Inhibition of severe acute respiratory syndrome virus replication by small interfering RNAs in mammalian cells - PubMed (original) (raw)

Inhibition of severe acute respiratory syndrome virus replication by small interfering RNAs in mammalian cells

Zhi Wang et al. J Virol. 2004 Jul.

Abstract

Severe acute respiratory syndrome (SARS) is an acute respiratory infectious disease that spread worldwide in early 2003. The cause was determined as a novel coronavirus (CoV), SARS-associated CoV (SARS-CoV), with a single-stranded, plus-sense RNA. To date, no effective specific treatment has been identified. To exploit the possibility of using RNA interference as a therapeutic approach to fight the disease, plasmid-mediated small interfering RNAs (siRNAs) were generated to target the SARS-CoV genome. The expression of siRNAs from two plasmids, which specifically target the viral RNA polymerase, effectively blocked the cytopathic effects of SARS-CoV on Vero cells. These two plasmids also inhibited viral replication as shown by titer assays and by an examination of viral RNA and protein levels. Thus, our results demonstrated the feasibility of developing siRNAs as effective anti-SARS drugs.

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Figures

FIG. 1.

FIG. 1.

Schematic representation of the sites of the six siRNA target sequences against genomic RNA of SARS-CoV (strain HKU-39849).

FIG. 2.

FIG. 2.

CPE of SARS-CoV on Vero cells. Vero cells were transfected with various plasmids and then infected with 1 × 102 TCID50 of SARS-CoV. CPE were first observed after 20 h, and the pictures were taken at 24 h postinfection. In panels A and B are mock-infected cells without virus and cells infected with SARS-CoV, respectively. In panels C to I, the cells were infected by virus after pretransfection with pSR (C), pNS (D), pSR01 (E), 1.5 μg of pSR02 (F), 3 μg of pSR02 (G), 1.5 μg of pSR03 (H), and 3 μg of pSR03 (I).

FIG. 3.

FIG. 3.

Effect of siRNA on SARS-CoV replication. The virus-containing supernatants were collected 24 h after virus infection of Vero cells pretransfected with or without 3 μg of various plasmids, as indicated. The supernatants were used for the titer assays. Virus titers were determined from three independent experiments in triplicate, and data shown are the means ± standard deviations. An asterisk indicates that the titer values from pSR02- and pSR03-transfected cells compared to the titer from untreated cells are statistically significant with a P value of 0.0005 (t test). pSR02 and pSR03: siRNAs against SARS-CoV.

FIG. 4.

FIG. 4.

Reduction of SARS-CoV protein levels by siRNA. Vero cells were transfected with 3 μg of various plasmids and then infected with 1 × 102 TCID50 of SARS-CoV. Total cellular proteins were harvested at 24 h postinfection. Western blot analysis was performed with antisera against N protein, 3CL proteinase, or anti-β-actin antibodies. p SR01, pSR02, and pSR03: siRNAs against SARS-CoV. This experiment was repeated three times, and the results of a typical experiment are shown.

FIG. 5.

FIG. 5.

Attenuation of SARS-CoV mRNA levels by siRNA. Vero cells pretransfected with no vector (-), empty vector (pSR), nonspecific siRNA plasmid (pNS), or plasmids expressing siRNA against SARS-CoV (pSR02 and pSR03) were infected with 1 × 102 TCID50 of SARS-CoV. Samples were collected at 18h postinfection; mRNA was extracted and reverse transcribed to cDNA. The cDNA was subjected to real-time PCR analysis by using gene-specific primers for SARS-CoV or GAPDH. The change (n_-fold) in SARS-CoV mRNA levels were presented as 2−ΔΔ_Ct normalized to GAPDH (an endogenous reference) and relative to a virus sample (an experimental control). Data are from triplicate experiments and are shown as the means ± standard deviations. Three independent experiments were performed, and similar results were obtained.

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