Small interfering RNA inhibits SARS-CoV nucleocapsid gene expression in cultured cells and mouse muscles

Abstract

SARS-CoV is a newly identified coronavirus that causes severe acute respiratory syndrome (SARS). Currently, there is no effective method available for prophylaxis and treatment of SARS-CoV infections. In the present study, the influence of small interfering RNA (siRNA) on SARS-CoV nucleocapsid (N) protein expression was detected in cultured cells and mouse muscles. Four siRNA expression cassettes driven by mouse U6 promoter targeting SARS-CoV N gene were prepared, and their inhibitory effects on expression of N and enhanced green fluorescence protein (EGFP) fusion protein were observed. A candidate siRNA was proved to down-regulate N and EGFP expression actively in a sequence-specific manner. The expression vector of this siRNA was constructed and confirmed to reduce N and EGFP expression efficiently in both cultured cells and adult mouse muscles. Our findings suggest that the siRNA should provide the basis for prophylaxis and therapy of SARS-CoV infection in human.

Figure 1

Preparation of PCR‐based siRNA expression cassettes. (A) Two‐step PCR procedure for the siRNA expression cassettes was described in the text, the PCR products consist of mouse U6 promoter, short hairpin DNA and terminator sequence, six thymidines (T6). (B) The siRNA expression cassettes were prepared by two rounds of PCR. Lanes 1–4: PCR products of siRNA expression cassettes of N186, N388, N587 and N811, respectively. Lane 5: PCR product of muN388 expression cassettes. Lane 6: PCR product of S1184 expression cassettes. Lane 7: PCR product of mouse U6 promoter.

Figure 2

Inhibition of SARS‐CoV N protein expression by siRNA expression cassettes. The 293T cells were co‐transfected with N‐EGFP expression plasmid and various PCR products. The SARS‐CoV N protein expression was measured. (A) N protein expression was detected by Western blot at 48 h post‐transfection. The upper panel shows N protein expression (lane 1 shows N expression in cells transfected with pN‐EGFP alone, other lanes show that in cells co‐transfected with pN‐EGFP and various PCR products, U6P denotes PCR product of mouse U6 promoter only). The lower panel shows β‐actin expression (as an endogenous control). (B) At 48 h post‐transfection, the total RNA was isolated and subjected to reverse transcription and real‐time PCR analysis. The mRNA levels of cells transfected with plasmid pN‐EGFP along with various PCR products were quantitated relative to that in the cells transfected with plasmid pN‐EGFP alone.

Figure 3

Inhibition of SARS‐CoV N protein expression by siRNA expression vector. The N388 siRNA expression vector was constructed and its influence on the N‐EGFP expression was detected in cultured 293T cells. (A) The PCR product of N388 siRNA expression cassette was inserted into the vector pMD‐18T, and the resulting plasmid was used as siRNA expression vector. (B) 293T cells were co‐transfected with plasmid pN‐EGFP and N388 siRNA expression vector, along with HBcAg expression plasmid as an unrelated control. The images show the EGFP expression at 48 h post‐transfection. The left panel shows the cells under a bright field. (C) The expression of N protein and HBcAg were detected by Western blot at 48 h post‐transfection. Lane 1: 293T cells transfected with 0.3 μg pN‐EGFP, 0.3 μg pCI‐HBc and 0.6 μg pUC18. Lane 2: 293T cells transfected with 0.3 μg pN‐EGFP, 0.3 μg pCI‐HBc and 0.3 μg pU6‐shN388. Lane 3: 293T cells transfected with 0.3 μg pN‐EGFP, 0.3 μg pCI‐HBc and 0.6 μg pU6‐shN388. In fluorescence and Western blot analysis, plasmid pUC18 was used to standardize the plasmid dosage for transfection. The experiment was repeated three times, and similar results were obtained.

Figure 4

Silencing of N‐EGFP expression by siRNA in mouse muscles. Mice were injected with N‐EGFP expression plasmid and the siRNA expression vector in both tibialis anterior muscle. The N and EGFP expression was detected at day 4, 8, 12 and 16 post‐injection. (A) The muscles were isolated and frost slices were prepared, and EGFP expression was visualized under fluorescence microscope. The upper panel shows EGFP expression in mice injected with plasmid pN‐EGFP along with siRNA expression vector at various time post‐injection. The lower panel shows EGFP expression in mice injected with plasmid pN‐EGFP along with pUC18. (B) The muscles were isolated and N protein expression was detected by Western blot at different times. The upper panel indicates N protein expression in mouse muscles injected with plasmid pN‐EGFP along with: (1) pUC18 or (2) siRNA expression vector. The lower panel shows β‐actin expression as a loading control. The plasmid pUC18 used was to standardize the plasmid dosage for gene delivery in vivo. This figure shows the results of one representative experiment of two.