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. 2007 May;81(10):5121-31.
doi: 10.1128/JVI.01511-06. Epub 2007 Mar 14.

Small interfering RNAs against the TAR RNA binding protein, TRBP, a Dicer cofactor, inhibit human immunodeficiency virus type 1 long terminal repeat expression and viral production

Helen S Christensen  1 , Aïcha Daher, Kaitlin J Soye, Lisa B Frankel, Marina R Alexander, Sébastien Lainé, Sylvie Bannwarth, Chi L Ong, Sean W L Chung, Shahan M Campbell, Damian F J Purcell, Anne Gatignol
Affiliations

Small interfering RNAs against the TAR RNA binding protein, TRBP, a Dicer cofactor, inhibit human immunodeficiency virus type 1 long terminal repeat expression and viral production

Helen S Christensen et al. J Virol. 2007 May.

Abstract

RNA interference (RNAi) is now widely used for gene silencing in mammalian cells. The mechanism uses the RNA-induced silencing complex, in which Dicer, Ago2, and the human immunodeficiency virus type 1 (HIV-1) TAR RNA binding protein (TRBP) are the main components. TRBP is a protein that increases HIV-1 expression and replication by inhibition of the interferon-induced protein kinase PKR and by increasing translation of viral mRNA. After HIV infection, TRBP could restrict the viral RNA through its activity in RNAi or could contribute more to the enhancement of viral replication. To determine which function will be predominant in the virological context, we analyzed whether the inhibition of its expression could enhance or decrease HIV replication. We have generated small interfering RNAs (siRNAs) against TRBP and found that they decrease HIV-1 long terminal repeat (LTR) basal expression 2-fold, and the LTR Tat transactivated level up to 10-fold. In the context of HIV replication, siRNAs against TRBP decrease the expression of viral genes and inhibit viral production up to fivefold. The moderate increase in PKR expression and activation indicates that it contributes partially to viral gene inhibition. The moderate decrease in micro-RNA (miRNA) biogenesis by TRBP siRNAs suggests that in the context of HIV replication, TRBP functions other than RNAi are predominant. In addition, siRNAs against Dicer decrease viral production twofold and impede miRNA biogenesis. These results suggest that, in the context of HIV replication, TRBP contributes mainly to the enhancement of virus production and that Dicer does not mediate HIV restriction by RNAi.

