HIV-1 NC Protein Modifies Opening and Closing Kinetics of TAR RNA Hairpin

Abstract

Retroviral nucleocapsid (NC) proteins are nucleic acid chaperones that play a key role in the viral life cycle, including reverse transcription, where NC destabilizes the transactivation response RNA (TAR RNA) hairpin. To quantify the interaction of HIV-1 NC and TAR RNA, we used optical tweezers to exert tension upon the free ends of the individual TAR hairpin, forcing the hairpin open and then allowing it to close. In the absence of NC, the TAR hairpin opens and closes at forces that depend upon the hairpin stability, as well as the rate of pulling and relaxation. Surprisingly, once saturated with NC, the opening forces increase, suggesting an apparent stabilization of the structure. However, the higher opening rate in the presence of NC is only observed at high pulling rates, while extrapolation of the measured TAR opening rate vs pulling rate to infinitely slow pulling yields ∼1000-fold faster opening with NC. This ability of NC to facilitate opening of the TAR hairpin is equivalent to ∼4 kcal/mol decrease in the TAR opening barrier. The stronger pulling rate dependence of TAR opening in the presence of NC indicates that fewer TAR base pairs unzip prior to complete hairpin stem destabilization. Specifically, of the 24 TAR hairpin stem base pairs it is sufficient to unzip 12 bp in the absence of but only 6 bp in the presence of NC to cause complete TAR unfolding. This result quantitatively characterizes the ability of NC to moderately destabilize every nucleic acid base pair.