Molecular Basis of the pH-Controlled Maturation of the Tick-Borne Encephalitis Flavivirus.

Flaviviruses are enveloped viruses causing high public concerns. Their maturation spans several cellular compartments having different pH. Thus, complex control mechanisms are in place to avoid premature maturation. Here we report the dynamical behavior at neutral and acidic pH of the precursor of the membrane fusion protein E of tick-borne encephalitis, showing the different stabilizations of the E dimer and the role played by the small fusion-assisting protomer (pr). The comprehension, at atomic resolution, of the fine regulation of viral maturation will be fundamental to the development of efficient strategies against emerging viral threats.

Tick-borne encephalitis nonstructural protein NS1 expressed in E. coli retains immunological properties of the native protein.

There is evidence that flaviviral NS1 glycoprotein plays an important role in the pathology of tick-borne encephalitis (TBE) and NS1-specific antibodies are detected in the blood of patients with TBE. This makes NS1 a good target for the development of therapeutic inhibitors and NS1 could be an important biomarker for the early diagnosis of TBE in vaccinated individuals. Eukaryotic expression systems are mainly used to produce recombinant tick-borne encephalitis virus (TBEV) NS1. The expression of TBEV NS1 proteins in eukaryotic cells was successful, but there were some limitations. Several attempts have also been made to obtain the NS1 protein in Escherichia coli cells; however, they were unsuccessful due to the low solubility of the recombinant protein and improper folding. In this study, using Trx-tag as a fusion partner, soluble Trx-fused TBEV NS1 protein was first produced in the E. coli BL21 strain. In addition, insoluble Trx-fused TBEV NS1 protein was obtained when cultivation conditions were changed to increase the productivity. The insoluble TBEV NS1 obtained from inclusion bodies was solubilized using chaotropic reagents and successfully refolded using dialysis. Both soluble variant and successfully refolded from inclusion bodies variant showed immunological properties similar to the native TBEV NS1 protein and were recognized by specific monoclonal antibodies (mAbs), immune ascetic fluid in ELISA, western blot, and competitive analysis.

An Absolutely Conserved Tryptophan in the Stem of the Envelope Protein E of Flaviviruses Is Essential for the Formation of Stable Particles.

The major envelope protein E of flaviviruses contains an ectodomain that is connected to the transmembrane domain by the so-called "stem" region. In mature flavivirus particles, the stem is composed of two or three mostly amphipathic α-helices and a conserved sequence element (CS) with an undefined role in the viral life cycle. A tryptophan is the only residue within this region which is not only conserved in all vector-borne flaviviruses, but also in the group with no known vector. We investigated the importance of this residue in different stages of the viral life cycle by a mutagenesis-based approach using tick-borne encephalitis virus (TBEV). Replacing W421 by alanine or histidine strongly reduced the release of infectious virions and their thermostability, whereas fusion-related entry functions and virus maturation were still intact. Serial passaging of the mutants led to the emergence of a same-site compensatory mutation to leucine that largely restored these properties of the wildtype. The conserved tryptophan in CS (or another big hydrophobic amino acid at the same position) is thus essential for the assembly and infectivity of flaviviruses by being part of a network required for conferring stability to infectious particles.

Dynamics and Extent of Non-Structural Protein 1-Antibody Responses in Tick-Borne Encephalitis Vaccination Breakthroughs and Unvaccinated Patients.

Tick-borne encephalitis (TBE) has a substantial impact on human public health in many parts of Europe and Asia. Effective inactivated purified whole-virus vaccines are in widespread use in TBE-endemic countries. Nevertheless, vaccination breakthroughs (VBTs) with manifest clinical disease do occur, and their specific serodiagnosis was shown to be facilitated by the detection of antibodies to a non-structural protein (NS1) that is produced during virus replication. However, recent data have shown that NS1 is also present in the current inactivated vaccines, with the potential of inducing corresponding antibodies and obscuring a proper interpretation of NS1-antibody assays for diagnosing VBTs. In our study, we quantified anti-virion and anti-NS1 antibody responses after vaccination as well as after natural infection in TBE patients, both without and with a history of previous TBE vaccination (VBTs). We did not find significant levels of NS1-specific antibodies in serum samples from 48 vaccinees with a completed vaccination schedule. In contrast, all TBE patients mounted an anti-NS1 antibody response, irrespective of whether they were vaccinated or not. Neither the dynamics nor the extent of NS1-antibody formation differed significantly between the two cohorts, arguing against substantial NS1-specific priming and an anamnestic NS1-antibody response in VBTs.

