Location: Foreign Animal Disease Research2010 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
Identification of Classical Swine Fever (CSF) viral mechanisms involved in induction of disease, generalization of infection, tissue tropism, transmission, immunogenicity, and virulence remain to be defined. Our hypothesis is that Classical Swine Fever Virus (CSFV) determinants of virulence, host restriction and host response are linked to specific interactions between viral proteins and cell proteins that occur upon infection. Our objective is to characterize the molecular basis of CSFV-host cell interactions by identifying swine macrophage proteins that interact with virus proteins during infection. The effects of these interactions on virulence, attenuation, and induction of protection will be studied in swine. This project is aimed to develop science-based information that could result in the generation of tools (e.g. vaccines and/or anti-virals) to reduce the number and severity of CSF outbreaks should they occur in the United States. We have determined that interactions between CSFV Core protein and host cell proteins are important for the life cycle of the virus in vivo. We were able to identify host cell proteins involved with the cellular SUMOylation pathway, SUMO-1 and UBC9, a SUMO-1 conjugating enzyme that interact with the Core protein. Five highly conserved lysine residues were identified as putative SUMOylation sites. These data shows a clear correlation between the disruption of Core protein binding to SUMO-1 and UBC9 and CSFV attenuation. Overall these data suggest that the interaction of Core with the cellular SUMOylation pathway plays a significant role in the CSFV growth cycle in vivo. No technologies have been transferred so far. This project will produce basic information about the biology of CSFV-host cell interactions that may be used by industry or other scientists for designing of novel CSFV control tools such as antivirals. A manuscript has been sent for publication to Virology (Elsevier). The manuscript has been entitled: “Effects of the interactions of Classical Swine Fever Virus Core protein with proteins of the SUMOylation pathway on virulence in swine” D. P. Gladue, et al. Monitoring was accomplished through Conference calls.