The DeStefano Lab

Retroviral Recombination and Replication 

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Research in my laboratory has focused on studying the role of HIV-reverse transcriptase (RT) and nucleocapsid protein (NC) in the processes of retroviral recombination and replication. We also analyze basic properties of these proteins including how they interact with specific nucleic acid sequences involved in replication and how the activities of the proteins as defined in the test tube, function in cellular replication. Other projects are aimed at isolating nucleic acid inhibitors (aptamers) that can bind very tightly to RT and inhibit its function. We also study poliovirus replication, specifically by examining the polymerase (3Dpol) and 3AB proteins. Recombination is one of the mechanisms that retroviruses like HIV use to generate genetic diversity. By producing genetic variants viruses are able to circumvent the host immune response and escape drug therapies. Currently we work on several aspects of recombination, including the basic mechanism(s) by which recombination occurs, NC’s role in stimulating recombination, and the role of specific viral sequences in recombination. Our basic approach has been to use in vitro systems that mimic recombination and replication in the cell to understand the processes. Our group also collaborates with those of Drs. Eric Arts and Matteo Negroni on a project to study how intersubtype recombinants arise (Baird et al., 2006). These are HIV viruses that form by recombination between two different subgroups (A and D for example to produce an A/D recombinant). Intersubtype recombinants are becoming more prevalent, especially in Asia and Africa. This could complicate attempts to produce effective vaccines and drugs as therapies developed against the more common B and C type viruses may not be effective against other types or intersubtypes. Another goal has been to produce in vitro replication systems that more closely mimic what occurs in the cell. Such systems could potentially be used to test reverse transcription and recombination inhibitors. Recently conditions that allow the production of near genome length DNA synthesis products have been uncovered in the lab (Anthony and DeStefano, 2007). This is a major step as previous systems yielded mostly small products. Another recent highlight is the discovery of primer-template sequences that bind HIV-RT with high affinity (DeStefano and Cristofaro, 2006). The sequences closely resembled the HIV polypurine tract (ppt) sequence which is pivotal for genome replication. The results suggested that HIV-RT has co-evolved with this region to ensure tight binding and efficient replication. We are currently analyzing other retroviruses to see if all RTs evolved to bind their cognate ppt sequence tightly. In addition, this information was used to develop a small nucleic acid inhibitor of HIV-RT that we are currently testing to see if it can inhibit viral infection of cells. Recent highlights of our poliovirus work include the finding that 3AB is a nucleic acid chaperone protein (DeStefano and Titlope, 2006). Chaperones like HIV NC protein, help nucleic acids fold properly and in the case of NC, are involved in several steps of the virus life cycle. Protein 3AB is important for anchoring poliovirus replication complexes to internal cell membranes (3A portion) and serving as the “protein primer” for genome replication (3B portion). It has been studied for several years and our lab was the first to recognize its chaperone activity. Site-directed mutagenesis is currently by used to map the chaperone function of the protein. By studying unique aspects of the HIV and poliovirus life cycle we hope to contribute to new therapies that exploit these unique functions.  

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