Nucleotides within the anticodon stem are important for optimal use of tRNALys,3 as the primer for HIV-1 reverse transcription

HIV-1 utilizes tRNALys,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 tRNALys 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 tRNALys,3, which shares many bases with mammalian tRNALys,3, and demonstrated that infectious virus was obtained only if the provirus was co-transfected with the plasmid encoding E. coli tRNALys,3. In the current study we have mutated E. coli tRNALys,3 so that nucleotides within the stem of the anticodon stem–loop were made identical to mammalian tRNALys,3. Analysis of the complementation revealed that the modified E. coli tRNALys,3 (E. coli tRNALys,3-MA) complemented 3–5 times more efficiently than E. coli tRNALys,3. Mutation of nucleotides within the anticodon stem region of E. coli tRNALys,3-MA that differed from E. coli tRNALys,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 tRNALys,3 are important for the preference of tRNALys,3 as the primer for HIV-1 reverse transcription.