Cell culture
Cell lines
A549 (CCL-185, American Type Culture Collection (ATCC)), BSR T7/5 (ref. 55) (BHK-21 cells that constitutively express the T7 RNA polymerase, RRID:CVCL_RW96) and HEK293T cells (CRL-3216, ATCC) were cultured in DMEM (31966021, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; F7524, Merck) and 1% penicillin–streptomycin (pen–strep; 15140122, Thermo Fisher Scientific). Vero cells (CCL-81, ATCC) were cultured in DMEM supplemented with 5% FBS and 1% pen–strep. HEp-2 cells (CCL-23, ATCC) were cultured in MEM (42360032, Thermo Fisher Scientific) with 10% FBS and 1% pen–strep. All cells were maintained at 37 °C and 5% CO 2 . Cell lines used in this study were confirmed to be mycoplasma negative.
Primary cells
Primary human nasal epithelial cells (HNECs) from healthy donors (EP51AB, Epithelix) were cultured in an air–liquid interface (ALI) transwell system, as previously described56. In brief, HNECs were expanded to log phase in PneumaCult Ex Plus medium (05040, StemCell Technologies). Passage 1 cells were cryopreserved in 78% Ham’s F12 (51651C, Merck/Sigma), 10% heat-inactivated FBS, 2% HEPES (pH 7.2) and 10% DMSO, then stored at −135 °C. After thawing, cells were expanded with medium, replaced every 1–2 days, then seeded at a density of 7 × 104 cells per well on PET transwells (CLS3470, Corning; 0.4-μm pores, 6.5 mm in diameter) with 100 μl apical and 650 μl basolateral PneumaCult Ex Plus medium. After 48–72 h, confluent monolayers were airlifted by aspirating media and adding PneumaCult ALI medium (05001, StemCell Technologies) supplemented with 100 U ml−1 penicillin, 0.1 mg ml−1 streptomycin (P4333, Sigma), 0.48 μg ml−1 hydrocortisone (07926, StemCell Technologies) and 4 μg ml−1 heparin (07980, StemCell Technologies) to only the basolateral chambers. Medium was replaced every 2–3 days; from 2 weeks post-airlift, apical surfaces were washed 1–2 times weekly with 200 μl HBSS (14025050, Gibco) to remove mucus. Cultures were maintained at 37 °C with 5% CO 2 .
Nasal wash samples
Nasal wash samples from individuals infected with RSV were obtained from participants enrolled in a previously described controlled human infection model (CHIM) study57. That study, conducted independently of the present work, involved inoculation of healthy adults with RSV-A Memphis-37b58. Nasal wash samples were collected at defined time points, frozen and stored for subsequent analysis. For the present study, nasal wash samples collected from a single participant on study days 4 and 5, which showed detectable nasal viral load as determined by quantitative PCR with reverse transcription (RT–qPCR), were utilized.
RSV protein samples
RSV Nrings without tag were purified by co-expression in Escherichia coli and co-purification with GST–P CTD (amino acids 161–241), using the GST tag, as previously described19. When specified, the GST tag was removed by thrombin cleavage. For NMR measurements, 15N-labelled P CTD was expressed in E. coli from P CTD cloned into the pGEX-4T3 plasmid, and purified as previously described59. For expression and purification of all the GST–P fragments, the corresponding P sequences were cloned into pGEX-4T3 plasmid, and the proteins were expressed in E. coli. Purifications were performed as previously described19.
Screening of DARPins binding to RSV vRNP
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