Simulations Help Unravel Flavivirus Envelope Structure and Function

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Over 40 flaviviruses worldwide are human pathogens. These include dengue virus (DENV), responsible for more than 400 million infections per year, and emerging infectious diseases such as Zika virus (ZIKV). The flavivirus nucleocapsid core is encapsulated by a phospholipid bilayer, within which are embedded envelope and membrane proteins. Low pH conditions trigger conformational changes in the envelope protein and a dimer-to-trimer transition, leading to exposure of the hydrophobic fusion peptide (FP). Subsequent interaction of FPs with the host endosomal membrane initiates fusion, resulting in release of the RNA genome into the cell. The pre-fusogenic flaviviral membrane exhibits significant curvature and deformation, indicated by cryo-electron microscopy (cryo-EM) maps. Here, multiscale simulations of the complete mature flavivirus envelope particle have been used to refine the envelope structure in unprecedented detail. The final structures of DENV were morphologically consistent with cryo-EM maps and revealed the structural basis for membrane curvature, whilst interesting differences are highlighted in simulations of the more robust ZIKV envelope. Extra-membranous protein components are shown to serve as scaffolds which position transmembrane domains and help to determine lipid bilayer architecture, dependent upon specific electrostatic interactions with anionic lipids. The observed lipid-protein coupling in the mature virus has potential functional significance for pH-dependent envelope protein conformational changes, subsequently investigated by constructing a viable structure of the fusogenic flavivirus particle, guided by biophysical data, and simulating its interaction with models of late endosomal membranes. Clustering of trimeric envelope protein complexes was observed to be dependent upon the viral membrane lipid composition, whilst contact between the envelope protein FP tips and endosomal lipid bilayer induced “micro-budding” in the target membrane. Remarkably, many FPs could simultaneously interact with and embed themselves within the endosomal membrane, challenging traditional mechanistic views of viral fusion and resultant infective states.