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

T. Li, Y. Zhang, L. Fu, C. Yu, X. Li, Y. Li, X. Zhang, Z. Rong, Y. Wang, H. Ning, R. Liang, W. Chen, L. A. Babiuk, Z. Chang

Research output: Contribution to journalArticlepeer-review

51 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)751-761
Number of pages11
JournalGene Therapy
Volume12
Issue number9
DOIs
StatePublished - May 2005

Keywords

  • Coronavirus
  • Gene silence
  • Leader sequence
  • RNA interference
  • SARS
  • SARS-CoV
  • siRNA

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics

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