Efficient expression by an alphavirus replicon of a functional ribozyme targeted to human immunodeficiency virus type 1.
Intracellular applications of ribozymes have been limited partly by the availability of suitable high-expression systems. For RNA effectors, consideration of an RNA virus vector system for delivery and expression is reasonable. We show that alphavirus replicons can be highly efficient nonintegrating ribozyme-expressing vectors. Using a hammerhead ribozyme targeted to a highly conserved sequence in the U5 region of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat, we demonstrate that a full-length 8.3-kb Semliki Forest virus ribozyme (SFVRz) chimeric RNA maintains catalytic activity. SFVRz is packaged into viral particles, and these particles transduce mammalian cells efficiently. SFVRz-transduced BHK cells were found to produce large amounts of genomic and subgenomic forms of ribozyme-containing RNAs that are functional in cleaving a U5-tagged mRNA. The RNase protection assay shows that HIV-1 U5-chloramphenicol acetyltransferase mRNA expressed intracellularly from an RNA polymerase II promoter is quantitatively eliminated in SFVRz-transduced BHK cells.
Smith
S M
SM
Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892-0460, USA.
Maldarelli
F
F
Jeang
K T
KT
D000524:Alphavirus / Q000201:enzymology / Q000235:genetics; D000818:Animals; D016827:CD8 Antigens / Q000096:biosynthesis; D002384:Catalysis; D002460:Cell Line; D006224:Cricetinae; D015870:Gene Expression; D005822:Genetic Vectors; D016679:Genome, Viral; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D016337:RNA, Catalytic / Q000096:biosynthesis* / Q000235:genetics; D012093:Replicon; D012672:Semliki forest virus / Q000201:enzymology*; D013347:Subcellular Fractions
D016827:CD8 Antigens; D016337:RNA, Catalytic