Article:Tick-Borne Encephalitis Virus: A Structural View (6071267)

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This page is the ScienceSource HTML version of the scholarly article described at https://www.wikidata.org/wiki/Q56476080. Its title is Tick-Borne Encephalitis Virus: A Structural View and the publication date was 2018-06-28. The initial author is Lauri I. A. Pulkkinen.

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Converted JATS paper:

Journal Information

Title: Viruses

Tick-Borne Encephalitis Virus: A Structural View

  • Lauri I. A. Pulkkinen
  • Sarah J. Butcher
  • Maria Anastasina

1HiLIFE—Institute of Biotechnology, University of Helsinki, 00790 Helsinki, Finland; lauri.ia.pulkkinen@helsinki.fi (L.I.A.P.); sarah.butcher@helsinki.fi (S.J.B.)

2Faculty of Biological and Environmental Sciences, University of Helsinki, 00790 Helsinki, Finland

Publication date (epub): 6/2018

Publication date (collection): 7/2018

Abstract

Tick-borne encephalitis virus (TBEV) is a growing health concern. It causes a severe disease that can lead to permanent neurological complications or death and the incidence of TBEV infections is constantly rising. Our understanding of TBEV’s structure lags behind that of other flaviviruses, but has advanced recently with the publication of a high-resolution structure of the TBEV virion. The gaps in our knowledge include: aspects of receptor binding, replication and virus assembly. Furthermore, TBEV has mostly been studied in mammalian systems, even though the virus’ interaction with its tick hosts is a central part of its life cycle. Elucidating these aspects of TBEV biology are crucial for the development of TBEV antivirals, as well as the improvement of diagnostics. In this review, we summarise the current structural knowledge on TBEV, bringing attention to the current gaps in our understanding, and propose further research that is needed to truly understand the structural-functional relationship of the virus and its hosts.

Paper

1. Introduction

Tick-borne encephalitis virus (TBEV) is a major tick-borne viral pathogen of humans. Most TBEV infections are asymptomatic, but the symptomatic cases typically have neurological manifestations, such as meningitis, encephalitis, and meningoencephalitis and, together, are referred to as tick-borne encephalitis (TBE) [[1],[2]]. TBE is a severe disease that often results in life-long neurological complications and can lead to death [[1],[2]]. The morbidity and mortality of TBE varies depending on the viral subtype, these are the European, the Siberian, and the Far-Eastern (TBEV-Eu, TBEV-Sib, and TBEV-FE, respectively) [[1],[2],[3],[4]]. TBEV-Eu is associated with neurological sequelae in up to 10% of patients, with a 0.5–2% mortality rate, and TBEV-Sib patients are prone to develop prolonged infections with a 2–3% mortality rate, whereas TBEV-FE is associated with high rates of neurological sequelae, and up to 40% of cases are fatal [[1],[2]]. Interestingly, the amino acid sequence variation in the polyprotein is low: up to 2.2% within and up to 5.6% between subtypes [[5]]. Thus, the determinants of virulence could be limited to a handful of amino acid residues in the viral proteins and/or to variable non-coding regions in the viral genome, but have not been investigated in detail [[6],[7]]. Infection with any subtype is serious, but TBEV-FE infection is the most severe.

TBEV is endemic to Northern Eurasia and it has been estimated that there are at least 10,000 clinical cases annually, with probable underreporting [[2],[8],[9]]. The virus is usually transmitted by ticks of the Ixodideae family, but TBEV infections can also occur via the consumption of unpasteurized contaminated dairy products [[1],[10],[11]]. Despite the availability of efficient vaccines for disease prevention, the incidence of TBE is on the rise as vaccine coverage is insufficient for many risk groups [[2],[12],[13]]. Another significant factor behind the TBE rise is global climate change, increasing the ticks’ abundance and expanding their habitats [[14],[15]]. It is, therefore, likely that we will observe further emergence of TBEV in the upcoming decades, which calls for the development of specific antivirals for TBEV, to complement the palliative care now available [[1],[2]].

The three TBEV subtypes are members of the genus Flavivirus in the family Flaviviridae along with other important human pathogens, such as Zika virus (ZIKV), dengue virus (DENV), West Nile virus (WNV), and Japanese encephalitis virus (JEV) [[3],[4],[16],[17]]. The latter are transmitted by mosquitoes and have been extensively studied due to their significant health care threat. Tick-borne flaviviral pathogens, such as TBEV, Omsk haemorrhagic fever virus (OHFV), Powassan virus, and the emerging Alkhurma virus, have received significantly less attention compared to their mosquito-borne counterparts. Even though TBEV has been studied more than the other tick-borne flaviviruses, many of its characteristics are poorly understood. In particular, our understanding of structural details of TBEV infection is mostly based on extrapolations from the better-characterised mosquito-borne species.

The field has advanced recently with the publication of a high-resolution structure of TBEV virion alone and in complex with a neutralizing antibody, but our understanding of the details of TBEV structure and function still needs improvement [[18]]. In this review, we summarize current structural knowledge on TBEV, and highlight further avenues for research.

2. The Structure of TBEV Particles

TBEV has a ~11 kilobase-long positive-strand RNA (+RNA) genome that encodes a single polyprotein (UniProt: Q01299, P14336, and P07720) that is processed co- and post-transcriptionally into three structural proteins (SP) and seven non-structural proteins (nSP) [[19]]. Flaviviruses undergo maturation during their production, and infected cells produce at least three types of particles: immature non-infectious particles, partially-mature, and mature infectious particles (Figure 1A) [[18],[19],[20]]. The mature TBEV particles are smooth and have a diameter of 50 nm like other flaviviruses [[18],[21],[22],[23],[24],[25]]. The virion consists of a nucleocapsid (NC) surrounded by a membrane composed of host-derived lipids in which the viral envelope (E) and membrane (M) proteins are embedded (Figure 1B) [[18]]. The transmembrane domains of the E and M proteins distort the lipid envelope making it slightly angular [[18]]. This is a common flavivirus characteristic [[18],[21],[22],[23],[24],[25],[26]]. The NC is made up of multiple copies of the capsid protein (C) and a single copy of the genome [[19]]. Just as with the icosahedrally-symmetric cryo-EM reconstructions of other flaviviruses, the TBEV NC is not resolved as it does not follow this symmetry, but the E and M protein are seen to ‘coat’ the lipid bilayer in an organised fashion [[18],[21],[22],[23],[24],[25],[27]]. They form heterodimers and three E-M dimers constitute the asymmetric unit of the icosahedrally-symmetric TBEV virion [[18],[21],[22],[23],[24],[25]] (Figure 1C). The main building block of the virion is an E-M-M-E heterotetramer that is formed by head-to-tail dimerization of two E-M heterodimers (Figure 1D and Figure 2A) [[18]].

2.1. Envelope Proteins

The E glycoprotein (496 residues) is the major component of the mature TBEV particle and the X-ray structure of its ectodomains was the first flavivirus envelope protein structure solved. E consists of four domains, which are all visible in the cryo-EM reconstruction (Figure 2B) [[18],[28]]. The N-terminal domain I forms a β-barrel structure that is central to the protein [[18],[28]]. Domain II is elongated and consists of two areas of β-strands connected by loops and two short helices. It is the site of the dimerization interface, with a buried surface area of 14.9 nm2 at the interface [[18],[28]]. Additionally, it contains the only glycosylation site of the mature virus (Asn154), which has a role in egress from mammalian cells, as well as neurovirulence (Figure 2A) [[29],[30]]. In the cryo-EM reconstruction, a density corresponding to N-acetyl-d-glucosamine was observed attached to this residue [[18]]. At its tip, domain II also contains the highly-conserved fusion loop that is responsible for the fusion of the viral and host membranes in the final stages of TBEV entry (Figure 2A) [[18],[28],[31]]. The hydrophobic fusion loop (residues 100–109) is hidden from the aqueous environment in the hinge region between domains I and III of the other E protein in the dimer, as well as by the carbohydrate moiety of residue 154 [[18],[28]]. Domain III of the E protein has an immunoglobulin-like fold [[18],[23],[24],[25],[28],[32],[33],[34],[35]]. This domain has been proposed to function in the binding to host receptors, but no residues directly responsible for entry have been identified [[28],[36]]. Domain IV includes a stem region of three peripheral membrane helices (h1–h3) and a transmembrane region made up of two helices (h4 and h5) [[18]]. As the X-ray structure was of a cleaved ectodomain of E, domain IV was missing [[28]].

The M protein is made up of 75 residues and is therefore much smaller than the E protein. Correspondingly, it has a minor role compared to E in the mature particle, [[18]]. The M protein has one peripheral membrane helix (h1), two transmembrane helices (h2 and h3), and an N-terminal loop region that interacts with both E proteins in an E-M-M-E heterotetramer (Figure 2C) [[18]]. M is completely buried in the E-E interface, and presumably works as a ‘cement’ protein, strengthening the interaction of the E proteins [[18],[23]]. It probably also prevents the E proteins from moving into the fusogenic conformation before the virus encounters the low-pH environment of the endosome [[18],[23]]. The M protein is a remnant of its precursor prM (162 residues) that has a major role in the maturation of the TBEV particles (UniProt: Q01299, P14336, and P07720).

