Folding and Dimerization of Tick-Borne Encephalitis Virus Envelope Proteins prM and E in the Endoplasmic Reticulum (original) (raw)
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Journal of Virology, 2003
Flavivirus envelope proteins have been shown to play a major role in virus assembly. These proteins are anchored into cellular and viral membranes by their C-terminal domain. These domains are composed of two hydrophobic stretches separated by a short hydrophilic segment containing at least one charged residue. We investigated the role of the transmembrane domains of prM and E in the envelope formation of the flavivirus yellow fever virus (YFV). Alanine scanning insertion mutagenesis has been used to examine the role of the transmembrane domains of prM and E in YFV subviral particle formation. Most of the insertions had a dramatic effect on the release of YFV subviral particles. Some of these mutations were introduced into the viral genome. The ability of these mutant viruses to produce infectious particles was severely reduced. The alanine insertions did not affect prM-E heterodimerization. In addition, replacement of the charged residues present in the middle of the transmembrane domains had no effect on subviral particle release. Taken together, these data indicate that the transmembrane domains of prM and E play a crucial role in the biogenesis of YFV envelope. In addition, these data indicate some differences between the transmembrane domains of the hepaciviruses and the flaviviruses.
1996
The exposure of the flavivirus tick-borne encephalitis (TBE) virus to an acidic pH is necessary for virusinduced membrane fusion and leads to a quantitative and irreversible conversion of the envelope protein E dimers to trimers. To study the structural requirements for this oligomeric rearrangement, the effect of low-pH treatment on the oligomeric state of different isolated forms of protein E was investigated. Full-length E dimers obtained by solubilization of virus with the detergent Triton X-100 formed trimers at low pH, whereas truncated E dimers lacking the stem-anchor region underwent a reversible dissociation into monomers without forming trimers. These data suggest that the low-pH-induced rearrangement in virions is a two-step process involving a reversible dissociation of the E dimers followed by an irreversible formation of trimers, a process which requires the stem-anchor portion of the protein. This region contains potential amphipathic ␣-helical and conserved structural elements whose interactions may contribute to the rearrangements which initiate the fusion process.
Biochemical and Biophysical Research Communications, 2002
Bovine viral diarrhea virus (BVDV) is a pestivirus member of the Flaviviridae family, closely related to, and used as a surrogate model for the hepatitis C virus. Its envelope contains the E1 and E2 glycoproteins, disulfide linked into homo-and heterodimers. In this study, we investigate the role of disulfide bond formation in the folding, assembly, and stability of BVDV glycoproteins. We provide molecular evidence that intact disulfide bonds are critical for the acquirement of a stable conformation of E2 monomers. Forcing the E2 glycoproteins to adopt a reduced conformation either co-or post-translationally before assembly into dimers, determines their misfolding and degradation by proteasome. In contrast, dimerization of E2 glycoproteins results in a conformation resistant to reducing agents and degradation. Furthermore, inhibition of the ER-a-mannosidase activity leads to impairment of misfolded E2 degradation, demonstrating the involvement of this enzyme in targeting viral proteins towards proteasomal degradation. Ó
The structure of immature tick-borne encephalitis virus
Tick-borne encephalitis virus (TBEV) is a medically important flavivirus that poses a significant health threat in Europe and Asia. However, the structure of the immature form of TBEV remains unknown. Here, we employed state-of-the-art cryogenic electron microscopy (cryoEM) to determine the structure of the immature TBEV particle. The immature TBEV particle has a diameter of 56 nm and its surface glycoproteins are organised into spikes characteristic of immature flaviviruses. The cryoEM reconstructions of the whole virus and of the individual spike enabled us to build atomic models of the major viral components, the E and prM proteins. The insights obtained from our study provide a foundation for understanding the early stages of TBEV assembly and maturation. The pr domains of prM have a critical role in holding the heterohexameric prM3E3 spikes in metastable conformation. Destabilisation of the prM furin-sensitive loop at acidic pH facilitates its processing. The prM cleavage, the ...
