Determination of the secondary structure of minus strong-stop DNA and the mechanism of annealing involved in the first strand transfer in HIV-1

• Main Author: Chen, Yingying
• Language: ENG
• Published: École normale supérieure de Cachan - ENS Cachan 2012
• Subjects: [SDV:SA] Life Sciences/Agricultural sciences; [SDV:SA] Sciences du Vivant/Sciences agricoles; Strong-stop DNA; First strand transfer; Hiv-1; Reverse transcription; HIV-1 nucleocapsid protein
• Online Access: http://tel.archives-ouvertes.fr/tel-00777238; http://tel.archives-ouvertes.fr/docs/00/77/72/38/PDF/Chen2012.pdf

The 1st strand transfer, a crucial step of reverse transcription involving the HIV-1 nucleocapsid protein (NC), relies on base pairing of the r sequence of strong-stop DNA (ssDNA) with the 3' R sequence of viral RNA (3' UTR) which forms the TAR and polyA stem-loops. The r sequence can form the cTAR and cpolyA stem-loops. Therefore, the transfer relies probably on annealing of folded molecules. This process is not well known at the molecular and structural level. The tools of molecular biology and three DNA-targeted probes were used to get insights into the annealing process. Our results were the following: 1) in the absence of NC, the cTAR DNA folds into two distinct conformations in equilibrium; 2) NC slightly shifts the equilibrium toward one conformation and binds tightly the internal loop of the cTAR hairpin; 3) NC is required for the formation of heteroduplex of the full-length ssDNA and 3' UTR; 4) the annealing of ssDNA to 3' UTR can be initiated from different sites in the presence of 0.2 mM MgCl2; 5) the full-length ssDNA folds into two conformations in equilibrium in 0.2 mM MgCl2 but mainly into one conformation in 2 mM MgCl2 ; 6) NC preferentially binds to the single-stranded region between the cTAR and cpolyA hairpins in ssDNA. This binding site probably plays an important role in the annealing of complementary DNA and RNA hairpins. This study helps us to gain insights into the reverse transcription process and the associated genetic recombination.