2.2. Nucleocapsid

The flavivirus NCs do not follow the icosahedral symmetry of the E and M proteins so the signal is averaged out in the reconstruction process [[27]]. Therefore, the structure of the TBEV NC has not yet been determined [[18],[21],[22],[23],[24],[25],[26]]. It has been estimated that the molar ratio of E to C in a mature TBEV particle is close to 1:3, which would mean some 540 copies of C per virion [[37]]. The properties of the C protein have been investigated more thoroughly than the complete NC, and the structure of the C protein has been solved for three flaviviruses, DENV, ZIKV, and a variant of WNV, Kunjin virus (KUNV) [[38],[39],[40]]. These proteins share the same fold despite low sequence identity (Figure 3A) [[38],[39],[40],[41]]. Using the ZIKV C protein (wwPDB: 5YGH) as a template, we generated a homology model of TBEV C using the I-TASSER server (https://zhanglab.ccmb.med.umich.edu/I-TASSER/), which predicted a similar overall fold with a reliable confidence score (C-score = −0.77) (Figure 3B,C) [[42],[43],[44]].

The C protein of TBEV consists of 96 amino acid residues (UniProt: Q01299, P14336, and P07720) and it is most likely organized into four α-helices, α1–α4 (Figure 3B,C) [[38],[39],[40]]. The C protein forms antiparallel dimers with dimerization occurring between the corresponding α2 and α4 helices of the two subunits [[38],[39],[40]]. In each monomer, the helices α1–α3 are arranged in a bundle, and the two bundles of the dimer form a hydrophobic surface that is believed to interact with host membranes [[38],[39],[40]]. In KUNV and DENV the α1 helices differ in orientation to each other. In contrast, the N-terminus of ZIKV C is an extended loop resulting in a much shorter α1 [[38],[39],[40]]. The two α4 helices of the dimer form a surface that is rich in basic amino acids [[38],[39],[40]]. This is most likely the RNA-binding domain of the dimer, and it is believed that RNA-C binding occurs via non-specific electrostatic interactions [[38],[39],[40]]. When crystallized, the C protein dimers were arranged in oligomeric structures: dimers of dimers in KUNV, and trimers of dimers in ZIKV [[39],[40]]. In both cases, the authors observed channels in the middle of oligomers with RNA-binding α4 helices facing towards the channel interior. Therefore, they proposed that the formation of C oligomers can facilitate RNA packaging into the NC [[39],[40]]. However, as the environment of the protein crystal is different to the complex milieu of the cell or virion, it may be that the crystal packing is not biologically relevant. Additionally, no oligomerisation of the DENV C protein dimers was observed, which may be a result of a different method of structure determination (nuclear magnetic spectroscopy versus X-ray crystallography) [[38]]. Alternatively, the solution structure of the DENV C dimer may reflect a different functional state than RNA packing, as the C protein has other roles during flavivirus infection [[46],[47],[48],[49]].

3. Life Cycle

As is the case for other flaviviruses, the assembly of TBEV particles is complex and involves multiple maturation steps [[19]]. Some aspects of the process, like virion maturation, are structurally quite well characterised for many mosquito-borne flaviviruses but few data are available for TBEV [[50],[51],[52],[53],[54],[55],[56],[57]]. Especially, the early events of particle production remain to be elucidated. In addition, the TBEV life cycle has been mostly studied in mammalian cells, even though the tick is a central part of the biology of the virus. An overview of the TBEV life cycle is presented in Figure 4.

3.1. Entry

The entry process of flaviviruses occurs mainly via receptor-mediated endocytosis, but entry via micropinocytosis is also possible [[58],[59],[60]]. There are two major receptor candidates for TBEV in mammalian cells, laminin-binding protein (LBP) and the αVβ3 integrin but no receptor candidates in tick cells have been identified so far [[61],[62],[63]]. Studies using anti-idiotypic antibodies suggested that there could be other receptor candidates of as yet unknown identity [[61],[64],[65]]. In addition, under certain conditions it is possible to fuse TBEV with liposomes, hence, lipids could also be involved in binding [[66],[67]]. For other flaviviruses, various receptor candidates have been proposed, which could be also be relevant for TBEV (reviewed in [[68]]).

In addition to entry receptors, sensu stricto, it has been proposed that TBEV utilizes attachment factors that bind the virus on the cell surface without initiating endocytosis. The most prominent of these is heparan sulphate, a glycosaminoglycan that works as an attachment factor for multiple viruses [[69],[70],[71],[72],[73],[74]]. Even though utilisation of heparan sulphate is a commonly seen cell culture adaptation of TBEV, it is also present in some wild-type isolates. The binding between heparan sulphate and TBEV particles occurs via the E protein, and cell culture-associated adaptations mainly manifest as mutations that increase the positive charge of the E protein [[71],[73],[75]].

The carbohydrate moiety of the E protein has been shown to be dispensable for TBEV entry in cell culture [[30],[76]]. However, the lack of E glycosylation leads to reduced neuroinvasiveness in mice, which may indicate that interaction with a carbohydrate-binding protein has a role in TBEV attachment to neurons [[30]]. This is further supported by the observation that in a closely related tick-borne flavivirus, Louping ill virus, a mutation in the glycosylation site reduced neurovirulence [[77]]. The binding of DENV to its attachment factor, dendritic cell-specific ICAM3 grabbing nonintegrin, is mediated by the interaction of the carbohydrate recognition domain and the E protein Asn67 carbohydrate moieties of the virus [[78]]. Even though this residue is not glycosylated in TBEV, a similar interaction may occur using the glycosylated Asn154 instead.

TBEV can be endocytosed and cause infection, when bound by non-neutralising quantities of antibodies [[79]]. This phenomenon is known as antibody-dependent enhancement (ADE) and it has been shown in vitro for multiple flaviviruses (reviewed in [[80]]) and recently on the epidemiological level for DENV [[81]]. ADE is mediated by the binding of the virus-antibody complexes to Fcγ receptors in the host cell surfaces but, recently, a new ADE mechanism was identified for TBEV. This mode of entry is independent of Fcγ receptors and other cell surface proteins and is proposed to be mediated by antibody-mediated exposure of the E protein fusion loop which then binds directly to host membranes [[82]].

After the TBEV particles have entered the cells, the virions are localised inside endocytic vesicles. In the endosome, the pH progressively drops, which leads to major rearrangements in the virion. Mutagenesis studies of TBEV E proteins have implicated the protonation of His323 (and possibly His146) as the main pH detection mechanism, but the cryo-EM reconstruction of the virus implies that other histidines have pH-related roles as well. From a structural perspective, it seems likely that the protonation of histidines in the E and M proteins would cause them to repel each other, destabilising the heterotetramer and exposing the fusion loops. The residues implicated for this are His216 and His248 of the E protein and His7 and His17 of the M protein (Figure 5A) [[18],[23],[83]]. The fusion loops embed into the membrane of the endosome, possibly with the help of the detachment of the peripheral membrane helices of E from the viral envelope [[84],[85],[86]]. After binding to the membrane, E proteins trimerize via the interaction of the fusion loops [[84],[87]]. In the current model, this pre-fusion trimer undergoes a hairpin-like conformational change that brings the membranes of the endosome and the virus into close contact forming a post-fusion trimer (Figure 5B) [[88],[89]]. The post-fusion trimers are then stabilized by interactions between domains I and II, as well as the peripheral and transmembrane helices of the different subunits [[90],[91],[92],[93]]. The conformational change from a pre-fusion to a post-fusion trimer allows the fusion of the viral and endosomal membranes via a hemifusion intermediate. This leads to the release of the NC into the cytosol [[89],[94]]. Membrane fusion is dependent on the correct lipid composition, and cholesterol strongly enhances it [[66],[67],[87]]. After the NC has entered the cytosol, it disintegrates and releases the viral RNA. The events responsible to the uncoating of the TBEV RNA have not been elucidated, but for DENV, it has been shown that the dissociation of the NC requires non-degradative ubiquitination [[95]].

3.2. Replication and Translation

In infected cells, the TBEV genome is translated at the endoplasmic reticulum (ER) as a single polyprotein. The polyprotein is cleaved by viral and host enzymes to yield SPs that form the virion and nSPs that are responsible for genome replication, polyprotein processing and modulation of cellular functions (Figure 6) [[19]]. TBEV SPs have been studied in detail, but the current knowledge on flavivirus nSPs mostly comes from studies of mosquito-borne species. Most of the TBEV proteins are proposed to be either integral membrane proteins or to have membrane anchors, some of which are cleaved during polyprotein processing (UniProt: Q01299, P14336, and P07720) [[19]].

The mature C is a soluble cytoplasmic protein as its membrane anchor is cleaved but the prM and E proteins localise in the lumen of the ER, where they remain bound to the membrane by double-helical anchors that are typical to flaviviruses [[19]]. The cleavage of the C-terminal membrane anchor first from C and then from prM is sequential and strictly controlled. In Murray Valley encephalitis virus, YFV, and WNV, the perturbation of the cleavage order results in excessive formation of NC-deficient particles [[96],[97],[98],[99]]. For TBEV, however, the uncoupling of these events only affects particle production in tick cells [[97]].

