| The nucleocapsid (NC) protein from human immunodeficiency virus type-1 (HIV-1) functions as a nucleic acid chaperone protein and accelerates the annealing of 59-nt transactivation response region (TAR) RNA to the complementary TAR DNA during the minus-strand transfer step of reverse transcription. The present work is focused on probing the role of basic and aromatic residues of NC in mediating TAR RNA/DNA annealing. Alanine scanning mutagenesis studies show that residues K11, K26 and K41 are less important than K20 in facilitating the annealing reaction. Mutations of clusters of basic residues present in the N-terminal domain, proximal zinc finger and linker domain result in a severe defect in TAR RNA/DNA annealing and nucleic acid aggregation, albeit to varying extents, indicating that the contribution of basic residues in NC's chaperone function depends upon their position. The aromatic residues of NC (F16 and W37) were also found to be important for TAR RNA/DNA annealing and nucleic acid aggregation. The basic residues in the N-terminal domain were critical for binding of NC to an RNA hairpin (18-mer) resembling the apical region of TAR RNA. This is in contrast to the binding of NC to (TG)4, where the aromatic residues provide the largest contribution to the binding affinity.; The role of the different domains of HIV-1 NC in its chaperone function was also investigated by employing chemically synthesized peptides: NC(1-14), NC(15-35), NC(36-55), NC(1-35) and NC(29-55). The results suggest that the proximal zinc finger is more important than the distal zinc finger in mediating TAR RNA/DNA annealing. NC(1-14) by itself demonstrated dramatically reduced nucleic acid annealing and aggregation capability, and the presence of NC(15-35) in trans did not increase activity. In contrast, NC(1-35) displays annealing and aggregation activity that is similar to that of WT NC. Binding of NC Fragments to a single-stranded DNA oligonucleotide was shown to be modulated by zinc coordination to NC's CCHC motifs. Strategies to probe the nucleic acid binding kinetics and duplex destabilization activities of NCs from Rous sarcoma virus (RSV), moloney murine leukemia virus (MLV) and human T-cell leukemia virus (HTLV-1) were also explored. |
