HIV-1 Nucleocapsid Protein Binds to the Viral DNA Initiation Sequences and Chaperones their Kissing Interactions

The chaperone properties of the human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein (NC) are required for the two obligatory strand transfer reactions occurring during viral DNA synthesis. The second strand transfer relies on the destabilization and the subsequent annealing of the primer binding site sequences (PBS) at the 3′ end of the (−) and (+) DNA strands. To characterize the binding and chaperone properties of NC on the (−)PBS and (+)PBS sequences, we monitored by steady-state and time-resolved fluorescence spectroscopy as well as by fluorescence correlation spectroscopy the interaction of NC with wild type and mutant oligonucleotides corresponding to the (−)PBS and (+)PBS hairpins. NC was found to bind with high affinity to the loop, the stem and the single-stranded protruding sequence of both PBS sequences. NC induces only a limited destabilization of the secondary structure of both sequences, activating the transient melting of the stem only during its “breathing” period. This probably results from the high stability of the PBS due to the four G–C pairs in the stem. In contrast, NC directs the formation of “kissing” homodimers efficiently for both (−)PBS and (+)PBS sequences. Salt-induced dimerization and mutations in the (−)PBS sequence suggest that these homodimers may be stabilized by two intermolecular G–C Watson–Crick base-pairs between the partly self-complementary loops. The propensity of NC to promote the dimerization of partly complementary sequences may favor secondary contacts between viral sequences and thus, recombination and viral diversity.