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

摘要

Reverse transcription of human immunodeficiency virus type-1 (HTV-1) genomic 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 tRNA(3)(Lys), in their free form and in the HIV-1 RNA/tRNA(3)(Lys) binary complex. Extensive reactivity changes were observed in both molecules upon formation of the binary complex. In the viral RNA, reactivity changes occurred up to 69 nucleotides upstream and 72 nucleotides downstream 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 stem 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-stranded sequences of tRNA(3)(Lys), intramolecular viral helix, an intramolecular tRNA helix, and two intermolecular helices formed by the template/primer interaction. This junction probably highly constrains the tertiary 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 small 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. [References: 48]