title: Investigating the mechanism of the nucleocapsid protein chaperoning of the second strand transfer during HIV-1 DNA synthesis.
abstract: Conversion of the human immunodeficiency virus type 1 (HIV-1) genomic RNA into the proviral DNA by reverse transcriptase involves two obligatory strand transfers that are chaperoned by the nucleocapsid protein (NC). The second strand transfer relies on the annealing of the (-) and (+) copies of the primer binding site, (-)PBS and (+) PBS, which fold into complementary stem-loops (SLs) with terminal single-stranded overhangs. To understand how NC chaperones their hybridization, we investigated the annealing kinetics of fluorescently labelled (+)PBS with various (-)PBS derivatives. In the absence of NC, the (+)/(-)PBS annealing was governed by a second-order pathway nucleated mainly by the single-stranded overhangs of the two PBS SLs. The annealing reaction appeared to be rate-limited by the melting of the stable G.C-rich stem subsequent to the formation of the partially annealed intermediate. A second pathway nucleated through the loops could be detected, but was very minor. NC(11-55), which consists primarily of the zinc finger domain, increased the (-)/(+) PBS annealing kinetics by about sixfold, by strongly activating the interaction between the PBS loops. NC(11-55) also activated (-)/(+) PBS annealing through the single-strand overhangs, but by a factor of only 2. Full-length NC(1-55) further increased the (-)/(+)PBS annealing kinetics by tenfold. The NC-promoted (-)/(+)PBS mechanism proved to be similar with extended (-)DNA molecules, suggesting that it is relevant in the context of proviral DNA synthesis. These findings favour the notion that the ubiquitous role of NC in the viral life-cycle probably relies on the ability of NC to chaperone nucleic acid hybridization via different mechanisms.
authors.affiliation: Photophysique des interactions biomoléculaires, UMR 7175 CNRS, Institut Gilbert Laustriat, Faculté de Pharmacie, Université Louis Pasteur, Strasbourg 1, 74, Route du Rhin, 67401 ILLKIRCH Cedex, France.
authors.forename: Nick
authors.initials: N
authors.lastname: Ramalanjaona
authors.forename: Hugues
authors.initials: H
authors.lastname: de Rocquigny
authors.forename: Aurélie
authors.initials: A
authors.lastname: Millet
authors.forename: Damien
authors.initials: D
authors.lastname: Ficheux
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Darlix
authors.forename: Yves
authors.initials: Y
authors.lastname: Mély
mesh_terms: D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D018832:Molecular Chaperones; D009690:Nucleic Acid Conformation; D019590:Nucleocapsid Proteins / Q000502:physiology*; D048348:Reverse Transcription; D018406:Virus Physiological Phenomena
chemical_list: D018832:Molecular Chaperones; D019590:Nucleocapsid Proteins