Human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein (NC) is a nucleic acid chaperone that facilitates the remodeling of nucleic acids during various steps of the viral life cycle. Two main features of NC's chaperone activity are its abilities to aggregate and to destabilize nucleic acids. These functions are associated with NC's highly basic character and with its zinc finger domains, respectively. While the chaperone activity of HIV-1 NC has been extensively studied, less is known about the chaperone activities of other retroviral NCs. In this work, complementary experimental approaches were used to characterize and compare the chaperone activities of NC proteins from four different retroviruses: HIV-1, Moloney murine leukemia virus (MLV), Rous sarcoma virus (RSV), and human T-cell lymphotropic virus type 1 (HTLV-1). The different NCs exhibited significant differences in their overall chaperone activities, as demonstrated by gel shift annealing assays, decreasing in the order HIV-1 ϳ RSV > MLV Ͼ Ͼ HTLV-1. In addition, whereas HIV-1, RSV, and MLV NCs are effective aggregating agents, HTLV-1 NC, which exhibits poor overall chaperone activity, is unable to aggregate nucleic acids. Measurements of equilibrium binding to single-and double-stranded oligonucleotides suggested that all four NC proteins have moderate duplex destabilization capabilities. Single-molecule DNAstretching studies revealed striking differences in the kinetics of nucleic acid dissociation between the NC proteins, showing excellent correlation between nucleic acid dissociation kinetics and overall chaperone activity.

Retroviral nucleocapsid (NC) proteins are molecular chaperones that facilitate nucleic acid (NA) remodeling events critical in viral replication processes such as reverse transcription. Surprisingly, the NC protein from human T-cell leukemia virus type 1 (HTLV-1) is an extremely poor NA chaperone. Using bulk and single molecule methods, we find that removal of the anionic C-terminal domain (CTD) of HTLV-1 NC results in a protein with chaperone properties comparable with that of other retroviral NCs. Increasing the ionic strength of the solution also improves the chaperone activity of full-length HTLV-1 NC. To determine how the CTD negatively modulates the chaperone activity of HTLV-1 NC, we quantified the thermodynamics and kinetics of wild-type and mutant HTLV-1 NC/NA interactions. The wild-type protein exhibits very slow dissociation kinetics, and removal of the CTD or mutations that eliminate acidic residues dramatically increase the protein/DNA interaction kinetics. Taken together, these results suggest that the anionic CTD interacts with the cationic N-terminal domain intramolecularly when HTLV-1 NC is not bound to nucleic acids, and similar interactions occur between neighboring molecules when NC is NA-bound. The intramolecular N-terminal domain-CTD attraction slows down the association of the HTLV-1 NC with NA, whereas the intermolecular interaction leads to multimerization of HTLV-1 NC on the NA. The latter inhibits both NA/NC aggregation and rapid protein dissociation from singlestranded DNA. These features make HTLV-1 NC a poor NA chaperone, despite its robust duplex destabilizing capability. Nucleic acid (NA)5 chaperones are proteins that facilitate NA remodeling and annealing (1). Retroviral nucleocapsid proteins (NC) are essential NA chaperones (2-4) that facilitate many steps in the retroviral life cycle, including dimerization of the RNA genome (5-10), reverse transcription (11-14), annealing of the tRNA primer to the primer-binding site (7,(15)(16)(17)(18)(19)(20)(21)(22), and integration of viral DNA into the host genome (23-27). Previous work has shown that NCs from different retroviruses display a wide range of NA chaperone activities (28). A modelannealing reaction involving complementary trans-activation response element (TAR) RNA and DNA hairpins derived from the R region of the HIV-1 genome was used to characterize NCs from human immunodeficiency virus, type 1 (HIV-1), Rous sarcoma virus, murine leukemia virus, and human T-cell leukemia virus, type 1 (HTLV-1) (28). Surprisingly, the annealing activity of these NCs varies by 5 orders of magnitude; HIV-1 NC was the most efficient chaperone and HTLV-1 NC was the least efficient. A single molecule (SM) Förster resonance energy transfer (FRET) study confirmed the observation that HTLV-1 NC is a very poor NA chaperone (29).To determine the physical origin of these differences in retroviral NC chaperone activity, we measured the NA aggregation and duplex destabilization activity for each protein (28). Both of these capabilities, together with rap...

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deviations from single exponential relaxation, indicating two distinct phases. Of particular interest is the observation that the rapid phase has a rate that is 10-20 times faster than the bending rate observed in the IHF-H' complex. Thus, reducing the energetic cost of bending/kinking DNA speeds up the bending rate by nearly the same factor as the increase in binding affinity, indicating that the free energy of the transition state is lowered by the same amount as the free energy of the complex. These results support our earlier conclusion, that spontaneous bending of DNA is the first step in the recognition mechanism.