Inhibition of HIV-1 replication by RNA with a microRNA-like function

Human immunodeficiency virus type 1 (HIV-1) replication is suppressed by a small guide RNA (sgRNA) that targets the packaging signal of HIV-1 RNA. We unintentionally produced a plasmid with the reverse sequence of the sgRNA and its terminator (pR-Ψ-sgRNA-ter). Both sgRNA and R-Ψ-sgRNA suppress HIV-1, but the mechanism by which R-Ψ-sgRNA suppresses HIV is not clear. To evaluate whether the suppressive effect is caused by an RNA interference or microRNA (miRNA)-like mechanism, R-Ψ-sgRNA was synthesized in vitro and treated with the Dicer enzyme, an important enzyme for RNA interference and miRNA. The RNA was cleaved into fragments of approximately 24 nucleotides (nt). We analyzed the sequence of the RNA fragments and predicted the RNA secondary structure of R-Ψ-sgRNA to determine the region recognized by the Dicer enzyme. The lengths of the R-Ψ-sgRNA fragments ranged from 48 to 140 nt, and were predicted to form double strands, including mismatches, in this region. An HIV-1 p24 assay indicated that the R-Ψ-sgRNA fragments suppressed HIV-1 replication. These findings suggest that R-Ψ-sgRNA acts as a miRNA to inhibit HIV-1.