INITIATION OF REVERSE TRANSCRIPTION OF HIV-1 - SECONDARY STRUCTURE OFTHE HIV-1 RNA TRNA(3)(LYS) (TEMPLATE/PRIMER) COMPLEX/

Reverse transcription of human immunodeficiency virus type-1 (HTV-1) g enomic RNA is primed by tRNA(3)(Lys), whose 3' end 18 nucleotides are complementary to the viral primer binding site (PBS). We used chemical and enzymatic probes to test the conformation of the viral RNA and tR NA(3)(Lys), in their free form and in the HIV-1 RNA/tRNA(3)(Lys) binar y complex. Extensive reactivity changes were observed in both molecule s upon formation of the binary complex. In the viral RNA, reactivity c hanges occurred up to 69 nucleotides upstream and 72 nucleotides downs tream of the PBS. A secondary structure model of the HIV-1 RNA/tRNA(3) (Lys) complex accounting for all probing data has been constructed. It reveals an unexpectedly complex and compact pseudoknot-like structure in which most of the anticodon loop, the 3' strand of the anticodon s tem and the 5' part of the variable loop of tRNA(3)(Lys) interact with viral sequences 12 to 39 nucleotides upstream of the PBS. The core of the binary complex is a complex junction formed by two single-strande d sequences of tRNA(3)(Lys), intramolecular viral helix, an intramolec ular tRNA helix, and two intermolecular helices formed by the template /primer interaction. This junction probably highly constrains the tert iary structure of the HIV-1 RNA/tRNA(3)(Lys) complex. Compared to the structure of the free molecules, only the D arm of tRNA(3)(Lys) and sm all viral stem-loop downstream of the PBS are unaffected in the binary complex. Sequence comparison reveals that the main characteristics of the binary complex model are conserved among all HIV-1 isolates.