Effects of modifying the tRNA(3Lys) anticodon on the initiation of human immunodeficiency virus type 1 reverse transcription.
tRNA(3Lys) is a primer for reverse transcription in human immunodeficiency virus type 1 (HIV-1), and the anticodon of tRNA(3Lys) has been implicated in playing a role in both its placement onto the HIV-1 genome and its interaction with HIV-1 reverse transcriptase (RT). In this work, the anticodon in a tRNA(3Lys) gene was changed from UUU to CUA (tRNA(3Lys)Su+) or, in addition, G-73 was altered to A (tRNA(3Lys)Su+G73A). COS-7 cells were transfected with either wild-type or mutant tRNA(3Lys) genes, and both the wild-type and mutant tRNA(3Lys) produced were purified by using immobilized tRNA-specific hybridization probes. Each mutant tRNA(3Lys) was tested for its ability to prime reverse transcription in vitro, either alone or in competition with wild-type tRNA(3Lys). Short RT extensions of wild-type and mutant tRNALys could be distinguished from each other by their different mobilities in one-dimensional single-stranded conformation polymorphism polyacrylamide gel electrophoresis. These reverse transcription products show that heat-annealed tRNA(3Lys)Su+ has the same ability as heat-annealed wild-type tRNA(3Lys) to prime RT and competes equally well with wild-type tRNA(3Lys) for priming RT. tRNA(3Lys)Su+G73A has 60% of the wild-type ability to prime RT but competes poorly with wild-type tRNA(3Lys) for priming RT. However, the priming abilities of wild-type and mutant tRNA(3) are quite different when in vivo-placed tRNA is examined. HIV-1 produced in COS cells transfected with a plasmid containing both the HIV-1 proviral DNA and DNA coding for tRNA(3Lys)Su+ contains both endogenous, cellular wild-type tRNA(3Lys) and mutant tRNA(3Lys). When total viral RNA is used as the source of primer tRNA placed onto the genomic RNA in vivo, only wild-type tRNA(3Lys) is used as a primer. If the total viral RNA is first heated and exposed to hybridizing conditions, then both the wild-type and mutant tRNA(3Lys) act as primers for RT. These results indicate that the tRNA(3Lys)Su+ packaged into the virions is unable to act as a primer for RT, and a model is proposed to explain the disparate results between heat-annealed and in vivo-placed primer tRNA.
Huang
Y
Y
Lady Davis Institute for Medical Research and McGill AIDS Centre, McGill University, Montreal, Quebec, Canada.
Shalom
A
A
Li
Z
Z
Wang
J
J
Mak
J
J
Wainberg
M A
MA
Kleiman
L
L
D000926:Anticodon; D001483:Base Sequence; D015342:DNA Probes; D015497:HIV-1 / Q000235:genetics* / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D012313:RNA; D012346:RNA, Transfer, Amino Acyl / Q000235:genetics* / Q000302:isolation & purification; D012367:RNA, Viral; D014158:Transcription, Genetic / Q000235:genetics*; D014771:Virion; D019065:Virus Assembly
D000926:Anticodon; D015342:DNA Probes; C034477:RNA primers; D012346:RNA, Transfer, Amino Acyl; D012367:RNA, Viral; D012313:RNA