Identification of a human immunodeficiency virus type 1 that stably uses tRNALys1,2 rather than tRNALys,3 for initiation of reverse transcription.

摘要

HIV-1 virions contain approximately equal amounts of tRNALys,3 and tRNALys1,2, yet tRNALys,3 has been found to be exclusively used for initiation of reverse transcription. Since previous studies have shown that even if the primer binding site (PBS) was mutated to be complementary to tRNALys1,2, the virus did not stably use tRNALys1,2 to initiate reverse transcription, the virus must have evolved a mechanism for the exclusive use of tRNALys,3 to initiate reverse transcription. To investigate how HIV-1 discriminates tRNALys1,2 from tRNALys,3 for initiation of reverse transcription, two proviral genomes that contain nucleotide changes in U5 and a PBS to be complementary to regions of tRNALys1,2 were constructed. One genome contains 5 [HXB2(L12-AC)] nucleotides while another contains 15 [HXB2(L12-ACgg)] nucleotides in U5 complementary to the anticodon region of tRNALys1,2. Viruses derived from the transfection of the proviral genomes were infectious in SupT1 cells. Analysis of the endogenous reverse transcription reactions from viruses derived from HXB2 (L12-AC) and HXB2 (L12-ACgg) obtained from transfection revealed that both exclusively used tRNALys1,2 to initiate reverse transcription. Following extensive in vitro culture, though, sequence analysis of proviral genomes revealed that while the virus derived from HXB2(L12-AC) stably maintained a PBS complementary to tRNALys1,2, the virus derived from HXB2 (L12-ACgg) had reverted back to contain a PBS complementary to tRNALys,3. RNA modeling of the U5-PBS of the genome from HXB2(L12-AC) supports the conclusion that the fine specificity for discrimination between tRNALys,3 and tRNALys1,2 for use as a primer for HIV-1 reverse transcription resides in the structure of the U5-PBS region of the viral genome. Copyright 1999 Academic Press.