Specific Nucleic Acid Chaperone Activity of HIV-1 Nucleocapsid Protein Deduced from Hairpin Unfolding

RNA and DNA hairpin formation and disruption play key regulatory roles in a variety of cellular processes. The 59-nucleotide Transactivation Response (TAR) RNA hairpin facilitates the production of full-length transcripts of the HIV-1 genome. Yet the stability of this long, irregular hairpin becomes a liability during reverse transcription as 24 base pairs must be disrupted for strand transfer. Retroviral nucleocapsid (NC) proteins serve as nucleic acid chaperones that have been shown to both destabilize the TAR hairpin and facilitate strand annealing with its complementary DNA sequence. In contrast, during viral assembly, NC binds to RNA as a domain within the larger group-specific antigen (Gag) polyprotein, to facilitate packaging. Thus, the same domain functions with surprising differences in distinct contexts. In this work, we use optical tweezers to measure the stability of TAR alone and in the presence of Gag and NC. We find that while both Gag and NC destabilize the hairpin, only NC binding dramatically shifts the transition state. This data is matched to an energy landscape predicted from a simple theory of definite base pair destabilization. This method determines key binding sites on the TAR hairpin for each protein. Though both proteins bind to and weaken the hairpin, NC affects more sites along the upper loop compared to Gag. Destabilization of this loop shifts the transition state, increasing the natural rate of hairpin opening by ~10⁴, an effect not seen by Gag binding. Thus, Gag cleavage and NC release is an essential step critical to successful reverse transcription within the virion. These experiments illustrate a general method for measuring energy landscape transformation that yields key physical insights into multiple functions of hairpin-binding proteins.