Replacement of a Single Aromatic Residue in HIV-1 Nucleocapsid Protein Strongly Alters its Nucleic Acid Chaperone Activity

Abstract

The human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) protein plays an essential role in several stages of HIV-1 replication. One important function of HIV-1 NC is to act as a nucleic acid chaperone, in which the protein facilitates nucleic acid rearrangements important for reverse transcription and recombination. NC contains only 55 amino acids, with 15 basic residues and two zinc fingers, each having a single aromatic residue (F16 and W37). Despite its simple structure, HIV-1 NC appears to have optimal chaperone activity, including the ability to strongly aggregate nucleic acids, destabilize nucleic acid secondary structure, and facilitate rapid protein-nucleic acid interaction kinetics. Here we use single molecule DNA stretching experiments to measure the characteristics of wild type and mutant HIV-1 NC that are important for nucleic acid chaperone activity. This work allows us to directly relate HIV-1 NC structure with its function as a nucleic acid chaperone. By stretching single DNA molecules in the presence of these proteins, we measure the ability of the proteins to destabilize dsDNA, and when the protein is relaxed we determine the capability of the protein to facilitate nucleic acid annealing. We show that the single amino acid substitution W37A significantly slows down NC's DNA interaction kinetics, while retaining NC's helix-destabilization capabilities. In contrast, the substitution F16W results in a protein that strongly resembles wild type NC. Thus, elimination of a single aromatic residue strongly reduces NC's chaperone activity. The results of these studies are consistent with in vivo HIV-1 replication measurements, which show that the aromatic W37 residue is required for efficient retroviral replication.