Tick-borne Encephalitis Virus Envelope Research Articles

Molecular Detection and Phylogenetic Analysis of Tick-Borne Encephalitis Virus from Ticks Collected from Cattle in Kyrgyzstan, 2023.

Ticks are important vectors of the tick-borne encephalitis virus (TBEV). In Kyrgyzstan, the livestock farming trade and nomadic lifestyle enable tick-borne diseases to be imported from neighboring countries, but there are few relevant studies. In this study, we collected 40 ticks from cattle in Kyrgyzstan. Molecular marker analysis identified the ticks as Ixodes persulcatus (97.5%; n = 39) and Haemaphysalis punctata (2.5%; n = 1). Real-time PCR screening revealed two ticks to be positive for TBEV, but only one tick was amplified using nested PCR targeting the TBEV envelope (E) and non-structure 5 (NS5) gene. The obtained sequences belonged to the TBEV Siberian subtype and phylogenetic tree analysis results confirmed that the virus was related to the Bosnia strain. We also performed next-generation sequencing, which confirmed the TBEV Siberian subtype. Continuous research and surveillance of TBEV in Kyrgyzstan are required to provide further information on tick-borne diseases.

Antiviral activity of porphyrins and porphyrin-like compounds against tick-borne encephalitis virus: Blockage of the viral entry/fusion machinery by photosensitization-mediated destruction of the viral envelope

Tick-borne encephalitis virus (TBEV), the causative agent of tick-borne encephalitis (TBE), is a medically important flavivirus endemic to the European–Asian continent. Although more than 12,000 clinical cases are reported annually worldwide, there is no anti-TBEV therapy available to treat patients with TBE. Porphyrins are macrocyclic molecules consisting of a planar tetrapyrrolic ring that can coordinate a metal cation. In this study, we investigated the cytotoxicity and anti-TBEV activity of a large series of alkyl- or (het)aryl-substituted porphyrins, metalloporphyrins, and chlorins and characterized their molecular interactions with the viral envelope in detail. Our structure–activity relationship study showed that the tetrapyrrole ring is an essential structural element for anti-TBEV activity, but that the presence of different structurally distinct side chains with different lengths, charges, and rigidity or metal cation coordination can significantly alter the antiviral potency of porphyrin scaffolds. Porphyrins were demonstrated to interact with the TBEV lipid membrane and envelope protein E, disrupt the TBEV envelope and inhibit the TBEV entry/fusion machinery. The crucial mechanism of the anti-TBEV activity of porphyrins is based on photosensitization and the formation of highly reactive singlet oxygen. In addition to blocking viral entry and fusion, porphyrins were also observed to interact with RNA oligonucleotides derived from TBEV genomic RNA, indicating that these compounds could target multiple viral/cellular structures. Furthermore, immunization of mice with porphyrin-inactivated TBEV resulted in the formation of TBEV-neutralizing antibodies and protected the mice from TBEV infection. Porphyrins can thus be used to inactivate TBEV while retaining the immunogenic properties of the virus and could be useful for producing new inactivated TBEV vaccines.

Genetically engineered bacterial‐like particles induced specific cellular and humoral immunity as effective tick‐borne encephalitis virus vaccine

AbstractTick‐borne encephalitis (TBE) is a natural focal disease with fatal encephalitis induced by tick‐borne encephalitis virus (TBEV), seriously threatening human and public health. Protection of TBE depends on vaccination with inactivated vaccine, which requires high cost and multiple immunizations. Here, we construct genetically engineered bacterial‐like particles (BLPs) as an effective TBEV vaccine with simplified immunizations and improved immune efficacy. The TBEV BLPs involve the combination of the gram‐positive enhancer matrix from Lactococcus lactis, and TBEV envelope (E) protein expressed by genetically engineered recombinant baculovirus. The prepared TBEV BLPs can effectively stimulate the activation of dendritic cells to present the TBEV E proteins to T and B cells, leading to strong and durable cellular and humoral immune responses in mice. Surprisingly, the serum levels of specific IgG antibodies in mice remain about 106 at 6 months after the secondary immunization. Overall, the TBEV BLPs can be used as a potent vaccine candidate, laying the foundation for developing novel TBEV genetically engineered vaccines.

