RNA interference directed against viral and cellular targets inhibits human immunodeficiency Virus Type 1 replication - PubMed (original) (raw)

RNA interference directed against viral and cellular targets inhibits human immunodeficiency Virus Type 1 replication

Rama M Surabhi et al. J Virol. 2002 Dec.

Abstract

Human immunodeficiency virus type 1 (HIV-1) gene expression is regulated by both cellular transcription factors and Tat. The ability of Tat to stimulate transcriptional elongation is dependent on its binding to TAR RNA in conjunction with cyclin T1 and CDK9. A variety of other cellular factors that bind to the HIV-1 long terminal repeat, including NF-kappaB, SP1, LBP, and LEF, are also important in the control of HIV-1 gene expression. Although these factors have been demonstrated to regulate HIV-1 gene expression by both genetic and biochemical analysis, in most cases a direct in vivo demonstration of their role on HIV-1 replication has not been established. Recently, the efficacy of RNA interference in mammalian cells has been shown utilizing small interfering RNAs (siRNAs) to result in the specific degradation of host mRNAs and decreases the levels of their corresponding proteins. In this study, we addressed whether siRNAs directed against either HIV-1 tat or reverse transcriptase or the NF-kappaB p65 subunit could specifically decrease the levels of these proteins and thus alter HIV-1 replication. Our results demonstrate the specificity of siRNAs for decreasing the expression of these viral and cellular proteins and inhibiting HIV-1 replication. These studies suggest that RNA interference is useful in exploring the biological role of cellular and viral regulatory factors involved in the control of HIV-1 gene expression.

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Figures

FIG. 1.

FIG. 1.

Effect of siRNAs on inhibiting gene expression. (A) MAGI cells were transfected with 50 nM siRNAs directed against either the NF-κB p65 subunit (lanes 1 and 4), HTLV-1 tax (lanes 2 and 5), or Oligofectamine alone (OF, lanes 3 and 6). Cells were harvested at either 3 days (lanes 1 to 3) or 6 days (lanes 4 to 6) posttransfection, and the lysates (20 μg) were subjected to Western blot analysis using antibodies directed against p65 (top gel) or α-actin (lower gel). (B) MAGI cells were transfected with a 50 nM concentration of each of three siRNAs directed against HIV-1 tat (tat1, tat2, or tat3, lanes 1 to 3), p65 (lane 4), tax (lane 5), or Oligofectamine alone (lane 6) for 24 h. The cells were then transfected with 2 μg of a CMV expression vector expressing Tat-GFP. Cells were harvested 48 h later, and Western blot analysis was performed on these lysates (20 μg) with antibodies directed against GFP (top gel). The Western blot was reprobed with antibodies directed against p65 (middle gel) and α-actin (bottom gel).

FIG. 2.

FIG. 2.

Inhibition of HIV-1 replication by siRNAs. (A) MAGI cells were transfected with 50 nM concentrations of siRNAs directed against either HIV-1 tat (tat1, tat2, and tat3), p65, or HTLV-1 tax or with Oligofectamine alone (OF). Twenty-four hours later, the cells were infected with HIV-1 NL4.3 and cell culture supernatants were collected on day 1 (gray bars), 3 (white bars), or 6 (black bars) postinfection and were assayed for p24 antigen (Ag). The p24 antigen values are expressed as the percent values relative to those seen with Tax siRNA. The actual p24 antigen values in the culture supernatants are shown in the adjacent table. (B) MAGI cells transfected with various siRNAs for 24 h prior to HIV-1 infection were split on the 3rd day postinfection, and culture supernatants were collected on day 10 (day 13 postinfection) and were assayed for p24 antigen. The p24 antigen values were expressed as the percentage relative to that seen with tax siRNA. (C) MAGI cells were transfected with siRNAs directed against HIV-1 tat (tat1, lane 1; tat2, lane 2; and tat3, lane 3), p65 (lane 4), HTLV-1 tax (lane 5), or Oligofectamine alone (OF, lane 6) for 24 h and were then infected with HIV-1 for 6 days. Whole-cell lysates from these cells were prepared and analyzed by Western blotting. The membrane was first probed with HIV-1-specific antisera and then reprobed with antibodies directed against p65 and α-actin. The positions of the HIV-1-specific proteins p24 antigen and p51/p66 (RT) are indicated. (D) RNA was prepared from the HIV-1-infected MAGI cells and was subjected to RT-PCR amplification to determine the relative levels of HIV-1 tat RNA expression. The α-actin RNA was also amplified in these samples as a control.

FIG. 3.

FIG. 3.

