LTR plasmid construction and reporter assay
LTR reporter plasmids were constructed by inserting nucleotides −208 to +64 relative to the transcriptional initiation site of HIV-1Lai, HIV-1Bal (B subtype), HIV-198IN17, HIV-198IN22, HIV-198CH01, HIV-1CM9 (C subtype), HIV-193TH64, HIV-192TH53, HIV-192TH51, and HIV-1KR25 (E subtype) into the reporter vector pGL3 (Promega BioSciences, www.promega.com) using Xho I and Hind III restriction enzyme sites. Sequences were aligned and analyzed with CLUSTAL W (www.ebi.ac.uk/clustalw/). The HIV-1Lai NFAT5 binding site-mutant (N5-Mut) reporter plasmid was created by standard PCR-based mutagenesis methods [34]. THP-1 cells (0.8×106/ml) were transfected with 0.3 µg/ml LTR wild-type (WT) or mutated reporter plasmids in combination with 0.03 µg/ml Renilla luciferase (pRL-TK) control vector using Effectene transfection reagent (Qiagen; www.qiagen.com). Cells were incubated at 37°C for 16 hours after which they were stimulated with 10 µg/ml MTb CDC1551 lysate or left unstimulated for 8 hours. Reporter gene expression was quantitated by dual-luciferase reporter assay according to the manufacturer's protocol (Promega; www.promega.com).