The first translated nSP in flaviviruses is the NS1 protein that localises in the lumen of the ER (reviewed in [[100]]). It is a multi-functional protein that exists in dimeric and hexameric forms. As a dimer, NS1 has a role in replication whereas as a hexamer it is co-secreted with TBEV particles and modulates the complement system of the mammalian host. The immunomodulatory activity of NS1 results in reduced formation of membrane attack complexes and, therefore, prevents the destruction of infected cells [[100]]. It also reduces the inactivation of extracellular viruses by binding to the C4 component of the complement system [[101]]. NS2A and NS2B are both integral membrane proteins with roles in particle assembly. NS2A functions in replication and immunomodulation whereas NS2B is a co-factor for the NS3 protease [[102],[103],[104],[105],[106]]. The NS2B-NS3 complex has both protease and helicase activities and it is responsible for the viral enzyme-mediated cleavage of the polyprotein (reviewed in [[107]]). The helicase domain of NS3 has an ATPase activity that is regulated by the integral membrane protein NS4A [[108]]. NS4A is separated from NS4B by a signal sequence called the 2k peptide that directs NS4B to the ER membrane and is later cleaved off by the host signal peptidase. After 2k cleavage, NS4B remains integrated in the ER membrane where it performs multiple functions from replication complex formation to immunomodulation (reviewed in [[109]]). The flaviviral genome is replicated by the RNA-dependent RNA polymerase NS5, which has an immunomodulatory role as well (reviewed in [[110],[111]]). In addition to the nSPs, the C proteins of flaviviruses have multiple regulatory roles during the infection, including immunomodulation and the prevention of nucleosome formation [[46],[47],[48],[49]]. No TBEV nSP structures have been solved. The nSP structures available for other flaviviruses have been studied using X-ray crystallography of purified proteins, which makes it difficult to provide a full structural picture of flaviviral replication in the context of the infected cell [[112],[113],[114]].

The replication of TBEV occurs in a close contact with the ER membrane, which is extensively rearranged by NS1, NS2B, NS4A, and NS4B [[100],[104],[115],[116],[117],[118],[119],[120],[121],[122],[123]]. In tick-borne flaviviruses these rearranged ER membranes are observed in both tick and mammalian cells, but in tick cells the membrane rearrangements are less prominent. Corresponding to a slower rate of replication in the tick cells, fewer particles are also observed than in mammalian cells [[117],[124]]. The replication of the TBEV genomes occurs via a dsRNA intermediate in ER invaginations. The invaginations have ‘necks’ that connect to the cytosol, presumably allowing nucleotides to enter and the RNA genomes to exit [[19],[115],[117],[120],[121],[123]].

3.3. Assembly and Budding

The newly-synthesized viral genomes are encompassed by multiple copies of the C protein to form NCs. Based on the structures of the C proteins, it seems that the NCs are formed by electrostatic interactions between the C-terminal α4 helices of C and viral RNA [[38],[39],[40]]. This suggestion is corroborated by data showing C proteins of ZIKV and DENV bind various types of nucleic acids regardless of the sequence [[40],[125]]. Furthermore, recombinant DENV C protein dimers bind double-stranded DNA of various lengths, forming capsid-like particles (CLP) [[125]]. Overall, the packaging of flaviviral genomes is a robust process, as the C proteins can remain functional despite large-scale deletions [[41],[126],[127],[128]]. In a YFV-based reporter system, it was even noticed that assembly required either the α4 helix or the N-terminal basic residues, but not both [[41],[128]]. The TBEV C protein is similar to other flaviviruses: it can remain functional despite internal deletions, it binds various nucleic acids without signal specificity, and CLPs can be produced from purified C protein and nucleic acids [[126],[129]]. This suggests that the assembly of TBEV NCs is analogous to other flaviviruses.

As the C proteins can package RNA regardless of sequence, a spatial and temporal coupling of replication, translation, assembly, and budding has been proposed to explain how flaviviruses manage to specifically pack their genomes (reviewed in [[130]]). Several lines of evidence support this hypothesis: in DENV-infected cells budding into the ER lumen occurs directly opposite to or in close contact with the vesicular structures where the genome is replicated. In KUNV only actively-transcribed RNA is packaged [[131],[132],[133]]. Furthermore, many of the nSPs that localize at the sites of replication have also been implicated in particle assembly. Functional NS2A is required for the assembly of KUNV, DENV, and YFV particles, the transmembrane domains of NS2B and its binding partner are required for JEV particle formation, and NS3 has been implicated in particle assembly in YFV and KUNV [[104],[105],[134],[135],[136],[137],[138],[139]]. It is also possible that even though RNA-C protein binding is sequence-independent, the specificity of RNA packaging is mediated by genomic assembly signals that target one or multiple nSPs instead of C. However, the binding of nSPs to the only candidate for a flaviviral packaging signal, CCR1, has not been studied. Furthermore, the data supporting its role in assembly is indirect [[140]].

The particle and NC assembly processes do not solely rely on viral proteins. In JEV-infected cells, transmembrane domains of NS2B interact with the host factor SPCS1 to secure particle production and in DENV infections, the interaction of C protein and nucleolin is essential for formation of virions [[141],[142]]. Additionally, WNV particle production requires the presence of the host helicase DDX56 at the viral assembly sites [[143],[144]]. However, the host factors required by TBEV particle formation are not known. In contrast, it has been recently shown that the host protein viperin prevents TBEV assembly by promoting the production of non-infectious particles containing solely C protein and a membrane [[145]]. The detailed characterisation of other antiviral host factors in TBEV infection is outside the scope of this article and has been reviewed elsewhere [[146]].

Once the NCs have been assembled, they acquire their lipid envelopes by budding into the ER lumen. The budding, however, can occur without the presence of the NC, as the production of NC-deficient subviral particles is a normal part of flavivirus infections [[20]]. Fusion-competent subviral particles can also be produced by recombinantly expressing prM and E in cells, which implies that the budding process is mediated by the lateral interactions of these proteins [[66],[147],[148]]. The structural details of budding have not been elucidated, but it seems that the interaction of the prM and E protein transmembrane helices is required [[149]]. Although budding can occur without the assembly of NCs, the events need to be coupled as flavivirus-infected cells rarely produce mainly empty particles and naked NCs are generally not observed in infected cells [[19],[120],[121],[123],[124]].

3.4. Particle Maturation and Egress

The immature flavivirus particles formed by budding through the ER differ greatly from their mature, infectious forms. Even though the immature TBEV particle has not been structurally characterized, it is presumed to be similar to the flaviviruses for which the structure of this intermediate form is available [[50],[52],[56],[57]] (Figure 7). In the immature particles, the pr peptide has not been cleaved from M yet, and the particles consist of heterodimers of prM and E. The cryo-EM reconstructions of both naturally occurring and artificially induced immature flavivirus particles consistently reach lower resolution than those of mature virions. This implies flexibility that is not present in the mature virions [[24],[52],[56],[57]]. The immature particles are larger than the mature forms, which is due to the organisation of the prM-E dimers into trimeric spikes [[50],[52],[56],[57]]. In the immature particles, the pr peptides coat the fusogenic loops of the E proteins, preventing premature fusion [[50],[52],[56],[57],[150]]. In DENV and ZIKV, the prM is glycosylated in the area directly on top of the fusion loop (Asn69), which increases the hydrophilicity of the spike tip, presumably to prevent interaction with the ER membrane [[53],[57]]. In TBEV the glycosylated residue is Asn25 (UniProt: Q01299, P14336, and P07720). In the DENV and ZIKV pr structures, residues 25 and 69 are close together, indicating that the glycosylation of either could have a similar effect, which suggests that pr glycosylation in TBEV has the same role as in DENV and ZIKV [[53],[57]]. Each spike is stabilised by the interaction of the pr proteins at the tip and by the interactions between domains II and III of neighbouring E proteins. These connections, however, are not very strong, which may contribute to the lability of the immature particle [[53],[56]]. In most reconstructions of immature flavivirus particles, the NC density remains poorly resolved, but in the immature ZIKV particle, a density corresponding to C protein was observed under the trimeric spikes, suggesting partial organisation of the NC [[50],[52],[56],[57]]. Since this ordered density is not visible in the mature virion, it implies that during the maturation process the NC undergoes a conformational change [[24],[57]].

The current model for flavivirus maturation was first established with TBEV using biochemical and molecular biology methods [[151],[152],[153],[154],[155]]. Later, this model was supported via structural studies of mosquito-borne flaviviruses [[53],[54],[56]]. After the immature particles form by budding into the ER, they pass through the Golgi apparatus and the trans-Golgi network (TGN) [[19]]. In the TGN, the particles are exposed to low pH, which causes a major conformational change in the (prM-E)3 spikes. The spiky immature particles change into smooth ‘pre-mature’ particles as the trimeric spikes dissociate, and the prM-E dimers further dimerize, forming a structure similar to the mature particle. The only difference is the presence of the pr peptide, which is still localised on top of the fusion peptide [[53],[54],[56]]. The rearrangement of the spikes begins in one or more independent nucleation centres instead of occurring simultaneously across the particle, as this would lead to steric clashes [[55]]. Interestingly, this conformational change is reversible in DENV and irreversible in TBEV, indicating possible differences between the maturation of mosquito-borne and tick-borne flaviviruses [[54],[153]].

No specific pH-sensing residues have been implicated for maturation in TBEV but it is tempting to speculate that the same histidines that are needed for the conformational changes leading to membrane fusion would have a role in this process as well [[18],[83]]. Indeed, His244 in E and His98 in prM are needed for the formation of pre-mature DENV particles and they are presumably protonated during the maturation process [[156],[157]]. In TBEV, there are histidines in comparable positions in E and prM (His248 and His95, respectively) and it is, therefore, likely that they have similar roles in TBEV maturation (UniProt: Q01299, P14336, and P07720). Based on the structure of the mature TBEV particle, these residues may also function as pH sensors during fusion (His95 of prM is in position 7 in M after pr is cleaved) [[18]].