Effect of siRNAs on different steps in the HIV-1 life cycle. (A) MAGI cells were transfected with siRNAs directed against either HIV-1 tat (tat1, lane 1; tat2, lane 2; and tat3, lane 3), p65 (lane 4), HTLV-1 tax (lane 5), or Oligofectamine alone (OF, lane 6) for 24 h and were then infected with HIV-1 for another 24 h. Hirt lysates were prepared from these cells, and the cytoplasmic DNA was amplified with oligonucleotide primers to assay for either HIV-1-specific strong-stop (SS) (+43/+65 and +182/+158) (top gel), first-strand jump (−49/−30 and +93/+70) (second gel), full-length (FL) viral DNA (+96/+118 and +234/+212) (third gel), or the mitochondrial cytochrome c oxidase (CO) gene (bottom gel). As controls, cytoplasmic DNA from mock-infected (lane 7) and different amounts of NL4.3 plasmid DNA (20, 2, and 0.2 pg, lanes 8 to 10) were also amplified. (B) Chromosomal DNA was prepared from MAGI cells that were transfected with siRNAs directed against either HIV-1 tat (tat1, lane 1; tat2, lane 2; and tat3, lane 3), p65 (lane 4), HTLV-1 tax (lane 5), or Oligofectamine alone (OF, lane 6) for 24 h and were then infected with HIV-1 for 10 days. Portions of HIV-1 gag (top gel) and cellular α-actin (lower gel) genes were amplified as detailed in Materials and Methods. As controls, 1 ng (lane 7) and 4 ng (lane 8) of NL4.3 plasmid DNA were also amplified.

FIG. 4.

FIG. 4.

Transfection of tat and p65 siRNAs following HIV-1 infection. (A) MAGI cells were infected with HIV-1 for 12 h and were then transfected with siRNAs directed against either HIV-1 tat (tat1, tat2, or tat3), p65, HTLV-1 tax, or Oligofectamine alone (OF). Culture supernatants were collected at 6 days following siRNA transfection and were assayed for p24 antigen (Ag). (B) MAGI cells infected with HIV-1 for 6 days were split and were then transfected with siRNAs directed against HIV-1 tat (tat1, tat2, or tat3), p65, HTLV-1 tax, or Oligofectamine (OF) alone. Culture supernatants were collected at 3 and 6 days following siRNA transfection and were assayed for p24 antigen (Ag). In both parts of the figure, the p24 values are expressed as the percentage relative to that seen with tax siRNA.

FIG. 5.

FIG. 5.

Inhibition of HIV-1 replication by siRNAs specific for HIV-1 RT. (A) MAGI cells were transfected with siRNAs directed against HIV-1 RT (RT1 or RT2), HTLV-1 tax, or Oligofectamine (OF) alone for 24 h, and the cells were then infected with HIV-1. Culture supernatants were collected at 6 days following siRNA transfection and were assayed for p24 antigen levels and expressed as the percentage relative to that seen with tax siRNA. (B) Whole-cell extracts were prepared from the MAGI cells that were transfected with siRNAs directed against HIV-1 RT (RT1, lane 1; or RT2, lane 2), HTLV-1 tax (lane 3), or Oligofectamine alone (OF, lane 4) for 24 h and then were infected with HIV-1 for 6 days. Western blot analysis was performed with HIV-1-specific antisera, and then the blot was reprobed with antibodies against p65 and α-actin.

FIG. 6.

FIG. 6.

β-Galactosidase staining of MAGI cells. MAGI cells were transfected with 50 nM siRNAs directed against either HIV-1 tat, RT, p65, HTLV-1 tax, or Oligofectamine alone (OF). Twenty-four hours later, the cells were infected with HIV-1 NL4.3. (A) On day 6 postinfection, the cells were washed and stained for β-galactosidase as described in Materials and Methods and photographed. Cells infected with HIV-1 in the absence of other treatment (control) are also shown. (B) At the same time postinfection, RNA was prepared from these HIV-1-infected MAGI cells and subjected to RT-PCR amplification of HIV-1 leader sequences with oligonucleotide primers to generate a fragment extending from +31 to +259 in order to determine relative levels of HIV-1 mRNA expression. The α-actin mRNA was also amplified from these samples as a control.

FIG. 7.

FIG. 7.

SiRNAs directed against tat, RT, and p65 inhibit HIV-1 replication in Jurkat cells. Jurkat cells (n = 2 × 105) were transfected with Oligofectamine containing 100 pmol of siRNAs corresponding to either tat1, tat2, and tat3; p65 and RT1 and -2; Tax or mock transfected. After 16 h, the cells were washed, resuspended in RPMI medium, and infected with HIV-1 NL4.3, and p24 antigen levels were determined by ELISA at 3 days postinfection.

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References

    1. Alcami, J., T. Lain de Lera, L. Folgueira, M. A. Pedraza, J. M. Jacque, F. Bachelerie, A. R. Noriega, R. T. Hay, D. Harrich, R. B. Gaynor, J. L. Virelizier, and F. Arenzana-Seisdedos. 1995. Absolute dependence on kappa B responsive elements for initiation and Tat-mediated amplification of HIV transcription in blood CD4 T lymphocytes. EMBO J. 14:1552-1560. - PMC - PubMed
    1. Baeuerle, P. A., and D. Baltimore. 1996. NF-κB: ten years after. Cell 87:13-20. - PubMed
    1. Baldwin, A. S. 1996. The NF-κB and IκB proteins: new discoveries and insights. Annu. Rev. Immunol. 14:649-681. - PubMed
    1. Barboric, M., R. M. Nissen, S. Kanazawa, N. Jabrane-Ferrat, and B. M. Peterlin. 2001. NF-κB binds P-TEFb to stimulate transcriptional elongation by RNA polymerase II. Mol. Cell 8:327-337. - PubMed
    1. Bass, B. L. 2000. Double-stranded RNA as a template for gene silencing. Cell 101:235-238. - PubMed

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