title: 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.
authors.affiliation: Department of Microbiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & School of Basic Medicine, Peking Union Medical College, Beijing 100005, China.
authors.forename: Ying-Li
authors.initials: YL
authors.lastname: Cao
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.forename: Rong
authors.initials: R
authors.lastname: Guo
authors.forename: Fan
authors.initials: F
authors.lastname: Yang
authors.forename: Yun
authors.initials: Y
authors.lastname: Zhang
authors.forename: Shu-Hui
authors.initials: SH
authors.lastname: Wang
authors.forename: Li
authors.initials: L
authors.lastname: Liu
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D015536:Down-Regulation; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D019590:Nucleocapsid Proteins / Q000235:genetics* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D014709:Vero Cells
chemical_list: D019590:Nucleocapsid Proteins; D034741:RNA, Small Interfering