Background: Recent reports demonstrate that short interfering RNA duplexes (siRNA) targeting structural and regulatory genes of HIV act through Post Transcriptional Gene Silencing (PTGS). In plants siRNA targeting promoter regions induce transcriptional gene silencing (TGS). RNA-directed DNA methylation (RdDM) of the promoter region is thought to be essential for TGS induced by siRNAs. We explored the possibility of silencing HIV-1 gene expression via siRNA targeting regions within the 5’LTR. We targeted the NF-kB binding site in the HIV-1 as it is essential for gene expression, is highly conserved across subtypes, but its sequence is different from that in promoter regions of human genes.

Methods: siRNA homologous to a section of the NF-kB binding site was synthesized (NF-kB dsRNA). A siRNA targeting the promoter region of mismatch repair gene hMLH1 (hMLH1 dsRNA) was used as a control. Magic-5 cells were used as substrate for infection with 5 subtype B isolates: NL4-3, IIIB, RF and 2 clinical isolates. Virus production was measured by reverse transcriptase (RT) levels in supernatant. Protein expression and viral mRNA levels in cell lysates were also analyzed by Western blot and real time PCR respectively. Methylation status of proviral DNA was determined by bisulfite sequencing.

Results: Up to 1000 fold reductions in NL4-3 production were detected at day 8 in those cultures treated with NF-B dsRNA compared to controls. Similarly, RT activity of the 4 other isolates was reduced by between 50 and 400 fold in cells transfected with NF-kB dsRNA. Dramatic reductions in p24 expression and greater than 300 fold reductions of viral mRNA were seen in NF-kB dsRNA treated cultures. Without transfection of the NF-kB dsRNA, we observed methylation of cytosine residues in CpG motifs in the U3 region of 5’LTR. However, after transfection with NF-kB dsRNA cytosine residues of CpG sites in the 5’LTR and early coding region were heavily methylated.

Conclusions: We have shown for the first time de novo methylation associated with gene silencing induced by siRNA duplexes targeting the promoter region of HIV.