Preferential completion of human immunodeficiency virus type 1 proviruses initiated with tRNA3Lys rather than tRNA1,2Lys.
All retroviral genomes contain a nucleotide sequence designated as the primer binding site (PBS) which is complementary to the tRNA used for initiation of reverse transcription. For human immunodeficiency virus type 1 (HIV-1), all naturally occurring genomes have a PBS complementary to tRNA3Lys. However, within HIV-1 virions, there are approximately equal amounts of tRNA1Lys, tRNA2Lys, and tRNA3Lys. We have used an endogenous reverse transcription-PCR technique specific for the tRNA species within isolated HIV-1 virions to demonstrate that in addition to tRNA3Lys, tRNA1Lys and tRNA2Lys could be used for initiation of HIV-1 reverse transcription. Using a single-round infection assay which employed an HIV-1 genome with a gpt gene encoding xanthine-guanine phosphoribosyl transferase in place of the env gene, we generated cell lines resistant to mycophenolic acid. Analysis of the U5-PBS from single-cell clones revealed PBS complementary to tRNA3Lys, not tRNA1Lys or tRNA2Lys. A mutant HIV-1 genome was then created which would favor the completion of reverse transcription with tRNA1,2Lys. Using this provirus in the complementation system, we again found only genomes with a PBS complementary to tRNA3Lys from proviral DNA isolated from gpt-resistant single-cell colonies. Finally, infection of cells with a mutant HIV genome with a PBS complementary to tRNA1,2Lys resulted in gpt- resistant cell colonies which contained integrated provirions with a PBS complementary to tRNA1,2Lys. The results of these studies suggest that the selection of tRNA3Lys for initiation of HIV-1 reverse transcription occurs both at the initiation and at a postinitiation step in reverse transcription prior to integration of the proviral DNA.
Zhang
Z
Z
Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
Yu
Q
Q
Kang
S M
SM
Buescher
J
J
Morrow
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CD
D001483:Base Sequence; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D011533:Proviruses / Q000235:genetics*; D012357:RNA, Transfer, Lys / Q000378:metabolism*; D014158:Transcription, Genetic
D012357:RNA, Transfer, Lys