PSORALEN CROSSLINKING BETWEEN HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 RNA AND PRIMER TRNA(3)(LYS)

摘要

Initiation of reverse transcription is a crucial step of retroviral infection, In HIV-1, it involves hybridization of the 18 3'-terminal nucleotides of the primer tRNA(3)(Lys) to the primer binding site (PBS) of the viral RNA, Moreover, additional interactions between the two RNAs were recently evidenced [Isel at al, (1995) J. Mol. Biol, 247, 25269-25272], To get further information on the topology of the viral RNA/tRNA(3)(Lys) complex, we used psoralen to induce RNA-RNA crosslinking. A defined intermolecular crosslinked complex was obtained, The crosslinked regions were characterized by RNase TI digestion followed by bi-dimensional gel electrophoresis. The crosslinked residues (nucleotide mcm(5)S(2)U34 and U35 in the anticodon loop of tRNA(3)(Lys) and UCU154 in the viral RNA upstream of the PBS) were mapped using a retardation method coupled with random hydrolysis, The formation of this crosslink depends on the same elements that are required for the formation of the extended interactions between primer and template RNAs, i.e., the modified bases of the tRNA and a conserved A-rich loop located upstream of the PBS in the genomic RNA, Therefore, the present crosslinking data provide relevant information on the topology of the template/primer binary complex.