Inhibition of viral multiplication by hammerhead ribozymes targeted against the polymerase gene of mouse hepatitis virus.
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.
Maeda
A
A
Department of Laboratory Animal Science, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
Mizutani
T
T
Hayashi
M
M
Watanabe
T
T
Namioka
S
S
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D015977:Eukaryotic Initiation Factor-1 / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D006517:Murine hepatitis virus / Q000201:enzymology / Q000235:genetics / Q000502:physiology*; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D016337:RNA, Catalytic / Q000096:biosynthesis*; D012194:RNA-Directed DNA Polymerase / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D018894:Reverse Transcriptase Inhibitors; D014162:Transfection; D014779:Virus Replication
D017931:DNA Primers; D015977:Eukaryotic Initiation Factor-1; D016337:RNA, Catalytic; D011994:Recombinant Proteins; D018894:Reverse Transcriptase Inhibitors; D012194:RNA-Directed DNA Polymerase