Comprehensive N-glycosylation mapping of envelope glycoprotein from tick-borne encephalitis virus grown in human and tick cells

Tick-borne encephalitis virus (TBEV) is the causative agent of severe human neuroinfections that most commonly occur after a tick bite. N-Glycosylation of the TBEV envelope (E) glycoprotein is critical for virus egress in mammalian cells, but not in tick cells. In addition, glycans have been reported to mask specific antigenic sites from recognition by neutralizing antibodies. In this regard, the main purpose of our study was to investigate the profile of N-glycans linked to the E protein of TBEV when grown in human neuronal cells and compare it to the profile of virus grown in tick cells. Mass spectrometric analysis revealed significant differences in these profiles. High-mannose glycan with five mannose residues (Man5GlcNAc2), a complex biantennary galactosylated structure with core fucose (Gal2GlcNAc2Man3GlcNAc2Fuc), and a group of hybrid glycans with the composition Gal0-1GlcNAc1Man3-5GlcNAc2Fuc0-1 were confirmed as the main asparagine-linked oligosaccharides on the surface of TBEV derived from human neuronal cells. The observed pattern was supported by examination of the glycopeptides, providing additional information about the glycosylation site in the E protein. In contrast, the profile of TBEV grown in tick cells showed that paucimannose (Man3-4 GlcNAc2Fuc0-1) and high-mannose structures with five and six mannoses (Man5-6GlcNAc2) were major glycans on the viral surface. The reported results complement existing crystallography and cryoelectron tomography data on the E protein structure and could be instrumental for designing carbohydrate-binding antiviral agents active against TBEV.

Recombinant Fusion Protein Joining E Protein Domain III of Tick-Borne Encephalitis Virus and HSP70 of Yersinia pseudotuberculosis as an Antigen for the TI-Complexes.

Domain III (DIII) of the tick-borne encephalitis virus (TBEV) protein E contains epitopes, which induce antibodies capable of neutralizing the virus. To enhance the immunogenicity of this protein, which has a low molecular weight, the aim of the present work was to express, isolate, and characterize a chimeric protein based on the fusion of the bacterial chaperone HSP70 of Yersinia pseudotuberculosis and EIII (DIII + stem) as a prospective antigen for an adjuvanted delivery system, the tubular immunostimulating complex (TI-complex). The chimeric construction was obtained using pET-40b(+) vector by ligating the respective genes. The resulting plasmid was transformed into DE3 cells for the heterologous expression of the chimeric protein, which was purified by immobilized metal affinity chromatography (IMAC). ELISA, differential scanning calorimetry, intrinsic fluorescence, and computational analysis were applied for the characterization of the immunogenicity and conformation of the chimeric protein. Mice immunization showed that the chimeric protein induced twice the number of anti-EIII antibodies in comparison with EIII alone. In turn, the incorporation of the HSP70/EIII chimeric protein in the TI-complex resulted in a twofold increase in its immunogenicity. The formation of this vaccine construction was accompanied by significant conformational changes in the chimeric protein. Using HSP70 in the content of the chimeric protein represents an efficient means for presenting the main antigenic domain of the TBEV envelope protein to the immune system, whereas the incorporation of this chimeric protein into the TI-complex further contributes to the development of a stronger immune response against the TBEV infection.

Viral Fusion Peptides Incorporated in Monoolein Membranes: Secondary Structure and Lipid Phase Behavior

