Public health concems have emerged regarding use of smallpox as a bioterrorist weapon since mostAmericans are no longer immune. Poxviruses subvert the complement system via the expression ofregulatory proteins. In variola, vaccinia and ectromelia, the proteins are called SPICE (for smallpox inhibitorof complement enzymes), VCP (vaccinia virus complement control protein) and EMICE (an uncharactedzedanalog in ectromelia that we have labeled 'ectromelia inhibitor of complement enzymes'). These secretedvirulence factors down-regulate complement activation by mimicking the functional repertoire of a family ofhost proteins called the Regulators of Complement Activation (RCA). The viral proteins are also structurallyrelated to their host counterparts.
Specific Aims : 1. To characterize the complement inhibitory profile ofSPICE compared to its human counterparts. We will identify the principal complement-evading activity ofSPICE and this will become a target for neutralization. These assessments will take place with the nativesoluble protein as well as after it attaches to cells via either its heparin-binding site(s) or by addition of ananchor. 2. To determine the complement regulatory sites of SPICE. These experiments will take advantageof the functional profiles (defined in Aim 1) and the sequences of active sites of RCA proteins that arehomologous to corresponding regions of SPICE, VCP, and EMICE. These two sets of data provide a logicalstrategy for a mutational analysis to locate the active sites. 3. To characterize the complement regulatoryactivity of EMICE. This mousepox protein has not been evaluated for its complement inhibitory (virulence)activity. It is about 90% identical to SPICE and VCP. We will first characterize its regulatory activity forhuman and mouse complement. Second, we will assess its role in vivo as a virulence factor by infectingsensitive and resistant mouse strains with the ectromelia virus deleted of its complement regulator. Theproposed experiments should provide novel information relative to the pathogenesis of poxvirus infections ofman and mouse. Additionally, these results will serve as a guide to produce a less toxic small pox vaccineand to identify a viral target for mAb treatment of variola infection.
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