Potent and specific inhibition of human immunodeficiency virus type 1 replication by RNA interference

Abstract

Synthetic small interfering RNAs (siRNAs) have been shown to induce the degradation of specific mRNA targets in human cells by inducing RNA interference (RNAi). Here, we demonstrate that siRNA duplexes targeted against the essential Tat and Rev regulatory proteins encoded by human immunodeficiency virus type 1 (HIV-1) can specifically block Tat and Rev expression and function. More importantly, we show that these same siRNAs can effectively inhibit HIV-1 gene expression and replication in cell cultures, including those of human T-cell lines and primary lymphocytes. These observations demonstrate that RNAi can effectively block virus replication in human cells and raise the possibility that RNAi could provide an important innate protective response, particularly against viruses that express double-stranded RNAs as part of their replication cycle.

FIG. 1.

(A) Schematic representation of part of the HIV-1 genome. HIV-1 tat and rev exons are depicted with black and grey boxes, respectively, while the env open reading frame is shown as a white box (not to scale). The structure and target coordinates of the siRNAs specific for tat and rev are depicted. Boxed residues were mutated by transversion for use as control duplexes in some experiments. (B) siRNAs directed against tat and rev specifically inhibit Tat and Rev protein expression. Human 293T cells were cotransfected with 50 ng of both the pcTat and pcRev expression plasmid in the absence of RNA oligonucleotides (N) or in the presence of 0.1 μM antisense oligonucleotides (AS) or 0.1 μM siRNA duplexes (Si). Cell lysates were prepared 60 h posttransfection, and protein expression was assayed by Western analysis with polyclonal antiTat and antiRev antisera. Western blots were also probed with antibodies directed against the endogenous TAP/NXF-1 protein, which here served as an internal control for both loading and specificity.

FIG. 2.

(A) siRNAs directed against HIV-1 tat inhibit HIV-1 Tat but not BIV Tat function. Human 293T cells were transfected with 100 ng of pTAR/CAT or pbTAR/CAT, 50 ng of the internal control plasmid pBC12/CMV/β-Gal, and 0.1 ng of the indicated effector plasmid. siRNA oligonucleotides or siRNA duplexes were cotransfected with the reporter and expression plasmids as described in Materials and Methods. Cells were harvested 48 h after transfection, and induced CAT activities were determined. The data shown represent averages for three experiments with the standard deviations indicated by error bars. (B) siRNAs directed against HIV-1 rev specifically inhibit HIV-1 Rev but not HTLV Rex function. 293T cells were transfected with 25 ng of the pDM128/RRE or pDM128/RxRE reporter plasmid, 50 ng of pBC12/CMV/β-Gal, and 1 ng of the indicated effector plasmid. siRNA oligonucleotides or siRNA duplexes were cotransfected with the reporter and expression plasmids as described in Materials and Methods. Cells were harvested 48 h after transfection, and induced CAT and β-galactosidase activities were determined. The data shown represent averages for three experiments with the standard deviations indicated by error bars.

FIG. 3.

Inhibition of HIV-1 replication in human cells by RNAi. 293T cells were transfected with CD4 and CCR5 expression plasmids and siRNA duplexes (si) or antisense RNAs (as) (0.1 μM each). After 48 h, CD4⁺ CCR5⁺ cells were infected overnight with 50 ng of p24 antigen of the luciferase reporter virus NL-luc-ADA or the wild-type virus NL-ADA. Viral gene expression was determined by luciferase activity after 48 h of infection (A), while viral replication was measured by p24 Gag antigen production 60 h posttransfection (B). 293T cells lacking CD4 and CCR5 served as the negative control (NEG), while CD4⁺ CCR5⁺ 293T cells infected in the absence of siRNAs served as the positive control (POS). A low level of carryover of the virus stock used to infect the cells explains the background level of p24 Gag seen in panel B (vertical dashed line).

FIG. 4.

Analysis of HIV-1 RNA expression in human cells reveals that tat and rev siRNAs can reduce the expression of all three classes of HIV mRNA. Human 293T cells were cotransfected with siRNA duplexes (si) or with antisense RNAs (as) (0.1 μM each) and the CD4 and CCR5 expression plasmids as indicated at the top of the lanes. CD4⁺ CCR5⁺ cells were then infected overnight with 50 ng of p24 antigen. After 48 h of infection, total RNA was extracted from infected 293T cells and subjected to Northern analysis. The approximate sizes of the three classes of HIV-1 transcripts are indicated on the right. 18S rRNA, stained with ethidium bromide, is shown at the bottom of the figure as a loading control.

FIG. 5.

siRNAs directed against tat and rev can initiate the degradation of HIV-1 genomic RNA prior to reverse transcription. Human 293T cells were transfected with siRNA duplexes (si) or with antisense RNAs (as). CD4⁺ CCR5⁺ cells were infected overnight with 50 ng of p24 antigen. After 48 h, total DNA was extracted from infected 293T cells, denatured in 0.2 M NaOH, spotted onto a nylon membrane, and probed with a ³²P-labeled HIV-1-specific probe as described in Materials and Methods. As a control for loading, equivalent amounts of DNA were probed with a ³²P-labeled β-globin probe. 293T cells treated with 0.1 μM AZT, the reverse transcriptase inhibitor (2), are also shown (+AZT). DNA isolated from mock-infected 293T cells served as the negative control (NEG), while CD4⁺ CCR5⁺ 293T cells infected in the absence of siRNAs served as the positive control (POS).

FIG. 6.

siRNA inhibition of HIV-1 replication in T cells. (A) Jurkat T cells were transfected with a 12 μM concentration of each of the indicated siRNA duplex or single-stranded RNA oligonucleotides. Mock-transfected Jurkat T cells served as the positive control (POS), while the two 3-nt mismatch siRNAs shown in Fig. 1A served as the control (siRNA-M). After 24 h, transfected cells were infected overnight with VSV-G-pseudotyped NL-ADA. Viral production was determined by measurement of p24 Gag production at 48 h after infection. The data shown represent averages for three experiments with the standard deviations indicated by error bars. (B) Human PBMCs were transfected with the wild-type or mutant forms of the Tat and Rev siRNAs by electroporation and were infected with the R5-tropic NL-ADA strain 24 h later. Progeny virus production was measured 48 h after infection by p24 ELISA.