Ribozyme-mediated decrease in mumps virus nucleocapsid mRNA level and progeny in infected vero cells.
The effects of endogenously expressed ribozymes directed to the mumps virus nucleocapsid (NP) mRNA were studied during viral infection. To this end, eukaryotic expression vectors encoding ribozymes or controls of passive hybridization effects were constructed and used to transfect mumps permissive Vero cells. Transcripts spanning trans-acting ribozymes of the hammerhead and hairpin types were designed to hydrolyze the first 5'GUC-3' sequence downstream from the initiation site and to hybridize to a 16 base sequence containing the putative cleavage site. Control vectors encoded mutated and catalytically inactive forms of the ribozymes or a 16 base antisense version of the target sequence. When stably expressed in cells, both ribozymes and passive control RNAs reduced viral yields. A ribozyme-mediated effect on viral growth was, however, observed, as both ribozyme types reduced viral titers by approximately 80%, well above the highest inhibition level of approximately 35% found when noncatalytic RNAs were expressed. In addition, levels of NP mRNA were generally lower in cells expressing catalytic RNAs, supporting the observed inhibition of viral growth. Although cleavage in vitro of a synthetic analog of the NP mRNA was demonstrated using RNAs isolated from ribozyme-expressing cells, in vivo cleavage products were not detectable despite the use of sensitive methods, possibly because of degradation phenomena. We also suggest here that additional controls should be conducted when semicompetitive RT-PCR methods are used to evaluate intracellular cleavage by ribozymes, as the results may depend on the initial target RNA concentration.
Albuquerque-Silva
J
J
Applied Genetics, University of Brussels, Belgium.
Milican
F
F
Bollen
A
A
Houard
S
S
D000818:Animals; D001483:Base Sequence; D002522:Chlorocebus aethiops; D017931:DNA Primers; D009109:Mumps virus / Q000187:drug effects* / Q000235:genetics; D019590:Nucleocapsid Proteins / Q000235:genetics*; D016337:RNA, Catalytic / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D014709:Vero Cells
D017931:DNA Primers; D019590:Nucleocapsid Proteins; D016337:RNA, Catalytic; D012333:RNA, Messenger