In vitro evidence for the interaction of tRNA(3)(Lys) With U3 during the first strand transfer of HIV-1 reverse transcription

Over the course of its evolution, HIV-1 has taken maximum advantage of its tRNA(3)(Lys) primer by utilizing it in several steps of reverse transcripti on. Here, we have identified a conserved nonanucleotide sequence in the U3 region of HIV-1 RNA that is complementary to the anticodon stem of tRNA(3)( Lys). In order to test its possible role in the first strand transfer react ion, we applied an assay using a donor RNA corresponding to the 5'-part and an acceptor RNA spanning the 3'-part of HIV-1 RNA. In addition, we constru cted two acceptor RNAs in which the nonanucleotide sequence complementary t o tRNA(3)(Lys) was either substituted (S) or deleted (Delta). We used eithe r natural tRNA(3)(Lys) or an 18 nt DNA as primer and measured the efficienc y of (-) strand strong stop DNA transfer in the presence of wild-type, S or Delta acceptor RNA. Mutations in U3 did not decrease the transfer efficien cy when reverse transcription was primed with the 18mer DNA, However, they significantly reduced the strand transfer efficiency in the tRNA(3)(Lys)-pr imed reactions, This reduction was also observed in the presence of nucleoc apsid protein. These results suggest that tRNA(3)(Lys) increases (-) strand strong stop transfer by interacting with the U3 region of the genomic RNA, Sequence comparisons suggest that such long range interactions also exist in other lentiviruses.