EFFECTS OF MODIFYING THE TRNA(3)(LYS) ANTICODON ON THE INITIATION OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 REVERSE TRANSCRIPTION

tRNA(3)(Lys) is a primer for reverse transcription in human immunodefi ciency virus type 1 (HIV-1), and the anticodon of tRNA(3)(Lys) has bee n implicated in playing a role in both its placement onto the HIV-1 ge nome and its interaction viith HIV-1 reverse transcriptase (RT), In th is work, the anticodon in a tRNA(3)(Lys) gene was changed from UUU to CUA (tRNA(3)(Lys)Su(+)) or, in addition, G-73 was altered to A (tRNA(3 )(Lys)Su(+)G73A). COS-7 cells were transfected with either wild-type o r mutant tRNA(3)(Lys) genes, and both the. wild-type and mutant tRNA(3 )(Lys) produced were purified by using immobilized tRNA-specific hybri dization probes. Each mutant tRNA(3)(Lys), was tested for its ability to prime reverse transcription in vitro, either alone or in competitio n with wild-type tRNA(3)(Lys), Short RT extensions of wild-type and mu tant tRNA(3)(Lys) could be distinguished from each other by their diff erent mobilities in one-dimensional single-stranded conformation polym orphism polyacrylamide gel electrophoresis, These reverse transcriptio n products show that heat-annealed tRNA(3)(Lys)Su(+) has the same abil ity as heat-annealed wild-type tRNA(3)(Lys) to prime RT and competes e qually well with wild-type tRNA(3)(Lys) for priming RT, tRNA(3)(Lys)Su (+)G73A has 60% of the wild-type ability to prime RT but competes poor ly with wild-type tRNA(3)(Lys) for priming RT, However, the priming ab ilities of wild-type and mutant tRNA(3)(Lys) are quite different when in vivo-placed tRNA is examined, HIV-1 produced in COS cells transfect ed with a plasmid containing both the HIV-1 proviral DNA and DNA codin g for tRNA(3)(Lys)Su(+) contains both endogenous, cellular wild-type t RNA(3)(Lys) and mutant tRNA(3)(Lys). When total viral RNA is used as t he source of primer tRNA placed onto the genomic RNA in vivo, only wil d-type tRNA(3)(Lys) is used as a primer. If the total viral RNA is fir st heated and exposed to hybridizing conditions, then both the wild-ty pe and mutant tRNA(3)(Lys) act as primers for RT, These results indica te that the tRNA(3)(Lys)Su(+) packaged into the virions is unable to a ct as a primer for RT, and a model is proposed to explain the disparat e results between heat-annealed and in vivo-placed primer tRNA.