Next-Generation Sequencing Reveals HIV-1-Mediated Suppression of T Cell Activation and RNA Processing and Regulation of Noncoding RNA Expression in a CD4(+) T Cell Line

Chang, Stewart T.; Sova, Pavel; Peng, Xinxia; Weiss, Jeffrey; Law, G. Lynn; Palermo, Robert E.; Katze, Michael G.

SUP-T1 cells were obtained from American Type Culture Collection (CRL-1942) and propagated in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (HyClone), penicillin (100 U/ml), streptomycin (100 g/ml), and GlutaMAX-I. HIV-1 LAI strain (catalog no. 2522) was obtained from the NIH AIDS Research and Reference Reagent Program (Germantown, MD) and propagated in SUP-T1 cells. U373-MAGI-CXCR4 CEM cells were obtained from M. Emerman through the AIDS Research and Reference Reagent Program, and the virus titer in these cells was measured by the protocol of Deminie and Emerman (39) . Typical titers reached 10 7 infectious units per ml. Infections were carried out at a multiplicity of infection (MOI) of 5 and performed in triplicate. Mock-infected samples received SUP-T1 cell conditioned medium and were also performed in triplicate. The infectious dose was optimized to achieve 100% infected cells at 24 hpi with 50% cell viability as measured by trypan blue exclusion assay. Infected cells were visualized by immunofluorescence assay with rabbit HIV-1 SF2 p24 antiserum kindly provided by BioMolecular Technologies through the AIDS Research and Reference Reagent Program.