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

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

Author information

1
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.
2
From the LBPA, ENS Cachan, CNRS, Université Paris-Saclay, 61 avenue du Président Wilson, 94235 Cachan cedex, France.
3
the Laboratoire de Biophotonique et Pharmacologie, UMR-CNRS 7213, Faculté de Pharmacie, Université de Strasbourg, 74 route du Rhin, 67401 Illkirch-Cedex, France.
4
From the LBPA, ENS Cachan, CNRS, Université Paris-Saclay, 61 avenue du Président Wilson, 94235 Cachan cedex, France, fosse@ens-cachan.fr.

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).

KEYWORDS:

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

PMID:
26668324
PMCID:
PMC4751388
DOI:
10.1074/jbc.M115.708099
[Indexed for MEDLINE]
Free PMC Article

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