Ribozyme mediated destruction of influenza A virus in vitro and in vivo

Abstract

Short catalytic RNAs with inherent, specific endoribonuclease activity, called ribozymes, have recently been shown to exist in nature. According 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 hammerhead 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 hammerhead ribozyme is considerably more efficient at this target site than the hairpin ribozyme. Mg²⁺ dependent hammerhead ribozyme-mediated cleavage reactions were enhanced at higher temperature and in presence of spermidine, but catalytic activities were retained also in cellular extract S-100 or nuclear extracts at physiological temperatures. Recombinant plasmids derived from transfection vector pSV2-neo were engineered to allow the expression of specific ribozymes under the control of SV40 early promoter or SV40 early+ late promoters. These plasmids were introduced by transfection into COS cells, and their expression and enzymatic activities were analyzed in stable cell lines after selection of neomycin-re-sistance. Several permanent ribozyme-express-ing clones were established and characterized: ribozyme coding DNA sequences and synthesis of ribozyme RNA molecules in the transfected cells were determined 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 coinciding 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.