Mutational analysis of the tRNA(3)(Lys)/HIV-1 RNA (primer/template)complex

摘要

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 tRNA(3)(Lys), We previously proposed a secondary structure model of this complex based on chemical and enzymatic probing, In this model, tRNA(3)(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 tRNA(3)(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 tRNA(3)(Lys)/mutant viral RNA complexes support the previously proposed model, Furthermore, they indicate that destroying the complementarity between the anticodon of tRNA(3)(Lys) and the so-called viral 'A-rich loop' destabilizes all four helices of the extended tRNA(3)(Lys)/HIV-1 RNA interactions.