The annealing mechanism of HIV-1 reverse transcription primer onto the viral genome

Reverse transcription of HIV-1 viral RNA uses human tRNALys3 as a primer. The first step of viral replication is thus the annealing of the primer tRNA onto the primer-binding site located in the 5' leader region of the viral RNA. This involves large rearrangements of both RNA structures and requires the chaperone activity of the viral nucleocapsid protein. We have developped a novel approach to analyse dynamically such RNA refolding events, using heteronuclear NMR spectroscopy of mixtures of 15N-labelled and unlabelled large RNA fragments (up to 50 kDa). We have thus been able to characterise the detailed mechanisms of both heat- and nucleocapsid-mediated annealing, and to identify previously unknown key steps. The role played by the nucleocapsid is two-fold: it facilitates strand exchange at the level of the tRNA acceptor stem, presumably via its basic N- and C-terminal extensions and it unlocks the highly stable tertiary interactions at the level of the TΨC loop, most likely by specific interactions involving its two zinc knuckles