293freestyle medium

293F suspension cells were cultured in 293Freestyle medium (Gibco BRL Life Technologies) at 37°C in 10% CO₂ at 125 rpm. HEK293S (GntI^−/−) cells were cultured in suspension as described above but supplemented with 2% heat-inactivated fetal bovine serum (FBS). Adherent CD4⁺ CCR5⁺ TZM-bl HeLa and HEK293T cells were grown to confluence in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 22.7 mM HEPES (Gibco BRL Life Technologies), and 50 μg/ml gentamicin (Sigma) at 37°C in 5% CO₂. Monolayers were disrupted with 0.25% trypsin in 1 mM EDTA (Sigma). For pseudoviruses, HEK293T cells were seeded in 10 ml at 2 × 10⁶ cells/ml in 10-cm² dishes. Twenty-four hours later, plasmids expressing the Env protein of interest and the pSG3ΔEnv backbone (obtained from the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH) were cotransfected by using a 3:1 ratio of PEI-Max 40,000 (Polysciences). Cultures were incubated for 48 h at 37°C, filtered through a 0.45-μm filter, and frozen in 20% FBS. Mutant Env plasmids were made with the QuikChange Lightning kit (Stratagene).

293 T cells were from an in-house stock originally from the Mount Sinai School of Medicine. 293 Freestyle cells were purchased from Invitrogen (ThermoFisher Cat#R79007) and cultured in 293 Freestyle medium (Gibco®). Hep-2 cells were purchased directly from ATCC (with a HeLa contamination disclaimer) (ATCC, Cat#CCL-23). Composite CHO cells expressing preF^C were generated and cloned under contract with Abzena Ltd. preF^C was cloned into the pcDNA 3.1 Zeo plasmid and used for transient transfections. Motavizumab, AM14, 5C4 anti-F primary antibodies were gifts from Jason McClellan (UT Austin) and Barney Graham, or purchased from Creative Biolabs (Shirley, NY). D25 and MPE8 were provided by Jason McClellan and Barney Graham. The anti-mouse and human secondary antibodies were purchased from GE Healthcare Life Sciences and Jackson ImmunoResearch Laboratories, Inc. respectively. HRP Rat anti-mouse IgG2a (BD Pharmingen) was used in the conformational sandwich ELISA. Biotin-SP (long spacer) Affinipure goat anti-mouse IgG, Fc gamma fragment specific (Jackson Immunochemicals) was used in the ELISPOT assays. The RSV-luciferase virus was obtained from Dr. Martin Moore (Emory University).