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siRNA targeting the Leader sequence of SARS-CoV inhibits virus replication

Gene Therapy volume 12, pages 751–761 (2005)Cite this article

Abstract

SARS-CoV (the SARS-Associated Coronavirus) was reported as a novel virus member in the coronavirus family, which was the cause of severe acute respiratory syndrome. Coronavirus replication occurs through a unique mechanism employing Leader sequence in the transcripts when initiating transcription from the genome. Therefore, we cloned the Leader sequence from SARS-CoV(BJ01), which is identical to that identified from SARS-CoV(HKU-39849), and constructed specific siRNA targeting the Leader sequence. Using EGFP and RFP reporter genes fused with the cloned SARS-CoV Leader sequence, we demonstrated that the siRNA targeting the Leader sequence decreased the mRNA abundance and protein expression levels of the reporter genes in 293T cells. By stably expressing the siRNA in Vero E6 cells, we provided data that the siRNA could effectively and specifically decrease the mRNA abundance of SARS-CoV genes as analyzed by RT-PCR and Northern blot. Our data indicated that the siRNA targeting the Leader sequence inhibited the replication of SARS-CoV in Vero E6 cells by silencing gene expression. We further demonstrated, via transient transfection experiments, that the siRNA targeting the Leader sequence had a much stronger inhibitory effect on SARS-CoV replication than the siRNAs targeting the Spike gene or the antisense oligodeoxynucleotides did. This report provides evidence that targeting Leader sequence using siRNA could be a powerful tool in inhibiting SARS-CoV replication.

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Acknowledgements

This work was supported by a special grant (fd0311) from Tsinghua University 985 Program and the grants from 973 Project (2001CB510006, 2002CB513000) and the Chinese National Academic Foundation (No. 39970369, 30070703 and 30030050). We would like to thank Dr Yang Shi at Harvard University for kindly providing the pBS/U6 and pBS/U6/GFP-RNAi vectors. We specially thank Dr Anming Meng (Tsinghua University) for providing pDsRed1.1/RFP vectors. We thank the Genome Institute of Singapore for proving whole genome clones and S gene expression vector, which were used as references in our study. We thank Dr Q Zhu (Beijing Institute of Microbiology and Epidemiology, AIMMS, China) for providing SARS-CoV (BJ01) virus strain.

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Authors and Affiliations

  1. Department of Biological Sciences and Biotechnology, Institute of Biomedicine, Tsinghua University, Beijing, China

    T Li, Y Zhang, X Li, Y Li, X Zhang, Z Rong, Y Wang, H Ning & Z Chang

  2. Institute of Microbiology and Epidemiology, AMMS, Beijing, China

    L Fu, C Yu & W Chen

  3. Vaccine & Infectious Disease Organization (VIDO), University of Saskatchewan, Saskatoon, SK, Canada

    R Liang & L A Babiuk

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Li, T., Zhang, Y., Fu, L. et al. siRNA targeting the Leader sequence of SARS-CoV inhibits virus replication. Gene Ther 12, 751–761 (2005). https://doi.org/10.1038/sj.gt.3302479

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