Poster abstracts

Poster number 42 submitted by Mithun Mitra

Probing the Role of Functional Domains of HIV-1 Nucleocapsid Protein in its Nucleic Acid Chaperone Function

Mithun Mitra (Chemistry, The Ohio State University), Daniel G Mullen (Chemistry, University of Minnesota), George Barany (Chemistry, University of Minnesota), Karin Musier-Forsyth (Chemistry, The Ohio State University)

Abstract:
The nucleocapsid protein from human immunodeficiency virus type-1 (HIV-1) functions as a nucleic acid chaperone protein by facilitating nucleic acid conformational changes to form the most thermodynamically stable arrangement. The nucleic acid chaperone activity of NC depends on its nucleic acid aggregating ability and weak duplex destabilizing activity. During the minus-strand transfer step of reverse transcription, NC facilitates the annealing of highly structured transactivation response region (TAR) RNA to the complementary TAR DNA. In this work, the role of different functional domains of NC in facilitating 59-nt TAR RNA/DNA annealing was probed by employing chemically synthesized peptides derived from full-length (55 amino acids) HIV-1 NC: NC(1-14), NC(15-35), NC(36-55), NC(1-35) and NC(29-55). All of these peptides, except NC(1-35), displayed significantly reduced annealing kinetics even when present at ~50-fold higher concentration than WT. The truncated NC constructs generally bind more weakly to single-stranded oligonucleotides, consistent with the loss of either electrostatic or hydrophobic contacts. In addition, the binding of the NC constructs was found to be slightly modulated by zinc-coordination to the CCHC motifs. Interestingly, NC(1-35), displayed annealing kinetics equal to that of full-length NC. NC(1-35) also displayed nucleic acid binding and aggregation activity that was indistinguishable from that of WT NC. Thus, we conclude that the N-terminal zinc finger flanked by the N-terminus and linker domains represents the minimal sequence necessary and sufficient for chaperone function. In contrast, a 14-residue peptide derived from the highly basic N-terminal domain of NC, which is known to be the major contributor to the nucleic acid aggregating activity of NC, was unable to effectively aggregate nucleic acids. Moreover, addition of NC(1-14) to NC(15-35) in trans does not stimulate aggregation or annealing activity of either construct. Therefore, covalent continuity of the N-terminal 35 amino acids of NC is critical for chaperone function.

Keywords: HIV-1, Nucleocapsid, Chaperone