Half-life determination of Rev-regulated transcripts.

<p>(A) Expression of Rev-dependent <i>gag</i> (unspliced) and <i>env</i> (single spliced) mRNAs in HeLa-tTA cells. HeLa-tTA cells were transiently cotransfected with the Rev-deficient doxycyline-regulated proviral construct pUHC-HXB2Δ<i>rev</i> and Rev expression plasmid or the parental control vector pBC12/CMV. Transcription of viral RNA was blocked where indicated by 100 ng/ml doxycycline treatment at 5 h post-transfection. Subsequently, the viral RNA levels were quantified at 24 h posttransfection by real-time PCR. Results for viral RNA copies were adjusted to the level of endogenous <i>gapdh</i> mRNA. (B) Inhibition of replication by doxycycline treatment. HeLa-tTA cells were transiently cotransfected with pUHC-HXB2Δ<i>rev,</i> pBC12/CMV/SEAP (internal control) and the indicated Rev constructs. The accumulation of p24^Gag antigen in the culture supernatants was quantified by ELISA at 48 h post-transfection. All values were adjusted for transfection efficiency to the SEAP activity present in each supernatant. Viral expression was blocked when the culture medium was supplemented with 100 ng/ml doxycycline. (C) Effect of the indicated Rev mutants on the half-life of unspliced full-length (<i>gag</i>) HIV-1 RNA. Transfection experiments were performed in HeLa-tTA cells using pUHC-HXB2Δ<i>rev</i> DNA and the indicated <i>trans</i>-activator constructs. Total RNA was isolated and viral <i>gag</i> RNA copy numbers were quantified after transcriptional pulse at the indicated time points by real-time PCR and adjusted to the endogenous <i>gapdh</i> mRNA level in each sample. The relative RNA levels are show in comparison to the respective initial level (after transcriptional pulse) at time point 0 h (set to 1.0).