Figure 1.

Forskolin induces transcriptional activation of HIV-1 LTR and upregulation of CXCR4. (A) The molecular sequence of CREs in the LTR of reference HIV-1 strains and in the 5′ UTR of the CXCR4 gene. TRANSFAC analysis identifies the presence of a lone CRE in the CXCR4 promoter-5′ UTR and the HIV-1 LTRs. The sequences are compared with a classical consensus CRE in the T-lymphocyte SH2D2A promoter, and the variant base pairs are marked in red. (B) With forskolin stimulation (the range of concentrations included 0, 10, 25, 50, 75, and 100 µm), the HIV-1 LTRs are activated and drive luciferase gene expression to different extents. The LAI-LTR was the most inducible (6.2-fold), followed by the YU2-LTR (2.9-fold) and the 89.6 LTR (1.9-fold). (C) After site-directed mutagenesis of LTR-luc constructs, the activity of 100 µm forskolin-stimulated TG LAI-LTR, YU2-LTR, and 89.6 LTR was reduced by 81%, 83%, and 64%, respectively. (D) The biphasic curve of CXCR4 transcription in response to 100 µm forskolin is characterized by an initial peak at 6–9 hours (4-fold upregulation) followed by a trough at 15 hours (1.92-fold upregulation) and a smaller peak at 18 hours (2.8-fold upregulation) after stimulation. Although transcript levels declined at 15 hours, they remained elevated in comparison with untreated samples. Results are normalized to RPLPO expression and expressed in linear scale as fold over untreated. Asterisks indicate significance of P < 0.05 using Student’s t–test.