A , Mutations of S16 and S46 of Tat reduce its ability to induce viral production. HLM1 is a HeLa derived cells containing one copy of integrated HIV-1 proviral genome with a Tat-defective mutation. Various Flag-tagged Tat (WT Tat, Tat S16A, Tat S46A or Tat S16,46A) expression vectors were used for HLM-1 transfections. Cells were cultured in complete media in absence of G418 for 14 days. The supernatants were collected at Day 0, 1, 2, 7 and 14, and analyzed for p24 by ELISA assay. B , Mutations of S16 and S46 of Tat inhibit Tat phosphorylation during one round of viral replication. HLM1 cells were transfected with vectors expressing Flag-tagged Tat (WT Tat, Tat S16A, Tat S46A or Tat S16,46A). At 48 hours post transfection the cells were labeled with (32P)-orthophosphate for 2 hours with 1 μM okadaic acid. Flag-Tat was immunoprecipitated from whole cell extracts with anti-Flag antibodies and resolved by 15% Tris-Tricine SDS-PAGE. The gel was dried and exposed to Phosphor Imager screen. Lane 1, Wt Tat. Lane 2, Tat S16A. Lane 3, Tat S46A. Lane 4, Tat S16,46A. Lane 5, mock-transfected cells. The picture is an autoradiogram.