Figure 6

Inhibition of HIV-1 Nef chimera replication and endogenous SFK activation by DQBS. A) CEM-T4 cells (1 × 10⁴ per well of a 96-well plate) were infected with wild-type HIV-1 NL4-3, a Nef-defective mutant (ΔNef), or the indicated HIV-1 Nef chimeras in a final culture volume of 200 μl. Input virus for HIV-1 ΔNef was increased by ten-fold relative to wild-type to compensate for the reduced infectivity and replication of Nef-defective virus in CEM-T4 cells [41]. DQBS was added to the cultures to final concentrations of 0.3 and 1.0 μM, and viral replication was determined by p24 ELISA 10 days later. Data are expressed as the mean percent of HIV-1 replication observed in control cultures incubated with the carrier solvent (0.1% DMSO) ± S.D. from duplicate experiments performed in triplicate. B) CEM-T4 cells were infected with wild-type or Nef-defective (ΔNef) HIV-1 NL4-3 in the presence of the indicated concentrations of DQBS or the carrier solvent (DMSO). SFK proteins were immunoprecipitated from infected cell lysates and immunoblotted with an antibody specific for the phosphorylated activation loop tyrosine (pY418) common to all Src-family members. Controls blots were performed on the cell extracts for HIV-1 Gag proteins (p55, p40, p24), Nef, as well as actin. Blots from uninfected, untreated cells were also included as a negative control (No virus).