After the conformational change, the maturation is completed by the cleavage of the pr peptide from prM by the host protease, furin. In the immature particle, the prM furin cleavage site is inaccessible. After the pH-mediated conformational change, it is exposed and the pr peptide is cleaved. However, it still remains bound to the E-M-M-E heterotetramer at the acidic pH of the TGN. Therefore, it still obscures the fusion loop, preventing premature fusion with the TGN membranes. The pr peptides can only dissociate from the virion after it exits the cell via endocytosis and reaches the neutral extracellular milieu. The pr dissociation primes the virion for fusion, thus rendering it infectious [[53],[54],[156],[157]].

The maturation process in flaviviruses is not always complete, which leads to the production of immature and partially mature particles by the infected cells. The fully immature particles are non-infectious because they are incapable of fusion, but the partially mature particles can infect new cells [[152],[153]]. The partially mature particles are structurally and antigenically heterogenous. Their production has been suggested to act as an immune evasion strategy (reviewed in [[158]]) and as a way to increase the range of tissue tropisms (reviewed in [[159]]).

For TBEV, the process of maturation and egress has mainly been studied in mammalian cells, but limited evidence shows there may be differences between the mammalian and tick systems. The glycosylation of E protein is required for egress in mammalian, but not in tick, cells [[29],[30],[76]]. Additionally, in tick cells blocking the transport from the ER to the Golgi apparatus did not reduce virus production [[30]]. In some electron microscopy studies, it has been reported that the entire process of flavivirus assembly and maturation in tick and mosquito cells differs from mammalian cells. In these reports, pre-formed NCs have been observed associated with various host cell structures like phagosomes to acquire their membranes via budding through the plasma membrane [[117],[160]]. These findings have not, however, been confirmed with other approaches.

4. Future Perspectives

It is clear that many aspects of TBEV biology remain unknown, even though there have been considerable advances in flavivirus research in recent years. The TBEV life cycle is complex, and we can better understand viral assembly, maturation, and entry by the structural characterisation of the different intermediate forms of the virus particles. The TBEV NC is a tempting target for study: it is difficult to approach but can provide important knowledge about uncoating and assembly, the two most enigmatic stages of the flavivirus life cycle. Understanding NC assembly may help to answer the question of how TBEV specifically packages its genome despite the apparent sequence-agnosticism of the C protein.

The multi-functional nSPs of TBEV are critical for infection and could be determinants of virulence, which makes them important targets for structural and functional studies. They would provide essential information about TBEV genome replication, particle assembly, virus-host interactions, and immune evasion. We can decipher the nSPs’ mechanistic roles in infection by combining structural and in situ approaches. These studies could also yield novel drug targets, as exemplified by the current development of nucleoside analogues that block the function of the NS5 protein and reduce TBEV neurovirulence in vivo [[161],[162]].

The virulence factors responsible for the different TBEV subtype pathologies have not yet been comprehensibly examined. Tissue tropism could explain the clinical differences between the TBEV subtypes. Therefore, this variation may be investigated by studying TBEV entry into different cell types. A number of residues that vary across TBEV subtypes have been localised to the E protein, which is responsible for receptor interaction [[5]]. Hence, by identifying the TBEV receptor(s) and studying interactions with the virus we may better explain TBEV pathogenicity.

Finally, despite its obvious importance to TBEV biology, the virus has been poorly studied in ticks. Although research in mammalian systems is warranted, it needs to be combined with investigations in tick systems for a more complete understanding of TBEV biology and emergence.

Acknowledgements

Acknowledgments

The writing of this article was supported by a University of Helsinki Research Foundation PhD grant and a Doctoral Programme on Microbiology and Biotechnology fellowship to L.I.A.P. and an Academy of Finland grant (275199) to S.J.B. This project has received funding from the European Union’s Horizon 2020 research innovation programme under the Marie Sklodowska-Curie Actions grant agreement no. 799929 to M.A. and ViBrant ITN grant agreement 765042 to S.J.B.

