Skip to main content
Springer Nature - PMC COVID-19 Collection logoLink to Springer Nature - PMC COVID-19 Collection
. 2006;33(2):141–148. doi: 10.1385/MB:33:2:141

Identification of effective siRNA blocking the expression of SARS viral envelope E and RDRP genes

Bo Meng 1, Yue-woon Lui 2, Shi Meng 1, Changxiu Cao 2, Yinghe Hu 1,
PMCID: PMC7090727  PMID: 16757801

Abstract

A cell-based assay was developed to screen small interference RNA (siRNA) to block the expression of two genes of the severe acute respiratory syndrome (SARS) virus. These two genes encode RNA-dependent RNA polymerase (RDRP) and envelope E protein. The RDRP plays an essential role in viral RNA replication where envelope E protein is involved in envelope formation and virus assembly. The RDRP and envelope E genes, based on published sequences, have been synthesized and cloned into mammalian expression vectors. In addition, four siRNA sites for the RDRP gene and two siRNA sites for envelope E gene were designed and tested. The siRNA or short hairpin RNA (shRNA) expression cassettes were co-transfected with the SARS viral RDRP or envelope E expression vectors into NIH 3T3 cells. The expression levels of RDRP and envelope E genes were examined by reverse transcription followed by quantitative real-time polymerase chain reaction (PCR). Two of the siRNA expression cassettes for RDRP successfully inhibited the expression of the gene, whereas both of the siRNA expression cassettes for envelope E decreased approx 90% of the envelope E gene expression. The siRNA and shRNA for one of the siRNA sites of the RDRP gene were also tested, and it was found that both inhibited exogenous RDRP expression in a dose-dependent manner. These siRNA molecules could be used to examine the function of these genes in SARS virus replication and assembly. Furthermore, these molecules could potentially be developed into therapeutic agents for the treatment of patients with SARS.

Index Entries: RNAi, SARS, RNA-dependent RNA polymerase (RDRP), envelope E.

References

  • 1.Lassus P., Opitz-Araya X., Lazebnik Y. Requirement for caspase-2 in stress-induced apoptosis before mitochondrial permeabilization. Science. 2002;297:1352–1354. doi: 10.1126/science.1074721. [DOI] [PubMed] [Google Scholar]
  • 2.Kong X. C., Barzaghi P., Ruegg M. A. Inhibition of synapse assembly in mammalian muscle in vivo by RNA interference. EMBO Rep. 2004;5:183–188. doi: 10.1038/sj.embor.7400065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Kamath R. S., Ahringer J. Genome-wide RNAi screening in Caenorhabditis elegans. Methods. 2003;30:313–321. doi: 10.1016/S1046-2023(03)00050-1. [DOI] [PubMed] [Google Scholar]
  • 4.Boutros M., Kiger A. A., Armknecht S., et al. Genome-wide RNAi analysis of growth and viability in Drosophila cells. Science. 2004;303:832–835. doi: 10.1126/science.1091266. [DOI] [PubMed] [Google Scholar]
  • 5.Paddison P. J., Silva J. M., Conklin D. S., et al. A resource for large-scale RNA-interference-based screens in mammals. Nature. 2004;428:427–431. doi: 10.1038/nature02370. [DOI] [PubMed] [Google Scholar]
  • 6.Berns K., Hijmans E. M., Mullenders J., et al. A large-scale RNAi screen in human cells identifies new components of the p53 pathway. Nature. 2004;428:431–437. doi: 10.1038/nature02371. [DOI] [PubMed] [Google Scholar]
  • 7.Poy M. N., Eliasson L., Krutzfeldt J., et al. A pancreatic islet-specific microRNA regulates insulin secretion. Nature. 2004;432:226–230. doi: 10.1038/nature03076. [DOI] [PubMed] [Google Scholar]
  • 8.Soutschek J., Akinc A., Bramlage B., et al. Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature. 2004;432:173–178. doi: 10.1038/nature03121. [DOI] [PubMed] [Google Scholar]
  • 9.Zhang Y., Boado R. J., Pardridge W. M. in vivo knockdown of gene expression in brain cancer with intravenous RNAi in adult rats. J. Gene Med. 2003;5:1039–1045. doi: 10.1002/jgm.449. [DOI] [PubMed] [Google Scholar]
  • 10.Lewis D. L., Hagstrom J. E., Loomis A. G., et al. Efficient delivery of siRNA for inhibition of gene expression in postnatal mice. Nat. Genet. 2002;32:107–108. doi: 10.1038/ng944. [DOI] [PubMed] [Google Scholar]
  • 11.McCaffrey A. P., Meuse L., Pham T. T., et al. RNA interference in adult mice. Nature. 2002;418:38–39. doi: 10.1038/418038a. [DOI] [PubMed] [Google Scholar]
  • 12.Rota P. A., Oberste M. S., Monroe S. S., et al. Characterization of a novel coronavirus associated with severe acute respiratory syndrome. Science. 2003;300:1394–1399. doi: 10.1126/science.1085952. [DOI] [PubMed] [Google Scholar]
  • 13.Marra M. A., Jones S. J., Astell C. R., et al. The Genome sequence of the SARS-associated coronavirus. Science. 2003;300:1399–1404. doi: 10.1126/science.1085953. [DOI] [PubMed] [Google Scholar]
  • 14.Qin E. D., Zhu Q. Y., Yu M. A complete sequence and comparative analysis of a SARS-associated virus (Isolate BJ01) Chi. Sci. Bull. 2003;48:941–948. doi: 10.1360/03wc0186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Zhang J., Meng B., Liao D., et al. De novo synthesis of PCR templates for the development of SARS diagnostic assay. Mol. Biotechnol. 2003;25:107–112. doi: 10.1385/MB:25:2:107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Livak K. J., Schmittgen T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C (T) method. Methods. 2001;25:402–408. doi: 10.1006/meth.2001.1262. [DOI] [PubMed] [Google Scholar]
  • 17.Elbashir S. M., Harborth J., Lendeckel W., et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 2001;411:494–498. doi: 10.1038/35078107. [DOI] [PubMed] [Google Scholar]
  • 18.Zamore P. D., Tuschl T., Sharp P. A., et al. RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell. 2000;101:25–33. doi: 10.1016/S0092-8674(00)80620-0. [DOI] [PubMed] [Google Scholar]
  • 19.Ui-Tei K., Naito Y., Takahashi F., et al. Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference. Nucleic Acids Res. 2004;32:936–948. doi: 10.1093/nar/gkh247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Amarzguioui M., Prydz H. An algorithm for selection of functional siRNA sequences. Biochem. Biophys. Res. Commun. 2004;316:1050–1058. doi: 10.1016/j.bbrc.2004.02.157. [DOI] [PubMed] [Google Scholar]

Articles from Molecular Biotechnology are provided here courtesy of Nature Publishing Group

RESOURCES