Transcriptional gene silencing of HIV-1 through promoter targeted RNA is highly specific

We have previously reported induction of transcriptional gene silencing (TGS) of HIV-1 by short hairpin RNA (shRNA) expressed in MOLT-4 cells. The shRNA (termed shPromA) targets the highly conserved tandem NFκB binding sequences of the HIV-1 promoter. Recent articles have reported that TGS mediated by promoter-targeted siRNAs was exclusively the result of sequence non-specific off-target effects. Specifically, several mismatched siRNAs to the target promoter sequences were reported to also induce significant TGS, suggesting TGS was a consequence of off-target effects. Here, following extensive investigation, we report that shPromA induces sequence specific transcriptional silencing in HIV-1 infection in MOLT-4 cells, while four shRNA variants, mismatched by 2-3 nucleotides, fail to suppress viral replication. We confirm similar levels of shRNA expression from the U6 promoter and the presence of processed/cleaved siRNAs for each construct in transduced MOLT-4 cells. HIV-1 sequence specific shPromA does not suppress HIV-2, which has an alternate NFκB binding sequence. As a result of the unique sequence targeted, shPromA does not induce downregulation of other NFκB driven genes, either at the mRNA or protein level. Furthermore, we confirmed shPromA does not have sequence non-specific off-target effects through unaltered expression of CD4, CXCR4 and CC R5, which are used for viral entry. Additionally, shPromA does not alter PKR, IFN levels, and three downstream mediators of IFNα response genes. Our data clearly shows that shPromA achieved highly specific TGS of HIV-1, demonstrating that effective TGS can be induced with minimal off-target effects.