Proteomic Profiling of HIV Infection in T Cells

A population of 500 × 10⁶ SupT1 cells (lymphoblastic T cell line) was cultured in RPMI 1640 medium with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Invitrogen) (Fig. 1). Isotope-labeled amino acids (¹³C₆-L-lysine, ¹³C₆¹⁵N₄-L-arginine, Cambridge Isotope Laboratories (CIL), Andover, MA) were included in the heavy (H) SILAC medium at 100 mg/l, while normal Arginine and Lysine were used in the light (L) SILAC medium. Heavy or light SILAC labeling was achieved by culturing the cells in the two media (H and L) for a minimum of 2 weeks to allow for at least 5 cell divisions. H-labeled cells were Mock infected, while the L-labeled cells were infected with an HIVeGFP/VSV-G virus at 3 μg/10⁶ cells. Infection (both Mock and with an HIVeGFP vector) was carried out by spinoculation for 30 min at 1500 g in presence of 5 μg/ml polybrene. As previously described^{18 (link)}, this allowed reaching a quasi-universal infection. Cells were then washed and further incubated. The HIVeGFP viral vector used expresses GFP instead of the viral protein env. At multiple time points post-infection, cells were collected and processed for analysis of HIV life cycle progression and normalized by the 24 h time point as in^{18 (link)}, as well as for transcriptome, proteome and phosphoproteome as detailed below (Supplementary File S10).