Structural Insights into the HIV-1 Minus-strand Strong-stop DNA.
Fiche publication
Date publication
février 2016
Journal
The Journal of biological chemistry
Auteurs
Membres identifiés du Cancéropôle Est :
Pr MELY Yves
Tous les auteurs :
Chen Y, Maskri O, Chaminade F, René B, Benkaroun J, Godet J, Mély Y, Mauffret O, Fossé P
Lien Pubmed
Résumé
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).
Mots clés
Binding Sites, Codon, Terminator, DNA, Recombinant, chemistry, DNA, Single-Stranded, chemistry, DNA, Viral, chemistry, Electrophoretic Mobility Shift Assay, HIV-1, metabolism, Kinetics, Models, Molecular, Molecular Weight, Mutation, Nucleic Acid Conformation, Nucleic Acid Hybridization, Nucleocapsid Proteins, chemistry, Phylogeny, Protein Conformation, RNA, Viral, chemistry, gag Gene Products, Human Immunodeficiency Virus, chemistry
Référence
J. Biol. Chem.. 2016 Feb;291(7):3468-82