Dynamics of the HIV-1 reverse transcription complex during initiation of DNA synthesis

Initiation of human immunodeficiency virus-1 (HIV-1) reverse transcription requires formation of a complex containing the viral RNA (vRNA), tRNA(3)(Ly s) and reverse transcriptase (RT). The vRNA and the primer tRNA(3)(Lys) for m several intermolecular interactions in addition to annealing of the prime r 3' end to the primer binding site (PBS). These interactions are crucial f or the efficiency and the specificity of the initiation of reverse transcri ption. However, as they are located upstream of the PBS, they must unwind a s DNA synthesis proceeds. Here, the dynamics of the complex during initiati on of reverse transcription was followed by enzymatic probing. Our data rev ealed reciprocal effects of the tertiary structure of the vRNA . tRNA(3)(Ly s) complex and reverse transcriptase (RT) at a distance from the polymeriza tion site, The structure of the initiation complex allowed RT to interact w ith the template strand up to 20 nucleotides upstream from the polymerizati on site, Conversely, nucleotide addition by RT modified the tertiary struct ure of the complex at 10-14 nucleotides from the catalytic site. The viral sequences became exposed at the surface of the complex as they dissociated from the tRNA following primer extension. However, the counterpart tRNA seq uences became buried inside the complex. Surprisingly, they became exposed when mutations prevented the intermolecular interactions in the initial com plex, indicating that the fate of the tRNA depended on the tertiary structu re of the initial complex.