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

J Biol Chem. 2016 Feb 12;291(7):3468-82. doi: 10.1074/jbc.M115.708099. Epub 2015 Dec 14.


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).

Keywords: DNA structure; DNA-protein interaction; human immunodeficiency virus (HIV); nucleic acid structure; reverse transcription.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Codon, Terminator*
  • 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
  • Electrophoretic Mobility Shift Assay
  • HIV-1 / metabolism*
  • Kinetics
  • Models, Molecular*
  • Molecular Weight
  • Mutation
  • Nucleic Acid Conformation
  • Nucleic Acid Hybridization
  • Nucleocapsid Proteins / chemistry*
  • Nucleocapsid Proteins / metabolism
  • Phylogeny
  • Protein Conformation
  • RNA, Viral / chemistry
  • RNA, Viral / metabolism
  • gag Gene Products, Human Immunodeficiency Virus / chemistry*
  • gag Gene Products, Human Immunodeficiency Virus / metabolism


  • Codon, Terminator
  • DNA, Recombinant
  • DNA, Single-Stranded
  • DNA, Viral
  • NCP7 protein, Human immunodeficiency virus 1
  • Nucleocapsid Proteins
  • RNA, Viral
  • gag Gene Products, Human Immunodeficiency Virus