Vero e6

The USA reference strain SARS-CoV-2 USA-WA 1/2020 (BEI resources NR-52281) was propagated in African green monkey kidney cells (Vero-E6 ATCC CRL-1586) as described previously [36 (link)]. Handling of SARS-CoV-2 was carried out under strict BSL3 biosafety protocols at the Center for Food Safety BSL3 laboratory. Briefly, one- or two-day-old 90% confluent cells were used to prepare virus stocks using a multiplicity of infection of 0.01. Infection media consisted of DMEM supplemented with 2% heat-inactivated fetal bovine serum and 1% antibiotic–antimycotic cocktail. Harvesting the virus was carried out at 72 h post-infection. Infected cells were collected from the flasks and centrifuged at 450× g for 5 min at 4 °C to pellet the cell debris, while supernatants containing the virus were ultrafiltered through Amicon 100 KDa Ultra-15 centrifugal devices (MilliporeSigma, St. Louis, MO, USA) immediately after harvest to concentrate the virus 10 times, while semi-purifying it from cell culture lysates [37 (link)]. The latter would also overcome the buffering effect of infection media in virus stocks on NaOCl. An aliquot of the virus was immediately titrated as described below. The original viral titer generated was ~7 log₁₀ TCID₅₀/mL, while the ultrafiltered virus titer was ~8 log₁₀ TCID₅₀/mL.

Human HEK293T (American Type Culture Collection [ATCC]), HeLa (NIH HIV Reagent Program, Division of AIDS, NIAID, NIH from Dr. Richard Axel), HEK293T-ACE2 (BEI resources), and VeroE6 (ATCC) cells were cultured in complete medium (Dulbecco’s Modified Eagle Medium [DMEM, ThermoFisher Scientific] supplemented with 10% fetal bovine serum [FBS, ThermoFisher Scientific], 1% Penicillin-Streptomycin [ThermoFisher Scientific] and 1% L-glutamine [ThermoFisher Scientific]). A549-ACE2 (BEI resources) cells were maintained in complete medium with 50 ng/mL blasticidin S HCl (ThermoFisher Scientific). HEK293T-ACE2 stably expressing pQCXIP-BST2 or pQCXIP, HEK293T-ACE2-BST2-ATG5KO and HEK293T-ACE2-BST2-LentiCRISPRv2 cells were grown in complete medium with 1 μg/mL of puromycin (ThermoFisher Scientific). A549-ACE2 cells stably expressing pQCXIP-BST2 or pQCXIP were maintained in complete medium with 50 ng/mL blasticidin S HCl and 1 μg/mL of puromycin. Viability of cells was measured after each transfection. Only cells with viabilities > 90% were considered for further experiments.

Vero cells (CCL-81) were obtained originally from the International Reagent Resource and frozen stocks prepared. Those stocks screened negative for mycoplasma contamination and were used between total passage level 35 and 41. Virus (isolate USA-WA1/2020) was acquired through BEI Resources (product NR-52281) and amplified in Vero C1008 (Vero E6) cell culture. Vero E6 cells (ATCC CRL-1568) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with glucose, L-glutamine, sodium pyruvate, 5% fetal bovine serum (FBS) and antibiotics. Inoculation of Vero E6 cells with SARS-CoV-2 was carried out directly in DMEM containing (1%) FBS. The identity of the virus as SARS-CoV-2 was established by shotgun sequencing and confirmation with a database on sequence data for the virus. Medium harvested from infected cells 3–4 days after inoculation was clarified by centrifugation, supplemented with FBS to 10% and frozen to -80°C in aliquots. All virus propagation occurred in a BSL-3 laboratory setting. We measured all values for virus concentration using a standard plaque assay. Results are presented in number of infectious virus (plaque forming units per mL).

Vero E6 (CRL-1586; American Type Culture Collection) were cultured Dulbecco’s Modified Eagle’s Medium (DMEM,Corning) supplemented with 10% fetal bovine serum (FBS, Atlanta Biologics) and 1% nonessential amino acids (NEAA,Corning). SARS-CoV-2, isolate USA-WA1/2020 19 (BEI resources #NR52281), a gift from Dr. Mark Mulligan at the NYU Langone Vaccine Center was amplified once in Vero E6cells. All experiments with SARS-CoV-2 were conducted in the NYU Grossman School of Medicine ABSL3 facility by personnel equipped with powered air-purifying respirators.

Vero E6 (CRL-1586, American Type Culture Collection (ATCC) and 293T (ATCC) were cultured at 37°C in Dulbecco’s Modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES pH 7.3, 1 mM sodium pyruvate, and 100 U/mL of penicillin–streptomycin. 293T was cultured in DMEM medium. 293T-Spike and 293T-ACE2 were cultured in DMEM medium containing 100 μg/mL Zeocin. Expi293F was maintained in Expi293™ Expression Medium (ThermoFisher, Cat# A1435103). The SARS-CoV-2 spike pseudotyped HIV-1 backboned virus were packaged in 293T cells after transfecting pNL4-3.luc.RE and pcDNA3.1-spike plasmids (WT, Alpha, Beta, Gamma, Delta, Omicron BA.1 and Omicron B.2). The SARS-CoV-2 live virus variants (WT, Alpha, Beta, Delta, Omicron BA.1) ordered from BEI Resources and propagated VeroE6 cells. The mouse ACE2 adapted SAR-CoV-2 virus (Beta variants) gene recovered by the reverse genetics was produced in VeroE6 cells. All work with infectious SARS-CoV-2 was performed in Institutional Biosafety Committee approved BSL3 facilities using appropriate positive pressure air respirators and protective equipment.