The Pestivirus Glycoprotein E Is Anchored in Plane in the Membrane via an Amphipathic Helix*

Erns is a structural glycoprotein of pestiviruses found to be attached to the virion and tomembraneswithin infected cells via its COOH terminus, although it lacks a hydrophobic anchor sequence. The COOH-terminal sequence was hypothesized to fold into an amphipathic -helix. Alanine insertion scanning revealed that the ability of the Erns COOH terminus to bind membranes is considerably reduced by the insertion of a single amino acid at a wide variety of positions. Mutations decreasing the hydrophobicity of the apolar face of the putative helix led to reduction of membrane association. Proteinase K protection assays showed that Erns translated in vitro in the presence of microsomal membranes was protected, whereas a mutant with an artificial transmembrane region and a short cytosolic tag was shortened by the protease treatment. A tag fused to the COOH terminus of wild type Erns was not accessible for antibodies within digitonin-permeabilized cells, but the variant with the tag located downstream of the artificial transmembrane region was detected under the same conditions. These results are in accordance with themodel that theCOOH-terminalmembrane anchor of Erns represents an amphipathic helix embedded in plane into the membrane. The integrity of the membrane anchor was found to be important for recovery of infectious virus.