A mechanism for plus-strand transfer enhancement by the HIV-1 nucleocapsidprotein during reverse transcription

The HIV-1 nucleocapsid protein (NC) functions as a nucleic acid chaperone d uring the plus-strand transfer step in reverse transcription by facilitatin g annealing of the primer binding site (PBS) sequence in the short plus-str and strong-stop DNA fragment [(+) SSDNA] to a complementary site located ne ar the 3' end of the minus-strand DNA [(-) PBS DNA]. To investigate the mec hanism by which NC performs this function, we have prepared an 1s-nucleotid e (-) PBS DNA for nuclear magnetic resonance (NMR) based structural and NC binding studies. The (-) PBS DNA forms a stable hairpin (T-m similar to 42 +/- 5 degrees C) that contains a five-residue loop and a bulged thymine in a guanosine-cytosine-rich stem. Addition of substoichiometric amounts of NC results in significant broadening and reductions in NMR signal intensities of the Watson-Crick base-paired imino protons and a reduction by 20 degree s C in the upper temperature at which the imino proton signals are detectab le, consistent with destabilization of the structure. The results suggest t hat inefficient annealing in the absence of NC may be due to the intrinsic stability of an internal (-) PBS DNA hairpin and that NC facilitates strand transfer by destabilizing the hairpin and exposing stem nucleotides for ba se pairing with the PBS sequence in (+) SSDNA.