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Absolute quantification of HIV-LTR in PBMC. Genomic DNA was extracted using the QIAamp DNA Mini Kit (Qiagen). HIV-LTR sense, anti-sense primer sequences and probes were previously described [14] (Applied Biosystems). For absolute quantification of HIV-LTR, AICAR control plasmid was constructed by cloning the 121 bp HIV-LTR amplicon into pCR 4-TOPO (Invitrogen). Plasmid copy number was quantified as described [15].
Plasmid constructs and luciferase assay. To create plasmid pLTR-GFP, a 450-bp fragment corresponding to the HIV-1 LTR-promoter region was isolated from the plasmid pLTR/CAT (AIDS reagent project/program EVA centralized facility) and sub-cloned into the promoter-less plasmid pEGFP-1. Similarly, the HIV-1 LTR-luciferase reporter (pLTR-Luc) was generated by cloning the LTR-encoding promoter fragment into plasmid pGL3-basic vector (Promega, WI, USA). Plasmid pcDNA1-p65 and the Luciferase measurement were previously described [9].
Immunoprecipitation and immunoblotting. Cells were routinely analyzed 48 h post-transfection. Immunoprecipitation and immunoblotting were performed essentially as described [12].