The SARS-CoV-2 protein ORF8 functions as a mimic of histone H3 to disrupt host cell epigenetic regulation.
A549ACE cells ACE2-expressing A549 cells were generated as previously described3. A549ACE2 cells were grown in RPMI-1640 with 10% FBS and 1% penicillin-streptomycin and were maintained free of mycoplasma. Cells were infected at an MOI of 1 and fixed or lysed at 24 or 48 h after infection. HEK293T cells HEK293T cells were obtained from the American Type Culture Collection (ATCC), cultured in DMEM (with 4.5 g L–1 glucose, l-glutamine and sodium pyruvate) supplemented with 10% FBS (Sigma-Aldrich, F2442-500ML) and 1% penicillin-streptomycin (Gibco, 15140122) and maintained free of mycoplasma. Calcium phosphate transfection was used to introduce plasmid DNA encoding GFP, ORF8 and mutant ORF8 into HEK293T cells. For immunocytochemistry experiments, cells were plated on poly(d-lysine)-coated coverslips. Cells were washed 24 h after transfection with culture medium and fixed or pelleted and flash frozen 48 h after transfection. Cells were fixed using 4% paraformaldehyde (PFA) in PBS for 8 min. To pellet cells, cells were detached from the culture plate using TrypLE Express (Gibco, 12605010) dissociation reagent, spun down for 5 min at 180 g and flash frozen in liquid nitrogen. iAT2 cells Generation of human-derived induced alveolar epithelial type II-like (iAT2) cells was performed as described44. To maintain a stable and pure culture of the iAT2 cell line, SFTPCtdTomato+ cells were sorted and serially passaged every 14 d. Cells were grown in organoid format using 90% Matrigel with a cell density of 400 cells per µl. Cells were fed using CK+DCI medium + Rock inhibitor for the first 48 h after splitting and then changed to K+DCI medium for 5 d followed by CK+DCI medium for 7 d. Every 14 d, alveolosphere organoids were passaged, organoids were released from Matrigel using 2 mg ml–1 Dispase for 1 h at 37 °C and single cells were then generated using 0.05% trypsin for 15 min at 37 °C. Cell number and viability were assessed using Trypan blue, and cells were finally…
Kee, Department Of Genetics At The Perelman School Of Medicine At The University Of Pennsylvania, Philadelphia, Epigenetics Institute At The Perelman School Of Medicine At The University Of Pennsylvania, Thudium, Renner, David M., Department Of Microbiology At The Perelman School Of Medicine At The University Of Pennsylvania, Penn Center For Research On Coronaviruses, Other Emerging Pathogens At The Perelman School Of Medicine At The University Of Pennsylvania