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.

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.

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.

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.

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.

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.

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.

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.