Figure 1

Discordance between the Transactivation and cytokine induction functions of Tat. (A) Rescue of the Tat-defective provirus. HLM-1 cells harbor an integrated provirus defective for Tat and produce large quantities of virus when complemented with a functional Tat protein. HLM-1 cells were incubated with B- or C-Tat (5 μg/ml) in complete medium or without Tat. Following Tat transfection, culture medium was collected at 24, 48 and 72 h and the levels of the viral structural protein, p24, secrerted into the medium was estimated using an antigen – capture assay. Data for the 72 h time point are presented here. Tat proteins (from subtypes -B and C) synthesized using a previously reporetd protocol were competent for the transactivation property. Tat proteins prepared using the protocol described in the present report were also transactivation competent (data not presented) (B) Induction of TNF-α secretion from primary monocytes. B- and C-Tat proteins prepared as above and as described in the present report were included for a comparative analysis. Monocytes freshly isolated from peripheral blood were incubated with the Tat proteins at two different concentrations (20 or 200 ng/ml) for 30 or 60 min. Cells were washed three times to remove Tat, resuspended in complete medium and incubated for 24 h. The amount of TNF-α secreted into the culture medium was assessed using a commericial kit following the manufacturer's instructions (R&D Systems). Tat proteins prepared using the protocol reported in the present manuscript successfully induced cytokine production from the monocytes in a dose-dependent manner. In contrast, Tat proteins prepared using reverse-phase chromatography failed in the induction. Note that the same Tat proteins were transactivation-competent and activated expression of a Tat-defective provirus (panel A above) and reporter genes under the viral LTR (data not presented).