Initiation of HIV-2 reverse transcription: a secondary structure model of the RNA-tRNA(Lys3) duplex

Human immunodeficiency virus type 2 (HIV-2) reverse transcription is initia ted from cellular tRNA(Lys3) partially annealed to the RNA viral genome at the primer binding site (PBS), This annealing involves interactions between two highly structured RNA molecules, In contrast to HIV-1, in which the re verse transcription initiation complex has been thoroughly studied, there i s still little information regarding a possible model to describe the secon dary structure of the template-primer complex in HIV-2. To determine whethe r HIV-2 RNA sequences flanking the PBS may specifically interact with the n atural primer tRNA, we performed site-directed mutagenesis and enzymatic fo otprinting, An RNA fragment corresponding to the HIV-2 U5 RNA domain and tR NA(Lys3) were probed either in their free form or in the binary complex. Im portant reactivity changes to nucleases were obtained upon complex formatio n. In addition to the canonical contacts between the viral PBS and the 3 ' end acceptor stem of tRNA(Lys3), we identified two additional interacting d omains: (i) the U-rich region of the anticodon loop with the A-rich sequenc e of the internal loop within the U5-prePBS region; (ii) nucleotides 48-54 from the T PsiC domain of tRNA(Lys3) and the 240-247 region of viral U5-RNA , In view of these experimental data and sequence comparison between differ ent HIV-2 isolates, we propose a model for the secondary structure of the H IV-2 template-primer initiation complex.