The annealing mechanism of HIV-1 reverse transcription primer onto the viral genome.
Reverse transcription of human immunodeficiency virus-1 viral RNA uses human tRNA(3)(Lys) 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 developed a novel approach to analyze dynamically such RNA refolding events using heteronuclear NMR spectroscopy of mixtures of (15)N-labeled and unlabeled large RNA fragments (up to 50 kDa). We have thus been able to characterize the detailed mechanisms of both heat- and nucleocapsid-mediated annealing and to identify previously unknown key steps. The role played by the nucleocapsid is 2-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 T Psi C loop, most likely by specific interactions involving its two zinc knuckles.
Tisné
Carine
C
Laboratoire de Cristallographie et Résonance Magnétique Nucléaire Biologiques, UMR 8015 CNRS, 4 avenue de l'Observatoire, 75006 Paris, France.
Roques
Bernard P
BP
Dardel
Frédéric
F
D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D016679:Genome, Viral; D054303:HIV Reverse Transcriptase / Q000378:metabolism / Q000502:physiology*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011107:Polylysine / Q000737:chemistry; D012313:RNA / Q000737:chemistry; D013696:Temperature; D015032:Zinc / Q000737:chemistry
D017931:DNA Primers; D011107:Polylysine; D012313:RNA; D054303:HIV Reverse Transcriptase; D015032:Zinc