Conformational Changes of the Flavivirus E Glycoprotein
Structure, 2004
ies. These functions are frequently distributed among several different viral proteins, but in the flaviviruses, 1 Department of Biological Sciences Lilly Hall the envelope glycoprotein (E) participates in all these functions. After entering cells by receptor-mediated en-915 West State Street Purdue University docytosis, fusion with the host membrane is initiated by low pH induced conformational change of the E protein West Lafayette, Indiana 47907 2 Hawaii Biotech Inc. (Heinz and Allison, 2000), followed by release of the genomic RNA into the cytoplasm. The genomic RNA is 99-193 Aiea Heights Drive Suite 200 translated into a polyprotein in which capsid, precursor membrane (prM), and E structural proteins reside in the Aiea, Hawaii 96701 3 Division of Biology 156-29 N-terminal region. The polyprotein is processed on the endoplasmic reticulum membrane into individual pro-California Institute of Technology Pasadena, California 91125 teins by both viral and cellular proteases and assembled together with the genomic RNA into immature particles (Mackenzie and Westaway, 2001). These contain 60 asymmetric trimers of prM-E heterodimers (Zhang et al., Summary 2003b). A substantial portion of the prM protein covers E, thereby protecting it against premature fusion while Dengue virus, a member of the Flaviviridae family, has passing through the acidic environment of the transa surface composed of 180 copies each of the enve-Golgi network (TGN) (Guirakhoo et al., 1992). The 100 lope (E) glycoprotein and the membrane (M) protein. N-terminal amino acids of the prM protein (the prepep-The crystal structure of an N-terminal fragment of E tide, pr) are then released by furin cleavage (Stadler et has been determined and compared with a previously al., 1997) in the final step of maturation. This cleavage described structure. The primary difference between induces a rearrangement of the E proteins and activates these structures is a 10؇ rotation about a hinge relating the virus to be fusogenic. In the mature virus, E proteins the fusion domain DII to domains DI and DIII. These exist as homodimers that lie on the viral membrane, two rigid body components were used for independent arranged as 30 "rafts" organized into a herringbone patfitting of E into the cryo-electron microscopy maps of tern (Figure 1). Each raft contains three parallel dimers, both immature and mature dengue viruses. The fitted with the center of the raft coincident with an icosahedral E structures in these two particles showed a difference 2-fold axis (Kuhn et al., 2002; Mukhopadhyay et al., 2003; of 27؇ between the two components. Comparison of Zhang et al., 2003a). the E structure in its postfusion state with that in the A large tryptic fragment of the E protein (sE, also immature and mature virions shows a rotation approxreferred to here as the ectodomain of E) of TBEV (resiimately around the same hinge. Flexibility of E is appardues 1-395) was the first flavivirus component whose ently a functional requirement for assembly and infecatomic structure was determined (Rey et al., 1995). The tion of flaviviruses. structure of sE consists of three domains: domain I (DI), the N-terminal, but structurally central, domain; domain Introduction II (DII), the fusion (or dimerization) domain containing the hydrophobic fusion peptide (residues 98-110) (Allison et Many flaviviruses, such as dengue (DEN), yellow fever al., 2001); and domain III (DIII), the putative receptor (YFV), West Nile (WNV), and tick-borne encephalitis virus binding domain (Bhardwaj et al., 2001). The structure of (TBEV), are arthropod-borne, human pathogens. They a DEN serotype 2 sE dimer (DEN sE Harvard, DEN sE(H)) can cause various diseases, including jaundice, encephhas also been determined both with and without n-octylalitis, hemorrhagic fevers, and shock syndrome (Lin--D-glucoside (-OG) detergent bound to sE (Modis et denbach and Rice, 2001). Currently, there are no efficaal., 2003). The difference between the two DEN sE(H) cious antiviral drugs against flavivirus infections, and structures is a rearrangement of the "kl" hairpin (Figure vaccines are only available for YFV, TBEV, and Japanese 1), which is "open" on binding -OG. This hairpin forms encephalitis virus (JEV). Flaviviruses can be subdivided part of the "hinge" that connects domains DI and DII. into those transmitted by ticks, by mosquitoes, and Other structures of sE from flaviviruses, formed by treatthose that apparently lack an arthropod vector (Kuno et ment with acid and lipid to produce the postfusion state, al., 1998). have been reported very recently (Bressanelli et al., In membrane-containing viruses, the external struc-2004; Modis et al., 2004). These structures show a trimer tural proteins function to bind to cellular receptors and in which the fusion peptides of each monomer are at to interact with host cell membranes for fusion and subone end and domains DI and DIII are at the other. The "stem-anchor," C-terminal 100 amino acids of DEN E are absent in sE, but they have been identified in virions
Scientific Reports, 2017
