Retroviral Integrase Enzymes and AIDS Pathogenesis

Summary: Our laboratory studies the mechanism of retroviral integration. The goal is to understand how a copy of the retrovirus genome is permanently inserted into the DNA of an infected cell. Our major focus is the integrase enzyme of HIV-1 and other retroviruses. We use a wide variety of molecular biological and biochemical techniques to analyze the structure and function of this critical viral enzyme. These studies may lead to new ways to prevent or treat AIDS and also are relevant to cancer.



Details: Our laboratory focuses on the integrase enzyme of human immunodeficiency virus (HIV), avian sarcoma viruses, and visna virus. Integrase is responsible for inserting (or integrating) retroviral DNA into the DNA of an infected cell, an event that makes HIV infection permanent and leads to the acquired immunodeficiency syndrome (AIDS). Retroviral integration is also relevant to certain other human diseases, some animal models of cancer, and gene therapy. The overall goal is to understand the mechanism of retroviral integration so as to develop new ways to prevent or treat AIDS. We routinely clone, express, and purify mutant or chimeric integrase proteins that are analyzed in a variety of biochemical assays to map regions of the enzyme that interact with its various substrates.



Several years ago, I participated in the first purification of an enzymatically active retroviral integrase from a bacterial expression system and developed an assay that is used throughout the world to study this enzyme. After establishing my laboratory, we created the first set of functional chimeric lentiviral integrases by making novel proteins partly from HIV and partly from a sheep virus; these studies identified the parts of integrase that interact with viral DNA and with human DNA, while also identifying the parts of viral DNA that interact with integrase. We then characterized the largest set (to that point) of HIV integrases derived from infected patients. These studies identified an amino acid in the middle of the protein that likely contacts human DNA during integration. More recently, we made several mutations at the position identified by the human samples to yield novel integrases that were improved at acting on viral DNA but impaired at acting on cellular DNA, the first mutants to separate the two biologically relevant actions of integrase in this way. We are now working to exploit these properties to define the cellular-DNA and viral-DNA binding sites on the integrase protein, which would improve models of integration and identify precise new antiviral targets. In another project, we are trying to stimulate the ability of integrase to nick and damage DNA (an activity we first described a decade ago) for a novel antiviral strategy of using integrase to destroy viral DNA. Thus, our focus on integrase has contributed to the current model of retroviral integration and will enhance efforts to target specific actions or domains of integrase for clinical therapy.