References

  1. P. BogovicF. StrleTick-borne encephalitis: A review of epidemiology, clinical characteristics, and managementWorld J. Clin. Cases2015343010.12998/wjcc.v3.i5.43025984517
  2. P. TabaE. SchmutzhardP. ForsbergI. LutsarU. LjøstadA. MyglandI. LevchenkoF. StrleI. SteinerEAN consensus review on prevention, diagnosis and management of tick-borne encephalitisEur. J. Neurol.2017241214-e6110.1111/ene.1335628762591
  3. G. GrardG. MoureauR.N. CharrelJ.J. LemassonJ.P. GonzalezP. GallianT.S. GritsunE.C. HolmesE.A. GouldX. de LamballerieGenetic characterization of tick-borne flaviviruses: New insights into evolution, pathogenetic determinants and taxonomyVirology2007361809210.1016/j.virol.2006.09.01517169393
  4. D.M. HeinzeE.A. GouldN.L. ForresterRevisiting the clinal concept of evolution and dispersal for the tick-borne flaviviruses by using phylogenetic and biogeographic analysesJ. Virol.2012868663867110.1128/JVI.01013-1222674986
  5. M. EckerS.L. AllisonT. MeixnerF.X. HeinzSequence analysis and genetic classification of tick-borne encephalitis viruses from Europe and AsiaJ. Gen. Virol.19998017918510.1099/0022-1317-80-1-1799934700
  6. G. WallnerC.W. MandlC. KunzF.X. HeinzThe flavivirus 3’-noncoding region: Extensive size heterogeneity independent of evolutionary relationships among strains of tick-borne encephalitis virusVirology199521316917810.1006/viro.1995.15577483260
  7. G. WallnerC.W. MandlM. EckerH. HolzmannK. StiasnyC. KunzF.X. HeinzCharacterization and complete genome sequences of high- and low-virulence variants of tick-borne encephalitis virusJ. Gen. Virol.1996771035104210.1099/0022-1317-77-5-10358609469
  8. J. SussTick-borne encephalitis in Europe and beyond—The epidemiological situation as of 2007Euro Surveill.2008131891610.2807/ese.13.26.18916-en18761916
  9. O.D. MantkeC. EscadafalM. NiedrigM. PfefferTick-borne encephalitis in Europe, 2007 to 2009Euro Surveill.2011161997610.2807/ese.16.39.19976-en21968423
  10. H. HolzmannS.W. AberleK. StiasnyP. WernerA. MischakB. ZainerM. NetzerS. KoppiE. BechterF.X. HeinzTick-borne encephalitis from eating goat cheese in a mountain region of AustriaEmerg. Infect. Dis.2009151671167310.3201/eid1510.09074319861072
  11. N. HudopiskM. KorvaE. JanetM. SimetingerM. Grgič-VitekJ. GubenšekV. NatekA. KraigherF. StrleT. Avšič-ŽupancTick-borne encephalitis associated with consumption of raw goat milk, Slovenia, 2012Emerg. Infect. Dis.20131980680810.3201/eid1905.12144223697658
  12. H. KollaritschV. ChmelíkI. DontsenkoA. GrzeszczukM. KondrusikV. UsonisA. LakosThe current perspective on tick-borne encephalitis awareness and prevention in six Central and Eastern European countries: Report from a meeting of experts convened to discuss TBE in their regionVaccine2011294556456410.1016/j.vaccine.2011.04.06121549781
  13. A. LehrerM. HolbrookTick-borne encephalitis vaccinesJ. Bioterror. Biodef.20112011Suppl. 12157252610.4172/2157-2526.S1-00323997980
  14. N.K. TokarevichA.A. TroninO.V. BlinovaR.V. BuzinovV.P. BoltenkovE.D. YurasovaJ. NurseThe impact of climate change on the expansion of Ixodes persulcatus habitat and the incidence of tick-borne encephalitis in the north of European RussiaGlob. Health Action20114844810.3402/gha.v4i0.844822028678
  15. R.S. OstfeldJ.L. BrunnerClimate change and Ixodes tick-borne diseases of humansPhilos. Trans. R. Soc. B Biol. Sci.20153702014005110.1098/rstb.2014.005125688022
  16. G. MoureauS. CookP. LemeyA. NougairedeN.L. ForresterM. KhasnatinovR.N. CharrelA.E. FirthE.A. GouldX. De LamballerieNew insights into flavivirus evolution, taxonomy and biogeographic history, extended by analysis of canonical and alternative coding sequencesPLoS ONE201510e011784910.1371/journal.pone.011784925719412
  17. M.W. GauntA.A. SallX. de LamballerieA.K. FalconarT.I. DzhivanianE.A. GouldPhylogenetic relationships of flaviviruses correlate with their epidemiology, disease association and biogeographyJ. Gen. Virol.2001821867187610.1099/0022-1317-82-8-186711457992
  18. T. FüzikP. FormanováD. RůžekK. YoshiiM. NiedrigP. PlevkaStructure of tick-borne encephalitis virus and its neutralization by a monoclonal antibodyNat. Commun.2018943610.1038/s41467-018-02882-029382836
  19. B.D. LindenbachC.L. MurrayH.-J. ThielC.M. RiceFlaviviridaeFields VirologyD. KnipeP. HowleyLippincott Williams & WilkinsPhiladelphia, PA, USA2013
  20. T.J. SmithW.E. BrandtJ.L. SwansonJ.M. McCownE.L. BuescherPhysical and biological properties of dengue-2 virus and associated antigensJ. Virol.197055245324195055
  21. R.J. KuhnW. ZhangM.G. RossmannS.V. PletnevJ. CorverE. LenchesC.T. JonesS. MukhopadhyayP.R. ChipmanE.G. StraussStructure of dengue virus: Implications for flavivirus organization, maturation, and fusionCell200210871772510.1016/S0092-8674(02)00660-811893341
  22. S. MukhopadhyayB.S. KimP.R. ChipmanM.G. RossmannR.J. KuhnStructure of West Nile virusScience200330224810.1126/science.108931614551429
  23. X. ZhangP. GeX. YuJ.M. BrannanG. BiQ. ZhangS. ScheinZ.H. ZhouCryo-EM structure of the mature dengue virus at 3.5-Å resolutionNat. Struct. Mol. Biol.20122010511010.1038/nsmb.246323241927
  24. D. SirohiZ. ChenL. SunT. KloseT.C. PiersonM.G. RossmannR.J. KuhnThe 3.8 Å resolution cryo-EM structure of Zika virusScience201635246747010.1126/science.aaf531627033547
  25. X. WangS.H. LiL. ZhuQ.G. NianS. YuanQ. GaoZ. HuQ. YeX.F. LiD.Y. XieNear-atomic structure of Japanese encephalitis virus reveals critical determinants of virulence and stabilityNat. Commun.201781410.1038/s41467-017-00024-628446752
  26. W. ZhangB. KaufmannP.R. ChipmanR.J. KuhnM.G. RossmannMembrane curvature in flavivirusesJ. Struct. Biol.2013183869410.1016/j.jsb.2013.04.00523602814
  27. Y. ZhangV.A. KostyuchenkoM.G. RossmannStructural analysis of viral nucleocapsids by subtraction of partial projectionsJ. Struct. Biol.200715735636410.1016/j.jsb.2006.09.00217064936
  28. F.A. ReyF.X. HeinzC. MandlC. KunzS.C. HarrisonThe envelope glycoprotein from tick-borne encephalitis virus at 2 Å resolutionNature199537529129810.1038/375291a07753193
  29. A. GotoK. YoshiiM. ObaraT. UekiT. MizutaniH. KariwaI. TakashimaRole of the N-linked glycans of the prM and E envelope proteins in tick-borne encephalitis virus particle secretionVaccine2005233043305210.1016/j.vaccine.2004.11.06815811651
  30. K. YoshiiN. YanagiharaM. IshizukaM. SakaiH. KariwaN-linked glycan in tick-borne encephalitis virus envelope protein affects viral secretion in mammalian cells, but not in tick cellsJ. Gen. Virol.2013942249225810.1099/vir.0.055269-023824303
  31. S.L. AllisonJ. SchalichK. StiasnyW. MandlF.X. HeinzMutational evidence for an internal fusion peptide in flavivirus envelope protein EJ. Virol.2001754268427510.1128/JVI.75.9.4268-4275.200111287576
  32. R. KanaiK. KarK. AnthonyL.H. GouldM. LedizetE. FikrigW.A. MarascoR.A. KoskiY. ModisCrystal structure of West Nile virus envelope glycoprotein reveals viral surface epitopesJ. Virol.200680110001100810.1128/JVI.01735-0616943291
  33. M. MukherjeeK. DuttaM.A. WhiteD. CowburnR.O. FoxNMR solution structure and backbone dynamics of domain III of the E protein of tick-borne Langat flavivirus suggests a potential site for molecular recognitionProtein Sci.2006151342135510.1110/ps.05184400616731969
  34. D.E. VolkL. ChavezD.W.C. BeasleyA.D.T. BarrettM.R. HolbrookD.G. GorensteinStructure of the envelope protein domain III of Omsk hemorrhagic fever virusVirology200635118819510.1016/j.virol.2006.03.03016647096
  35. D.E. VolkF.J. MayS.H.A. GandhamA. AndersonJ.J. Von LindernD.W.C. BeasleyA.D.T. BarrettD.G. GorensteinStructure of yellow fever virus envelope protein domain IIIVirology2009394121810.1016/j.virol.2009.09.00119818466
  36. C.W. MandlS.L. AllisonH. HolzmannT. MeixnerF.X. HeinzAttenuation of tick-borne encephalitis virus by structure-based site-specific mutagenesis of a putative flavivirus receptor binding siteJ. Virol.2000749601960910.1128/JVI.74.20.9601-9609.200011000232
  37. O. ScheinostV. ChmelikX. HeinzSpecificities of human CD4+ T cell responses to an inactivated flavivirus vaccine and infection: correlation with structure and epitope predictionJ. Virol.2014887828784210.1128/JVI.00196-1424789782
  38. L. MaC.T. JonesT.D. GroeschR.J. KuhnC.B. PostSolution structure of dengue virus capsid protein reveals another foldProc. Natl. Acad. Sci. USA20041013414341910.1073/pnas.030589210114993605
  39. T. DoklandM. WalshJ.M. MackenzieA.A. KhromykhK. EeS. WangWest Nile Virus Core Protein: Tetramer Structure and Ribbon FormationStructure2004121157116310.1016/j.str.2004.04.02415242592
  40. Z. ShangH. SongY. ShiJ. QiG.F. GaoCrystal structure of the capsid protein from Zika virusJ. Mol. Biol.201843094896210.1016/j.jmb.2018.02.00629454707
  41. C.G. PatkarC.T. JonesY. ChangR. WarrierR.J. KuhnFunctional requirements of the yellow fever virus capsid proteinJ. Virol.2007816471648110.1128/JVI.02120-0617526891
