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Medical Research Institute
700 Ellicott Street
Buffalo, NY 14203 - 1102
Structural characterization of proteins from SARS coronavirus (SARS-CoV) and mouse hepatitis virus (MHV); identification and characterization of virus:host protein interactions involved in animal to human transmission; enzymatic mechanism of light emission and pH-regulation of luciferases from marine dinoflagellates; sensing cellular oxygen levels by prolyl-hyroxylases.
SARS-CoV and MHV replication
The first cases of Severe Acute Respiratory Syndrome (SARS) originated in November 2002 and by March 2003, the infectious vector was identified as a positive-stranded RNA coronavirus (SARS-CoV). The genome of the SARS-CoV was quickly sequenced and is comprised of approximately 30 kilobases. From this large viral genome, SARS-CoV has been predicted to encode 15 mature non-structural replicase proteins, 4 envelope proteins and 7 accessory non-structural proteins. Currently, there is little known about the function of these proteins. Our research focuses on the non-structural proteins that combine to form a replication complex that is the central engine driving viral reproduction.
Virus•host protein interactions
Many of the world’s most severe pandemics have resulted when an animal virus acquired the ability to infect humans. The “Spanish flu” of 1918 (500,000 deaths in the US and millions worldwide), the “Asian flu” of 1957 (70,000 deaths in the US), the “Hong Kong flu” of 1968 (34,000 deaths in the US), and HIV (tens of millions of deaths worldwide) are all thought to have evolved from animal viruses. Current concern over avian flu illustrates the continuing threat posed by new human viruses emerging from reservoirs of endemic animal viruses.
We propose to identify and characterize virus•host protein:protein interactions that are important in establishing infection in a new host species. Formation of new cell surface virus•host protein complexes is an important first step towards cross-species infection. It is our hypothesis that simutaneous changes in intracellular virus•host complexes are also necessary. Successful adaptation to a new host not only requires receptor binding, but post-entry optimization of viral replication, virion assembly and transmission (K.V. Holmes Science, September 2005, 309, 1822-1823).
Many different organisms exist with the ability to emit light. All use a luciferase enzyme that catalyzes the oxidation of a luciferin substrate. The Gonyaulax polyedra luciferase (LCF) (recently renamed Lingulodinium polyedrum) system is unique in its use of a luciferin binding protein (LBP) that sequesters the substrate, a novel tetrapyrrole, until cellular conditions trigger its release. The activity of the LCF and the binding affinity of the LBP for the luciferin are tightly controlled by pH. The biochemistry of the Gonyaulax system has been described but has raised many questions regarding the composition of the LCF active site, the mechanism of light emission, cooperativity between luciferase domains and pH regulation of the enzyme conformation and activity. We seek to use the three-dimensional structures of LCF and the LCF:luciferin complex to understand this unique system and gain insight into the mechanism of bioluminescence.
Appropriate responses to decreased environmental oxygen (hypoxia) are imperative for mammalian survival. These responses govern a wide range of physiological processes, from the maintenance of ventilation, cardiac output, and cellular ATP levels to the production of various mitogenic, immunological, and vasoactive substances. Numerous studies have shown that many of these responses to hypoxia involve alterations in gene expression. Prolyl-hydroxylases (PHXs) are enzymes that play an important role in the regulation of the hypoxic response. Molecular oxygen is required for PHX activity and provides for the “sensing” of oxygen levels in cells. We are interested in crystallographic analysis of PHX structures and developing small molecule inhibitors of their activity.
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