Characterization and in vitro assembly of tick-borne encephalitis virus C protein.

Tick-borne encephalitis virus (TBEV), a member of flaviviruses, represents a serious health threat by causing human encephalitis mainly in central and eastern Europe, Russia, and northeastern Asia. As no specific therapy is available, there is an urgent need to understand all steps of the TBEV replication cycle at the molecular level. One of the critical events is the packaging of flaviviral genomic RNA by TBEV C protein to form a nucleocapsid. We purified recombinant TBEV C protein and used a combination of physical-chemical approaches, such as size-exclusion chromatography, circular dichroism, NMR spectroscopies, and transmission electron microscopy, to analyze its structural stability and its ability to dimerize/oligomerize. We compared the ability of TBEV C protein to assemble in vitro into a nucleocapsid-like structure with that of dengue C protein.

Backbone Free Energy Estimator Applied to Viral Glycoproteins.

Earlier analysis of the Protein Data Bank derived the distribution of rotations from the plane of a protein hydrogen bond donor peptide group to the plane of its acceptor peptide group. The quasi Boltzmann formalism of Pohl-Finkelstein is employed to estimate free energies of protein elements with these hydrogen bonds, pinpointing residues with a high propensity for conformational change. This is applied to viral glycoproteins as well as capsids, where the 90th+ percentiles of free energies determine residues that correlate well with viral fusion peptides and other functional domains in known cases and thus provide a novel method for predicting these sites of importance as antiviral drug or vaccine targets in general. The method is implemented at https://bion-server.au.dk/hbonds/ from an uploaded Protein Data Bank file.

Molecular Basis of a Protective/Neutralizing Monoclonal Antibody Targeting Envelope Proteins of both Tick-Borne Encephalitis Virus and Louping Ill Virus.

Tick-borne encephalitis virus (TBEV) and louping ill virus (LIV) are members of the tick-borne flaviviruses (TBFVs) in the family Flaviviridae which cause encephalomeningitis and encephalitis in humans and other animals. Although vaccines against TBEV and LIV are available, infection rates are rising due to the low vaccination coverage. To date, no specific therapeutics have been licensed. Several neutralizing monoclonal antibodies (MAbs) show promising effectiveness in the control of TBFVs, but the underlying molecular mechanisms are yet to be characterized. Here, we determined the crystal structures of the LIV envelope (E) protein and report the comparative structural analysis of a TBFV broadly neutralizing murine MAb (MAb 4.2) in complex with either the LIV or TBEV E protein. The structures reveal that MAb 4.2 binds to the lateral ridge of domain III of the E protein (EDIII) of LIV or TBEV, an epitope also reported for other potently neutralizing MAbs against mosquito-borne flaviviruses (MBFVs), but adopts a unique binding orientation. Further structural analysis suggested that MAb 4.2 may neutralize flavivirus infection by preventing the structural rearrangement required for membrane fusion during virus entry. These findings extend our understanding of the vulnerability of TBFVs and other flaviviruses (including MBFVs) and provide an avenue for antibody-based TBFV antiviral development.IMPORTANCE Understanding the mechanism of antibody neutralization/protection against a virus is crucial for antiviral countermeasure development. Tick-borne encephalitis virus (TBEV) and louping ill virus (LIV) are tick-borne flaviviruses (TBFVs) in the family Flaviviridae They cause encephalomeningitis and encephalitis in humans and other animals. Although vaccines for both viruses are available, infection rates are rising due to low vaccination coverage. In this study, we solved the crystal structures of the LIV envelope protein (E) and a broadly neutralizing/protective TBFV MAb, MAb 4.2, in complex with E from either TBEV or LIV. Key structural features shared by TBFV E proteins were analyzed. The structures of E-antibody complexes showed that MAb 4.2 targets the lateral ridge of both the TBEV and LIV E proteins, a vulnerable site in flaviviruses for other potent neutralizing MAbs. Thus, this site represents a promising target for TBFV antiviral development. Further, these structures provide important information for understanding TBFV antigenicity.

