Location: Foreign Animal Disease Research2012 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:
During FY 2012 we studied the six amino acids, called Cysteines, which are harbored by Classical Swine Fever Virus (CSFV) E1 structural protein. E1, along with Erns and E2, is one of the three envelope glycoproteins of CSFV. In infected cells, E2 forms heterodimers, which are proteins formed by two polypeptide chains, with E1 that are formed by disulfide bridges between cysteine residues. This interaction may play a role in virus assembly and virus entry. The role of these residues in the formation of E1-E2 heterodimers and their effect on CSFV viability in vitro and in vivo remain unclear. Six cysteines residues of E1 are found in CSFV strain Brescia. It was observed that recombinant viruses harboring individual cysteine-to-serine substitutions within the E1 envelope protein were viable and still allow for interactions and formation of heterodimers with E2 protein. These single mutant viruses retained virulence in swine. However, a double mutant harboring Cys24Ser and Cys94Ser substitutions within the E1 protein altered formation of E1-E2 heterodimers in infected cells. This recombinant virus, E1detla Cys24/94v, showed delayed growth kinetics in primary swine macrophage cultures and was attenuated in swine. Furthermore, despite the observed diminished growth in vitro, infection with E1delta Cys24/94v protected swine from challenge with virulent CSFV strain Brescia at 3 and 28 days post infection. Although still undetermined, the interaction between E1 and E2 plays a critical unknown role in the cycle of CSFV. Determination and assessing the role of interactions between CSFV proteins are key for understanding virus virulence. In FY 2013 we will explore the interaction between host proteins and the virus nonstructural protein p7 and complete the characterization of host proteins interacting with structural protein Core Publications for FY2012 include: Fernández-Sainz I, Holinka LG, Gladue D, O'Donnell V, Lu Z, Gavrilov BK, Risatti GR, Borca MV. (2011). Substitution of specific cysteine residues in the E1 glycoprotein of classical swine fever virus strain Brescia affects formation of E1-E2 heterodimers and alters virulence in swine. Journal of Virology 85:7264-72. B.K. Gavrilov, K. Rogers, I.J. Fernandez-Sainz, L.G. Holinka, M.V. Borca, and G.R. Risatti. (2011) Effects of Glycosylation on Antigenicity and Immunogenicity of Classical Swine Fever Virus Envelope Proteins. Virology 420:135-145 D.P. Gladue, L.G. Holinka, E. Largo, I. Fernandez Sainz, V. O’Donnell, R. Baker-Branstetter, Z. Lu, X. Ambroggio, G.R. Risatti, J.L. Nieva, and M.V. Borca. (2012) Classical Swine Fever Virus p7 protein is a viroporin involved in virulence. D.P. Gladue, L.G. Holinka, E. Largo, I. Fernandez Sainz, V. O’Donnell, R. Baker-Branstetter, Z. Lu, X. Ambroggio, G.R. Risatti, J.L. Nieva, and M.V. Borca. Journal of Virology 86:6778-91.