New insight into flavivirus maturation from structure/function studies of the yellow fever virus envelope protein complex.

IMPORTANCE: All enveloped viruses enter cells by fusing their envelope with a target cell membrane while avoiding premature fusion with membranes of the producer cell-the latter being particularly important for viruses that bud at internal membranes. Flaviviruses bud in the endoplasmic reticulum, are transported through the TGN to reach the external milieu, and enter other cells via receptor-mediated endocytosis. The trigger for membrane fusion is the acidic environment of early endosomes, which has a similar pH to the TGN of the producer cell. The viral particles therefore become activated to react to mildly acidic pH only after their release into the neutral pH extracellular environment. Our study shows that for yellow fever virus (YFV), the mechanism of activation involves actively knocking out the fusion brake (protein pr) through a localized conformational change of the envelope protein upon exposure to the neutral pH external environment. Our study has important implications for understanding the molecular mechanism of flavivirus fusion activation in general and points to an alternative way of interfering with this process as an antiviral treatment.

Novel requirements for HAP2-mediated gamete fusion in Tetrahymena.

The ancestral gamete fusion protein, HAP2, catalyzes sperm-egg fusion in a broad range of taxa dating to the last eukaryotic common ancestor. Remarkably, HAP2 orthologs are structurally related to the class II fusogens of modern-day viruses, and recent studies make clear that these proteins utilize similar mechanisms to achieve membrane merger. To identify factors that may regulate HAP2 activity, we screened mutants of the ciliate Tetrahymena thermophila for behaviors that mimic Δhap2 knockout phenotypes in this species. Using this approach, we identified two new genes, GFU1 and GFU2, whose products are necessary for the formation of membrane pores during fertilization and show that the product of a third gene, namely ZFR1, may be involved in pore maintenance and/or expansion. Finally, we propose a model that explains cooperativity between the fusion machinery on apposed membranes of mating cells and accounts for successful fertilization in T. thermophila's multiple mating type system.

The prefusion structure of herpes simplex virus glycoprotein B.

Cell entry of enveloped viruses requires specialized viral proteins that mediate fusion with the host membrane by substantial structural rearrangements from a metastable pre- to a stable postfusion conformation. This metastability renders the herpes simplex virus 1 (HSV-1) fusion glycoprotein B (gB) highly unstable such that it readily converts into the postfusion form, thereby precluding structural elucidation of the pharmacologically relevant prefusion conformation. By identification of conserved sequence signatures and molecular dynamics simulations, we devised a mutation that stabilized this form. Functionally locking gB allowed the structural determination of its membrane-embedded prefusion conformation at sub-nanometer resolution and enabled the unambiguous fit of all ectodomains. The resulting pseudo-atomic model reveals a notable conservation of conformational domain rearrangements during fusion between HSV-1 gB and the vesicular stomatitis virus glycoprotein G, despite their very distant phylogeny. In combination with our comparative sequence-structure analysis, these findings suggest common fusogenic domain rearrangements in all class III viral fusion proteins.

A glycerophospholipid-specific pocket in the RVFV class II fusion protein drives target membrane insertion.

The Rift Valley fever virus (RVFV) is transmitted by infected mosquitoes, causing severe disease in humans and livestock across Africa. We determined the x-ray structure of the RVFV class II fusion protein Gc in its postfusion form and in complex with a glycerophospholipid (GPL) bound in a conserved cavity next to the fusion loop. Site-directed mutagenesis and molecular dynamics simulations further revealed a built-in motif allowing en bloc insertion of the fusion loop into membranes, making few nonpolar side-chain interactions with the aliphatic moiety and multiple polar interactions with lipid head groups upon membrane restructuring. The GPL head-group recognition pocket is conserved in the fusion proteins of other arthropod-borne viruses, such as Zika and chikungunya viruses, which have recently caused major epidemics worldwide.

Prevalence of Equine Hepacivirus Infections in France and Evidence for Two Viral Subtypes Circulating Worldwide.

Like hepatitis C virus (HCV) in humans, the newly identified equine hepacivirus (NPHV) displays a predominating liver tropism that may evolve into chronic infections. The genomes of the two viruses share several organizational and functional features and are phylogenetically closest amongst the Hepacivirus genus. A limited amount of data is available regarding the spread of hepacivirus infections in horses. In this study, we asked whether in a more representative sample the prevalence and distribution of NPHV infections in France would resemble that reported so far in other countries. A total of 1033 horses sera from stud farms throughout France were analysed by qRT-PCR to determine the prevalence of ongoing NPHV infections and viral loads; in positive samples, partial sequences of NPHV's genome (5'UTR, NS3 and NS5B genes) were determined. Serum concentrations of biliary acids, glutamate dehydrogenase (GLDH) and L-gamma-glutamyl transferase (γ-GT) were measured for most horses. We detected NPHV infections in 6.2% of the horses, a prevalence that reached 8.3% in thoroughbreds and was significantly higher than in other breeds. The presence of circulating virus was neither significantly associated with biological disturbances nor with clinical hepatic impairment. Our phylogenetic analysis was based on both neighbour-joining and maximum-likelihood approaches. Its result shows that, like almost everywhere else in the world so far, two major groups of NPHV strains infect French domestic horses. Based on genetic distances, we propose a classification into two separate NPHV subtypes. Viral loads in the serum of horses infected by the main subtype were, in average, four times higher than in those infected by the second subtype. We hypothesize that amino acid substitutions in the palm domain of NS5B between NPHV subtypes could underlie viral phenotypes that explain this result.

