Enhanced gene silencing of HIV-1 specific siRNA using microRNA designed hairpins

Abstract

Post-transcriptional inhibition of HIV-1 replication can be achieved by RNA interference (RNAi). The cellular expression of short interfering RNA (siRNA) or short hairpin RNA (shRNA) homologous to regions of the HIV-1 genome decreases viral replication by the selective degradation of targeted RNA. Here, we demonstrate that another class of noncoding regulatory RNA, termed microRNA (miRNA), can be used to deliver antiviral RNAi. By incorporating sequences encoding siRNA targeting the HIV-1 transactivator protein tat into a human miR-30 pre-microRNA (pre-miRNA) backbone, we were able to express tat siRNA in cells. The tat siRNA delivered as pre-miRNA precursor was 80% more effective in reducing HIV-1 p24 antigen production than tat siRNA expressed as conventional shRNA. Our results confirm the utility of expressing HIV-1 specific siRNA through a miR-30 precursor stem-loop structure and suggest that this strategy can be used to increase the antiviral potency of RNAi.

Figure 1

Schematic representation of predicted short hairpin tat and pre-micro mir-30/tat RNA sequences. Underlined arrows indicate sense and antisense tat sequences embedded in a shRNA or a miR-30 precursor RNA backbone. Bold letters in miA-tat and miB-tat represent stem–loop sequences derived from pre-miR-30. G:U wobbles are indicated with a star.

Figure 2

HIV-1 p24 antigen production in culture supernatant of 293 cells transfected with HIV-1_NL4.3 and the indicated tat siRNA expression plasmids determined by p24 immunoassay 48 h post-transfection; luc indicates a shRNA expression plasmid targeting luciferase. (A) Absolute p24 antigen in nanograms per milliliter of culture supernatant. (B) Antiviral potency of the various tat siRNA expression plasmids relative to control plasmid luc shown as fold inhibition. Values represent averages of three independent experiments, with the range indicated.

Figure 2

HIV-1 p24 antigen production in culture supernatant of 293 cells transfected with HIV-1_NL4.3 and the indicated tat siRNA expression plasmids determined by p24 immunoassay 48 h post-transfection; luc indicates a shRNA expression plasmid targeting luciferase. (A) Absolute p24 antigen in nanograms per milliliter of culture supernatant. (B) Antiviral potency of the various tat siRNA expression plasmids relative to control plasmid luc shown as fold inhibition. Values represent averages of three independent experiments, with the range indicated.

Figure 3

Reduction of HIV-1 p24 antigen in cell-free supernatant and cell lysate determined by western blot analysis. 293 cells were co-transfected with HIV-1_NL4.3 and the indicated tat siRNA expression vectors. The control plasmid luc expresses a luciferase hairpin siRNA. Tubulin served as a loading control.

Figure 4

Reduction of total intracellular HIV-1 RNA determined by real-time PCR. Variation of RNA input was normalized by quantifying GAPDH expression by real-time PCR. To convert threshold cycles to copy number, an external standard curve was created with known copy numbers of HIV-1_NL4.3. Values represent averages of three independent experiments, with the range indicated.

Figure 5

Northern blot analysis of tat shRNA, pre-miRNA and siRNA expression from 293 cells co-transfected with HIV-1_NL4.3 and the indicated DNA constructs. Total cellular RNA was extracted from 293 cells 48 h after transfection and separated on a 15% polyacrylamide–7 M urea gel. Hybridization was performed using a ³²P-labeled DNA oligonucleotide probe complementary to the tat sense strand. (A) Northern blot was exposed for 1 h. (B) Detection of processed tat siRNA effector molecules after 10 h of exposure.