type Culture Collection). A monoclonal ACE2-expressing A549 cell line (A549-ACE2) was a kind gift from Brad Rosenberg. Monoclonal ACE2-expressing STAT1-knockout A549 cells (A549-ACE2ΔSTAT1) were generated as previously described (44 (link), 45 (link)). All cell lines were cultured under humidified 5% CO₂ conditions in 10% vol/vol fetal bovine serum (FBS, Thermo Fisher Scientific) and 100 I.U. penicillin and 100 µg/mL streptomycin (Pen/Strept, Corning) in Dulbecco’s Modified Eagle Medium (DMEM, Corning). Cells were confirmed negative for mycobacteria monthly (Lonza).

HEK-293T cells (HEK-293T/17; ATCC CRL-11268) were maintained in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% fetal bovine serum (Gibco), 100 units/mL penicillin, and 0.1 mg/mL streptomycin at 37 °C in a 5% CO₂ humidified atmosphere. One day prior to transfection, 4000 cells were seeded in 384-well flat (F) bottom μClear plates (Greiner, Bio One GmbH) coated with fibronectin (BD Biosciences). Transfection of HEK-293T cells was performed at 80% confluence using 25 ng of the total mixed plasmid(s) with 150 nL of Fugene 6 (Promega) reagent per well. For fixed and variable donor BRET saturation assays, HEK-293T were co-transfected with acceptor YFP- and donor Nluc-tagged genes of interest at 11 ratios ranging from 243:1 to 1:243 (Supplementary Fig. S2). After 48 h, protein expression was quantitatively assessed using fluorescence microscopy and bioluminescence readout. If indicated, cells were treated at 500 U/mL with recombinant human IFN-beta 1a (IFNβ) (PBL Assay Science, Cat 11415-1), or with 0.46 to 3000 nM of rapamycin (Seleckchem).

Human carcinoma epithelial cells (HeLa, ATCC CCL2™), human epithelial kidney cells (HEK-293T/17, ATCC CRL-11268™) are part of the collection of our laboratory and were cultured as previously described [17 (link)]. Virus infection was performed in 6- or 96-well plate dishes during indicated times at 37°C and using different viruses at a multiplicity of infection (MOI) of 1. Thailand virus, referred as Tha (isolate 8743THA), is a field strain of RABV isolated in Thailand from a human bitten by a dog (EVAg collection, Ref-SKU: 014V-02106). SAD-B19 virus (SAD) is a vaccine strains of RABV (EVAg collection, Ref-SKU: 014V-02283). A recombinant Tha virus mutated on the positions 77, 100, 104, 110 of the matrix protein (Th4M) was used as previously described [19 (link)].

BHK-21 (baby hamster kidney; ATCC CCL-10), A549 (human lung carcinoma; ATCC CCL-185), HEK 293T/17 (ATCC CRL-11268), and HEK 293 (human embryonic kidney; ATCC CRL-1573) cells were grown in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% antibiotic/antimycotic, 1% L-glutamine, and 1% sodium pyruvate at 37°C (Gibco Life Technologies) in a 5% CO₂ incubator.