  42. Y. ZhangI-TASSER server for protein 3D structure predictionBMC Bioinform.200894010.1186/1471-2105-9-4018215316
  43. A. RoyA. KucukuralY. ZhangI-TASSER: A unified platform for automated protein structure and function predictionNat. Protoc.2010572573810.1038/nprot.2010.520360767
  44. J. YangR. YanA. RoyD. XuJ. PoissonY. ZhangThe I-TASSER suite: Protein structure and function predictionNat. Methods2014127810.1038/nmeth.321325549265
  45. X. RobertP. GouetDeciphering key features in protein structures with the new ENDscript serverNucleic Acids Res.20144232032410.1093/nar/gku31624753421
  46. S. SangiambutP. KeelapangJ. AaskovC. PuttikhuntW. KasinrerkP. MalasitN. SittisombutMultiple regions in dengue virus capsid protein contribute to nuclear localization during virus infectionJ. Gen. Virol.2008891254126410.1099/vir.0.83264-018420804
  47. T.M. ColpittsS. BarthelP. WangE. FikrigDengue virus capsid protein binds core histones and inhibits nucleosome formation in human liver cellsPLoS ONE20116e2436510.1371/journal.pone.002436521909430
  48. R. BhuvanakanthamM.L. NgWest Nile virus and dengue virus capsid protein negates the antiviral activity of human Sec3 protein through the proteasome pathwayCell. Microbiol.2013151688170610.1111/cmi.1214323522008
  49. G.H. SamuelM.R. WileyA. BadawiZ.N. AdelmanK.M. MylesYellow fever virus capsid protein is a potent suppressor of RNA silencing that binds double-stranded RNAProc. Natl. Acad. Sci. USA2016113138631386810.1073/pnas.160054411327849599
  50. Y. ZhangJ. CorverP.R. ChipmanW. ZhangS.V. PletnevD. SedlakT.S. BakerJ.H. StraussR.J. KuhnM.G. RossmannStructures of immature flavivirus particlesEMBO J.2003222604261310.1093/emboj/cdg27012773377
  51. Y. ZhangW. ZhangS. OgataD. ClementsJ.H. StraussT.S. BakerR.J. KuhnM.G. RossmannConformational changes of the flavivirus E glycoproteinStructure2004121607161810.1016/j.str.2004.06.01915341726
  52. Y. ZhangB. KaufmannP.R. ChipmanR.J. KuhnM.G. RossmannStructure of immature West Nile virusJ. Virol.2007816141614510.1128/JVI.00037-0717376919
  53. L. LiS.M. LokI.M. YuY. ZhangR.J. KuhnJ. ChenM.G. RossmannThe flavivirus precursor membrane-envelope protein complex: Structure and maturationScience20083191830183410.1126/science.115326318369147
  54. I.-M. YuW. ZhangH.A. HoldawayL. LiV.A. KostyuchenkoP.R. ChipmanR.J. KuhnM.G. RossmannJ. ChenStructure of the immature dengue virus at low pH primes proteolytic maturationScience20083191834183710.1126/science.115326418369148
  55. P. PlevkaA.J. BattistiJ. JunjhonD.C. WinklerH.A. HoldawayP. KeelapangN. SittisombutR.J. KuhnA.C. StevenM.G. RossmannMaturation of flaviviruses starts from one or more icosahedrally independent nucleation centresEMBO Rep.20111260260610.1038/embor.2011.7521566648
  56. V.A. KostyuchenkoQ. ZhangJ.L. TanT.-S. NgS.-M. LokImmature and mature dengue serotype 1 virus structures provide insight into the maturation processJ. Virol.2013877700770710.1128/JVI.00197-1323637416
  57. V.M. PrasadA.S. MillerT. KloseD. SirohiG. BudaW. JiangR.J. KuhnM.G. RossmannStructure of the immature Zika virus at 9 Å resolutionNat. Struct. Mol. Biol.20172418418610.1038/nsmb.335228067914
  58. H.M. Van Der SchaarM.J. RustC. ChenH. Van Der Ende-MetselaarJ. WilschutX. ZhuangJ.M. SmitDissecting the cell entry pathway of dengue virus by single-particle tracking in living cellsPLoS Pathog.20084e100024410.1371/journal.ppat.100024419096510
  59. E.G. AcostaV. CastillaE.B. DamonteAlternative infectious entry pathways for dengue virus serotypes into mammalian cellsCell. Microbiol.2009111533154910.1111/j.1462-5822.2009.01345.x19523154
  60. L. SuksanpaisanT. SusantadD.R. SmithCharacterization of dengue virus entry into HepG2 cellsJ. Biomed. Sci.2009161710.1186/1423-0127-16-1719272179
  61. E.V. ProtopopovaA.V. SorokinS.N. KonovalovaA.V. KachkoS.V. NetesovV.B. LoktevHuman laminin binding protein as a cell receptor for the tick-borne encephalitis virusZent. Bakteriol.199928963263810.1016/S0934-8840(99)80021-8
  62. A.A. MalyginE.I. BondarenkoV.A. IvanisenkoE.V. ProtopopovaG.G. KarpovaV.B. LoktevC-terminal fragment of human laminin-binding protein contains a receptor domain for Venezuelan equine encephalitis and tick-borne encephalitis virusesBiochemistry2009741328133610.1134/S000629790912005019961413
  63. B.N. ZaitsevF. BenedettiA.G. MikhaylovD.V. KorneevS.K. SekatskiiT. KarakouzP.A. BelavinN.A. NetesovaE.V. ProtopopovaS.N. KonovalovaForce-induced globule-coil transition in laminin binding protein and its role for viral-cell membrane fusionJ. Mol. Recognit.20142772773810.1002/jmr.239925319621
  64. D.G. MaldovG. KarganovaA. TimofeevTick-borne encephalitis virus interaction with the target cellsArch. Virol.199212732132510.1007/BF013095941456894
  65. J. KopeckýL. GrubhofferV. KovářL. JindrákD. VokurkováA putative host cell receptor for tick-borne encephalitis virus identified by anti-idiotypic antibodies and virus affinoblottingIntervirology19994291610.1159/00002495410393498
  66. J. CorverA. OrtizS.L. AllisonJ. SchalichF.X. HeinzJ. WilschutMembrane fusion activity of tick-borne encephalitis virus and recombinant subviral particles in a liposomal model systemVirology2000269374610.1006/viro.1999.017210725196
  67. K. StiasnyF.X. HeinzEffect of membrane curvature-modifying lipids on membrane fusion by tick-borne encephalitis virusJ. Virol.2004788536854210.1128/JVI.78.16.8536-8542.200415280462
  68. M. Perera-LecoinL. MeertensX. CarnecA. AmaraFlavivirus entry receptors: An updateViruses20136698810.3390/v601006924381034
  69. E. TrybalaT. BergstroD. SpillmannB. SvennerholmS.J. FlynnP. RyanInteraction between Pseudorabies Virus and Heparin/Heparan Sulfate. Pseudorabies virus mutants differ in their interaction with heparin/heparan sulfate when altered for specific glycoprotein C heparin-binding domainJ. Biol. Chem.19982735047505210.1074/jbc.273.9.50479478954
  70. J.M. SmitB. WaartsK. KimataB. WilliamR. BittmanJ. WilschutW.B. KlimstraAdaptation of alphaviruses to heparan sulfate: Interaction of sindbis and Semliki Forest viruses with liposomes containing lipid-conjugated heparinJ. Virol.200276101281013710.1128/JVI.76.20.10128-10137.200212239287
  71. H. KroschewskiS.L. AllisonF.X. HeinzC.W. MandlRole of heparan sulfate for attachment and entry of tick-borne encephalitis virusVirology20033089210010.1016/S0042-6822(02)00097-112706093
  72. M. KaliaV. ChandraS.A. RahmanD. SehgalS. JameelHeparan sulfate proteoglycans are required for cellular binding of the hepatitis E Virus ORF2 capsid protein and for viral infectionJ. Virol.200983127141272410.1128/JVI.00717-0919812150
  73. L.I. KozlovskayaD.I. OsolodkinA.S. ShevtsovaL.I. RomanovaY.V. RogovaT.I. DzhivanianV.N. LyapustinG.P. PivanovaA.P. GmylV.A. PalyulinGAG-binding variants of tick-borne encephalitis virusVirology201039826227210.1016/j.virol.2009.12.01220064650
  74. S.M. De BoerJ. KortekaasC.A.M. de HaanP.J.M. RottierR.J.M. MoormannB.J. BoschHeparan sulfate facilitates Rift Valley fever virus entry into the cellJ. Virol.201286137671377110.1128/JVI.01364-1223015725
  75. C.W. MandlH. KroschewskiS.L. AllisonR. KoflerH. HolzmannT. MeixnerF.X. HeinzAdaptation of tick-borne encephalitis virus to BHK-21 cells results in the formation of multiple heparan sulfate binding sites in the envelope protein and attenuation in vivoJ. Virol.2001755627563710.1128/JVI.75.12.5627-5637.200111356970
  76. G. WinklerF.X. HeinzC. KunzStudies on the glycosylation of flavivirus E proteins and the role of carbohydrate in antigenic structureVirology198715923724310.1016/0042-6822(87)90460-02441520
  77. W.R. JiangA. LoweS. HiggsH. ReidE.A. GouldSingle amino acid codon changes detected in louping ill virus antibody-resistant mutants with reduced neurovirulenceJ. Gen. Virol.19937493193510.1099/0022-1317-74-5-9318388021
  78. E. PokidyshevaY. ZhangA.J. BattistiC.M. Bator-KellyP.R. ChipmanC. XiaoG.G. GregorioW.A. HendricksonR.J. KuhnM.G. RossmannCryo-EM reconstruction of dengue virus in complex with the carbohydrate recognition domain of DC-SIGNCell200612448549310.1016/j.cell.2005.11.04216469696
  79. R. PhillpottsJ. StephensonJ. PorterfieldAntibody-dependent enhancement of tick-borne encephalitis virus infectivityJ. Gen. Virol.1985661831183710.1099/0022-1317-66-8-18312991448
  80. K.A. DowdT.C. PiersonAntibody-mediated neutralization of flaviviruses: A reductionist viewVirology201141130631510.1016/j.virol.2010.12.02021255816
  81. L.C. KatzelnickL. GreshM.E. HalloranJ.C. MercadoG. KuanA. GordonA. BalmasedaE. HarrisAntibody-dependent enhancement of severe dengue disease in humansScience2017683692993210.1126/science.aan683629097492
  82. D. HaslwanterD. BlaasF.X. HeinzK. StiasnyA novel mechanism of antibody-mediated enhancement of flavivirus infectionPLoS Pathog.201713e100664310.1371/journal.ppat.100664328915259
