Synthetic human tRNA(UUULys3) and natural bovine tRNA(UUULys3) 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(UUULys3) were synthesized by in vitro transcription using SP6 RNA polymerase. Bovine(b) tRNA(SUULys3) 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(UUULys3) 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(UUULys3) contributes to the interaction with HIV-1 RT. Escherichia coli tRNA(UUULys) and tRNA(UUCGlu2) 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(SUULys3) and h-tRNA(UUULys3) 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(UUULys) and tRNA(UUCGlu2), 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(UUULys3) and b-tRNA(SUULys3) 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.
Boehringer Mannheim GmbH, Research Center Penzberg, F.R.G.
S
S
Weiss
B
B
König
H J
HJ
Müller
H
H
Seidel
R S
RS
Goody
D000818:Animals; D001482:Base Composition / Q000235:genetics; D001483:Base Sequence; D002417:Cattle; D004252:DNA Mutational Analysis; D004279:DNA, Viral / Q000096:biosynthesis*; D005813:Genes, Synthetic / Q000235:genetics; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D016133:Polymerase Chain Reaction; D012313:RNA / Q000378:metabolism*; D012357:RNA, Transfer, Lys / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*
D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; C034477:RNA primers; D012357:RNA, Transfer, Lys; D012367:RNA, Viral; D012313:RNA; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase