Structure of the antiviral miRNAs based on pre-miRNAs D^wt and E^wt. Upper panel: The r/t5 and ldr9 siRNAs were incorporated into the D^wt backbone, which produces the guide strand from the 5′-side of the hairpin. The guide strand is marked in grey. We modified the passenger strand to mimic structural features (mismatches, bulges and thermodynamic stability) of the natural pre-miRNA. The 19-nt antiviral siRNAs were positioned either at the 5′ (D^r/t5-19 and D^ldr9-19) or 3′-end (D^r/t5-19' and D^ldr9-19') of the original miRNA and the length of the siRNAs was extended at the 3′-end to 23-nt (D^r/t5-23 and D^ldr9-23). Lower panel: The r/t5 siRNA was similarly incorporated into the E^wt pre-miRNA, which produces the guide strand from the 3′-side of the hairpin. The structure of the original shRNAs sh^r/t5 and sh^ldr9 are presented. HIV-1 sequences are blue, mature wild-type miRNA sequences are red, pre-miRNA sequences are black, Watson–Crick base pairs are shown with dashes and G•U wobbles with dots.

Expression and processing of siRNA from a miRNA versus an shRNA construct. We titrated 39–2500 ng sh^r/t5 construct in 293T cells and compared that with 2500 ng D^r/t5 construct. Northern blot analysis was performed on total cellular RNA. M, RNA size marker (nt) is shown on the left. Expression of siRNAs by 2500 ng sh^r/t5 was set at 100% and relative siRNA expression levels are indicated below the blot. Ethidium bromide staining of the 5S rRNA serves as sample loading control.

Northern blot analyses of siRNAs derived from antiviral miRNA constructs. 293T cells were transfected with the indicated 1, 2, 3 and 4 antiviral miRNA constructs and siRNAs were detected in total cellular RNA using 19-nt complementary LNA oligonucleotide probes. Different miRNA transcripts were analysed: (A) A^pol47, (B) B^pol1, (C) C^gag5, (D) D^r/t5 and (E) E^ldr9. As negative controls, pBS or miRNA constructs targeting another gene were used. The original shRNA constructs were used as positive controls, showing the mature siRNAs. Ethidium bromide staining of 5S rRNA serves as sample loading control. M, RNA size marker (nt) is shown on the left, the probe used, is shown on the right.

Schematic of the mir-17-92 cluster and design of the antiviral miRNAs. (A) Genomic organization of the mir-17-92 polycistron (upper panel) and predicted secondary structure of the mir-17-19b transcript (A^wt-E^wt, lower panel). Mature miRNAs are indicated in red; CMV, cytomegalovirus immediate early promoter; pA, SV40 polyadenylation signal. (B) Scheme of the HIV-1 proviral genome and position of the target sequences (upper panel) used for the design of the antiviral miRNA polycistron (lower panel). Guide strands of the siRNA against HIV-1 are blue. Numbers between the pre-miRNAs represent the length of the flanks (in nucleotides). Original miRNA names were replaced with the letters A–E and target genes are indicated in superscript.

Inhibition of luciferase reporters and HIV-1 production by individual antiviral miRNAs. (A) Luciferase reporters (100 ng) were co-transfected in 293T cells with 20 ng antiviral miRNA construct and 1 ng pRL. The original shRNAs sh^r/t5 and sh^ldr9 were used as positive controls and non-matching miRNAs as negative controls. Firefly and renilla luciferase expression was measured 2 days post-transfection. Normalized firefly luciferase expression in the presence of pBS was set at 100%. Mean values obtained in three independent experiments are shown (±SE). FL, firefly luciferase; RL, renilla luciferase. (B) 239T cells were co-transfected with 150 ng HIV-1 molecular clone LAI, 1 ng pRL and 20 ng of the indicated antiviral miRNA constructs. sh^r/t5 and sh^ldr9 constructs were used as positive controls, D^wt and E^wt as negative controls. CA-p24 levels in the culture supernatant and renilla luciferase expression were measured 2 days post-transfection. Normalized CA-p24 expression in the presence of pBS was set at 100%. Error bars represent the standard deviation in four independent experiments.

Inhibition of luciferase reporters and HIV-1 production by antiviral polycistronic miRNAs. (A) Knockdown efficiencies of the antiviral miRNA A^pol47 expressed from 1-, 2- and 6-hairpin transcripts: A^pol47, A^pol47B^wt and A^pol47A^wt-E^wt. Left panel: 293T cells were co-transfected with 100 ng Luc-A^pol47, 10 ng of different hairpin RNA constructs and 1 ng pRL. Two days post-transfection, firefly and renilla luciferase expression was measured. Normalized firefly luciferase expression with pBS was set at 100%. The A^wt-E^wt construct was used as negative control and sh^pol47 as positive control. Right panel: 293T cells were co-transfected with 150 ng pLAI, 1 ng pRL and 5 ng of the hairpin constructs. Normalized CA-p24 level in the absence of inhibitor was set at 100%. FL, firefly luciferase; RL, renilla luciferase. (B) Luciferase knockdown efficiencies of 1, 2, 3 and 4 antiviral miRNA transcripts. 293T cells were co-transfected with 100 ng of the indicated luciferase reporter, 10 ng miRNA construct and 1 ng pRL. Two days post-transfection, firefly and renilla luciferase expression was measured. The A^wt-E^wt construct and a non-matching miRNA were used as negative controls. The original shRNA constructs sh^pol47, sh^pol1, sh^gag5, sh^r/t5 and sh^ldr9 were used as positive controls. (C) Inhibition of HIV-1 production by the miRNA constructs. 293T cells were co-transfected with 150 ng pLAI, 1 ng pRL and 5 ng 1, 2, 3 or 4 miRNA constructs. Two days post-transfection, CA-p24 levels in the culture supernatant and renilla luciferase expression were measured. Normalized CA-p24 level in the absence of inhibitor was set at 100%. Mean values obtained in four independent experiments are shown (±SE).

Inhibition of HIV-1 replication in SupT1 cells transduced with lentiviral vectors expressing antiviral miRNAs A^pol47, B^pol1, C^gag5, D^r/t5, E^ldr9 and ACDE. Untransduced SupT1 cells and cells transduced with the unrelated miRNA N were used as negative controls. Cells expressing an extended triple shRNA construct (e-shRNA 3) serve as positive control. SupT1 cells stably expressing miRNAs A^pol47, B^pol1, C^gag5, D^r/t5, E^ldr9 and ACDE were infected with HIV-1 and virus replication was monitored for 7 days by measuring CA-p24 in the culture supernatant. One representative experiment is shown, similar results were obtained in three independent experiments.