  83. R. FritzK. StiasnyF.X. HeinzIdentification of specific histidines as pH sensors in flavivirus membrane fusionJ. Cell Biol.200818335336110.1083/jcb.20080608118936253
  84. K. StiasnyS.L. AllisonJ. SchalichX. HeinzF.X. HeinzMembrane interactions of the tick-borne encephalitis virus fusion protein E at low pH membrane interactions of the tick-borne encephalitis virus fusion protein E at low pHJ. Virol.2002763784379010.1128/JVI.76.8.3784-3790.200211907218
  85. K. StiasnyC. KösslJ. LepaultF. ReyF. HeinzCharacterization of a structural intermediate of flavivirus membrane fusionPLoS Pathog.20073e2010.1371/journal.ppat.003002017305426
  86. B. KaufmannP.R. ChipmanH.A. HoldawayS. JohnsonD.H. FremontR.J. KuhnM.S. DiamondM.G. RossmannCapturing a flavivirus pre-fusion intermediatePLoS Pathog.20095e100067210.1371/journal.ppat.100067219956725
  87. J. PanC.B. LaiW.R.P. ScottS.K. StrausSynthetic fusion peptides of tick-borne encephalitis virus as models for membrane fusionBiochemistry20104928729610.1021/bi901789520000438
  88. D.E. KleinJ.L. ChoiS.C. HarrisonStructure of a dengue virus envelope protein late-stage fusion intermediateJ. Virol.2013872287229310.1128/JVI.02957-1223236058
  89. X. ZhangJ. ShengS.K. AustinT.E. HoornwegJ.M. SmitR.J. KuhnM.S. DiamondM.G. RossmannStructure of acidic pH dengue virus showing the fusogenic glycoprotein trimersJ. Virol.20158974375010.1128/JVI.02411-1425355881
  90. S.L. AllisonK. StiasnyK. StadlerC.W. MandlF.X. HeinzMapping of functional elements in the stem-anchor region of tick-borne encephalitis virus envelope protein EJ. Virol.1999735605561210364309
  91. S. BressanelliK. StiasnyS.L. AllisonE.A. SturaS. DuquerroyJ. LescarF.X. HeinzF.A. ReyStructure of a flavivirus envelope glycoprotein in its low-pH-induced membrane fusion conformationEMBO J.20042372873810.1038/sj.emboj.760006414963486
  92. R. FritzJ. BlazevicC. TaucherK. PangerlF.X. HeinzK. StiasnyThe Unique Transmembrane hairpin of flavivirus fusion protein E is essential for membrane fusionJ. Virol.2011854377438510.1128/JVI.02458-1021325407
  93. K. StiasnyS. KiermayrA. BernhartF.X. HeinzThe membrane-proximal “stem” region increases the stability of the flavivirus E protein postfusion trimer and modulates its structureJ. Virol.2013879933993810.1128/JVI.01283-1323804648
  94. L.H. ChaoD.E. KleinA.G. SchmidtJ.M. PeñaS.C. HarrisonSequential conformational rearrangements in flavivirus membrane fusioneLife20143e0438910.7554/eLife.0438925479384
  95. L.A. BykN. IglesiasF. De MaioL. GebhardM. RossiV. GamarnikDengue virus genome uncoating requires ubiquitinationmBio20167e00804-1610.1128/mBio.00804-1627353759
  96. M. LobigsE. LeeInefficient signalase cleavage promotes efficient nucleocapsid incorporation into budding flavivirus membranesJ. Virol.20047817818610.1128/JVI.78.1.178-186.200414671099
  97. S. SchraufC.W. MandlL. Bell-SakyiT. SkernExtension of flavivirus protein C differentially affects early RNA synthesis and growth in mammalian and arthropod host cellsJ. Virol.200983112011121010.1128/JVI.01025-0919692461
  98. M. LobigsE. LeeM.L. NgM. PavyP. LobigsA flavivirus signal peptide balances the catalytic activity of two proteases and thereby facilitates virus morphogenesisVirology2010401808910.1016/j.virol.2010.02.00820207389
  99. L.A. VanBlarganK.A. DavisK.A. DowdD.L. AkeyJ.L. SmithT.C. PiersonContext-dependent cleavage of the capsid protein by the West Nile virus protease modulates the efficiency of virus assemblyJ. Virol.2015898632864210.1128/JVI.01253-1526063422
  100. M. RastogiN. SharmaS.K. SinghFlavivirus NS1: A multifaceted enigmatic viral proteinVirol. J.20161313110.1186/s12985-016-0590-727473856
  101. P. AvirutnanA. FuchsR.E. HauhartP. SomnukeS. YounM.S. DiamondJ.P. AtkinsonAntagonism of the complement component C4 by flavivirus nonstructural protein NS1J. Exp. Med.201020779380610.1084/jem.2009254520308361
  102. W.J. LiuH.B. ChenX.J. WangH. HuangA.A. KhromykhAnalysis of adaptive mutations in Kunjin virus replicon RNA reveals a novel role for the flavivirus nonstructural protein NS2A in inhibition of beta interferon promoter-driven transcriptionJ. Virol.200478122251223510.1128/JVI.78.22.12225-12235.200415507609
  103. X. WangW.J. LiuX.J. WangV.V. MokhonovP. ShiInhibition of interferon signaling by the New York 99 strain and Kunjin subtype of West Nile virus involves blockage of STAT1 and STAT2 activation by nonstructural proteinsJ. Virol.2005791934194210.1128/JVI.79.3.193415650219
  104. J.Y. LeungG.P. PijlmanN. KondratievaJ. HydeJ.M. MackenzieA.A. KhromykhRole of nonstructural protein NS2A in flavivirus assemblyJ. Virol.2008824731474110.1128/JVI.00002-0818337583
  105. X. XieS. GayenC. KangZ. YuanP.-Y. ShiMembrane topology and function of dengue virus NS2A proteinJ. Virol.2013874609462210.1128/JVI.02424-1223408612
  106. X.-D. LiC.-L. DengH.-Q. YeH.-L. ZhangQ.-Y. ZhangD.-D. ChenP.-T. ZhangP.-Y. ShiZ.-M. YuanB. ZhangTransmembrane domains of NS2B contribute to both viral RNA replication and particle formation in Japanese encephalitis virusJ. Virol.2016905735574910.1128/JVI.00340-1627053551
  107. D. LuoS.G. VasudevanJ. LescarThe flavivirus NS2B-NS3 protease-helicase as a target for antiviral drug developmentAntivir. Res.201511814815810.1016/j.antiviral.2015.03.01425842996
  108. S.A. ShiryaevA.V. ChernovA.E. AleshinT.N. ShiryaevaA.Y. StronginNS4A regulates the ATPase activity of the NS3 helicase: A novel cofactor role of the non-structural protein NS4A from West Nile virusJ. Gen. Virol.2009902081208510.1099/vir.0.012864-019474250
  109. J. ZmurkoJ. NeytsK. DallmeierFlaviviral NS4b, chameleon and jack-in-the-box roles in viral replication and pathogenesis, and a molecular target for antiviral interventionRev. Med. Virol.20152520522310.1002/rmv.183525828437
  110. A.D. DavidsonNew insights into flavivirus nonstructural protein 5Advances in Virus ResearchElsevier Inc.Cambridge, MA, USA2009Volume 74411019780123785879
  111. S.M. BestThe many faces of the flavivirus NS5 protein in antagonism of type I interferon signalingJ. Virol.201791e01970-1610.1128/JVI.01970-1627881649
  112. P. ErbelN. SchieringA. D’ArcyM. RenatusM. KroemerS.P. LimZ. YinT.H. KellerS.G. VasudevanU. HommelStructural basis for the activation of flaviviral NS3 proteases from dengue and West Nile virusNat. Struct. Mol. Biol.20061337237310.1038/nsmb107316532006
  113. L. AkeyC. BrownS. DuttaJ. KnowerskiJ. JoseT. JurkiwJ. DelPropostoC. OgataG. SkiniotisR. KuhnFlavivirus NS1 structures reveal surfaces for associations with membranes and the immune systemScience201434356757110.1126/science.124774924505133
  114. Y. ZhaoT.S. SohJ. ZhengK.W.K. ChanW.W. PhooC.C. LeeM.Y.F. TayK. SwaminathanT.C. CornvikS.P. LimA crystal structure of the dengue virus NS5 protein reveals a novel inter-domain interface essential for protein flexibility and virus replicatioPLoS Pathog.201511e100468210.1371/journal.ppat.100468225775415
  115. T. BílýM. PalusL. EyerJ. ElsterováM. VancováD. RůžekElectron tomography analysis of tick-borne encephalitis virus infection in human neuronsSci. Rep.201551074510.1038/srep1074526073783
  116. B.D. LindenbachC.M. RiceGenetic interaction of flavivirus nonstructural proteins NS1 and NS4A as a determinant of replicase functionJ. Virol.1999734611462110233920
  117. F. ŠeniglL. GrubhofferJ. KopeckyDifferences in maturation of tick-borne encephalitis virus in mammalian and tick cell lineIntervirology20064923924810.1159/00009147116491019
  118. S. MillerS. KastnerJ. Krijnse-LockerS. BühlerR. BartenschlagerThe non-structural protein 4A of dengue virus is an integral membrane protein inducing membrane alterations in a 2K-regulated mannerJ. Biol. Chem.20072828873888210.1074/jbc.M60991920017276984
  119. S. YounT. LiB.T. McCuneM.A. EdelingD.H. FremontI.M. CristeaM.S. DiamondEvidence for a genetic and physical interaction between nonstructural proteins NS1 and NS4B that modulates replication of West Nile virusJ. Virol.2012867360737110.1128/JVI.00157-1222553322
  120. L. MiorinI. Romero-BreyP. MaiuriS. HoppeJ. Krijnse-LockerR. BartenschlagerA. MarcelloThree-dimensional architecture of tick-borne encephalitis virus replication sites and trafficking of the replicated RNAJ. Virol.2013876469648110.1128/JVI.03456-1223552408
  121. M. HiranoK. YoshiiM. SakaiR. HasebeO. IchiiH. KariwaTick-borne flaviviruses alter membrane structure and replicate in dendrites of primary mouse neuronal culturesJ. Gen. Virol.20149584986110.1099/vir.0.061432-024394700
  122. P.H. KaufusiJ.F. KelleyR. YanagiharaV.R. NerurkarInduction of endoplasmic reticulum-derived replication-competent membrane structures by West Nile virus non-structural protein 4BPLoS ONE20149e8404010.1371/journal.pone.008404024465392
  123. C. YuK. AchaziL. MöllerJ.D. SchulzkeM. NiedrigR. BückerTick-borne encephalitis virus replication, intracellular trafficking, and pathogenicity in human intestinal Caco-2 cell monolayersPLoS ONE20149e9695710.1371/journal.pone.009695724820351
