Class II enveloped viruses

International audienceA number of viruses transport their genomic material from cell to cell enclosed within a lipid bilayer that is in turn encased within a symmetric protein shell. This review focuses in a group of RNA viruses that have this type of virions. This group includes several of important human pathogenic viruses, such as the hepatitis C virus, dengue virus, chikungunya virus, rubella virus and the bunyaviruses. The best studied are the flaviviruses and the alphaviruses, which have a β-sheet rich class II viral fusion protein used for entry into susceptible cells. We extend here the class II concept to encompass symmetric viruses in which the envelope proteins are derived from a precursor polyprotein containing two transmembrane glycoproteins arranged in tandem. The first glycoprotein acts as chaperone for the folding of the second one, which carries the membrane fusion function. Since the bunyaviruses, included here, are very similar to the class I arenaviruses in other respects, this analysis highlights the patchwork nature of the various viral functional modules acting at different stages of the virus cycle, which appear assembled from genes of different origins

Alphavirus structure: activation for entry at the target cell surface

International audienceA wealth of new data about the 3D organization of alphavirus particles was obtained in the last few years. This includes the crystal structures of the envelope glycoprotein complexes at neutral and at acid pH, as well as electron microscopy reconstructions of intact virions at neutral pH to resolutions between 7 Å and 4 Å. The combination has provided unprecedented detail in the description of the alphavirus virion. This review surveys the main features discovered and the implications for the biology of the virus, in particular for the process of disassembly of the glycoprotein shell during entry. The major outstanding questions in this area are also identified and discussed

Structural rearrangements between portal protein subunits are essential for viral DNA translocation

Transport of DNA into preformed procapsids is a general strategy for genome packing inside virus particles. In most viruses, this task is accomplished by a complex of the viral packaging ATPase with the portal protein assembled at a specialized vertex of the procapsid. Such molecular motor translocates DNA through the central tunnel of the portal protein. A central question to understand this mechanism is whether the portal is a mere conduit for DNA or whether it participates actively on DNA translocation. The most constricted part of the bacteriophage SPP1 portal tunnel is formed by twelve loops, each contributed from one individual subunit. The position of each loop is stabilized by interactions with helix alpha-5, which extends into the portal putative ATPase docking interface. Here, we have engineered intersubunit disulfide bridges between alpha-5s of adjacent portal ring subunits. Such covalent constraint blocked DNA packaging, whereas reduction of the disulfide bridges restored normal packaging activity. DNA exit through the portal in SPP1 virions was unaffected. The data demonstrate that mobility between alpha-5 helices is essential for the mechanism of viral DNA translocation. We propose that the alpha-5 structural rearrangements serve to coordinate ATPase activity with the positions of portal tunnel loops relative to the DNA double helix

Structure and interactions at the viral surface of the envelope protein E1 of Semliki Forest virus

International audienceSemliki Forest virus (SFV) is enveloped by a lipid bilayer enclosed within a glycoprotein cage made by glycoproteins E1 and E2. E1 is responsible for inducing membrane fusion, triggered by exposure to the acidic environment of the endosomes. Acidic pH induces E1/ E2 dissociation, allowing E1to interact with the target membrane, and, at the same time, to rearrange into E1 homotrimers that drive the membrane fusion reaction. We previously reported a preliminary C alpha trace of the monomeric E1 glycoprotein ectodomain and its organization on the virus particle. We also reported the 3.3 angstrom structure of the trimeric, fusogenic conformation of E1. Here, we report the crystal structure of monomeric E1 refined to 3 angstrom resolution and describe the amino acids involved in contacts in the virion. These results identify the major determinants for the E1/E2 icosahedral shell formation and open the way to rational mutagenesis approaches to shed light on SFV assembly

CryoEM Structure and Assembly Mechanism of a Bacterial Virus Genome Gatekeeper

International audienceNumerous viruses package their dsDNA genome into preformed capsids through a portal gatekeeper that is subsequently closed. We report the structure of the DNA gatekeeper complex of bacteriophage SPP1 (gp612gp1512gp166) in the post-DNA packaging state at 2.7 Å resolution obtained by single particle cryo-electron microscopy. Comparison of the native SPP1 complex with assembly-naïve structures of individual components uncovered the complex program of conformational changes leading to its assembly. After DNA packaging, gp15 binds via its C-terminus to the gp6 oligomer positioning gp15 subunits for oligomerization. Gp15 refolds its inner loops creating an intersubunit β-barrel that establishes different types of contacts with six gp16 subunits. Gp16 binding and oligomerization is accompanied by folding of helices that close the portal channel to keep the viral genome inside the capsid. This mechanism of assembly has broad functional and evolutionary implications for viruses of the prokaryotic tailed viruses-herpesviruses lineage

Crystal Structure of the Pestivirus Envelope Glycoprotein E(rns) and Mechanistic Analysis of Its Ribonuclease Activity.

