title: HIV-1 Pr55Gagbinds genomic and spliced RNAs with different affinity and stoichiometry.
abstract: The HIV-1 Pr55Gagprecursor specifically selects genomic RNA (gRNA) from a large variety of cellular and spliced viral RNAs (svRNAs), however the molecular mechanisms of this selective recognition remains poorly understood. To gain better understanding of this process, we analyzed the interactions between Pr55Gagand a large panel of viral RNA (vRNA) fragments encompassing the main packaging signal (Psi) and its flanking regions by fluorescence spectroscopy. We showed that the gRNA harbors a high affinity binding site which is absent from svRNA species, suggesting that this site might be crucial for selecting the HIV-1 genome. Our stoichiometry analysis of protein/RNA complexes revealed that few copies of Pr55Gagspecifically associate with the 5' region of the gRNA. Besides, we found that gRNA dimerization significantly impacts Pr55Gagbinding, and we confirmed that the internal loop of stem-loop 1 (SL1) in Psi is crucial for specific interaction with Pr55Gag. Our analysis of gRNA fragments of different length supports the existence of a long-range tertiary interaction involving sequences upstream and downstream of the Psi region. This long-range interaction might promote optimal exposure of SL1 for efficient Pr55Gagrecognition. Altogether, our results shed light on the molecular mechanisms allowing the specific selection of gRNA by Pr55Gagamong a variety of svRNAs, all harboring SL1 in their first common exon.
authors.affiliation: a Université de Strasbourg, CNRS, Architecture et Réactivité de l'ARN , Strasbourg , France.
authors.forename: Serena
authors.initials: S
authors.lastname: Bernacchi
authors.affiliation: a Université de Strasbourg, CNRS, Architecture et Réactivité de l'ARN , Strasbourg , France.
authors.forename: Ekram W
authors.initials: EW
authors.lastname: Abd El-Wahab
authors.affiliation: a Université de Strasbourg, CNRS, Architecture et Réactivité de l'ARN , Strasbourg , France.
authors.forename: Noé
authors.initials: N
authors.lastname: Dubois
authors.affiliation: b Centre for Virology, Burnet Institute , Melbourne , Victoria , Australia.|c Department of Biochemistry and Molecular Biology , Monash University , Clayton , Victoria , Australia.
authors.forename: Marcel
authors.initials: M
authors.lastname: Hijnen
authors.affiliation: a Université de Strasbourg, CNRS, Architecture et Réactivité de l'ARN , Strasbourg , France.
authors.forename: Redmond P
authors.initials: RP
authors.lastname: Smyth
authors.affiliation: b Centre for Virology, Burnet Institute , Melbourne , Victoria , Australia.|c Department of Biochemistry and Molecular Biology , Monash University , Clayton , Victoria , Australia.|d School of Medicine, Faculty of Health, Deakin University , Geelong , Victoria , Australia.|e Commonwealth Scientific and Industrial Research Organization, Livestock Industries, Australian Animal Health Laboratory , Geelong , Victoria , Australia.
authors.forename: Johnson
authors.initials: J
authors.lastname: Mak
authors.affiliation: a Université de Strasbourg, CNRS, Architecture et Réactivité de l'ARN , Strasbourg , France.
authors.forename: Roland
authors.initials: R
authors.lastname: Marquet
authors.affiliation: a Université de Strasbourg, CNRS, Architecture et Réactivité de l'ARN , Strasbourg , France.
authors.forename: Jean-Christophe
authors.initials: JC
authors.lastname: Paillart
mesh_terms: D001665:Binding Sites; D016679:Genome, Viral; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D055029:Inverted Repeat Sequences; D007700:Kinetics; D009154:Mutation; D009690:Nucleic Acid Conformation; D011498:Protein Precursors / Q000378:metabolism*; D012326:RNA Splicing; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism
chemical_list: D011498:Protein Precursors; D012367:RNA, Viral; D016601:RNA-Binding Proteins; C059767:p55 gag precursor protein, Human immunodeficiency virus 1
keywords: Fluorescence spectroscopy; HIV-1; Pr55Gag; genomic RNA selection; high affinity binding site; protein-RNA interaction; stoichiometry