Assessment of NS1 protein as an early diagnostic marker for Kyasanur forest disease virus.

BACKGROUND & OBJECTIVES: Due to the emergence of Kyasanur forest disease (KFD) virus to new regions in India, there is an urgent need to develop an early diagnostic system, which is cost-effective and can be efficiently used with minimum paraphernalia. The non-structural-1 (NS1) protein is known to be an early diagnostic marker for flaviviruses. Furthermore, NS1 antigen capture ELISA kits developed using bacterially expressed dengue NS1 protein are commercially available. METHODS: Based on the data available on dengue virus, West Nile virus and other flaviviruses, bacterially expressed Kyasanur forest disease virus (KFDV) NS1 protein and polyclonal serum raised against the NS1 protein in mice and rabbit were used to develop an antigen capture ELISA for early diagnosis of the virus. The feasibility of this ELISA was further tested using in silico predictions. RESULTS: KFDV NS1 gene was cloned, expressed and confirmed by SDS-PAGE and western blotting. An antigen detection ELISA was standardized and sensitivity and specificity was tested with other flaviviruses. KFDV acute phase 43 samples were tested and only two were found to be positive for KFDV NS1 antigen. Superimposition of KFDV NS1 and TBEV NS1 revealed a root mean square distance (RMSD) of ~0.79 Å covering 1220 backbone atoms. This implies that the structures are very similar in terms of 3D fold. The identity of amino acid composition between these proteins was 73.4% and similarity was 92.9%, as revealed from the pairwise comparison. INTERPRETATION & CONCLUSION: The study points out that the half-life, expression and secretion levels of KFDV NS1 protein are not sufficient enough for its use as early diagnostic marker. The protein may have to be expressed in eukaryotic host to counter the lack of glycosylation in bacterial plasmid based expression of proteins. Hence, bacterially expressed KFDV NS1 protein may not be an ideal early diagnostic marker for the virus.

Engineering of Chimeric Protein Based on E Protein Domain III of Tick- Borne Encephalitis Virus and OmpF Porin of Yersinia pseudotuberculosis.

BACKGROUND: Tick-borne encephalitis poses a serious public health threat in the endemic regions. The disease treatment is restricted to symptomatic therapy, so great expectations are in the development of the prophylactic and therapeutic vaccines. The domain III of E protein of the tickborne encephalitis virus is the main antigenic domain which includes virus-specific epitopes recognized by neutralizing antibodies. OBJECTIVES: The main objective of this study was to design, express, isolate and characterize the chimeric protein based on the fusion of domain III of E protein of the tick-borne encephalitis virus and bacterial porin OmpF from Yersinia pseudotuberculosis. METHODS: The chimeric gene was obtained by the PCR based fusion method from two fragments containing overlapping linker sequences. Resulting plasmids were transformed into BL21(DE3) pLysS electrocompetent cells for subsequent heterologous protein expression. All recombinant proteins were purified using immobilized metal affinity chromatography under denaturing conditions. The identity of the chimeric protein was confirmed by MALDI-TOF mass spectrometry and immunoblot analysis. The content of antibodies against the EIII protein was estimated in mice blood serum by ELISA. RESULTS: The bacterial partner protein was used for decreasing toxicity and increasing immunogenicity of antigen. The chimeric protein was successfully expressed by the Escherichia coli cells. The purified protein was recognized with immunoblots by anti-E protein of tick-borne encephalitis virus monoclonal antibodies. Furthermore, the protein was able to elicit antibody response against domain III of E protein in immunized mice. CONCLUSION: The newly obtained chimeric antigen could be valuable for the development of the preventing tick-borne encephalitis subunit vaccines.

A comparative analysis on the physicochemical properties of tick-borne encephalitis virus envelope protein residues that affect its antigenic properties.

This work is dedicated to the study of the variability of the main antigenic envelope protein E among different strains of tick-borne encephalitis virus at the level of physical and chemical properties of the amino acid residues. E protein variants were extracted from then NCBI database. Four amino acid residues properties in the polypeptide sequences were investigated: the average volume of the amino acid residue in the protein tertiary structure, the number of amino acid residue hydrogen bond donors, the charge of amino acid residue lateral radical and the dipole moment of the amino acid residue. These physico-chemical properties are involved in antigen-antibody interactions. As a result, 103 different variants of the antigenic determinants of the tick-borne encephalitis virus E protein were found, significantly different by physical and chemical properties of the amino acid residues in their structure. This means that some strains among the natural variants of tick-borne encephalitis virus can potentially escape the immune response induced by the standard vaccine.

