Membrane binding and bending in Ebola VP40 assembly and egress

Abstract

Lipid-enveloped viruses contain a lipid bilayer coat that protects their genome and helps to facilitate entry into the host cell. Filoviruses are lipid-enveloped viruses that have up to 90% clinical fatality and include Marbug (MARV) and Ebola (EBOV). These pleomorphic filamentous viruses enter the host cell through their membrane-embedded glycoprotein and then replicate using just seven genes encoded in their negative-sense RNA genome. EBOV budding occurs from the inner leaflet of the plasma membrane (PM) and is driven by the matrix protein VP40, which is the most abundantly expressed protein of the virus. VP40 expressed in mammalian cells alone can trigger budding of filamentous virus-like particles (VLPs) that are nearly indistinguishable from authentic EBOV. VP40, such as matrix proteins from other viruses, has been shown to bind anionic lipid membranes. However, how VP40 selectively interacts with the inner leaflet of the PM and assembles into a filamentous lipid enveloped particle is mostly unknown. This article describes what is known regarding VP40 membrane interactions and what answers will fill the gaps.

Keywords: Ebola; VP40; filovirus; membrane binding; phosphatidylserine; phosphoinositides; plasma membrane.

FIGURE 1

Chemical structures of lipids found in the PM cytoplasmic leaflet. The inner leaflet of the PM has a relatively high concentration of PE and PS and it is known to be enriched in cholesterol and PI. Additionally, PIPs including PI(4)P, PI(4,5)P₂, and PI(3,4,5)P₃ are found in low abundance in the PM inner leaflet but have crucial roles in regulating peripheral protein localization and function at the membrane interface. Structures are shown at physiological pH.

FIGURE 2

VP40 structural analysis. VP40 is a dimer (Bornholdt et al., 2013) that is the building block for VP40 filament formation. Dimerization is mediated by a hydrophobic N-terminal domain interface (Bornholdt et al., 2013). The dimeric structure revealed a robust cationic patch exposed on one face of the dimer (Bornholdt et al., 2013) Mutation of Lys residues in this C-terminal domain cationic patch greatly reduced PM localization and budding in cells and is surmised to mediate membrane binding (Bornholdt et al., 2013). Adjacent to the cationic patch is a hydrophobic loop in the C-terminal domain that mediates membrane insertion of VP40, an important step for VP40 PM binding and membrane curvature generation (Adu-Gyamfi et al., 2013; Soni et al., 2013).

FIGURE 3

Plasma membrane binding model for VP40. Considering previous studies and available data on VP40 structure and membrane-binding properties a simplified model of VP40 association with the PM is proposed. The VP40 dimer, which contains a large cationic solvent exposed patch in the C-terminal domain likely mediates the association of VP40 with the highly anionic interface of the PM. VP40 interactions with PS (Ruigrok et al., 2000; Scianimanico et al., 2000; Adu-Gyamfi et al., 2013; Soni et al., 2013) and perhaps other lipids such as PIPs induce a conformational change of VP40 rearranging the dimers into a zigzagging hexamer (Bornholdt et al., 2013). The VP40 dimers can interconnect through the CTD to form linear hexamers as well (Bornholdt et al., 2013). These hexamers have “sticky” ends that can further interact to form long VP40 filaments (Bornholdt et al., 2013). Lateral interactions between VP40 filaments are also possible and can occur between dimers and hexamers (Bornholdt et al., 2013). Note, in this model a VP40 monomer would not robustly interact with the PM (Bornholdt et al., 2013) presumably due to weak affinity or lack of transport to the PM. Hydrophobic interactions between the more liberated (Bornholdt et al., 2013) C-terminal domain hydrophobic loop (Jasenosky et al., 2001; Adu-Gyamfi et al., 2013; Soni et al., 2013) and the PM hydrophobic core act to increase the membrane residence time of VP40. As VP40 assembles on the inner leaflet of the PM, the C-terminal hydrophobic residues are able to penetrate ~8.1Å. VP40 oligomers are able to induce budding (negative membrane curvature generation; Soni et al., 2013) of a filamentous particle that eventually will undergo scission and be released.