A chimeric tRNA(Lys3)-ribozyme inhibits HIV replication following virion assembly

Co-localization of ribozymes with their appropriate target is one method utilized to increase their effectiveness in vivo. Effective antiviral ribozymes will likely rely on mechanisms which direct the ribozyme to the genomic or subgenomic RNAs. Exploiting the fact that a specific host cellular tRNA primer is bound by viral proteins and co-packaged with viral genomes in newly synthesized virions, ribozymes were fused to the 3'-terminus of tRNA(Lys3) in an attempt to direct their activity to cleave the HIV-1 genome. This chimeric ribozyme is catalytically active in vitro, and is efficiently recognized and bound by HIV-1 reverse transcriptase with affinities similar to tRNA(Lys3). The intragenic RNA polymerase III promoter entity of the tRNA allows for high levels of expression of the tRNA-RBZ and the preferential localization of transcript within the cytoplasm in transfected cells. This ribozyme was effective in reducing the infectivity of a viral stock which was produced from transiently transfected cells bearing the chimeric gene. These results demonstrate the feasibility of using tRNAs as a means of co-localizing ribozymes with their viral genomic RNA targets. The possibility exists to fuse stable RNAs to ribozymes as a means of increasing their stability and localizing them to their appropriate target sites.