International audiencePestiviruses, which belong to the Flaviviridae family of RNA viruses, are important agents of veterinary diseases causing substantial economical losses in animal farming worldwide. Pestivirus particles display three envelope glycoproteins at their surface: E(rns), E1, and E2. We report here the crystal structure of the catalytic domain of E(rns), the ribonucleolytic activity of which is believed to counteract the innate immunity of the host. The structure reveals a three-dimensional fold corresponding to T2 ribonucleases from plants and fungi. Cocrystallization experiments with mono- and oligonucleotides revealed the structural basis for substrate recognition at two binding sites previously identified for T2 RNases. A detailed analysis of poly-U cleavage products using (31)P-NMR and size exclusion chromatography, together with molecular docking studies, provides a comprehensive mechanistic picture of E(rns) activity on its substrates and reveals the presence of at least one additional nucleotide binding site

Purification and Crystallization Reveal Two Types of Interactions of the Fusion Protein Homotrimer of Semliki Forest Virus

The fusion proteins of the alphaviruses and flaviviruses have a similar native structure and convert to a highly stable homotrimer conformation during the fusion of the viral and target membranes. The properties of the alpha- and flavivirus fusion proteins distinguish them from the class I viral fusion proteins, such as influenza virus hemagglutinin, and establish them as the first members of the class II fusion proteins. Understanding how this new class carries out membrane fusion will require analysis of the structural basis for both the interaction of the protein subunits within the homotrimer and their interaction with the viral and target membranes. To this end we report a purification method for the E1 ectodomain homotrimer from the alphavirus Semliki Forest virus. The purified protein is trimeric, detergent soluble, retains the characteristic stability of the starting homotrimer, and is free of lipid and other contaminants. In contrast to the postfusion structures that have been determined for the class I proteins, the E1 homotrimer contains the fusion peptide region responsible for interaction with target membranes. This E1 trimer preparation is an excellent candidate for structural studies of the class II viral fusion proteins, and we report conditions that generate three-dimensional crystals suitable for analysis by X-ray diffraction. Determination of the structure will provide our first high-resolution views of both the low-pH-induced trimeric conformation and the target membrane-interacting region of the alphavirus fusion protein

Extensive flavivirus E trimer breathing accompanies stem zippering of the post‐fusion hairpin

International audienceFlaviviruses enter cells by fusion with endosomal membranes through a rearrangement of the envelope protein E, a class II membrane fusion protein, into fusogenic trimers. The rod-like E subunits bend into "hairpins" to bring the fusion loops next to the C-terminal transmembrane (TM) anchors, with the TM-proximal "stem" element zippering the E trimer to force apposition of the membranes. The structure of the complete class II trimeric hairpin is known for phleboviruses but not for flaviviruses, for which the stem is only partially resolved. Here, we performed comparative analyses of E-protein trimers from the tick-borne encephalitis fla-vivirus with sequential stem truncations. Our thermostability and antibody-binding data suggest that the stem "zipper" ends at a characteristic flavivirus conserved sequence (CS) that cloaks the fusion loops, with the downstream segment not contributing to trimer stability. We further identified a highly dynamic behavior of E trimers C-terminally truncated upstream the CS, which, unlike fully stem-zippered trimers, undergo rapid deuterium exchange at the trimer interface. These results thus identify important "breathing" intermediates in the E-protein-driven membrane fusion process

Conformational change and protein-protein interactions of the fusion protein of Semliki Forest virus

International audienceFusion of biological membranes is mediated by specific lipid- interacting proteins that induce the formation and expansion of an initial fusion pore. Here we report the crystal structure of the ectodomain of the Semliki Forest virus fusion glycoprotein E1 in its low- pH- induced trimeric form. E1 adopts a folded- back conformation that, in the final post- fusion form of the full- length protein, would bring the fusion peptide loop and the transmembrane anchor to the same end of a stable protein rod. The observed conformation of the fusion peptide loop is compatible with interactions only with the outer leaflet of the lipid bilayer. Crystal contacts between fusion peptide loops of adjacent E1 trimers, together with electron microscopy observations, suggest that in an early step of membrane fusion, an intermediate assembly of five trimers creates two opposing nipple- like deformations in the viral and target membranes, leading to formation of the fusion pore

Evolution and activation mechanism of the flavivirus class II membrane-fusion machinery

We thank the staff at beamlines PX1 and PX2 at SOLEIL synchrotron (St. Aubin, France) and at PX beamlines at the ESRF (Grenoble, France); A. Haouz and the staff at the crystallographic facility at Institut Pasteur; F. Agou from the Chemogenomic and Biological Screening platform at Institut Pasteur; P. Guardado Calvo and I. Fernandez for helping for the MALS experiments.International audienceThe flavivirus envelope glycoproteins prM and E drive the assembly of icosahedral, spiky immature particles that bud across the membrane of the endoplasmic reticulum. Maturation into infectious virions in the trans-Golgi network involves an acid-pH-driven rearrangement into smooth particles made of (prM/E)2 dimers exposing a furin site for prM cleavage into "pr" and "M". Here we show that the prM "pr" moiety derives from an HSP40 cellular chaperonin. Furthermore, the X-ray structure of the tick-borne encephalitis virus (pr/E)2 dimer at acidic pH revealed the E 150-loop as a hinged-lid that opens at low pH to expose a positively-charged prbinding pocket at the E dimer interface, inducing (prM/E)2 dimer formation to generate smooth particles in the Golgi. Furin cleavage is followed by lid-closure upon deprotonation in the neutral-pH extracellular environment, expelling pr while the 150-loop takes the relay in fusion loop protection, thus revealing the elusive flavivirus mechanism of fusion activation