Role of the Structure of the Top Half of HIV-1 cTAR DNA on the Nucleic Acid Destabilizing Activity of the Nucleocapsid Protein NCp7.

摘要

The viral nucleic acid chaperone protein NCp7 of HIV-1 assists the two obligatory strand transfers required for the conversion of the genomic RNA into double-stranded DNA by reverse transcriptase. The first strand transfer necessitates the annealing of the early product of cDNA synthesis, the minus strand strong stop DNA (ss-cDNA) to the 3' end of the genomic RNA. The hybridization reaction involves regions containing imperfect stem-loop (SL) structures, namely the TAR RNA at the 3' end of the genomic RNA and the complementary sequence cTAR at the 3' end of ss-cDNA. To pursue the characterization of the interaction between NCp7 and cTAR DNA, we investigated by absorbance, steady-state and time-resolved fluorescence spectroscopy, the interaction of NCp7 with wild-type and mutated DNAs representing the top half of cTAR. NCp7 was found to activate the transient melting of this cTAR DNA structure but less efficiently than that of cTAR lower half. The NCp7-induced destabilization of cTAR top half is dependent upon the three nucleotides bulging out of the stem, which thus represent a melting initiation site. In contrast, despite its ability to bind NCp7, the top loop does not play any significant role in NCp7-mediated melting. Thermodynamic data further suggest that NCp7-mediated destabilization of this cTAR structure correlates with the free energy changes afforded by destabilizing motifs like loops and bulges within the SL secondary structure. Interestingly, since NCp7 melts only short double-stranded sequences, destabilizing motifs need to be regularly positioned along the genomic sequence in order to promote strand transfer and thus genetic recombination during proviral DNA synthesis.