Mutational analysis of the tRNA3Lys/HIV-1 RNA (primer/template) complex

Abstract

Retroviruses use a specific tRNA, whose 3′ end is complementary to the 18 nucleotides of the primer binding site (PBS), to prime reverse transcription. Previous work has shown that initiation of HIV-1 reverse transcription is a specific process, in contrast with the subsequent elongation phase. HIV-1 reverse transcriptase (RT) specifically recognizes the complex formed by the viral RNA and tRNA₃^Lys. We previously proposed a secondary structure model of this complex based on chemical and enzymatic probing. In this model, tRNA₃^Lys extensively interacts with the genomic RNA. Here, we have combined site-directed mutagenesis and structural probing to test crucial aspects of this model. We found that the complex interactions between tRNA₃^Lys and HIV-1 RNA, and the intra-molecular rearrangements did not depend on the presence of upstream and downstream viral sequences. Indeed, a short RNA template, encompassing nucleotides 123–217 of the HIV-1 Mal genome, was able, together with the primer tRNA, to adopt the same structure as longer viral RNA fragments. This model primer/template is thus amenable to detailed structural and functional studies. The probing data obtained on the tRNA₃^Lys/mutant viral RNA complexes support the previously proposed model. Furthermore, they indicate that destroying the complementarity between the anticodon of tRNA₃^Lys and the so-called viral ‘A-rich loop’ destabilizes all four helices of the extended tRNA₃^Lys/HIV-1 RNA interactions.