Location: Foreign Animal Disease Research
Project Number: 8064-32000-060-09-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jan 1, 2015
End Date: Dec 31, 2019
The objective of this collaborative research project is to assess the role of specific Classical Swine Fever Virus (CSFV) proteins in the process of virus replication and virulence. Specifically, this project is focused in the interaction between virus proteins and host cell membranes and its effect in virus replication in cell cultures and virulence during the infection in swine.
Functional genomics will be used to analalyze Classical Swine Fever Virus (CSFV) proteins with specific motifs indicating the possible interaction with cell membranes. These proteins are expected to play critical roles in the different steps of the virus replication cycle as well as in the process of disease production. The role of these selected proteins will be analyzed by reverse genetics. Recombinant viruses will be designed and developed having altered the areas of the proteins under study harboring the motifs allowing the putative interaction with the cell membranes. These recombinant viruses will be used to assess the significance of the introduced mutations in assays performed both in cell cultures and in infected animals. In addition, the assessment of the presence of interaction between cell membranes and the viral proteins harboring the predicted motifs will be performed using in vitro methodologies. CSFV proteins and derived peptides will be assessed for their insertion, oligomeric assembly and pore formation in model membranes. These models comprise: i) lipid monolayers (measurements of lateral pressure in Langmuir troughs); ii) unilamellar vesicles with defined diameters (LUV) prepared according to the extrusion method (measurements of water-membrane partitioning, lipid-mixing and aqueous contents release through spectrofluorimetric methods), iii) Giant Unilamellar Vesicles prepared according to the electroformation method (establishment of pore size through confocal microscopy analyses), and iv) Suspended planar phospholipid bilayers (evaluation of ion channel activity by electrophysiological measurements). These complementary approaches will be used to define sequence and lipid requirements for the function of the CSFV proteins. Insertion into membranes implies acquisition of defined secondary structure. Thus, circular dichroism measurements will be additionally carried out to follow membrane-partitioning coupled structuring. Identificiation and characterization of viral proteinsinteracting with membranes in the host cells will be an important goal in this project. Understanding these interactions and their role in the mechanism of virus replication and disease production may be used fopr developing novel tools for controlling virus infection in vivo.