MAPPING FEATURES OF HIV-1 INTEGRASE NEAR SELECTED SITES ON VIRAL AND TARGET DNA-MOLECULES IN AN ACTIVE ENZYME-DNA COMPLEX BY PHOTO-CROSS-LINKING

The virally encoded integrase protein carries out retroviral integrati on, and to do so, it must make specific interactions with both viral a nd target DNA sequences. The retroviral integrase has three domains: a n amino-terminal region of about 50 amino acids that contains a zinc f inger-like motif, a tightly folded, phylogenetically conserved core do main that contains the active site, and a carboxy-terminal domain that can bind DNA in a nonspecific manner. The complete roles of the amino -and carboxyl-terminal domains have not yet been determined, but they appear to participate in multimerization and nonspecific or target DNA binding, respectively. The number and identity of integrase's DNA bin ding sites have been difficult to determine by conventional mutagenesi s studies. In this report, we describe a photo-cross-linking approach to address these issues. Our findings suggest that HIV-1 integrase con tacts with conserved features of the viral DNA end are likely to be me diated by residues in two peptides within the conserved core domain. A dditional cross-links were seen between viral DNA and the carboxytermi nal DNA binding domain. Numerous sites in integrase, including peptide s in each of the three domains, could be cross-linked to target DNA fe atures. Integrase is known to function as a multimer, and it remains t o be determined which specific contacts are in cis or trans with respe ct to the active site.