CRITICAL CONTACTS BETWEEN HIV-1 INTEGRASE AND VIRAL-DNA IDENTIFIED BYSTRUCTURE-BASED ANALYSIS AND PHOTO-CROSS-LINKING

Analysis of the crystal structure of HIV-I integrase reveals a cluster of lysine residues near the active site. Using site-directed mutagene sis and photo-crosslinking we find that Lys156 and Lys159 are critical for the functional interaction of integrase with viral DNA. Mutation of Lys156 or Lys159 to glutamate led to a loss of both 3' processing a nd strand transfer activities irt vitro while maintaining the ability to interact with nonspecific DNA and support disintegration, However, mutation of both residues to glutamate produced a synergistic effect e liminating nearly all nonspecific DNA interaction and disintegration a ctivity, In addition, virus containing either of these changes was rep lication-defective at the step of integration, Photo-crosslinking, usi ng 5-iododeoxyuracil-substituted oligonucleotides, suggests that Lys15 9 interacts at the N7 position of the conserved deoxyadenosine adjacen t to the scissile phosphodiester bond of viral DNA. Sequence conservat ion throughout retroviral integrases and certain bacterial transposase s (e.g. Tn10/IS10) supports the premise that within those families of polynucleotidyl transferases, these residues are strategic for DNA int eraction.