Lipid Binding of the Amphipathic Helix Serving as Membrane Anchor of Pestivirus Glycoprotein E(rns)

Pestiviruses express a peculiar protein named E(rns) representing envelope glycoprotein and RNase, which is important for control of the innate immune response and persistent infection. The latter functions are connected with secretion of a certain amount of E(rns) from the infected cell. Retention/secretion of E(rns) is most likely controlled by its unusual membrane anchor, a long amphipathic helix attached in plane to the membrane. Here we present results of experiments conducted with a lipid vesicle sedimentation assay able to separate lipid-bound from unbound protein dissolved in the water phase. Using this technique we show that a protein composed of tag sequences and the carboxyterminal 65 residues of E(rns) binds specifically to membrane vesicles with a clear preference for compositions containing negatively charged lipids. Mutations disturbing the helical folding and/or amphipathic character of the anchor as well as diverse truncations and exchange of amino acids important for intracellular retention of E(rns) had no or only small effects on the proteins membrane binding. This result contrasts the dramatically increased secretion rates observed for E(rns) proteins with equivalent mutations within cells. Accordingly, the ratio of secreted versus cell retained E(rns) is not determined by the lipid affinity of the membrane anchor.