Nonstructural Protein 1 of Tick-Borne Encephalitis Virus Induces Oxidative Stress and Activates Antioxidant Defense by the Nrf2/ARE Pathway.

BACKGROUND: Infection with tick-borne encephalitis virus (TBEV) causes pathological changes in the central nervous system. However, the possible redox alterations in the infected cells that can contribute to the virus pathogenicity remain unknown. OBJECTIVE: In the current study we explored the ability of TBEV nonstructural protein 1 (NS1) to induce oxidative stress and activate antioxidant defense via the nuclear factor (erythroid-derived-2)-like 2/antioxidant response element (Nrf2/ARE) pathway. METHODS: HEK 293T cells were transfected with plasmid encoding NS1 protein, and the production of reactive oxygen species (ROS) was measured using oxidation-sensitive dyes, the activation of the ARE promoter was estimated using a reporter plasmid, and the expression of phase II detoxifying enzymes was quantified by measuring their mRNA levels using RT-qPCR. RESULTS: A high level of ROS production was detected in cells transfected with NS1-expressing plasmid. In addition, this protein activated the promoter with an ARE and upregulated the transcription of ARE-dependent genes that encode phase II enzymes. CONCLUSION: TBEV NS1 protein both triggers ROS production and activates a defense Nrf2/ARE pathway. These data suggest that a role of redox-mediated processes in TBEV-induced damage of the central nervous system should also be explored. These data can contribute to a better understanding of TBEV pathogenicity, further improvement of TBE treatment, and the development of vaccine candidates against this infection.

Development of a bio-analytical strategy for characterization of vaccine particles combining SEC and nanoES GEMMA.

Commonly used methods for size and shape analysis of bionanoparticles found in vaccines like X-ray crystallography and cryo-electron microscopy are very time-consuming and cost-intensive. The nano-electrospray (nanoES) gas-phase electrophoretic mobility macromolecular analyzer (GEMMA), belonging to the group of ion mobility spectrometers, was used for size determination of vaccine virus particles because it requires less analysis time and investment (no vacuum system). Size exclusion chromatography (SEC) of viral vaccines and production intermediates turned out to be a good purification/isolation method prior to GEMMA, TEM (transmission electron microscopy) and AFM (atomic force microscopy) investigations, as well as providing a GEMMA analysis-compatible buffer. Column materials and different elution buffers were tested for optimal vaccine particle yield. We used a Superdex 200 column with a 50 mM ammonium acetate buffer. In addition, SEC allowed the removal of process-related impurities from the virions of interest. A sample concentrating step or a detergent addition step was also investigated. As a final step of our strategy SEC-purified or untreated vaccine-nanoparticles were further analyzed: (a) by immunological detection with a specific polyclonal antibody (dot blot) to verify the biological functionality, (b) by GEMMA to provide the size of the particles at atmospheric pressure and (c) by AFM and (d) TEM to obtain both size and shape information. The mean diameter of inactivated tick-borne encephalitis virions (i.e. vaccine particles) determined by GEMMA measurement was 46.6 ± 0.5 nm, in contrast to AFM and TEM images providing diameters of about 58 ± 4 and 52 ± 5 nm, respectively.

The membrane-proximal "stem" region increases the stability of the flavivirus E protein postfusion trimer and modulates its structure.

The flavivirus fusion protein E contains a "stem" region which is hypothesized to be crucial for driving fusion. This sequence element connects the ectodomain to the membrane anchor, and its structure in the trimeric postfusion conformation is still poorly defined. Using E trimers of tick-borne encephalitis virus with stem truncations of different lengths, we show that the N-terminal part of the stem increases trimer stability and also modulates the trimer structure outside the stem interaction site.

NS2B/3 proteolysis at the C-prM junction of the tick-borne encephalitis virus polyprotein is highly membrane dependent.

