Enhanced expression and HIV-1 inhibition of chimeric tRNA(Lys3)-ribozymes under dual U6 snRNA and tRNA promoters.
We previously demonstrated that chimeric tRNA(Lys3)-ribozymes targeting the primer binding site of HIV produced virions with reduced infectivity. To further enhance the anti-HIV efficiency of these ribozymes by increasing their level of transcription, we designed several tRNA(Lys3) promoter variants and compared their expression levels from the internal tRNA(Lys3) promoters and also from an exogenous human U6 snRNA promoter. The dual U6/tRNA promoter constructs gave rise to much higher levels of expression than constructs that used only an internal tRNA promoter. The most abundant expression is produced when a U6 promoter drives a chimeric tRNA(Lys3)-ribozyme containing a mutation in the tRNA B box. As detected by fluorescent in situ hybridization, transcripts from a construct with the tRNA promoter alone localized strictly to the cytoplasm, whereas transcripts from dual U6/tRNA promoter were present in both the cytoplasm and the nucleus. Inhibition of HIV-1 correlates well with expression levels of the chimeric constructs. The results presented demonstrate that U6 and tRNA promoters can be placed in tandem for high-level expression of small RNA therapeutic transcripts.
Chang
Zongli
Z
Division of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA.
Westaway
Shawn
S
Li
Shirley
S
Zaia
John A
JA
Rossi
John J
JJ
Scherer
Lisa J
LJ
D002460:Cell Line; D004258:DNA Polymerase III / Q000378:metabolism; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D011401:Promoter Regions, Genetic; D016337:RNA, Catalytic / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000235:genetics*; D012357:RNA, Transfer, Lys / Q000235:genetics*; D014779:Virus Replication / Q000235:genetics
D016337:RNA, Catalytic; D012342:RNA, Small Nuclear; D012357:RNA, Transfer, Lys; C405486:U6 small nuclear RNA; D004258:DNA Polymerase III