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) to initiate reverse transcription. A novel HIV-1 mutant which stably utilizes 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(Met) 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 alter the U5-PBS structure and disrupt the U5-PBS-tRNA(Met) interaction of the virus derived from HXB2(Met-AC). All of the mutant viruses were infectious following transfection and coculture with SupT1 cells. Analysis of the initiation of reverse transcription revealed that some of the mutants were impaired compared to HXB2(Met-AC). The genetic stability of the PBS from each virus was determined following in vitro culture. Two mutant proviral constructs, one predicted to completely disrupt the stem-loop structure in U5 and 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 mutants maintained a PBS complementary to tRNA(Met) after in vitro culture, although all contained multiple nucleotide substitutions within the U5-PBS from the starting proviral clones. Most interestingly, a viral mutant containing a 32-nucleotide deletion between nucleotides 142 and 173, encompassing regions in U5 which interact with tRNA(Met), maintained a PBS complementary to tRNA(Met) following in vitro culture. All of the proviral clones recovered from this mutant, however, contained an additional 19-nucleotide insertion in U5. RNA modeling of the U5-PBS from this mutant demonstrated that the additional mutations present in U5 following culture restored RNA structures similar to those modeled from HXB2(Met-AC). These results provide strong genetic evidence that multiple sequence and structural elements in U5 in addition to the PBS are involved in the interaction with the tRNA used for initiation of reverse transcription.