Biochemical and pharmacological characterizations of the structure and function of human immunodeficiency virus type 1 integrase and its role during early steps of the viral life cycle

The insertion of the double-stranded DNA copy of viral genome into the host chromosome by the viral enzyme integrase is an essential step in retroviral life cycle. Integration requires proper interaction between integrase and the viral and target DNA. Functional domains of HIV-1 integrase involved in the protein-DNA interactions have not been well characterized due to the lack of structural information on full-length integrase. We are using a biochemical approach to map the functional domains and residues of HIV-1 integrase responsible for viral and target DNA binding by converting full-length HIV-1 integrase into a site-specific chemical nuclease. Using site-directed mutagenesis, ten mutant proteins each with a modifiable Cys in the putative DNA-binding domain were prepared. All mutants retained functional DNA-binding domains as revealed by enzymatic assays in vitro. The extent of modification by chemical nuclease was monitored by mass spectrometry. After modification, the site-specific chemical nuclease was bound with radiolabeled model DNA substrates and DNA cleavage should occur at sites in close proximity to the modified residue. Functional domains and residues of HIV-1 integrase involved in viral or target DNA interaction can be deduced by analyzing DNA-scission patterns of a series of mutants. Because of its critical role in establishing infection, integrase is an attractive target for anti-retroviral therapy. We investigated the mechanism of inhibition of integrase by dicaffeoylquinic acid derivatives. Our results suggested that these compounds cause irreversible inhibition by interacting with a conserved region in the core domain of integrase. During infection, integrase exists as part of a large nucleoprotein complex and mutations of the integrase gene cause defects at multiple steps of viral replication. The effect of Cys to Ser mutations of HIV-1 integrase on viral replication was examined. Our results showed that C130 of HIV-1 integrase plays an important role in the initiation of reverse transcription through physical interaction with reverse transcriptase. Studies on the structure and function of integrase and the additional roles it plays during viral replication will provide a better understanding of the molecular mechanism of viral replication and aid us in the development of novel anti-HIV agents.