  124. D.K. OfferdahlD.W. DorwardB.T. HansenM.E. BloomA Three-dimensional comparison of tick-borne flavivirus infection in mammalian and tick cell linesPLoS ONE20127e4791210.1371/journal.pone.004791223112871
  125. C. LópezL. GilL. LazoI. MenéndezE. MarcosJ. SánchezI. ValdésV. FalcónM.C. De La RosaG. MárquezIn vitro assembly of nucleocapsid-like particles from purified recombinant capsid protein of dengue-2 virusArch. Virol.200915469569810.1007/s00705-009-0350-819305942
  126. R.M. KoflerF.X. HeinzC.W. MandlCapsid protein C of tick-borne encephalitis virus tolerates large internal deletions and is a favorable target for attenuation of virulenceJ. Virol.2002763534354310.1128/JVI.76.7.3534-3543.200211884577
  127. P. SchlickC. TaucherB. SchittlJ.L. TranR.M. KoflerW. SchuelerA. von GabainA. MeinkeC.W. MandlHelices 2 and 3 of West Nile virus capsid protein are dispensable for assembly of infectious virionsJ. Virol.2009835581559110.1128/JVI.02653-0819297470
  128. A.A. KhromykhE.G. WestawayRNA binding properties of core protein of the flavivirus KunjinArch. Virol.199614168569910.1007/BF017183268645104
  129. S. KiermayrR.M. KoflerC.W. MandlF.X. HeinzP. MessnerIsolation of capsid protein dimers from the tick-borne encephalitis flavivirus and in vitro assembly of capsid-like particlesJ. Virol.2004788078808410.1128/JVI.78.15.8078-8084.200415254179
  130. S. Apte-SenguptaD. SirohiR.J. KuhnCoupling of replication and assembly in flavivirusesCurr. Opin. Virol.2014913414210.1016/j.coviro.2014.09.02025462445
  131. A.A. KhromykhA.N. VarnavskiP.L. SedlakE.G. WestawayCoupling between replication and packaging of flavivirus RNA: evidence derived from the use of DNA-based full-length cDNA clones of Kunjin virusJ. Virol.2001754633464010.1128/JVI.75.10.4633-4640.200111312333
  132. S. WelschS. MillerI. Romero-BreyA. MerzC.K.E. BleckP. WaltherS.D. FullerC. AntonyJ. Krijnse-LockerR. BartenschlagerComposition and three-dimensional architecture of the dengue virus replication and assembly sitesCell Host Microbe2009536537510.1016/j.chom.2009.03.00719380115
  133. J. JunjhonJ.G. PenningtonT.J. EdwardsR. PereraJ. LanmanR.J. KuhnUltrastructural characterization and three-dimensional architecture of replication sites in dengue virus-infected mosquito CellsJ. Virol.2014884687469710.1128/JVI.00118-1424522909
  134. B.M. KümmererC.M. RiceMutations in the yellow fever virus nonstructural protein NS2A selectively block production of infectious particles mutations in the yellow fever virus nonstructural protein NS2A selectively block production of infectious particlesJ. Virol.2002764773478410.1128/JVI.76.10.4773-4784.200211967294
  135. W.J. LiuH.B. ChenA.A. KhromykhMolecular and functional analyses of Kunjin virus infectious cDNA clones demonstrate the essential roles for NS2A in virus assembly and for a nonconservative residue in NS3 in RNA replicationJ. Virol.2003777804781310.1128/JVI.77.14.7804-7813.200312829820
  136. G.P. PijlmanN. KondratievaA.A. KhromykhTranslation of the flavivirus Kunjin NS3 gene in cis but not Its RNA sequence or secondary structure is essential for efficient RNA packagingJ. Virol.200680112551126410.1128/JVI.01559-0616971441
  137. C.G. PatkarR.J. KuhnYellow fever virus NS3 plays an essential role in virus assembly independent of its known enzymatic functionsJ. Virol.2008823342335210.1128/JVI.02447-0718199634
  138. S. VoßmannJ. WieselerR. KerberB.M. KümmererA basic cluster in the N terminus of yellow fever virus NS2A contributes to infectious particle productionJ. Virol.2015894951496510.1128/JVI.03351-1425694595
  139. X. XieJ. ZouC. PuttikhuntZ. YuanP.-Y. ShiTwo distinct sets of NS2A molecules are responsible for dengue virus RNA synthesis and virion assemblyJ. Virol.2015891298131310.1128/JVI.02882-1425392211
  140. A. Groat-CarmonaS. OrozcoP. FriebeA. PayneL. KramerE. HarrisA novel coding-region RNA element modulates infectious dengue virus particle production in both mammalian and mosquito cells and regulates viral replication in Aedes aegypti mosquitoesVirology201243251152610.1016/j.virol.2012.06.02822840606
  141. C.A. BalinskyH. SchmeisserS. GanesanK. SinghT.C. PiersonK.C. ZoonNucleolin interacts with the dengue virus capsid protein and plays a role in formation of infectious virus particlesJ. Virol.201387130941310610.1128/JVI.00704-1324027323
  142. L. MaF. LiJ.-W. ZhangW. LiD.-M. ZhaoH. WangR.-H. HuaZ.-G. BuHost factor SPCS1 regulates the replication of Japanese encephalitis virus through interactions with transmembrane domains of NS2BJ. Virol.201810.1128/JVI.00197-1829593046
  143. Z. XuR. AndersonT.C. HobmanThe capsid-binding nucleolar helicase DDX56 is important for infectivity of West Nile virusJ. Virol.2011855571558010.1128/JVI.01933-1021411523
  144. C.R. ReidT.C. HobmanThe nucleolar helicase DDX56 redistributes to West Nile virus assembly sitesVirology201750016917710.1016/j.virol.2016.10.02527821284
  145. K. VondersteinE. NilssonP. HubelL. Nygård SkalmanA. UpadhyayJ. PastoA. PichlmairR. LundmarkA.K. ÖverbyViperin targets flavivirus virulence by inducing assembly of non-infectious capsid particlesJ. Virol.201710.1128/JVI.01751-17
  146. T. CarlettiM.K. ZakariaA. MarcelloThe host cell response to tick-borne encephalitis virusBiochem. Biophys. Res. Commun.201749253354010.1016/j.bbrc.2017.02.00628167278
  147. I. FerlenghiM. ClarkeT. RuttanS.L. AllisonJ. SchalichF.X. HeinzS.C. HarrisonF.A. ReyS.D. FullerMolecular organization of a recombinant subviral particle from tick-borne encephalitis virusMol. Cell2001759360210.1016/S1097-2765(01)00206-411463384
  148. I.C. LorenzJ. KartenbeckA. MezzacasaS.L. AllisonF.X. HeinzA. HeleniusIntracellular assembly and secretion of recombinant subviral particles from tick-borne encephalitis virusJ. Virol.2003774370438210.1128/JVI.77.7.4370-4382.200312634393
  149. J. BlazevicH. RouhaV. BradtF.X. HeinzK. StiasnyMembrane anchors of the structural flavivirus proteins and their role in virus assemblyJ. Virol.2016906365637810.1128/JVI.00447-1627147734
  150. F.X. HeinzK. StiasnyG. Püschner-AuerH. HolzmannS.L. AllisonC.W. MandlC. KunzStructural changes and functional control of the tick-borne encephalitis virus glycoprotein E by the heterodimeric association with protein prMVirology199419810911710.1006/viro.1994.10138259646
  151. V.B. RandolphG. WinklerV. StollarAcidotropic amines inhibit proteolytic processing of flavivirus prM proteinVirology199017445045810.1016/0042-6822(90)90099-D2154882
  152. F. GuirakhooF.X. HeinzC.W. MandlH. HolzmannC. KunzFusion activity of flaviviruses: Comparison of mature and immature (prM-containing) tick-borne encephalitis virionsJ. Gen. Virol.1991721323132910.1099/0022-1317-72-6-13231710648
  153. K. StadlerS.L. AllisonJ. SchalichF.X. HeinzProteolytic activation of tick-borne encephalitis virus by furinJ. Virol.199771847584819343204
  154. S. ElshuberS.L. AllisonF.X. HeinzC.W. MandlCleavage of protein prM is necessary for infection of BHK-21 cells by tick-borne encephalitis virusJ. Gen. Virol.20038418319110.1099/vir.0.18723-012533715
  155. S. ElshuberC.W. MandlResuscitating mutations in a furin cleavage-deficient mutant of the flavivirus tick-borne encephalitis virusJ. Virol.200579118131182310.1128/JVI.79.18.11813-11823.200516140758
  156. A. ZhengM. UmashankarM. KielianIn Vitro and In vivo studies identify important features of dengue virus pr-E protein interactionsPLoS Pathog.20106e100115710.1371/journal.ppat.100115720975939
  157. A. ZhengF. YuanL.M. KleinfelterM. KielianA toggle switch controls the low pH-triggered rearrangement and maturation of the dengue virus envelope proteinsNat. Commun.20145387710.1038/ncomms487724846574
  158. F.X. HeinzK. StiasnyThe Antigenic structure of Zika virus and its relation to other flaviviruses: implications for infection and immunoprophylaxisMicrobiol. Mol. Biol. Rev.201781e00055-1610.1128/MMBR.00055-1628179396
  159. F.A. ReyK. StiasnyF.X. HeinzFlavivirus structural heterogeneity: Implications for cell entryCurr. Opin. Virol.20172413213910.1016/j.coviro.2017.06.00928683393
  160. T. HaseP.L. SummersK.H. EckelsW.B. BazeAn electron and immunoelectron microscopic study of dengue-2 virus infection of cultured mosquito cells: Maturation eventsArch. Virol.19879227329110.1007/BF013174843813888
  161. L. EyerJ.J. ValdésV.A. GilR. NenckaH. HřebabeckýM. ŠálaJ. SalátJ. ČernýM. PalusE. De ClercqNucleoside inhibitors of tick-borne encephalitis virusAntimicrob. Agents Chemother.2015595483549310.1128/AAC.00807-1526124166
  162. L. EyerH. KondoD. ZouharovaM. HiranoJ.J. ValdésM. MutoT. KastlS. KobayashiJ. HaviernikM. IgarashiEscape of tick-borne flavivirus from 2′-C-methylated nucleoside antivirals is mediated by a single conservative mutation in NS5 that has a dramatic effect on viral fitnessJ. Virol.201791e01028-1710.1128/JVI.01028-1728814513
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