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 ΔG nuc. 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.
Bibliographical noteFunding Information:
We thank Mr Donald G. Johnson and Ms Catherine V. Hixson (both of NIH, Frederick, MD) for their assistance in preparing the recombinant NC proteins, Dr Penny Beuning and Ms Abbey Rosen for synthesis of the RNA oligonucleotides, and Drs Mark Williams (Northeastern University) and Judith G. Levin (NIH, Bethesda) for many helpful discussions and critical reading of the manuscript. This research was supported by NIH grant AI43231 (K.M.-F.) and by NIH predoctoral training grant T32 GM08277 awarded to M.R.S.H. R.J.G. acknowledges support from the National Cancer Institute, NIH, under contract number N01-CO-12400 with SAIC-Frederick.
- Chaperone activity
- HIV-1, human immunodeficiency virus type 1
- NC protein
- NC, nucleocapsid protein
- Nucleic acid annealing
- Primer tRNA
- SSHS NC, a mutant form of NC wherein all six cysteine residues have been changed to serine
- Zinc fingers