Phasing of the Triatoma virus diffraction data using a cryo-electron microscopy reconstruction.

The blood-sucking reduviid bug Triatoma infestans, one of the most important vector of American human trypanosomiasis (Chagas disease) is infected by the Triatoma virus (TrV). TrV has been classified as a member of the Cripavirus genus (type cricket paralysis virus) in the Dicistroviridae family. This work presents the three-dimensional cryo-electron microscopy (cryo-EM) reconstruction of the TrV capsid at about 25 A resolution and its use as a template for phasing the available crystallographic data by the molecular replacement method. The main structural differences between the cryo-EM reconstruction of TrV and other two viruses, one from the same family, the cricket paralysis virus (CrPV) and the human rhinovirus 16 from the Picornaviridae family are presented and discussed.

On the fitting of model electron densities into EM reconstructions: a reciprocal-space formulation.

A fast method for fitting model electron densities into EM reconstructions is presented. The methodology was inspired by the molecular-replacement technique, adapted to take into account phase information and the symmetry imposed during the EM reconstruction. Calculations are performed in reciprocal space, which enables the selection of large volumes of the EM maps, thus avoiding the bias introduced when defining the boundaries of the target density.

Molecular organization of a recombinant subviral particle from tick-borne encephalitis virus.

The tick-borne encephalitis (TBE) flavivirus contains two transmembrane proteins, E and M. Coexpression of E and the M precursor (prM) leads to secretion of recombinant subviral particles (RSPs). In the most common form of these RSPs, analyzed at a 19 A resolution by cryo-electron microscopy (cryo-EM), 60 copies of E pack as dimers in a T = 1 icosahedral surface lattice (outer diameter, 315 A). Fitting the high-resolution structure of a soluble E fragment into the RSP density defines interaction sites between E dimers, positions M relative to E, and allows assignment of transmembrane regions of E and M. Lateral interactions among the glycoproteins stabilize this capsidless particle; similar interactions probably contribute to assembly of virions. The structure suggests a picture for trimer association under fusion-inducing conditions.

The Fusion glycoprotein shell of Semliki Forest virus: an icosahedral assembly primed for fusogenic activation at endosomal pH.

Semliki Forest virus (SFV) has been extensively studied as a model for analyzing entry of enveloped viruses into target cells. Here we describe the trace of the polypeptide chain of the SFV fusion glycoprotein, E1, derived from an electron density map at 3.5 A resolution and describe its interactions at the surface of the virus. E1 is unexpectedly similar to the flavivirus envelope protein, with three structural domains disposed in the same primary sequence arrangement. These results introduce a new class of membrane fusion proteins which display lateral interactions to induce the necessary curvature and direct budding of closed particles. The resulting surface protein lattice is primed to cause membrane fusion when exposed to the acidic environment of the endosome.

Atomic structure of the major capsid protein of rotavirus: implications for the architecture of the virion.

The structural protein VP6 of rotavirus, an important pathogen responsible for severe gastroenteritis in children, forms the middle layer in the triple-layered viral capsid. Here we present the crystal structure of VP6 determined to 2 A resolution and describe its interactions with other capsid proteins by fitting the atomic model into electron cryomicroscopic reconstructions of viral particles. VP6, which forms a tight trimer, has two distinct domains: a distal beta-barrel domain and a proximal alpha-helical domain, which interact with the outer and inner layer of the virion, respectively. The overall fold is similar to that of protein VP7 from bluetongue virus, with the subunits wrapping about a central 3-fold axis. A distinguishing feature of the VP6 trimer is a central Zn(2+) ion located on the 3-fold molecular axis. The crude atomic model of the middle layer derived from the fit shows that quasi-equivalence is only partially obeyed by VP6 in the T = 13 middle layer and suggests a model for the assembly of the 260 VP6 trimers onto the T = 1 viral inner layer.

Structural polymorphism of the major capsid protein of rotavirus.

