title: Structural Insights into the HIV-1 Minus-strand Strong-stop DNA.
abstract: An essential step of human immunodeficiency virus type 1 (HIV-1) reverse transcription is the first strand transfer that requires base pairing of the R region at the 3'-end of the genomic RNA with the complementary r region at the 3'-end of minus-strand strong-stop DNA (ssDNA). HIV-1 nucleocapsid protein (NC) facilitates this annealing process. Determination of the ssDNA structure is needed to understand the molecular basis of NC-mediated genomic RNA-ssDNA annealing. For this purpose, we investigated ssDNA using structural probes (nucleases and potassium permanganate). This study is the first to determine the secondary structure of the full-length HIV-1 ssDNA in the absence or presence of NC. The probing data and phylogenetic analysis support the folding of ssDNA into three stem-loop structures and the presence of four high-affinity binding sites for NC. Our results support a model for the NC-mediated annealing process in which the preferential binding of NC to four sites triggers unfolding of the three-dimensional structure of ssDNA, thus facilitating interaction of the r sequence of ssDNA with the R sequence of the genomic RNA. In addition, using gel retardation assays and ssDNA mutants, we show that the NC-mediated annealing process does not rely on a single pathway (zipper intermediate or kissing complex).
authors.affiliation: From the LBPA, ENS Cachan, CNRS, Université Paris-Saclay, 61 avenue du Président Wilson, 94235 Cachan cedex, France, the School of Life Sciences, East China Normal University, 500 Dongchuan Road, Shanghai 200241, China, and.
authors.forename: Yingying
authors.initials: Y
authors.lastname: Chen
authors.affiliation: From the LBPA, ENS Cachan, CNRS, Université Paris-Saclay, 61 avenue du Président Wilson, 94235 Cachan cedex, France.
authors.forename: Ouerdia
authors.initials: O
authors.lastname: Maskri
authors.affiliation: From the LBPA, ENS Cachan, CNRS, Université Paris-Saclay, 61 avenue du Président Wilson, 94235 Cachan cedex, France.
authors.forename: Françoise
authors.initials: F
authors.lastname: Chaminade
authors.affiliation: From the LBPA, ENS Cachan, CNRS, Université Paris-Saclay, 61 avenue du Président Wilson, 94235 Cachan cedex, France.
authors.forename: Brigitte
authors.initials: B
authors.lastname: René
authors.affiliation: From the LBPA, ENS Cachan, CNRS, Université Paris-Saclay, 61 avenue du Président Wilson, 94235 Cachan cedex, France.
authors.forename: Jessica
authors.initials: J
authors.lastname: Benkaroun
authors.affiliation: the Laboratoire de Biophotonique et Pharmacologie, UMR-CNRS 7213, Faculté de Pharmacie, Université de Strasbourg, 74 route du Rhin, 67401 Illkirch-Cedex, France.
authors.forename: Julien
authors.initials: J
authors.lastname: Godet
authors.affiliation: the Laboratoire de Biophotonique et Pharmacologie, UMR-CNRS 7213, Faculté de Pharmacie, Université de Strasbourg, 74 route du Rhin, 67401 Illkirch-Cedex, France.
authors.forename: Yves
authors.initials: Y
authors.lastname: Mély
authors.affiliation: From the LBPA, ENS Cachan, CNRS, Université Paris-Saclay, 61 avenue du Président Wilson, 94235 Cachan cedex, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Mauffret
authors.affiliation: From the LBPA, ENS Cachan, CNRS, Université Paris-Saclay, 61 avenue du Président Wilson, 94235 Cachan cedex, France, fosse@ens-cachan.fr.
authors.forename: Philippe
authors.initials: P
authors.lastname: Fossé
mesh_terms: D001665:Binding Sites; D018388:Codon, Terminator; D004274:DNA, Recombinant / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D004279:DNA, Viral / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D015497:HIV-1 / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008970:Molecular Weight; D009154:Mutation; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D019590:Nucleocapsid Proteins / Q000737:chemistry* / Q000378:metabolism; D010802:Phylogeny; D011487:Protein Conformation; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000737:chemistry* / Q000378:metabolism
chemical_list: D018388:Codon, Terminator; D004274:DNA, Recombinant; D004277:DNA, Single-Stranded; D004279:DNA, Viral; C071884:NCP7 protein, Human immunodeficiency virus 1; D019590:Nucleocapsid Proteins; D012367:RNA, Viral; D054301:gag Gene Products, Human Immunodeficiency Virus
keywords: DNA structure; DNA-protein interaction; human immunodeficiency virus (HIV); nucleic acid structure; reverse transcription