Analysis of localization, topology, membrane binding, secretion and function of the pestivirus structural glacoprotein Erns

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Pestiviruses belong to the family Flaviviridae and produce enveloped virions with a single stranded RNA genome of positive polarity. They have established fascinating mechanisms for establishment of long lasting persistence and are of major importance for veterinary medicine (Lindenbach et al, 2007).Pestivirus particles contain glycoprotein Erns that represents a crucial factor for virulence and persistence of pestivirus. It possesses RNase activity (Hulst & Moormann, 2001; Hulst et al, 1998; Schneider et al, 1993; Windisch et al, 1996), which we identified as virulence factor and cofactor for establishment of persistence (Meyer et al, 2002; Meyers et al, 1999; Tews et al, 2009; Meyers et al, 2007). Erns contains no typical membrane anchor and is partially secreted from infected cells (Magkouras et al, 2008; Mätzener et al, 2009; Rümenapf et al, 1993) whereas the overwhelming amount of the synthesized protein remains membrane bound within the ER or an ER-related compartment (Burrack et al, 2012; Tews & Meyers, 2007; Tews et al, 2009). The carboxyterminal region of Erns represents an unusual membrane anchor dar (Tews & Meyers, 2007; Tews et al, 2009), ensures the intracellular localization of the protein, the biologically important homodimerization and the processing of the Erns/E1 precursor. We could recently show that the Erns membrane anchor folds into an amphipathic helix in contact with a membrane. Unexpectedly the central part of this helix is located within the lipid bilayer. It contains a series of charged residues that could form a so-called charge zipper motif (Walther et al, 2013). The mechanism of Erns membrane anchoring and the function of the charge zipper motif in membrane anchoring will be analyzed with molecular techniques. Further structure analysis of the membrane bound anchor will be conducted in cooperation. The virulence factor function of Erns is dependent on the RNase activity and homodimer formation. We have isolated from animals a pseudorevertant that regained the ability to form dimers by a mutation in the middle of the membrane anchor. This finding raises questions concerning the primary sequence requirements and timely coordination of dimer formation, Erns/E1 processing and initiation of membrane binding, which will be investigated during the coming grant period. Erns secretion is hypothesized to be curcial for ist function as a virulence factor. We aim to analyze how the equilibrium between specific intracellular localization and secretion of the protein is achieved. Moreover, the role of the charge zipper motif for the insertion of the Erns carboxyterminus into the membrane and the influence of other viral proteins on Erns retention and recruitment into the virus particle will be investigated.