Rotaviruses are important human pathogens with a triple-layered icosahedral capsid. The major capsid protein VP6 is shown here to self-assemble into spherical or helical particles mainly depending upon pH. Assembly is inhibited either by low pH (<3.0) or by a high concentration (>100 mM) of divalent cations (Ca(2+) and Zn(2+)). The structures of two types of helical tubes were determined by electron cryomicroscopy and image analysis to a resolution of 2.0 and 2.5 nm. In both reconstructions, the molecular envelope of VP6 fits the atomic model determined by X-ray crystallography remarkably well. The 3-fold symmetry of the VP6 trimer, being incompatible with the helical symmetry, is broken at the level of the trimer contacts. One type of contact is maintained within all VP6 particles (tubes and virus), strongly suggesting that VP6 assemblies arise from different packings of a unique dimer of trimers. Our data show that the protonation state and thus the charge distribution are important switches governing the assembly of macromolecular assemblies.

Determinants in the envelope E protein and viral RNA helicase NS3 that influence the induction of apoptosis in response to infection with dengue type 1 virus.

One mechanism by which dengue (DEN) virus may cause cell death is apoptosis. In this study, we investigated whether the genetic determinants responsible for acquisition by DEN type 1 (DEN-1) virus of mouse neurovirulence interfere with the induction of apoptosis. Neurovirulent variant FGA/NA d1d was generated during the adaptation of the human isolate of DEN-1 virus strain FGA/89 to grow in newborn mouse brains and mosquito cells in vitro [Desprès, P. Frenkiel, M. -P. Ceccaldi, P.-E. Duarte Dos Santos, C. and Deubel, V. (1998) J. Virol., 72: 823-829]. Genetic determinants possibly responsible for mouse neurovirulence were studied by sequencing the entire genomes of both DEN-1 viruses. Three amino acid differences in the envelope E protein and one in the nonstructural NS3 protein were found. The cytotoxicity of the mouse-neurovirulent DEN-1 variant was studied in different target cells in vitro and compared with the parental strain. FGA/NA d1d was more pathogenic for mouse neuroblastoma cells and attenuated for human hepatoma cells. Changes in virus replicative functions and virus assembly may account, in a large part, for the differences in the induction of apoptosis. Our data suggest that identified amino acid substitutions in the envelope E protein and viral RNA helicase NS3 may influence DEN-1 virus pathogenicity by altering viral growth.

Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus.

We report the crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus, a major human pathogen, to 2.8-A resolution. This enzyme is a key target for developing specific antiviral therapy. The structure of the catalytic domain contains 531 residues folded in the characteristic fingers, palm, and thumb subdomains. The fingers subdomain contains a region, the "fingertips," that shares the same fold with reverse transcriptases. Superposition to the available structures of the latter shows that residues from the palm and fingertips are structurally equivalent. In addition, it shows that the hepatitis C virus polymerase was crystallized in a closed fingers conformation, similar to HIV-1 reverse transcriptase in ternary complex with DNA and dTTP [Huang H., Chopra, R., Verdine, G. L. & Harrison, S. C. (1998) Science 282, 1669-1675]. This superposition reveals the majority of the amino acid residues of the hepatitis C virus enzyme that are likely to be implicated in binding to the replicating RNA molecule and to the incoming NTP. It also suggests a rearrangement of the thumb domain as well as a possible concerted movement of thumb and fingertips during translocation of the RNA template-primer in successive polymerization rounds.

Dengue virus type 1 nonstructural glycoprotein NS1 is secreted from mammalian cells as a soluble hexamer in a glycosylation-dependent fashion.

Nonstructural glycoprotein NS1, specified by dengue virus type 1 (Den-1), is secreted from infected green monkey kidney (Vero) cells in a major soluble form characterized by biochemical and biophysical means as a unique hexameric species. This noncovalently bound oligomer is formed by three dimeric subunits and has a molecular mass of 310 kDa and a Stokes radius of 64.4 A. During protein export, one of the two oligosaccharides of NS1 is processed into an endo-beta-N-acetylglucosaminidase F-resistant complex-type sugar while the other remains of the polymannose type, protected in the dimeric subunit from the action of maturation enzymes. Complete processing of the complex-type sugar appears to be required for efficient release of soluble NS1 into the culture fluid of infected cells, as suggested by the repressive effects of the N-glycan processing inhibitors swainsonine and deoxymannojyrimicin. These results, together with observations related to the absence of secretion of NS1 from Den-infected insect cells, suggest that maturation and secretion of hexameric NS1 depend on the glycosylation status of the host cell.

The isolation of the ectodomain of the alphavirus E1 protein as a soluble hemagglutinin and its crystallization.

