Specific Basic and Aromatic Residues Optimize the Nucleic Acid Chaperone Activity of HIV-1 Nucleocapsid Protein

Abstract

As an efficient nucleic acid chaperone protein, the human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) protein facilitates the rearrangement of nucleic acid secondary structure during reverse transcription and recombination. The biophysical basis for NC's chaperone activity includes the ability to strongly aggregate nucleic acids, destabilize nucleic acid secondary structure, and facilitate rapid protein-nucleic acid interaction kinetics. HIV-1 NC contains only 55 amino acids, with 11 basic residues throughout its structure as well as two zinc fingers, each having a single aromatic residue (F16 and W37), which is highly conserved among retroviral NC proteins. Despite its simple structure, HIV-1 NC facilitates nucleic acid rearrangement efficiently. To determine the mechanism behind this high efficiency, we performed single-molecule DNA stretching experiments in the presence of wild-type and mutant HIV-NC. We measured the ability of the proteins to aggregate and destabilize dsDNA and determined their ability to facilitate nucleic acid annealing. We show that mutant F16W and F16W-W37F exhibit similar properties as wild-type NC. In contrast, the single aromatic substitutions W37A and F16A significantly reduce NC's rapid DNA interaction kinetics, while retaining a portion of its dsDNA destabilization capability. Furthermore, removing both aromatics leads to a complete loss of chaperone activity. Thus, we can conclude that both aromatic residues significantly contribute to HIV-1 NC's optimal chaperone activity. Moreover, experiments on basic residue mutants show that positively charged residues play a very important role in protein binding. While mutating basic residues in any region of the protein significantly reduces reannealing kinetics, residues in the zinc fingers and in the N-terminus are particularly important for rapid protein-nucleic acid interaction kinetics. This work was funded in part by Federal Funds from NCI, NIH under contract N01-CO-12400 (RJG).