Dengue and Zika are two of the most important human viral pathogens worldwide. In both cases, the envelope glycoprotein E is the main target of the antibody response. Recently, new complex quaternary epitopes were identified which are the consequence of the arrangement of the antiparallel E dimers on the viral surface. Such epitopes can be exploited to develop more efficient cross-neutralizing vaccines. Here we describe a successful covalent stabilization of E dimers from Dengue and Zika viruses in mammalian cells. Folding and dimerization of secretory E was found to be strongly dependent on temperature but independent of PrM co-expression. In addition, we found that, due to the close relationship between flaviviruses, Dengue and Zika viruses E proteins can form heterodimers and assemble into mosaic viral particles. Finally, we present new virus-free analytical platforms to study and screen antibody responses against Dengue and Zika, which allow for differentiation of epitopes restricted to specific domains, dimers and higher order arrangements of E. The Flaviviridae family includes some of the most important arthropod-borne human pathogens such as Dengue virus (DENV), Zika virus (ZIKV), West Nile virus (WNV) and Yellow fever virus (YFV) 1. Over the last three decades, DENV infections have increased at an unprecedented rate and are now one of the most important human infectious diseases worldwide, with an estimated annual incidence of 390 million cases, 100 million of which show clinical manifestations of the infection 2. Until recently, ZIKV infections were sporadic, mostly asymptomatic and restricted to specific regions in Africa and Southeast Asia 3. However, since the 2007 outbreak in the South Pacific islands, the virus has spread rapidly to a global scale that almost matches the distribution of dengue and is now associated with serious neurological and developmental pathologies 3, 4. Like all flaviviruses, DENV and ZIKV are enveloped viruses with a ≈11 Kb single-stranded, positive-sense RNA genome which codes for a single viral polyprotein that is processed into 10 mature viral proteins: 3 structural (Capsid (C), pre-membrane (PrM) and envelope glycoprotein (E)) and 7 non-structural (NS) proteins (NS1,-2A,-2B,-3,-4A,-4B and-5) 5. Of these, the E glycoprotein, a class II viral membrane fusion protein, covers almost the entire surface of the viral particle, serving pivotal functions during viral assembly and internalization 6. Based on the sequence of this antigenic protein, DENV is composed of 4 closely related serotypes (DENV1, DENV2, DENV3 and DENV4) while ZIKV has been shown to involve a single serotype 7. On the surface of the mature viral particle, E folds into an elongated rod-like structure forming 90 antiparallel homodimers, organized in 30 rafts, each composed of 3 parallel E dimers distributed in a herringbone-like configuration 8. The E protein ectodomain, also termed soluble E (sE), is formed by three different structural domains named DI, DII and DIII. DI has an 8-stranded β-barrel structure and is located at the center of the monomer with an axis parallel to the viral membrane 9. DII is formed by two coding segments that fold together in an elongated finger-like structure with a highly stable core composed of an antiparallel 5-stranded β-sheet and 2 α-helices from which an elongated 3-stranded β-sheet expands distally, forming two loops 10. The most distal one (cd loop)
Tyrosine 78 of premembrane protein is essential for assembly of West Nile virus
Journal of General …, 2009
Flavivirus premembrane (prM) protein plays an important role in conformational folding of the envelope (E) protein and protects it against premature fusion in acidic vesicles of the Golgi network. Currently, molecular determinants on the prM protein ectodomain which mediate critical steps during the flavivirus assembly process are poorly characterized. In this study, bioinformatics analysis and alanine scanning mutagenesis showed that the amino acid triplet valine 76, tyrosine 78 and glycine 79 is absolutely conserved among flavivirus prM ectodomains. Triple mutations engineered at these residues in prM ectodomain of West Nile virus (WNV) completely abrogated virus infectivity. Site-directed mutagenesis of prM protein revealed that tyrosine 78 of the amino acid triplet was required for virus infectivity and secretion. The mutation did not affect folding, post-translational modifications and trafficking of the prM and E proteins. Ultrastructural studies using transmission electron microscopy confirmed that virus particle formation was blocked by tyrosine 78 mutation. Specificity of assembly defect conferred by tyrosine 78 mutation was demonstrated by positive and negative trans complementation studies. Collectively, these results defined tyrosine 78 as a novel critical determinant present on prM protein ectodomain that is required for flavivirus assembly. Molecular dissection of prM protein function provides the crucial knowledge much needed in the elucidation of flavivirus particle formation. In order to elucidate the functional residues of prM protein involved in virus assembly, the E protein-binding domain was mapped using a yeast two-hybrid truncation study. Alanine mutagenesis was performed on conserved amino acids of the envelope-binding domain of prM protein selected through a bioinformatic analysis. Our study demonstrated that a triple mutation at the prM ectodomain completely abolished WNV infectivity in mammalian Supplementary material is available with the online version of this paper.