2011 Annual Report 1a.Objectives (from AD-416) Classical Swine Fever (CSF) is a highly contagious viral disease of swine. Controlling and eliminating the disease is dependant upon identification of CSF viral mechanisms involved in induction of disease generalization of infection, tissue tropism, host range, transmission, immunogenicity and strain virulence. Better understanding of these determinants will provide identification tools, vaccines and/or anti-virals. These determinants are linked to specific interactions between viral proteins with host cell proteins upon infection. To further characterize the molecular basis of CSFV and host-cell interactions ARS, PIADC and the University of Connecticut will identify swine macrophage proteins interacting with structural and non-structural CSFV proteins during infection. The effect(s) of these interactions on virulence, generalization of infection, tissue tropism, virus transmission, immunogenicity and induction of protection will be determined in swine. 1b.Approach (from AD-416) 1. A Yeast Two-Hybrid screening system will be used to identify cellular proteins, using a porcine macrophage expression cDNA library, currently available at ARS, PIADC, that interact with each of the Classical Swine Fever Virus proteins. 2. The University of Connecticut will conduct fine mapping of interacting host and viral proteins to identify specific binding residues or motifs mediating the interaction. 3. Mutant viruses harboring genetically modified binding motifs will be constructed and characterized in vitro and in vivo at ARS, PIADC. Particular emphasis will be placed on establishing the ability of mutant viruses to cause disease and to induce protection in swine, relative to parental virulent virus. 3.Progress Report 1. The identification of a cellular protein, IQGAP1, that plays a pivotal role as a regulator of the cytoskeleton interaction of CSFV core protein. It is shown that interaction between IQGAP and CSFV Core is critical for the process of virus virulence in swine. The areas of IQGAP1 involved in actually mediating the binding has been accurately mapped. Recombinant CSFV having those IQGAP1 areas mutated has shown a drastically altered virulence. Therefore, this interaction is an integral component in the CSFV life cycle, 2. Identification of conserved sequence elements in nucleotide-binding motifs that hydrolyze NTPs (nucleotide tri-phosphate) within the CSFV non-structural protein NS4B. These results clearly demonstrated the importance of NS4B in the process of replication of viral genome. Recombinant CSFV having mutated the critical areas previously mapped has shown a decreased to null ability to replicate in cell cultures. Both studies demonstrate the presence of areas of CSFV genome that can be targeted in order to alter the ability of the virus in replicating and/or producing disease in swine.