Figure 4

The interaction of Tat with human RNA depends on the RNA binding domain. A. Activity of wild-type HA-Tat (white bars) and HA-Tat K50S-K51G (RNA binding mutant), grey bars) in the LTR reporter cell line GHOST, measured by the proportion of GFP-positive cells (left panel) and mean GFP intensity (right panel). B. Immunoblotting for HA-Tat and β-actin in CEM cells 72 hours after infection with lentiviral vectors expressing either GFP alone, GFP and WT HA-Tat or GFP and HA-Tat-K50S-K51G. C. Immunoblotting for HA-Tat, TBP (nuclear protein) and Tubulin (cytoplasmic protein) in cytoplasmic (C) and nuclear (N) fractions of CEM cells expressing WT HA-Tat or HA-Tat-K50S-K51G. D. Amount of DNase-treated RNA purified from HA-Tat and control antibody IPs from CEM cells infected with WT-Tat or Tat K50S-K51G encoding lentivirus, as a percentage of the total RNA in the lysate. Data from 3 independent infections are shown. E. Enrichment (mean and SD) of the human mRNAs GAPDH, ACTIN, FADD and TNFRSF8 in HA-Tat immunoprecipitate from cells infected with WT HA-Tat or HA-Tat K50S-K51G encoding lentivirus, measured by Q-PCR.