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Elucidation of a Proteolytic Mechanism Required for Kaposi's Sarcoma-associated Herpesvirus Replication

出版	University of California, San Francisco, 2005
URL	http://books.google.com.hk/books?id=L8qMB53cMu4C&hl=&source=gbs_api

註釋Herpesviruses represent one of the most prevalent human pathogens worldwide. Infection results in a number of diseases ranging from cold sores to cancer. Yet despite the large clinical variation in disease states, the proteins expressed during the lytic cycle of all herpesviruses are highly conserved. A maturational serine protease is expressed late in viral replication and is required for production of properly assembled infectious progeny. In vitro structural and functional analysis has revealed the enzyme possesses a novel protein fold and is activated by dimerization. However, the mechanism of regulation of the enzyme both at a molecular level and in the context of viral replication is unknown. Positional scanning synthetic combinatorial libraries were used to define the substrate specificity of the protease encoded by Kaposi's Sarcoma-associated herpesvirus (KSHV). The strict specificity observed was used to guide the design and synthesis of a peptidyl-diphenylphosphonate inhibitor targeting the active site of the enzyme. Covalent modification of the active site serine of the enzyme by the transition-state analog inhibitor dramatically shifted the equilibrium toward dimeric enzyme illustrating the intimate communication between the spatially separate active sites and dimer interface of the protease. To elucidate the mechanism of stabilization and activation during catalysis, structural analysis was performed on purely monomeric (M197D), purely dimeric (inhibited) and an equilibrium mixture (uninhibited wild-type) by circular dichroism and nuclear magnetic resonance. A massive conformational change was observed upon loss of dimerization in which helices 5 and 6, at the interface and active site, respectively, become disordered, which destabilizes essential catalytic components. A disulfide bond was engineered into the protease at helix 6 that prevented the conformational change and subsequent inactivation of the enzyme. Addition of oxidant or reductant reversibly recovered or abolished enzymatic activity, respectively, even in a monomeric variant of the protease. A reduction in viral replication similar to currently available therapies targeting the viral polymerase was observed upon treatment of reactivated KSHV with the protease inhibitor. The work not only validated the virally-encoded protease as a potential therapeutic target, but also presented a mechanism by which protease inhibition may occur in vivo .