Triplex-forming MicroRNAs form stable complexes with HIV-1 provirus and inhibit its replication

One of the most fascinating discoveries in biology in recent years is unquestionably the identification of the family of small, noncoding RNAs known as microRNAs (miRNAs). Each miRNA targets multiple mRNA species through recognition of complementary sequences, typically located at multiple sites within the 3 untranslated region. In animals, single-stranded miRNA binds specific messenger RNA (mRNA) by a mechanism that is yet to be fully characterized. The bound mRNA remains untranslated resulting in reduced levels of the corresponding protein; however, if the sequence match between the miRNA and its target is precise, the bound mRNA can be degraded resulting in reduced levels of the corresponding transcript. Eukaryotic genes are also regulated by triplex formation between double helix and a third small RNA or DNA molecule. Thousands of triplex-forming (TF) islands in human genomes are mapped. However, the role of TF miRNAs within the hairpin structures of miRNA and the target mRNA has not been reported. We have explored TF complexes between human miRNAs (hsa-miR) that are complementary to human immunodeficiency virus (HIV)-1 and their antiviral potential as therapeutic agents.

We downloaded mature miRNA sequences from the human miRBase Sequence Database (http://microrna.sanger.ac.uk/sequences/), and computationally analyzed miRNAs that have significant homologies to HIV-1 genome (pNL 4-3 Accession #AF324493). We developed an algorithm to look for triplex-binding motifs (C+CG and T AT) and selected 4 miRNAs with 3 negative controls. TF stability was tested by using fluorophore-labeled duplexes connected by a single hexaethylene glycol moiety, representing HIV-1 proviral motifs, and black-hole quencher-1 labeled oligonucleotides, representing miRNA.

Fifty miRNAs were discovered that showed greater than 80% homology to HIV-1, of which 4 hsa-miR that exhibited an ability to form stable triplex with double stranded-HIV-1 sequences were selected. Three negative controls were used. The TF stability of the 4 hsa-miRs and the negative controls were confirmed and measured. Stably transfected Hela-CD4+ cell lines expressing each of the hsa-miR were developed. All 4 miRNAs exhibited a significant inhibition of HIV-1 as measured by HIV-1 p24 enzyme-linked immunosorbent assay (>90%; P>0.001) when compared with the 3 negative controls. By using immunohistochemical staining with triplex binding monoclonal antibodies, significant expression of TF miRNAs was detected in the cell lines, but not in the negative controls (P<0.001).

In this study, we demonstrated for the first time that besides the well-established post-transcriptional silencing based on mRNA degradation, miRNAs may be responsible for long-term latency of HIV-1 by TF, a different mechanism. We provide a possible molecular mechanism by which HIV-1 homologous miRNAs may impart resistance to HIV-1 and suggest a new miRNA-based therapeutic strategy for HIV-1.