Identification and characterization of three novel small interference RNAs that effectively down-regulate the isolated nucleocapsid gene expression of SARS coronavirus

Abstract

Nucleocapsid (N) protein of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is a major pathological determinant in the host that may cause host cell apoptosis, upregulate the proinflammatory cytokine production, and block innate immune responses. Therefore, N gene has long been thought an ideal target for the design of small interference RNA (siRNA). siRNA is a class of small non-coding RNAs with a size of 21-25nt that functions post-transcriptionally to block targeted gene expression. In this study, we analyzed the N gene coding sequences derived from 16 different isolates, and found that nucleotide deletions and substitutions are mainly located at the first 440nt sequence. Combining previous reports and the above sequence information, we create three novel siRNAs that specifically target the conserved and unexploited regions in the N gene. We show that these siRNAs could effectively and specifically block the isolated N gene expression in mammal cells. Furthermore, we provide evidence to show that N gene can effectively up-regulate M gene mediated interferon b (IFNb) production, while blocking N gene expression by specific siRNA significantly reduces IFNb gene expression. Our data indicate that the inhibitory effect of siRNA on the isolated N gene expression might be influenced by the sequence context around the targeted sites.

Figure 1

Sequence alignment of the N genes derived from 16 different SARS-CoV isolates.

Figure 2

Expression analysis of the isolated SARS-CoV N gene.(a) RT-PCR analysis on N gene expression. Total RNAs were isolated from pCMV-Myc (lane 2) or pCMV-Myc-Np (lane 3) transfected HEK293 cells and subjected RT-PCR. (b) Western blot analysis on N gene expression in HEK293 cells. HEK293 cells were transfected with either pCMV-Myc or pCMV-Myc-Np. After 48 h, the transfected cells were lysed. Equal amount of cell lysates were resolved onto 12% SDS-PAGE. The reaction products were probed with anti-Myc antibody. (c) Analysis on EGFP-N gene expression by fluorescence microscope. HEK293 cells were transfected with the either pEGFP-C or pEGFP-Np. (d) Cellular localization of N protein by immunostaining approach. HEK293 cells were transfected with pCMV-Myc-Np. The transfected cells were probed with either PBS (mock) or anti-Myc antibody as primary antibody.

Figure 3

RT-PCR analysis on siRNA mediated N gene inhibition.

Figure 4

The inhibitory effect of si-N213 on the isolated SARS-CoV N gene expression. (a) Western blot analysis on the effect of si-N213 on N gene expression. pCMV-Myc-Np was co-transfected with the increased doses of pBS/U6-si-N213 into HEK293 cells. After 48 h, the transfected cells were lysed. Equal amount of cell lysates were resolved onto 12% SDS-PAGE. The reaction products were probed with anti-Myc or anti-actin antibodies. (b) Quantitation of the individual band intensity detected in (a). (c) si-N213 but not si-M3 effectively inhibited EGFP-N gene expression. About 2 μg of pEGFP-N plasmid was co-transfected with increased doses of either si-M3 or si-N213. (d) The mean fluorescence intensity (MFI) of EGFP-Np fusion gene expression in the absence or presence of si-N213 was detected by flow cytometric analysis. SARS-CoV M gene specific siRNA, si-M3, was served as a non-specific control.

Figure 5

The inhibitory effect of si-N863 on the isolated SARS-CoV N gene expression. (a) Western blot analysis on the effect of si-N863 on N gene expression. pCMV-Myc-Np was co-transfected with the increased doses of pBS/U6-si-N863 into HEK293 cells. After 48 h, the transfected cells were lysed. Equal amount of cell lysates were resolved onto 12% SDS-PAGE. The reaction products were probed with anti-Myc or anti-actin antibodies. (b) Quantitation of the individual band intensity detected in (a). (c) si-N863 but not si-M3 effectively inhibited EGFP-N gene expression. About 2 μg of pEGFP-Np plasmid was co-transfected with increased doses of either si-M3 or si-N863. (d) The mean fluorescence intensity (MFI) of EGFP-Np fusion gene expression in the absence or presence of si-N863 was detected by flow cytometric analysis. SARS-CoV M gene specific siRNA, si-M3, was served as a non-specific control.

Figure 6

The inhibitory effect of si-N1240 on the isolated SARS-CoV N gene expression. (a) Western blot analysis on the effect of si-N1240 on N gene expression. pCMV-Myc-Np was co-transfected with the increased doses of pBS/U6-si-N1240 into HEK293 cells. After 48 h, the transfected cells were lysed. Equal amount of cell lysates were resolved onto 12% SDS-PAGE. The reaction products were probed with anti-Myc or anti-actin antibodies. (b) Quantitation of the individual band intensity detected in (a). (c) si-N1240 but not si-M3 effectively inhibited EGFP-N gene expression. About 2 μg of pEGFP-Np plasmid was co-transfected with increased doses of either si-M3 or si-N1240. (d) The mean fluorescence intensity (MFI) of EGFP-Np fusion gene expression in the absence or presence of si-N1240 was detected by flow cytometric analysis. SARS-CoV M gene specific siRNA, si-M3, was served as a non-specific control.

Figure 7

Real time quantitative RT-PCR (qRT-PCR) analysis on the inhibitory effect induced by N gene specific siRNA.

Figure 8

The inhibitory effect of N gene siRNAs on N gene mediated up-regulation of interferon β production.