Identification and characterization of genes which contribute to viral (poxvirus) pathogenesis and disease in their respective hosts
My long-term interests are in the identification and characterization of genes which contribute to viral (poxvirus) pathogenesis and disease in their respective hosts. Vertebrate (human) poxviruses encode a variety of genes designed to interfere with the immune response of the host. Each of the genes currently under investigation in the laboratory controls aspects of the host response which include inflammation, chemotaxis of immune cells and/or apoptosis. Mutations in the genes we examine attenuate the virus in animals, profoundly influence the ultimate outcome of the disease, effect host range of the virus, plaque phenotype in cell culture and "pock" color produced upon infection of embryonated eggs.
Currently, we are most interested in the family of viral encoded serine proteinase (serpin) inhibitors. Serpins are quite common in Nature but within viruses are only found in poxviruses. Orthopoxviruses such as vaccinia, cowpox and rabbitpox viruses encode three Serpins (SPI-1, 2 and 3). Two of these serpins, SPI-1 and SPI-2 (also called crmA) while >50% identical are biochemically distinct. SPI-2 prevents the influx of inflammatory cells into developing viral lesions and is known to inhibit the host interleukin-1ß converting enzyme (ICE), the prototypic member of the caspase family of proteinases. ICE not only regulates biogenesis of active interleukin-1 (IL-1), but over-expression of ICE has been shown to induce apoptosis. Cloned SPI-2 as expected, inhibits apoptosis in such systems. However inactivation of the SPI-2 gene within the virus itself does not generally affect apoptosis and has little effect on virulence. The related SPI-1 gene, has no effect on ICE or other caspases, inhibiting instead chymotrypsin-like enzymes such as cathepsin G, a constituent of neutrophils. While deletion of SPI-1 from the virus likewise has little effect on virulence, deletion of both SPI-1 and SPI-2 which has no effect on growth of the virus in cultured cells results in a completely attenuated virus in vivo. Determining how these two serpins act in concert in vivo in animal models is a major goal of the laboratory. A second major interest is the failure of serpins from two distinct genera, with similar biochemical functions to replace each other. The third orthopoxvirus serpin, SPI-3 and the SERP1 serpin of myxoma virus inhibit the same proteinases. Yet within their respective viruses, they can not replace each other in animal models and results in attenuation. These results suggest that other proteins influence the ultimate function of these serpins in vivo. Identifying these interacting proteins and how they serve to regulate function is another major goal of the laboratory.
We are also beginning to turn our attention to a second class of genes, those which regulate chemokines following infection. Chemokines are a class of cytokines which regulate leucocyte trafficking and inflammation. We are currently examining the properties of one such gene (p35), secreted from infected cells that efficiently binds most if not all ß-chemokines yet has no homology to any known chemokine receptor. A second (K2R) gene, appear to be a viral encoded chemokine receptor. How genes such as these influence pathogenesis is also a major interest of the laboratory.
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