Expression of vector-based small interfering RNA against West Nile virus effectively inhibits virus replication.
RNA interference is one of the effective emerging anti-viral strategies to inhibit virus infection in cells. In this study, a small interfering RNA expressing vector (pSilencer-NS5) targeting the NS5 gene of West Nile virus (WNV) was employed to target and destroy WNV transcripts. Real-time PCR revealed drastic reduction in WNV RNA transcripts in pSilencer-NS5-transfected Vero cells. The virus infectious titre was also significantly reduced by 90% as determined by plaque assays. The resulting decrease in virus replication was shown to be specific since both scrambled and nucleotide(s) mismatch siRNA against WNV NS5 gene did not have any effect on WNV productive yields. Furthermore, Western immunoblot analysis on the expression of viral NS5 and envelope (E) proteins showed significant down-regulation on the expression of viral NS5 and envelope (E) proteins in virus-infected cells that were pre-transfected with pSilencer-NS5. These data clearly supported the notion that the expression of vector-based siRNA against WNV NS5 gene is able to exert its silencing effect on WNV-infected cells without inducing cytotoxicity, hence holding promise in therapeutic treatment of this important emerging infectious disease.
Ong
S P
SP
Flavivirology Laboratory, Department of Microbiology, 5 Science Drive 2, National University of Singapore, 117597 Singapore, Singapore.
Choo
B G H
BG
Chu
J J H
JJ
Ng
M L
ML
D000818:Animals; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000737:chemistry; D015870:Gene Expression; D005822:Genetic Vectors; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence; D034622:RNA Interference; D020871:RNA Stability; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000096:biosynthesis; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis* / Q000235:genetics; D014779:Virus Replication / Q000235:genetics; D014902:West Nile virus / Q000235:genetics / Q000502:physiology*
C096041:NS5 protein, flavivirus; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012367:RNA, Viral; D014759:Viral Envelope Proteins; D017361:Viral Nonstructural Proteins