IL-27 inhibits HIV-1 infection in multi-type Immune cells. We have previously identified seven novel microRNAs (miRNAs) in IL-27-differentiated Macrophages and predicted that some of these miRNAs possess anti-viral properties. In this study, we assessed the anti-HIV properties of each miRNA. T cells and Macrophages were transfected with mimic miRNAs or control miRNA and were then infected with HIV-1. HIV-1 replication was monitored by a p24 HIV-1 antigen capture assay. One of the novel miRNAs, hsa-mir-7705, demonstrated a strong anti-HIV effect in Macrophages but not in T cells, and HIV-1 replication was suppressed by >95%.
A combination of multiple analysis using Microarrays, TargetScan, Immunoblotting, and RT-PCR assays revealed that hsa-mir-7705 targets Topoisomerase II alpha (TOP2a) mRNA, however endogenous protein expression was constitutively under detectable in Macrophages.
Condition media derived from has-mir-7705-transfected Macrophages dose-dependently inhibited HIV-1 infection in fresh Macrophages by nearly 100%, and this anti-HIV effect was diminished by incubating either the supernatants with B18R, a Type I IFN neutralization protein, or Macrophages with an IFNRα neutralization antibody, suggesting that hsa-mir-7705 exerts its anti-viral effect via Type-I IFNs. The hsa-mir-7705 mediated IFN-α production in Macrophage was detected in the range from 10 to 1900 pg/ml in a donor dependent manner. Knock-down of TOP2a mRNA using siRNA did not induce the IFN-α secretion from Macrophages, suggesting that IFN-α induction by hsa-mir-7705 is independent of a conventional miRNA mechanism.
To elucidate the mechanism of the IFN-α induction, using a series of variants of hsa-mir-7705, IFN-α inducing activity was studied. IFN-α induction was only detected by double-stranded hsa-mir-7705, and the induction required a minimum of a 23 nt length RNA with 5’ and 3’-end specific sequences through IRF activation.
Taken together, our data indicate that hsa-mir-7705 is a small noncoding RNA inducing IFN-α in a nucleotide structure/sequence dependent manner via a yet to be uncharacterized pathway.