Silencing SARS-CoV Spike protein expression in cultured cells by RNA interference

Abstract

The severe acute respiratory syndrome (SARS) has been one of the most epidemic diseases threatening human health all over the world. Based on clinical studies, SARS-CoV (the SARS-associated coronavirus), a novel coronavirus, is reported as the pathogen responsible for the disease. To date, no effective and specific therapeutic method can be used to treat patients suffering from SARS-CoV infection. RNA interference (RNAi) is a process by which the introduced small interfering RNA (siRNA) could cause the degradation of mRNA with identical sequence specificity. The RNAi methodology has been used as a tool to silence genes in cultured cells and in animals. Recently, this technique was employed in anti-virus infections in human immunodeficiency virus and hepatitis C/B virus. In this study, RNAi technology has been applied to explore the possibility for prevention of SARS-CoV infection. We constructed specific siRNAs targeting the S gene in SARS-CoV. We demonstrated that the siRNAs could effectively and specifically inhibit gene expression of Spike protein in SARS-CoV-infected cells. Our study provided evidence that RNAi could be a tool for inhibition of SARS-CoV.

Figure 1

Target sites and vector information. A: Locations of siRNA target sites. S1 and S2 represent the S1 and S2 regions of Spike protein respectively. The targeted sequences for siRNA design are listed. B: Schematic of vector for generating siRNA. pBS/U6 promoter, sequence generating siRNA hairpin and transcriptional terminal signal are shown as indicated. C: Diagram of predicted structure of siRNAs from pBS/U6/S‐RNA1 and pBS/U6/S‐RNA2. UUCAAGAGA was used to generate the hairpin loop, indicated as a cycle.

Figure 2

Inhibition of expression of the Spike protein. Transfected siRNAs as indicated inhibited Spike protein expression. The Spike protein tagged with HA at the C‐terminus (HA‐Spike) overexpressed in the cells, which were co‐transfected with the indicated plasmids, was detected by Western blot using anti‐HA antibody from the lysates of the cells. β‐Actin and EGFP were used as internal and positive controls.

Figure 3

Immunostaining for Spike protein in transfected cells. 293T cells were co‐transfected with pCMV‐Myc/HA‐Spike and pEGFP‐N1 vectors for 48 h in the presence of different siRNA plasmids as indicated. The cells were immunostained with anti‐HA antibody plus TRITC‐conjugated secondary antibody (red). The pictures were viewed with a Nikon TE300 microscope and captured with SPOT CCD software. The EGFP pictures were observed before immunostaining. The co‐transfected vectors are (a,b) empty vector pBS/U6, (c,d) pBS/U6/EGFP‐RNAi, (e,f) pBS/U6/S‐RNAi1, and (g,h) pBS/U6/S‐RNAi2.

Figure 4

Reduced mRNA accumulation of the S gene in cells overexpressing Spike protein. 293T cells were co‐transfected with the S gene expression vector (pCMV‐Myc/HA‐Spike) and the indicated plasmids for siRNA or control vectors. Cells were harvested after transfection for 2 days. RT‐PCR products for the S gene and the internal control, β‐actin, are shown as indicated molecular weights.

Figure 5

Dose‐dependent inhibition of S gene mRNA accumulation. The Vero E6 cells were transfected with the indicated plasmids for one day. Thereafter, the cells were infected with SARS‐CoV (BJ01) at 5×10⁵ MOI/ml. After 2 days of infection, the cells were harvested with Trizol (Promega) for RNA isolation in a P3 lab. RT‐PCR was performed to show the accumulation of S gene mRNA from the infected SARS‐CoV. Increasing amounts of siRNA vectors transfected are indicated. RT‐PCR products for the S gene and the internal control, β‐actin, are shown as indicated molecular weights.

Figure 6

Northern blot of S gene mRNA in the transfected cells. The Vero E6 cells were transfected with the indicated plasmids and treated with SARS‐CoV as in Fig. 5. The total RNA was subjected to Northern blot analysis with ³²P‐labeled probes amplified from SARS‐CoV cDNA (BJ01) and GAPDH. The abundance of S gene mRNA and GAPDH is indicated.

Figure 7

SARS‐CoV titration in the medium of the infected cells. The indicated Vero E6 cells were infected with SARS‐CoV for 2 h and then the virus was washed off with medium. The virus titrations were determined for the supernatant (medium) of the infected cells cultured for 72 h after infection. (+) indicates cells infected with SARS‐CoV, (−) indicates normal cultured cells.