Replication-competent HIV-1 was produced by transfecting 293T cells with plasmids containing full-length proviruses. Virus containing supernatants were clarified by low-speed centrifugation, passed through 0.45-μm filters, normalized for HIV-1 capsid (p24) antigen with an HIV-1 p24 enzyme-linked immunosorbent assay (ELISA) kit (XpressBio), and used to infect target cells.
MOLT-3 cells were infected in T25 flasks in 5 mL medium. FACS-sorted cells were infected in 96-well plates in 200 μL medium. PBMC were obtained from LRS chambers by Ficoll-Hypaque density gradient centrifugation and kept in RPMI 1640 medium supplemented with 15% human AB serum (Millipore Sigma). CD4+ T cells were purified from fresh PBMC by negative selection with an EasySep human CD4+ T cell enrichment kit (Stemcell Technologies). PBMC were seeded into 12-well plates in 2 mL medium and immediately stimulated with 2 μg/mL phytohemagglutinin (PHA) (Millipore Sigma). One day later, the PHA-containing medium was replaced by fresh medium, and the cells were infected in the presence of 20 U/mL interleukin 2 (Roche). CD4+ T cells were seeded into 12-well plates and immediately infected at a p24 concentration of 1 ng/mL. On day 3 after infection, the cells were stimulated with 2 μg/mL PHA. The next day, the PHA-containing medium was replaced by fresh medium containing 20 U/mL interleukin 2.
Virus replication was monitored by comparing Gag protein expression levels in infected cells by Western blotting using anti-HIV-1 capsid (CA) antibody 183-H12-5C and anti-actin and/or by measuring the accumulation of p24 antigen in the culture supernatants over time with an HIV-1 p24 ELISA kit (XpressBio).
MOLT-3 cells were infected in T25 flasks in 5 mL medium. FACS-sorted cells were infected in 96-well plates in 200 μL medium. PBMC were obtained from LRS chambers by Ficoll-Hypaque density gradient centrifugation and kept in RPMI 1640 medium supplemented with 15% human AB serum (Millipore Sigma). CD4+ T cells were purified from fresh PBMC by negative selection with an EasySep human CD4+ T cell enrichment kit (Stemcell Technologies). PBMC were seeded into 12-well plates in 2 mL medium and immediately stimulated with 2 μg/mL phytohemagglutinin (PHA) (Millipore Sigma). One day later, the PHA-containing medium was replaced by fresh medium, and the cells were infected in the presence of 20 U/mL interleukin 2 (Roche). CD4+ T cells were seeded into 12-well plates and immediately infected at a p24 concentration of 1 ng/mL. On day 3 after infection, the cells were stimulated with 2 μg/mL PHA. The next day, the PHA-containing medium was replaced by fresh medium containing 20 U/mL interleukin 2.
Virus replication was monitored by comparing Gag protein expression levels in infected cells by Western blotting using anti-HIV-1 capsid (CA) antibody 183-H12-5C and anti-actin and/or by measuring the accumulation of p24 antigen in the culture supernatants over time with an HIV-1 p24 ELISA kit (XpressBio).
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