Structural Insights into the HIV-1 Minus-strand Strong-stop DNA

• Published in: The Journal of biological chemistry 2016-02, Vol.291 (7), p.3468-3482
• Main Authors: Chen, Yingying, Maskri, Ouerdia, Chaminade, Françoise, René, Brigitte, Benkaroun, Jessica, Godet, Julien, Mély, Yves, Mauffret, Olivier, Fossé, Philippe
• Format: Article
• Language: English
• Subjects: Binding Sites; Codon, Terminator; DNA and Chromosomes; DNA structure; DNA, Recombinant - chemistry; DNA, Recombinant - isolation & purification; DNA, Recombinant - metabolism; DNA, Single-Stranded - chemistry; DNA, Single-Stranded - isolation & purification; DNA, Single-Stranded - metabolism; DNA, Viral - chemistry; DNA, Viral - isolation & purification; DNA, Viral - metabolism; DNA-protein interaction; Electrophoretic Mobility Shift Assay; gag Gene Products, Human Immunodeficiency Virus - chemistry; gag Gene Products, Human Immunodeficiency Virus - metabolism; HIV-1 - metabolism; human immunodeficiency virus (HIV); Human immunodeficiency virus 1; Kinetics; Lentivirus; Life Sciences; Models, Molecular; Molecular Weight; Mutation; Nucleic Acid Conformation; Nucleic Acid Hybridization; nucleic acid structure; Nucleocapsid Proteins - chemistry; Nucleocapsid Proteins - metabolism; Phylogeny; Protein Conformation; reverse transcription; RNA, Viral - chemistry; RNA, Viral - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M115.708099

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).