Genetic analysis of a unique human immunodeficiency virus type 1 (HIV-1) with a primer binding site complementary to tRNA(Met) supports a role for U5-PBS stem-loop RNA structures in initiation of HIV-1 reverse transcription

Human immunodeficiency virus type 1 (HIV-1) exclusively uses tRNA(3)(Lys) t o initiate reverse transcription. A novel HIV-1 mutant which stably utilize s tRNA(Met) rather than tRNA(3)(Lys) as a primer was previously identified [HXB2(Met-AC] (S.-M. Kang, Z. Zhang, and C. D. Morrow, J. Virol, 71:207-217 , 1997). Comparison of RNA secondary structures of the unique sequence (U5) -primer binding site (PBS) viral RNA genome alone or complexed with tRNA(Me t) Of HXB2(Met-AC) revealed structural motifs in common with the U5-PBS of the wild-type virus. In the current study, mutations were constructed to al ter the U5-PBS structure and disrupt the U5-PBS-tRNA(Met) interaction of th e virus derived from HXB2(Met-AC), All of the mutant viruses were infectiou s following transfection and coculture with SupT1 cells, Analysis of the in itiation of reverse transcription revealed that some of the mutants were im paired compared to HXB2(Met-AC). The genetic stability of the PBS from each virus was determined following in vitro culture. Two mutant proviral const ructs, one predicted to completely disrupt the stem-loop structure in U5 an d the other predicted to destabilize contact regions of U5 with tRNA(Met), reverted back to contain a PBS complementary to tRNA(3)(Lys). All other mut ants maintained a PBS complementary to tRNAMet after in vitro culture, alth ough all contained multiple nucleotide substitutions within the U5-PBS from the starling proviral clones. Most interestingly, a viral mutant containin g a 32-nucleotide deletion between nucleotides 142 and 173, encompassing re gions in U5 which interact with tRNA(Met), maintained a PBS complementary t o tRNA(Met) following in vitro culture, All of the proviral clones recovere d from this mutant, however, contained an additional 19-nucleotide insertio n in U5. RNA modeling of the U5-PBS from this mutant demonstrated that the additional mutations present in U5 following culture restored RNA structure s similar to those modeled from HXB2(Met-AC), These results provide strong genetic evidence that multiple sequence and structural elements in U5 in ad dition to the PBS are involved in the interaction with the tRNA used for in itiation of reverse transcription.