Infectious pancreatic necrosis virus RNA cleavage in vitro by hammerhead ribozymes and enhancement of ribozyme catalysis hy oligonucleotide facilitators

Infectious pancreatic necrosis virus (IPNV), an aquatic birnavirus, has a b isegmented double-stranded RNA genome consisting of a 3.2-kb A segment and a 2.9-kb B segment. To determine the function of IPNV's viral proteins and to study the effects of viral RNA cleavage by hammerhead ribozymes, we clon ed and sequenced the IPNV E1S strain of the A segment. After sequencing, we continued to study the virus pathogens inhibited by ribozyme cleavage and analyzed the cleavage of the virus RNA in vitro. The templates (VP2, 1220 b p) for in vitro transcription of S569 and S969 (substrates 569 and 969 bp i n length) were synthesized by polymerase chain reaction. The DNA templates of hammerhead ribozymes targeted different sites in the partial sense RNA o f IPNV. These templates were chemically synthesized RNAs prepared by runoff transcription of amplification products or synthetic DNA templates contain ing a T7 RNA polymerase promoter, and were used to characterize several pro perties of the cleavage reaction at 25 degrees C in 12 mM Mg2+. Under this condition (25 degrees C, 12 mM Mg2+), the hammerhead ribozymes formed an es timated fraction of product during the reaction of only 30% in cleaving lon g RNA substrates in vitro. Short DNA facilitators (12 or 24-mers) that bind adjacent to either the 3' or 5' end of the ribozyme enhanced the rate of c leavage of the long RNA substrates containing 569 and 969 nucleotides, resp ectively, in trans. The hammerhead ribozymes with 3'-end facilitators react ed more efficiently (i.e., 65%).