The replication of tick-borne encephalitis virus (TBEV), like that of all flaviviruses, is absolutely dependent on proteolytic processing. Production of the mature proteins C and prM from their common precursor requires the activity of the viral NS2B/3 protease (NS2B/3(pro)) at the C-terminus of protein C and the host signal peptidase I (SPaseI) at the N-terminus of protein prM. Recently, we have shown in cell culture that the cleavage of protein C and the subsequent production of TBEV particles can be made dependent on the activity of the foot-and-mouth disease virus 3C protease, but not on the activity of the HIV-1 protease (HIV1(pro)) (Schrauf et al., 2012). To investigate this failure, we developed an in vitro cleavage assay to assess the two cleavage reactions performed on the C-prM precursor. Accordingly, a recombinant modular NS2B/3(pro), consisting of the protease domain of NS3 linked to the core-domain of cofactor NS2B, was expressed in E. coli and purified to homogeneity. This enzyme could cleave a C-prM protein synthesised in rabbit reticulocyte lysates. However, cleavage was only specific when protein synthesis was performed in the presence of canine pancreatic microsomal membranes and required the prevention of signal peptidase I (SPaseI) activity by lengthening the h-region of the signal peptide. The presence of membranes allowed the concentration of NS2B/3(pro) used to be reduced by 10-20 fold. Substitution of the NS2B/3(pro) cleavage motif in C-prM by a HIV-1(pro) motif inhibited NS2B/3(pro) processing in the presence of microsomal membranes but allowed cleavage by HIV-1(pro) at the C-prM junction. This system shows that processing at the C-terminus of protein C by the TBEV NS2B/3(pro) is highly membrane dependent and will allow the examination of how the membrane topology of protein C affects both SPaseI and NS2B/3(pro) processing.

MicroRNA targeting of neurotropic flavivirus: effective control of virus escape and reversion to neurovirulent phenotype.

Neurotropic flaviviruses can efficiently replicate in the developing and mature central nervous systems (CNS) of mice causing lethal encephalitis. Insertion of a single copy of a target for brain-expressed microRNAs (miRNAs) in the 3' noncoding region (3'NCR) of the flavivirus genome (chimeric tick-borne encephalitis virus/dengue virus) abolished virus neurovirulence in the mature mouse CNS. However, in the developing CNS of highly permissive suckling mice, the miRNA-targeted viruses can revert to a neurovirulent phenotype by accumulating deletions or mutations within the miRNA target sequence. Virus escape from miRNA-mediated suppression in the developing CNS was markedly diminished by increasing the number of miRNA target sites and by extending the distance between these sites in the virus genome. Insertion of multiple miRNA targets into the 3'NCR altered virus neuroinvasiveness, decreased neurovirulence and neuroinflammatory responses, and prevented neurodegeneration without loss of immunogenicity. Although the onset of encephalitis was delayed, a small number of suckling mice still succumbed to lethal intracerebral infection with the miRNA-targeted viruses. Sequence analysis of brain isolates from moribund mice revealed that the viruses escaped from miRNA-mediated suppression exclusively through the deletion of miRNA targets and viral genome sequence located between the two miRNA targets separated by the greatest distance. These findings offer a general strategy to control the reversion of virus to a virulent phenotype: a simultaneous miRNA targeting of the viral genome at many different functionally important regions could prevent virus escape from miRNA-based attenuation, since a deletion of the targeted genomic sequences located between the inserted miRNA binding sites would be lethal for the virus.

A conserved region in the prM protein is a critical determinant in the assembly of flavivirus particles.

Flaviviruses are assembled to bud into the lumen of the endoplasmic reticulum (ER) and are secreted through the vesicle transport pathway, but the details of the molecular mechanism of virion assembly remain largely unknown. In this study, a highly conserved region in the prM protein was identified among flaviviruses. In the subviral particle (SP) system of tick-borne encephalitis virus (TBEV) and Japanese encephalitis virus, secretion of SPs was impaired by a mutation in the conserved region in the prM protein. Viral proteins were sparse in the Golgi complex and accumulated in the ER. Ultrastructural analysis revealed that long filamentous structures, rather than spherical SPs, were observed in the lumen of the ER as a result of the mutation. The production of infectious virions derived from infectious cDNA of TBEV was also reduced by mutations in the conserved region. Molecular modelling analysis suggested that the conserved region is important for the association of prM-envelope protein heterodimers in the formation of a spike of immature virion. These results are the first demonstration that the conserved region in the prM protein is a molecular determinant for the flavivirus assembly process.