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Figures

FIG. 1.
FIG. 1.
siRNAs against TRBP decrease exogenous TRBP expression. (A) Schematic representation of TRBP2 mRNA and location of the siRNAs. One microgram of each siRNA was run on a 2% agarose gel and visualized by ethidium bromide staining. MW indicates DNA molecular weight markers. (B) siRNAs against TRBP reduce the fluorescence of EGFP-TRBP. HeLa cells were cotransfected with 0.5 μg pEGFP-C1-TRBP2 and with 100 nM siRNA NS, siRNA567, siRNA571, and siRNA657, as indicated, by using FuGENE. At 48 h posttransfection, cells were fixed, mounted, and assayed for GFP expression by fluorescence detection. (C) Titration of EGFP-TRBP by FACS after cotransfection with siRNAs. HeLa cells were cotransfected with 0.5 μg pEGFP-C1-TRBP2 and with 3 (white bars), 7 (light gray bars), 14 (dark gray bars), or 18 (black bars) nM siRNA NS, siRNAGFP, siRNA567, siRNA571, or siRNA657, as indicated, by using Lipofectamine. Reporter expression is calculated as the percentage of EGFP-TRBP2 expression in the absence of siRNA. This result is the average of six independent experiments ± the standard error of the mean.
FIG. 2.
FIG. 2.
siRNAs against TRBP decrease endogenous protein expression. (A) Determination of TRBP mRNA stability. Jurkat cells were incubated with ActD (5 μg/ml) for 1, 2, 4, 6, 8, or 10 h. Five micrograms of total RNA was reverse transcribed and subjected to PCR amplification with specific primers for TRBP and c-myc. PCR products were quantified by densitometric scanning of the gel (Typhoon scanner). TRBP (open squares) and c-myc (solid circles) mRNA levels were expressed as percentages of the initial value and plotted against time after ActD treatment. The results are the means of two separate experiments. (B) siRNAs against TRBP decrease endogenous TRBPs. HeLa cells were transfected with 0 (lane 4), 20 (lanes 1 and 5), 40 (lanes 2 and 6), or 80 (lanes 3 and 7) nM siRNA-NS, siRNA567, siRNA571, or siRNA657, as indicated, by using FuGENE. Cells were transfected with 0.5 μg pCDNA3-TRBP1 (lane 8) or pCDNA3-TRBP2 (lane 9). Two hundred (lanes 1 to 7) or 20 (lanes 8 and 9) μg of cell extract was resolved by SDS-PAGE, analyzed by immunoblotting with an antibody against TRBP and exposed for 1 h or analyzed by immunoblotting with an antibody against actin and exposed for 1 min. The TRBP1 start codon is included within TRBP2 reading frame. This is a representative experiment of three that gave similar results. (C) siRNAs do not activate PKR. HeLa cells were transfected with 0 (lane 1) or 80 nM siRNA-NS (lane 2) or siRNA571 (lane 3) by using FuGENE. Two hundred micrograms of cell extract was resolved by SDS-PAGE; analyzed by immunoblotting with antibodies against phosphorylated PKR (top), PKR (middle), and actin (lower); and exposed for 1 min. This is a representative experiment of three that gave similar results.
FIG. 3.
FIG. 3.
siRNAs against TRBP decrease the expression of the HIV-1 LTR. (A) siRNAs against TAR and TRBP reduce HIV-1 LTR basal expression. HeLa cells were mock transfected (lane 1) or cotransfected with 0.05 μg of LTR-Luc and the indicated siRNAs at 80 nM (lanes 2 to 6) by using FuGENE. (B) siRNAs against Tat, TAR, and TRBP reduce HIV-1 LTR transactivated expression. HeLa cells were mock transfected (lane 1) or cotransfected with 0.05 μg of LTR-Luc (lanes 2 to 8), 0.01 μg of pCMV1-Tat, and the indicated siRNAs at 80 nM (lanes 3 to 8) by using FuGENE. Luciferase activity is the ratio of the luciferase level in the presence of the siRNA versus NS normalized to 100% for cells transfected with NS. Each value represents the average of five independent experiments ± the standard error of the mean. A representative quantitative PCR on the luciferase gene is shown at the bottom of each graph as a transfection control. nt, nucleotides.
FIG. 4.
FIG. 4.
siRNAs against TRBP decrease HIV-1 production in transfected HeLa cells. (A) HIV-1 RT activity in cell supernatants. HeLa cells were mock transfected or cotransfected with 0.5 μg of pNL4-3 and 14 nM siRNA-NS, siRNA-Tat1c, siRNA567, siRNA571, or siRNA657, as indicated, by using Lipofectamine in a T25 flask format. RT activity was calculated by densitometry with the software referred to in Materials and Methods. Each value represents the average of four independent experiments normalized as a percentage of the siRNA-NS RT value ± the standard error of the mean. (B) HIV protein expression in cell lysates. HeLa cells were transfected as described above. One hundred twenty micrograms of cell lysate was resolved by SDS-PAGE, analyzed by immunoblotting with an antibody against HIV-1, and exposed for 15 min. The values on the left are molecular sizes in kilodaltons. (C) Cellular protein expression in cell lysates. Two hundred micrograms of the above-described cell lysate was resolved by SDS-PAGE and analyzed by immunoblotting with antibodies against P-PKR, PKR, TRBP, and actin successively. The blots were exposed for 10 min for P-PKR, 1 min for PKR and TRBP, and 10 s for actin. The blots shown in panels B and C are representative data among four independent experiments.
FIG. 5.
FIG. 5.
siRNAs against TRBP decrease the production of various HIV strains in transfected HeLa cells. HeLa cells were cotransfected with 0.5 μg of pNL4-3; 1 μg each of pAD8, p89.6, pELI-1, pMAL-2, or pROD-10, as indicated; and 14 nM siRNA-NS (black bars) or TRBP 571 (gray bars), by using Lipofectamine in a six-well format. RT activity was calculated as described in the legend to Fig. 4. Each value represents the average of three independent experiments normalized as a percentage of the NS siRNA RT value ± the standard error of the mean.
FIG. 6.
FIG. 6.
siRNAs against Dicer decrease HIV-1 production in transfected HeLa cells. (A) siRNAs against Dicer decrease mRNA and protein levels. HeLa cells were mock transfected or transfected with 14 nM siRNA against NS or 4, 8, or 14 nM siRNA against Dicer, as indicated, by using Lipofectamine. (Top) Semiquantitative RT-PCR with primers that amplify Dicer or GAPDH mRNA as indicated. (Bottom) Two hundred micrograms of cell lysates was resolved by SDS-PAGE and analyzed by immunoblotting with antibodies against Dicer or actin, as indicated. The blots were exposed for 10 min for Dicer and 10 s for actin. (B) HIV-1 RT activity in cell supernatants. HeLa cells were mock transfected or cotransfected with 0.5 μg of pNL4-3; 14 nM siRNA against NS or Tat1c; or 4, 8, or 14 nM siRNA against Dicer, as indicated, by using Lipofectamine in a six-well format. RT activity was calculated as described in the legend to Fig. 4 and corrected for transfection efficiency. Each value represents the average of four independent experiments normalized as a percentage of the NS siRNA RT value ± the standard error of the mean.
FIG. 7.
FIG. 7.
Effects of siRNA against TRBP or Dicer on cell viability and RNAi. (A) siRNAs do not affect cell viability. HeLa cells were transfected with no siRNA (lane 1), 1.8 μg poly(I)·poly(C) (lane 2), or 14 nM siRNA against NS (lanes 3 and 4), siRNA against EGFP (lane 5) siRNA against Tat (lane 6), siRNA567 (lane 7), siRNA571 (lane 8), siRNA657 (lane 9), or siRNA against Dicer (lane 10). All siRNAs were generated from the Ambion siRNA construction kit, except siRNA against NS2 and siRNA against Dicer, which were purchased from QIAGEN. Cell viability was assessed by FACS analysis of 7-AAD-stained cells. The viable cell population of the mock-transfected cells was set at 100%. Each value represents the average of four independent experiments ± the standard error of the mean. (B) miRNA biogenesis is impaired in HeLa cells transfected with siRNA567, siRNA571, or siRNA against Dicer. (Top) Schematic representation of the vector-delivered EGFP pre-miRNA and the predicted structure of the fully processed EGFP miRNA. The EGFP sense sequence is highlighted. (Bottom) HeLa cells were cotransfected with 0 (lane 1) or 2 μg EGFP pre-miRNA vector (lanes 2 to 5) and 14 nM siRNA against NS (lane 2) siRNA567 (lane 3), siRNA571 (lane 4), or siRNA against Dicer (lane 5) in six-well plates. Expression of EGFP miRNA was determined by Northern blotting and quantified by densitometry. The results represent the average of three independent experiments ± the standard error of the mean.
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