In vitro evidence for the interaction of tRNA3Lys with U3 during the first strand transfer of HIV-1 reverse transcription

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Abstract
Over the course of its evolution, HIV-1 has taken maximum advantage of its tRNA3Lys primer by utilizing it in several steps of reverse transcription. Here, we have identified a conserved nonanucleotide sequence in the U3 region of HIV-1 RNA that is complementary to the anticodon stem of tRNA3Lys. In order to test its possible role in the first strand transfer reaction, 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 constructed two acceptor RNAs in which the nonanucleotide sequence complementary to tRNA3Lys was either substituted (S) or deleted (Δ). We used either natural tRNA3Lys or an 18 nt DNA as primer and measured the efficiency of (–) strand strong stop DNA transfer in the presence of wild-type, S or Δ acceptor RNA. Mutations in U3 did not decrease the transfer efficiency when reverse transcription was primed with the 18mer DNA. However, they significantly reduced the strand transfer efficiency in the tRNA3Lys-primed reactions. This reduction was also observed in the presence of nucleocapsid protein. These results suggest that tRNA3Lys 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.

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