PubMed2005
Reverse transcription of HIV-1 genomic RNA requires two obligatory strand transfers. During the first strand transfer reaction, the minus strand strong-stop DNA (ss-cDNA) is transferred by hybridization of complementary sequences located at the 3' ends of the ss-cDNA and genomic template, respectively. In HIV-1, the major components of ss-cDNA transfer are the terminally redundant structured TAR elements and the nucleocapsid protein NCp7, which actively chaperones the hybridization of cTAR DNA to TAR. In the present study, we investigated the annealing kinetics of TAR with fluorescently labelled cTAR derivatives both in the absence and in the presence of NC(12-55), a peptide that contains the finger and C-terminal domains of NCp7. The annealing of TAR with cTAR involves two second-order kinetic components that are activated by at least two orders of magnitude by NC(12-55). The NC-promoted activation of cTAR-TAR annealing was correlated with its ability to destabilize the lower half of TAR stem, in order to generate the single-stranded complementary regions for nucleating the duplex structures. The two kinetics components have been assigned to two different pathways. The rapid one does not lead to extended duplex formation but is associated with a limited annealing of the terminal bases of cTAR to TAR. On the other hand, extended duplex formation follows a slower pathway that is limited kinetically by the nucleation of residues located mainly within the central double-stranded segment of both cTAR and TAR stems. An alternative mechanism involving an interaction through TAR and cTAR loops has been observed but is a minor pathway in the present conditions.