Nucleotides within the anticodon stem are important for optimal use of tRNA(Lys,3) as the primer for HIV-1 reverse transcription.

HIV-1 utilizes tRNA(Lys,3) as the primer for initiation of reverse transcription. To further examine the tRNA sequence and structural requirements for primer selection, we developed a complementation system which required tRNA(Lys) to be provided in trans. We constructed an HIV-1 provirus in which the primer-binding site (PBS) was altered to be complementary to the 3' terminal 18-nucleotides of E. coli tRNA(Lys,3), which shares many bases with mammalian tRNA(Lys,3), and demonstrated that infectious virus was obtained only if the provirus was co-transfected with the plasmid encoding E. coli tRNA(Lys,3). In the current study we have mutated E. coli tRNA(Lys,3) so that nucleotides within the stem of the anticodon stem-loop were made identical to mammalian tRNA(Lys,3). Analysis of the complementation revealed that the modified E. coli tRNA(Lys,3) (E. coli tRNA(Lys,3)-MA) complemented 3-5 times more efficiently than E. coli tRNA(Lys,3). Mutation of nucleotides within the anticodon stem region of E. coli tRNA(Lys,3)-MA that differed from E. coli tRNA(Lys,3) revealed the importance of the nucleotide sequence for efficient use in reverse transcription. The results of our studies highlight that multiple regions of mammalian tRNA(Lys,3) are important for the preference of tRNA(Lys,3) as the primer for HIV-1 reverse transcription.