Format

Send to

Choose Destination
RNA Biol. 2011 Nov-Dec;8(6):1035-46. doi: 10.4161/rna.8.6.16264. Epub 2011 Nov 1.

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

Author information

1
Immunovirology Laboratory, St Vincent's Centre for Applied Medical Research; Darlinghurst, NSW Australia. k.suzuki@amr.org.au

Abstract

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 NF-kB 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 MOLT4 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 NF-kB binding sequence. As a result of the unique sequence targeted, shPromA does not induce down-regulation of other NF-kB 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 CCR5, which are used for viral entry. Additionally, shPromA does not alter PKR, IFN levels, and three downstream mediators of IFN-a 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.

PMID:
21955498
PMCID:
PMC3256422
DOI:
10.4161/rna.8.6.16264
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Taylor & Francis Icon for PubMed Central
Loading ...
Support Center