The highly basic 55 amino acid nucleocapsid protein (NC) that coats the HIV-1 genome has two zinc fingers that differ by five amino acids (strain pNL4-3). Previous work showed that NC's first finger (N-terminal) is primarily responsible for unwinding secondary structures (helix destabilizing activity), while the second (C-terminal) plays an accessory role. The amino acid differences between the fingers are (finger one to finger two): phenylalanine to tryptophan (F to W), asparagine to lysine (N to K), isoleucine to glutamine (I to Q), alanine to methionine (A to M), and asparagine to aspartic acid (N to D) at positions 16, 17, 24, 25, and 27 of finger one, respectively. To determine at an amino acid level the reason for the apparent distinction between the fingers, five point mutants were designed with amino acid residues in finger one incrementally replaced by those at corresponding locations in finger two. Each mutant was analyzed in annealing assays with unstructured and structured substrates. Three groupings emerged: (1) those similar to wild type (wt) levels (N17K, A25M), (2) those with diminished activity (I24Q, N27D), and (3) mutant F16W which had substantially greater helix destabilizing activity than wt NC. All mutants retained wt levels of the condensation/aggregation activity of NC. Unlike I24Q and others, N27D was defective in DNA binding. Only I24Q and N27D showed reduced strand transfer in in vitro recombination assays. Double and triple mutants F16W/I24Q, F16W/N27D, and F16W/I24Q/N27D all showed defects in DNA binding, strand transfer, and helix destabilization, suggesting that the I24Q and N27D mutations have a "dominant negative" effect and abolish the positive influence of F16W. Results show that amino acid differences at positions 24 and 27 contribute significantly to finger one's helix destabilizing activity and hence NC's chaperone activity. Preliminary results from in vivo experiments indicated that virus with the N27D mutation is infectious at near wt NC levels. This suggests that aggregation activity may be more important than helix destabilizing for viral viability. Results from two other forms of HIV-1 NC (NCp9 and NCp15) and NC proteins from Simian Immunodeficiency Virus and Murine Leukemia Virus are also reported.