Structure of the Membrane Anchor of Pestivirus Glycoprotein Erns, a Long Tilted Amphipathic Helix

Erns is an essential virion glycoprotein with RNase activity that suppresses host cellular innate immune responses upon being partially secreted from the infected cells. Its unusual C-terminus plays multiple roles, as the amphiphilic helix acts as a membrane anchor, as a signal peptidase cleavage site, and as a retention/secretion signal. We analyzed the structure and membrane binding properties of this sequence to gain a better understanding of the underlying mechanisms. CD spectroscopy in different setups, as well as Monte Carlo and molecular dynamics simulations confirmed the helical folding and showed that the helix is accommodated in the amphiphilic region of the lipid bilayer with a slight tilt rather than lying parallel to the surface. This model was confirmed by NMR analyses that also identified a central stretch of 15 residues within the helix that is fully shielded from the aqueous layer, which is C-terminally followed by a putative hairpin structure. These findings explain the strong membrane binding of the protein and provide clues to establishing the Erns membrane contact, processing and secretion.Author Summary: The Erns protein (envelope protein, RNase, secreted) of pestiviruses represents one of the most fascinating proteins in virology. Erns is not only an essential structural component of the virus particle but also an unspecific RNase. The latter activity is dispensable for pestivirus replication but represents a virulence factor involved in the establishment of lifelong persistent infection. These functions of Erns are connected with its repressive activity on the type I interferon response of the infected host probably depending on secretion of part of the protein synthesized within the infected cell followed by its distribution with the blood stream. To understand the mechanisms leading to an equilibrium between intracellular retention (for production of virus particles) and secretion (for repression of the innate immune response) the principles of Erns membrane binding need to be better understood. The recently published Erns crystal structure, however, is lacking the relevant carboxyterminal membrane anchor region. We report here structure analyses of the Erns membrane anchor bound to model membranes. This work was based on circular dichroism, nuclear magnetic resonance spectroscopy, and structure simulations, and revealed a new type of membrane anchor for a surface protein. These data will help to explain the unusual functions of Erns.