Coupled eukaryotic expression and biotinylation of functional pestivirus E2 envelope glycoprotein

FERNANDO MERWAISS; CECILIA CZIBENER; DIEGO E. ALVAREZ

Rosario

Congreso; SAIB 50th Annual Meeting; 2014

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Bovine viral diarrhea virus (BVDV) is a cattle pathogen belonging to the genus pestivirus within the family Flaviviridae, which causes a significant impact on the livestock industry worldwide. The virion has an outer lipid envelope bearing glycoproteins Erns, E1 and E2. Entry of BVDV into the host cell requires the interaction of the virus with a set of yet unidentified receptors and co-receptors. E2 mediates virus-receptor interactions and is the major antigen inducing neutralizing antibodies in an infected host. Thus, E2 is considered as an ideal target to be used in subunit vaccines. We developed an eukaryotic expression system that allows for the production of a soluble version of biotinylated-E2. Recombinant E2 is biotinylated in eukaryotic cells by co-expressing E2 fused to the 15 amino acid biotin acceptor peptide (BAP) and the bacterial biotin-protein ligase BirA, which specifically recognizes and attaches a biotin moiety to the single lysine residue of BAP. We demonstrate that soluble biotinylated-E2 is able to inhibit BVDV-induced cytopathic effect in cell culture, indicating that the recombinant protein is functional. We will discuss biotechnological applications of biotinylated-E2 in the design of subunit vaccines to differentiate infected from vaccinated animals, and in the identification of cell surface receptors for BVDV entry in immunoprecipitation assays.