Synthetic fusion peptides of tick-borne encephalitis virus as models for membrane fusion.

The fusion peptide of TBEV is a short segment of the envelope protein that mediates viral and host cell membrane fusion at acidic pH. Previous studies on the E protein have shown that mutations at L107 have an effect on fusogenic activity. Structural studies have also suggested that during the fusion process the E protein rearranges to form a trimer. In the present study, a number of short peptides were synthesized, and their structure/activity was examined: (1) monomers consisting of residues 93-113 of the wild-type E protein with Leu at position 107 (WT) and two mutants, namely, L107F and L107T; (2) a monomer consisting of residues 93-113 of the E protein with a C105A mutation (TFPmn); (3) a trimer consisting of three monomers described in (2), linked at the C-terminus via 1 Lys (TFPtr); (4) a monomer consisting of residues 93-113 of the E protein plus six additional Lys at the C-terminus; and (5) a trimer consisting of three monomers described in (3), linked via the side chain of the sixth lysine. The secondary structure content of all peptides was investigated using circular dichroism (CD). Approximately seven of the residues were in beta-strand conformation, in the presence of POPC/POPE/cholesterol. The structures did not depend on pH significantly. The fusogenicity of the peptides was measured by FRET and photon correlation spectroscopy. The data suggest that TFPtr is the most fusogenic at acidic pH and that the mutation from L107 to T reduces activity. Molecular dynamics simulations of WT, L107T, and L107F suggest that this reduction in activity may be related to the fact that the mutations disrupt trimer stability. Finally, tryptophan fluorescence experiments were used to localize the peptides in the membrane. It was found that WT, L107F, TFPmn, and TFPtr could penetrate better into the acyl chain region of the lipids than the other peptides tested. The implications of these results on the fusion mechanism of TBEV E protein will be presented.

Impact of quaternary organization on the antigenic structure of the tick-borne encephalitis virus envelope glycoprotein E.

The envelope protein E of flaviviruses mediates both receptor-binding and membrane fusion. At the virion surface, 180 copies of E are tightly packed and organized in a herringbone-like icosahedral structure, whereas in noninfectious subviral particles, 60 copies are arranged in a T=1 icosahedral symmetry. In both cases, the basic building block is an E dimer which exposes the binding sites for neutralizing antibodies at its surface. It was the objective of our study to assess the dependence of the antigenic structure of E on its quaternary arrangement, i.e., as part of virions, recombinant subviral particles, or soluble dimers. For this purpose, we used a panel of 11 E protein-specific neutralizing monoclonal antibodies, mapped to distinct epitopes in each of the three E protein domains, and studied their reactivity with the different soluble and particulate forms of tick-borne encephalitis virus E protein under nondenaturing immunoassay conditions. Significant differences in the reactivities with these forms were observed that could be related to (i) limited access of certain epitopes at the virion surface; (ii) limited occupancy of epitopes in virions due to steric hindrance between antibodies; (iii) differences in the avidity to soluble forms compared to the virion, presumably related to the flexibility of E at its domain junctions; and (iv) modulations of the external E protein surface through interactions with its stem-anchor structure. We have thus identified several important factors that influence the antigenicity of the flavivirus E protein and have an impact on the interaction with neutralizing antibodies.

Identification of specific histidines as pH sensors in flavivirus membrane fusion.

The flavivirus membrane fusion machinery, like that of many other enveloped viruses, is triggered by the acidic pH in endosomes after virus uptake by receptor-mediated endocytosis. It has been hypothesized that conserved histidines in the class II fusion protein E of these viruses function as molecular switches and, by their protonation, control the fusion process. Using the mutational analysis of recombinant subviral particles of tick-borne encephalitis virus, we provide direct experimental evidence that the initiation of fusion is crucially dependent on the protonation of one of the conserved histidines (His323) at the interface between domains I and III of E, leading to the dissolution of domain interactions and to the exposure of the fusion peptide. Conserved histidines located outside this critical interface were found to be completely dispensable for triggering fusion.