Inhibition of viral multiplication by hammerhead ribozymes targeted against the polymerase gene of mouse hepatitis virus.

Abstract

We designed and constructed two hammerhead ribozymes targeted against the polymerase gene of mouse hepatitis virus (MHV). They consisted of a 22-nucleotide (nt) ribozyme core sequence and antisense sequences of different lengths, 243-nt (S-ribozyme) and 926-nt (L-ribozyme). In cell-free reactions, the constructed ribozymes cleaved the target RNA at a specific site. Vectors that directed the expression of ribozymes by a promoter of human elongation factor 1 alpha were introduced into DBT cells, and the resulting several cell lines constitutively expressing the ribozymes were selected by Northern blot analysis and examined for intracellular multiplication of MHV. The production of infectious progeny virus particles was significantly reduced in the transfected cell lines expressing either S-ribozyme or L-ribozyme. Although the in vitro cleavage process of L-ribozyme was slower than that of S-ribozyme, no difference was observed in inhibitory effects on MHV multiplication between S- and L-ribozymes in the transfected cells.