Host tRNALYs3is the primer recruited by HIV-1 for initiating reverse transcription. When ribozymes targeted upstream of the primer binding site of the HIV-1 genome are tethered to the 3' end of the tRNALYs3, it is expected that the tRNA will carry the ribozymes into the virions. We made constructs that express chimeric tRNALYs3- ribozymes from internal tRNA promoters. These chimeras were also adjoined downstream of a U6 promoter, resulting in a hybrid U6/tRNA expression cassette with extragenic U6 and intragenic tRNA promoters. Juxtaposition of the tRNA and U6 promoters significantly enhanced expression levels compared to the tRNA promoter alone. A U6/tRNA expression cassette with two mutations in B box of the tRNA moiety gave rise to the highest expression level, suggesting that the mutations reduce the steric interference of the tRNA gene with the U6 promoter. Qualitative changes in the initiation site of one minor species of transcripts were observed. These may have resulted from steric interference between the U6 and tRNA promoter elements. In an anti-HIV assay, it was found that tRNA-ribozyme chimeras could be encapsulated into HIV virions leading to reduced infectivity.
The intracellular stability and processing of the chimeric tRNALYs3-ribozymes were also studied. Point mutations in B box were found to reduce the intracellular processing of the tRNA-ribozymes in a Wit analysis of the transcripts by Northern hybridization analysis. A deletion of one guanidine residue immediately upstream of the B box led to increased stability in a processing assay using cell extract with tRNA processing activities. Nevertheless, the transcripts from these constructs can still be exported to the cytoplasm as demonstrated by fluorescence in situ hybridization and can be packaged into the virions. Constructs with these changes were also found to inhibit HIV infectivity more efficiently. Changes in the tRNA sequence that enhance transcript stability could be useful in the design of other tRNA expression cassettes for RNA-based therapeutics. The tc/t analysis proposed could be useful for screening other changes in tRNA expression cassettes for increased transcript stability.
Microbiology and Molecular Genetics
John J. Rossi
William H.R. Langridge
Barry L. Taylor
Doctor of Philosophy (PhD)
Year Degree Awarded
Date (Title Page)
Library of Congress/MESH Subject Headings
DNA-binding protein -- Physiology; Transcription, genetic; RNA, transfer, lys; RNA polymerases.
Loma Linda University Libraries
This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.
Chang, Zongli, "Intracellular Traits of Chimeric tRNAlys3-ribozymes and their Inhibition of HIV" (2000). Loma Linda University Electronic Theses, Dissertations & Projects. 621.
Loma Linda University Electronic Theses and Dissertations
Loma Linda University. Del E. Webb Memorial Library. University Archives