Effective inhibition of herpes simplex virus 1 gene expression and growth by engineered RNase P ribozyme.
Using an in vitro selection procedure, we have previously isolated ribonuclease P (RNase P) ribozyme variants that efficiently cleave an mRNA sequence in vitro. In this study, an M1GS RNA variant was used to target the mRNA encoding human herpes simplex virus 1 (HSV-1) major transcription activator ICP4. The variant is about 15 times more efficient in cleaving the ICP4 mRNA sequence in vitro than the ribozyme derived from the wild type RNase P ribozyme. Moreover, the variant is also more effective in inhibiting viral ICP4 expression and growth in HSV-1-infected cells than the wild type ribozyme. A reduction of approximately 90% in the expression level of ICP4 and a reduction of 4000-fold in viral growth were observed in cells that expressed the variant. In contrast, a reduction of <10% in the ICP4 expression and viral growth was observed in cells that either did not express the ribozyme or produced a catalytically inactive ribozyme mutant. These results provide direct evidence that RNase P ribozyme variants can be highly effective in inhibiting HSV-1 gene expression and growth and furthermore, demonstrate the feasibility of developing highly effective RNase P ribozyme variants for anti-HSV applications by using in vitro selection procedures.
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Program in Infectious Diseases and Immunity, Program in Comparative Biochemistry, School of Public Health, 140 Warren Hall, University of California, Berkeley, CA 94720, USA.
Lee
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J
Kilani
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AF
Kim
J
J
Liu
F
F
D000818:Animals; D015153:Blotting, Western; D002213:Capsid / Q000378:metabolism; D036022:Capsid Proteins; D002460:Cell Line; D002522:Chlorocebus aethiops; D004722:Endoribonucleases / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D017874:Immediate-Early Proteins / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D016337:RNA, Catalytic / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D043262:Ribonuclease P; D018139:Simplexvirus / Q000235:genetics* / Q000254:growth & development; D013379:Substrate Specificity; D014709:Vero Cells; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
D036022:Capsid Proteins; C063128:ICP32-VP19c capsid protein, herpes simplex virus type 2; D017874:Immediate-Early Proteins; D016337:RNA, Catalytic; D012333:RNA, Messenger; D014764:Viral Proteins; C048925:herpes simplex virus, type 1 protein ICP4; D004722:Endoribonucleases; D043262:Ribonuclease P