Mechanistic Insights into the Kinetics of HIV-1 Nucleocapsid Protein-facilitated tRNA Annealing to the Primer Binding Site

Abstract

HIV-1 reverse transcriptase uses human tRNALys,3 as a primer to initiate reverse transcription. Prior to initiation, the 3′ 18 nucleotides of this tRNA are annealed to a complementary sequence on the RNA genome known as the primer binding site (PBS). Here, we show that the HIV-1 nucleocapsid protein (NC) enhances this annealing by approximately five orders of magnitude in vitro, decreasing the transition state enthalpy from approximately 20kcalmol−1 for the uncatalyzed reaction to 13kcalmol−1 for the NC-catalyzed process. Moreover, the annealing follows second-order kinetics, consistent with the nucleation of the intermolecular duplex being the rate-limiting step. This nucleation is preceded by melting of a small duplex region within the original structure, and is followed by much faster zipping of the rest of the 18 base-pair (bp) duplex. A tRNA mutational analysis shows that destabilization of the tRNA acceptor stem has only a minor effect on the annealing rate. In contrast, addition of bases to the 5′ end of tRNA that are complementary to its single-stranded 3′ end interferes with duplex nucleation and therefore has a much larger effect on the net reaction rate. Assuming that the apparent transition free energy of the annealing reaction, ΔG‡, is a sum of the melting (ΔGm) and nucleation (ΔGnuc) free energies, we show that NC affects both ΔGm and ΔGnuc. We estimate that ten to 100-fold of the overall rate enhancement is due to NC-induced destabilization of a 4 bp helix in the PBS, while the additional factor of 103–104 rate enhancement is a result of NC-facilitated duplex nucleation. The apparently similar effectiveness of wild-type and SSHS NC, a mutant that lacks the zinc finger structures, in facilitating the tRNA annealing reaction is most likely the result of the mutual cancellation of two factors: SSHS NC is less effective than wild-type NC as a duplex destabilizer, but more effective as a duplex nucleating agent.