SYNTHETIC HUMAN TRANSFER RNA(UUU)LYS3 AND NATURAL BOVINE TRANSFER RNA(SUU)LYS3 INTERACT WITH HIV-1 REVERSE-TRANSCRIPTASE AND SERVE AS SPECIFIC PRIMERS FOR RETROVIRAL CDNA SYNTHESIS

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(UUC)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.