Determination Of The Secondary Structure Of Minus Strong-stop DNA And The Mechanism Of Annealing Involved In The First Strand Transfer In HIV-1

The reverse transcription of the HIV-1 genome consists in a succession of steps allowing the conversion of the single stranded RNA genome in a double stranded DNA molecule. In the laboratory, we investigate the first strand transfer during which the strong stop DNA (ssDNA) migrates from the 5’end of the genome to the 3’end. Analysis of this step is important to gain insights into the reverse transcription process and the associated genetic recombination. Indeed, the strand transfer process is responsible for the recombination events that produce resistance to antiretroviral drugs that constitute a major problem in the anti-HIV-1 therapies. The main DNA and RNA sequences involved in the first strand transfer have been identified:these are the RNA sequences TAR and polyA and the complementary DNA sequences cTAR and cpolyA. The first strand transfer is facilitated by the HIV-1 nucleocapsid protein (NC). During the strand transfer, NC destabilizes the nucleic acid secondary structures and promotes their association. Nevertheless, the annealing process of the full-length ssDNA to the 3’end of the genomic RNA (3’UTR) is not known at the molecular and structural level. The main aim of my thesis is to better understand this annealing process. The tools of molecular biology and three DNA-targeted probes [potassium permanganate, DNase I and mung bean nuclease (MB)] will be use to achieve this goal.Firstly, the cTAR secondary structure was determined in the absence or in the presence of NC. Structural analysis using structural probes showed that the cTAR DNA folds into two stem-loops in equilibrium in the absence of NC. One conformation is named closed because the 5’and 3’ends of the cTAR DNA are paired, while the other conformation is named’Y’ conformation because the 5’and 3’ends of cTAR are unpaired. NC slightly destabilizes the lower stem and shifts the equilibrium toward the’Y’conformation. The MB footprinting results showed that in the presence of 7 mM MgCl2 (optimal concentration for reverse transcription and strand transfer in vitro) NC binds more strongly the internal loop than the apical loop of the cTAR hairpin. However, this preferential binding site has not been observed in the presence of 0.2 mM MgCl2 (intracellular concentration).Secondly, the annealing of the full-length wild-type ssDNA and its three mutations to the 3’UTR was investigated in the absence or in the presence of NC and in 0.2 mM and 2 mM MgCl2 (concentration required for reverse transcription and strand transfer in vitro). We have designed two full-length ssDNAs:ssDNA-L represents the ssDNA that is not annealed to the PBS region; ssDNA-S represents the ssDNA that is paired with the PBS region. We have shown that NC is required for the formation of heteroduplex of the full-length ssDNA and 3’ UTR. Our results suggest that the annealing of ssDNA to 3’UTR can be initiated from different sites in the presence of 0.2 mM MgCl2, whereas the initiation of annealing via the apical loops of TAR and cTAR hairpins plays an important role in the presence of 2 mM MgCl2.Finally, we have determined the secondary structures of the full-length ssDNA-S and ssDNA-L in the absence or in the presence of NC. Our results suggest that ssDNA folds mainly into one conformation in 2 mM MgCl2. Analysis of ssDNA by non-denaturing polyacrylamide gel electrophoresis suggests that ssDNA adopts two conformations in equilibrium in 0.2 mM MgCl2. The formation of two conformers of ssDNA may be due to the cTAR sequence that can form a long stem-loop or a short stem-loop in ssDNA. The closed and’Y’conformations of cTAR DNA are not formed in ssDNA. 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.