Modulation of HIV-1 replication by RNA interference

Abstract

RNA interference (RNAi) is the process by which double-stranded RNA (dsRNA) directs sequence-specific degradation of messenger RNA in animal and plant cells. In mammalian cells, RNAi can be triggered by 21-nucleotide duplexes of small interfering RNA (siRNA). Here we describe inhibition of early and late steps of HIV-1 replication in human cell lines and primary lymphocytes by siRNAs targeted to various regions of the HIV-1 genome. We demonstrate that synthetic siRNA duplexes or plasmid-derived siRNAs inhibit HIV-1 infection by specifically degrading genomic HIV-1 RNA, thereby preventing formation of viral complementary-DNA intermediates. These results demonstrate the utility of RNAi for modulating the HIV replication cycle and provide evidence that genomic HIV-1 RNA, as it exists within a nucleoprotein reverse-transcription complex, is amenable to siRNA-mediated degradation.

Figure 1

Small interfering RNAs inhibit late events in HIV replication by promoting degradation of HIV-1 RNA. a, HIV targets of siRNAs used in this study. Small interfering RNAs completely homologous to the target HIV sequence (HIV_NL-GFP) are shown in blue and those harbouring nucleotide mismatches are shown in yellow. b, Effect of siRNAs on HIV-1 particle production. c, Total and active (phosphorylated) PKR levels in siRNA-transfected Magi cells. d, Small interfering RNAs mediate sequence-specific HIV RNA degradation. The presence of HIV_NL-GFP or HIV_YU-2 RNA was determined by RT–PCR using HIV Nef-specific primers. Because of the GFP insertion in HIV_NL-GFP Nef, RNAs originating from HIV_NL-GFP are 710 nucleotides larger than those originating from HIV_YU-2. M, molecular weight marker (100 bp ladder, New England Biolabs). e, Effect of siRNAs on HIV expression in primary PBLs.

Figure 2

Small interfering RNAs block early events in HIV replication by promoting degradation of genomic HIV RNA. a, Experimental design. b, Levels of trypsin-resistant HIV gag p24 in siRNA-transfected cells. Dash indicates no siRNA transfected into the cells. c, Strategy for analysis of viral nucleic acid intermediates formed early after HIV infection. Major cDNA intermediates in viral reverse transcription are indicated. Blue line, viral RNA; red line, viral cDNA; open circles and squares, primer-binding sites for initiation of minus-strand synthesis and polypurine tracts for plus-strand synthesis, respectively. HIV-specific primers (green arrows) are shown next to the earliest cDNA intermediate they amplify. Integrated (proviral) HIV DNA was amplified using an HIV LTR-specific primer (Rc) and a primer directed to alu repeats (filled circles) within flanking cellular DNA. d, Effect of siRNAs on genomic viral RNA. e, Effect of siRNAs on formation of HIV-1 reverse transcription (RT) intermediates. f, Reduced levels of viral integration in siRNA-transfected cells.

Figure 3

Inhibition of HIV replication by siRNAs derived from plasmid DNA templates. a, Strategy for production of hairpin siRNAs from plasmid vectors. Linearization of each construct with BstBI and transfection into cells with a plasmid expressing T7 RNA polymerase (Pol) predicts the expression of a hairpin RNA with a 19-bp self-complementary Vif stem and non-base-paired loops of 3, 5 and 7 nucleotides. b, Effect of Vif hairpin siRNAs on HIV particle production. Tl ΔVif is identical to plasmids that express Vif hairpin except that it lacks self-complementary Vif sequences. c, Vif hairpin siRNAs promote degradation of HIV RNA. PCR products amplified from HIV_NL-GFP served as a control. d, Inhibition of HIV-1 expression by Vif hairpin siRNAs in primary PBLs.