title: Yeast tRNA(Phe) expressed in human cells can be selected by HIV-1 for use as a reverse transcription primer.
abstract: All naturally occurring human immune deficiency viruses (HIV-1) select and use tRNA(Lys,3) as the primer for reverse transcription. Studies to elucidate the mechanism of tRNA selection from the intracellular milieu have been hampered due to the difficulties in manipulating the endogenous levels of tRNA(Lys,3). We have previously described a mutant HIV-1 with a primer binding site (PBS) complementary to yeast tRNA(Phe) (psHIV-Phe) that relies on transfection of yeast tRNA(Phe) for infectivity. To more accurately recapitulate the selection process, a cDNA was designed for the intracellular expression of the yeast tRNA(Phe). Increasing amounts of the plasmid encoding tRNA(Phe) resulted in a corresponding increase in levels of yeast tRNA(Phe) in the cell. The yeast tRNA(Phe) isolated from cells transfected with the cDNA for yeast tRNA(Phe), or in the cell lines expressing yeast tRNA(Phe), were aminoacylated, indicating that the expressed yeast tRNA(Phe) was incorporated into tRNA biogenesis pathways and translation. Increasing the cytoplasmic levels of tRNA(Phe) resulted in increased encapsidation of tRNA(Phe) in viruses with a PBS complementary to tRNA(Phe) (psHIV-Phe) or tRNA(Lys,3) (wild-type HIV-1). Production of infectious psHIV-Phe was dependent on the amount of cotransfected tRNA(Phe) cDNA. Increasing amounts of plasmids encoding yeast tRNA(Phe) produced an increase of infectious psHIV-Phe that plateaued at a level lower than that from the transfection of the wild-type genome, which uses tRNA(Lys,3) as the primer for reverse transcription. Cell lines were generated that expressed yeast tRNA(Phe) at levels approximately 0.1% of that for tRNA(Lys,3). Even with this reduced level of yeast tRNA(Phe), the cell lines complemented psHIV-Phe over background levels. The results of these studies demonstrate that intracellular levels of primer tRNA can have a direct effect on HIV-1 infectivity and further support the role for PBS-tRNA complementarity in the primer selection process.
authors.affiliation: Department of Microbiology, University of Alabama at Birmingham, 35294, USA.
authors.forename: Nathan J
authors.initials: NJ
authors.lastname: Kelly
authors.forename: Casey D
authors.initials: CD
authors.lastname: Morrow
mesh_terms: D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics* / Q000378:metabolism; D005800:Genes, Fungal; D015497:HIV-1 / Q000235:genetics* / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D012313:RNA / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D012331:RNA, Fungal / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D012360:RNA, Transfer, Phe / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection; D014779:Virus Replication; D015003:Yeasts / Q000235:genetics* / Q000378:metabolism
chemical_list: D017931:DNA Primers; C034477:RNA primers; D012331:RNA, Fungal; D012360:RNA, Transfer, Phe; D012313:RNA; D012194:RNA-Directed DNA Polymerase; D000604:Amino Acyl-tRNA Synthetases