Identification of a human immunodeficiency virus type 1 that stably uses tRNA(Lys1,2) rather than tRNA(Lys,3) for initiation of reverse transcription

HIV-1 virions contain approximately equal amounts of tRNA(Lys,3) and tRNA(L ys1,2), yet tRNA(LyS,3) has been found to be exclusively used for initiatio n of reverse transcription. Since previous studies have shown that even if the primer binding site.(PBS) was mutated to be complementary to tRNA(Lys1, 2), the virus did not stably use tRNA(Lys1,2) to initiate reverse transcrip tion, the virus must have evolved a mechanism for the exclusive use of tRNA (Lys,3) to initiate reverse transcription. To investigate how HIV-1 discrim inates tRNA(Lys1,2) from tRNA(Lys,3) for initiation of reverse transcriptio n, two proviral genomes that contain nucleotide changes in U5 and a PBS to be complementary to regions of tRNA(Lys1,2) were constructed. One genome co ntains 5 [HXB2(L12-AC)] nucleotides while another contains 15 [HXB2(L12-ACg g)] nucleotides in U5 complementary to the anticodon region of tRNA(Lys1,2) . Viruses derived from the transfection of the proviral genomes were infect ious in SupT1 cells. Analysis of the endogenous reverse transcription react ions from viruses derived from HXB2 (L12-AC) and HXB2 (L12-ACgg) obtained f rom transfection revealed that both exclusively used tRNA(Lys1,2) to initia te reverse transcription. Following extensive in vitro culture, though, seq uence analysis of proviral genomes revealed that while the virus derived fr om HXB2(L12-AC) stably maintained a PBS complementary to tRNA(Lys1,2), the virus derived from HXB2 (L12-ACgg) had reverted back to contain a PBS compl ementary to tRNA(Lys,3). RNA modeling of the U5-PBS of the genome from HXB2 (L12-AC) supports the conclusion that the fine specificity for discriminati on between tRNA(LyS,3) and tRNA(Lys1,2) for use as a primer for HIV-1 rever se transcription resides in the structure of the U5-PBS region of the viral genome. (C) 1999 Academic Press.