Synthetic human tRNA uuuLys3 and natural bovine tRNA suuLys3 interact with HIV-1 reverse transcriptase and serve as specific primers for retroviral cDNA synthesis

Abstract

Full-length and 5′-truncated variants of human (h) tRNA uuu Lys3 were synthesized by in vitro transcription using SP6 RNA polymerase. Bovine(b) tRNA suu Lys3 was purified from calf liver. Both full-length tRNA species were shown to be biologically active in an aminoacylation assay. Gel retardation assays revealed that both full-length tRNA species, as well as a 5′-truncated h-tRNA uuu Lys3 molecule containing 24 nucleotides (nt at the 3′ end (Lys24), interact with human immunodeficiency virus (HIV)-1 reverse transcriptase (RT). Competition studies with these three tRNA species demonstrate that the 3′ end of h-tRNA uuu Lys3 contributes to the interaction with HIV-1 RT. Escherichia coli tRNA uuu Lys and tRNA uuu Glu2 were also able to interact with the enzyme, whereas unrelated RNA molecules such as E. coli 5S rRNA did not bind to RT. Both b-tRNA suu Lys3 and h-tRNA uuu Lys3 molecules, as well as the 5′-truncated variants, could be demonstrated to prime cDNA synthesis specifically using a HIV-1 RNA template, prepared by in vitro transcription, indicating that other viral or cellular proteins are not essential for this process. E. coli tRNA uuu Lys and tRNA uuc Glu2, although able to interact with HIV-1 RT, failed to prime retroviral transcription. Products of cDNA synthesis were characterized by polymerase chain reaction, demonstrating that at least 18 nt at the 3′ ends of h-tRNA uuu Lys3 and b-tRNA suu Lys3 are still present in the cDNA product, whereas the 5′ ends of both primer molecules were removed by the RNase H activity of HIV-1 RT.