A ribozyme targeted to RNA polymerase gene of infectious bursal disease virus effectively cleaves and inhibits expression of the viral gene product.
Five hammerhead-type ribozymes were designed and cloned to cleave infectious bursal disease virus (IBDV) RNA and inhibit protein synthesis from cloned full-length viral cDNA genes. Two ribozymes (R1 and R2) were directed to the large viral RNA segment gene and three ribozymes (R3, R4, and R5) were directed to the small viral RNA segment gene. Targets for the ribozymes were produced from cloned full-length coding regions of both small and large viral RNA segment genes. Ribozymes and their corresponding targets were synthesized as in vitro transcripts. Despite several attempts at different temperatures, no cleavage of viral RNA transcripts with four of the ribozymes (R1, R2, R3, and R5) was observed. One of the ribozymes, R4, was effective in cleaving the viral RNA polymerase gene transcripts in a magnesium-dependent manner. Ribozyme R4 caused 82% reduction in the synthesis of the viral RNA polymerase gene product. The inhibition was specific since there was no change in the rate of synthesis of the Escherichia coli beta-galactosidase protein. The results suggest that ribozyme R4 can be used as potential anti-IBDV agent. It was also demonstrated that the hammerhead-type ribozymes can cleave sites other than conventional GUC sequence motif as the cleavage site of ribozyme R4 had GUU motif which conformed to the NUX consensus motif.
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Department of Veterinary Pathobiology, School of Veterinary Medicine, Purdue University, West Lafayette, IN 47907-1175, USA.
Lin
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Wu
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D000818:Animals; D018175:Birnaviridae Infections / Q000821:virology*; D002645:Chickens; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism*; D007243:Infectious bursal disease virus / Q000201:enzymology* / Q000235:genetics*; D011201:Poultry Diseases / Q000821:virology; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D016337:RNA, Catalytic; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D012321:DNA-Directed RNA Polymerases