Alphaviruses are isometric enveloped viruses approximately 70 nm in diameter. The viral surface contains 80 glycoprotein spikes arranged in a T = 4 lattice. Each of these spikes consists of three heterodimers of the viral membrane proteins E1 (approximately 49 kDa) and E2 (approximately 51 kDa). Cryoelectron microscopic analyses have shown that the spikes form a protein shell on the viral surface. We have made an attempt to isolate biologically active protein fragments from this surface and to grow crystals from such fragments. To this end membrane proteins were extracted with Nonidet-P40 from the Semliki Forest alphavirus and the proteins were separated from detergent by centrifugation. A protein complex containing the E1 and E2 molecules in quantitative yield was obtained by this procedure. This complex has the following properties: It sediments at approximately 30S, it chromatographs with an apparent molecular mass of approximately 580,000 Da during gel filtration, it cannot be dissociated by either nonionic detergents or 6 M urea, and at acid pH it is a highly active hemagglutinin. The data indicate that this 30S hemagglutinin complex, which has not been hitherto described for alphaviruses, may represent a variant form of the protein lattice present on the alphavirus surface. Cleavage of this complex by subtilisin selectively removes carboxy-terminal sequences from the E1 and E2 proteins, which contain the cytoplasmic and transmembrane segments of the proteins and a small part of their ectodomain. The remaining ectodomains are called E1DeltaS and E2DeltaS. This proteolysis also leads to dissociation of the 30S complex. The cleavage products accumulate in the form of a heterodimer of the E1DeltaS and E2DeltaS proteins. Treatment of the heterodimer with PNGase F leads to rapid removal of carbohydrate from the E2DeltaS protein and a dissociation of the complex into the constituent molecules, which can be separated by chromatography. The finding that the heterodimer and the purified E1DeltaS protein both function as hemagglutinin at acid pH indicates that the E1 protein represents the alphavirus hemagglutinin. We have obtained crystals of the E1DeltaS protein and are currently in the process of determining the atomic structure of this protein by the isomorphous replacement method.

Crystallization and preliminary X-Ray analysis of rotavirus protein VP6.

As a first step to gain insight into the structure of the rotavirus virion at atomic resolution, we report here the expression, purification, and crystallization of recombinant rotavirus protein VP6. This protein has the property of polymerizing in the form of tubular structures in solution which have hindered crystallization thus far. Using a combination of electron microscopy and small-angle X-ray scattering, we found that addition of Ca2+ at concentrations higher than 100 mM results in depolymerization of the tubes, leading to an essentially monodisperse solution of trimeric VP6 even at high protein concentrations (higher than 10 mg/ml), thereby enabling us to search for crystallization conditions. We have thus obtained crystals of VP6 which diffract to better than 2.4 A resolution and belong to the cubic space group P4132 with a cell dimension a of 160 A. The crystals contain a trimer of VP6 lying along the diagonal of the cubic unit cell, resulting in one VP6 monomer per asymmetric unit and a solvent content of roughly 70%.

Comparison of two different DNA-binding modes of the NF-kappa B p50 homodimer.

Analysis of the NF-kappa B p50 homodimer bound to different DNA sequences shows that the protein can recognize half-site spacings of either three or four base pairs. The protein can maintain most of its DNA contacts by a relative reorientation of its two domains.

The envelope glycoprotein from tick-borne encephalitis virus at 2 A resolution.

The crystallographically determined structure of a soluble fragment from the major envelope protein of a flavivirus reveals an unusual architecture. The flat, elongated dimer extends in a direction that would be parallel to the viral membrane. Residues that influence binding of monoclonal antibodies lie on the outward-facing surface of the protein. The clustering of mutations that affect virulence in various flaviviruses indicates a possible receptor binding site and, together with other mutational and biochemical data, suggests a picture for the fusion-activating, conformational change triggered by low pH.

Structure of the NF-kappa B p50 homodimer bound to DNA.

The structure of a large fragment of the p50 subunit of the human transcription factor NF-kappa B, bound as a homodimer to DNA, reveals that the Rel-homology region has two beta-barrel domains that grip DNA in the major groove. Both domains contact the DNA backbone. The amino-terminal specificity domain contains a recognition loop that interacts with DNA bases; the carboxy-terminal dimerization domain bears the site of I-kappa B interaction. The folds of these domains are related to immunoglobulin-like modules. The amino-terminal domain also resembles the core domain of p53.

FFT method to compute solution X-ray scattering curves.

We present an efficient algorithm to compute X-ray intensities scattered by macromolecules in solution, from atomic positions found in crystal structures. The algorithm applies the Fast Fourier Transform to an electron density map created from the atomic coordinates and corrected for solvent density. We compute scattering curves for both allosteric forms of E. coli aspartate carbamoyltransferase. Calculated intensities are in agreement with the ones measured by Moody et al. which shows that the structures observed in solution in the presence or in the absence of a substrate analogue do correspond to those of two crystal forms analyzed by Lipscomb and collaborators .