RIBOZYME MEDIATED DESTRUCTION OF INFLUENZA-A VIRUS IN-VITRO AND IN-VIVO

Short catalytic RNAs with inherent, specific endoribonuclease activity , called ribozymes, have recently been shown to exist in nature. Accor ding to the structural models artificial ribozymes have been designed that can potentially hydrolyse any chosen target RNA sequence in trans at a specific site. We have constructed and characterized in vitro ha mmerhead and hairpin ribozymes designed to cleave viral RNA segment 5 of influenza A virus. Both ribozymes were functional under optimal in vitro conditions, but quantitative measurements indicate that the hamm erhead ribozyme is considerably more efficient at this target site tha n the hairpin ribozyme. Mg2+ dependent hammerhead ribozyme-mediated cl eavage reactions were enhanced at higher temperature and in presence o f spermidine, but catalytic activities were retained also in cellular extract S-100 or nuclear extracts at physiological temperatures. Recom binant plasmids derived from transfection vector pSV2-neo were enginee red to allow the expression of specific ribozymes under the control of SV40 early promoter or SV40 early+ late promoters. These plasmids wer e introduced by transfection into COS cells, and their expression and enzymatic activities were analyzed in stable cell lines after selectio n of neomycin-resistance. Several permanent ribozyme-expressing clones were established and characterized: ribozyme coding DNA sequences and synthesis of ribozyme RNA molecules in the transfected cells were det ermined and monitored by polymerase chain reactions. It was found that the highest levels (up to 70-80%) of resistance to influenza A virus strain X-31 super-infection was observed in COS cells transfected with plasmids containing SV40 early or SV40 early+late promoters coincidin g with relatively high and constitutive rates of ribozyme expression. These results suggest the feasibility of developing ribozymes designed against influenza virus to achieve therapeutic value. (C) 1994 Wiley- Liss, Inc.