The viral membrane fusion is mediated by fusion proteins located on the virion surface. The process involves major changes of the membrane curvatures leading to non-bilayer structures. For a better understanding of the way fusion proteins steer this process, we have studied the interaction of two different hydrophobic segments, HA2-FP and TBEV-FP, which are known as “fusion peptides”, with monoolein mesophases as a function of temperature and pressure at limited hydration. The fusion peptides are derived from the influenza virus hemagglutinin fusion protein (HA2-FP) and from the tick-borne encephalitis virus envelope glycoprotein E (TBEV-FP). The changes of the monoolein phase behavior upon binding the peptides have been determined by X-ray diffraction. FTIR spectroscopy has been used to analyze the concomitant secondary structures of the peptides. We have found that the interaction of the fusion peptides with monoolein as well as the change of the temperature and pressure dependent lipid phase behavior differs markedly depending on the fusion peptide. However, they both destabilize the fluid lamellar phase and favor phases with negative curvature, i.e. inverse bicontinuous cubic and inverse hexagonal phases. We could show a partial reversion of these peptide-induced phase changes by the application of high pressure, demonstrating that a less dense packing of the monoolein membranes achieved by the fusion peptides promotes the negative curvature of the membrane. Interestingly, the secondary structures of the fusion peptides appear unaffected by monoolein fluid−fluid phase transitions, suggesting that the fusion peptides are the structure dominant species in the fusion process of lipid membranes.

Structure of tick-borne encephalitis virus and its neutralization by a monoclonal antibody

Tick-borne encephalitis virus (TBEV) causes 13,000 cases of human meningitis and encephalitis annually. However, the structure of the TBEV virion and its interactions with antibodies are unknown. Here, we present cryo-EM structures of the native TBEV virion and its complex with Fab fragments of neutralizing antibody 19/1786. Flavivirus genome delivery depends on membrane fusion that is triggered at low pH. The virion structure indicates that the repulsive interactions of histidine side chains, which become protonated at low pH, may contribute to the disruption of heterotetramers of the TBEV envelope and membrane proteins and induce detachment of the envelope protein ectodomains from the virus membrane. The Fab fragments bind to 120 out of the 180 envelope glycoproteins of the TBEV virion. Unlike most of the previously studied flavivirus-neutralizing antibodies, the Fab fragments do not lock the E-proteins in the native-like arrangement, but interfere with the process of virus-induced membrane fusion.

Lipid Phase Control and Secondary Structure of Viral Fusion Peptides Anchored in Monoolein Membranes.

The fusion of lipid membranes involves major changes of the membrane curvatures and is mediated by fusion proteins that bind to the lipid membranes. For a better understanding of the way fusion proteins steer this process, we have studied the interaction of two different viral fusion peptides, HA2-FP and TBEV-FP, with monoolein mesophases as a function of temperature and pressure at limited hydration. The fusion peptides are derived from the influenza virus hemagglutinin fusion protein (HA2-FP) and from the tick-borne encephalitis virus envelope glycoprotein E (TBEV-FP). By using synchrotron X-ray diffraction, the changes of the monoolein phase behavior upon binding the peptides have been determined and the concomitant secondary structures of the peptides have been analyzed by FTIR spectroscopy. As main results we have found that the fusion peptides interact differently with monoolein and change the pressure and temperature dependent lipid phase behavior to different extents. However, they both destabilize the fluid lamellar phase and favor phases with negative curvature, i.e. inverse bicontinuous cubic and inverse hexagonal phases. These peptide-induced phase changes can partially be reversed by the application of high pressure, demonstrating that the promotion of negative curvature is achieved by a less dense packing of the monoolein membranes by the fusion peptides. Pressure jumps across the cubic-lamellar phase transition reveal that HA2-FP has a negligible effect on the rates of the cubic and the lamellar phase formation. Interestingly, the secondary structures of the fusion peptides appear unaffected by monoolein fluid-fluid phase transitions, suggesting that the fusion peptides are the structure dominant species in the fusion process of lipid membranes.

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.

Recombinant domains III of Tick-Borne Encephalitis Virus envelope protein in combination with dextran and CpGs induce immune response and partial protectiveness against TBE virus infection in mice

BackgroundE protein of tick-borne encephalitis virus (TBEV) and other flaviviruses is located on the surface of the viral particle. Domain III of this protein seems to be a promising component of subunit vaccines for prophylaxis of TBE and kits for diagnostics of TBEV.MethodsThree variants of recombinant TBEV E protein domain III of European, Siberian and Far Eastern subtypes fused with dextran-binding domain of Leuconostoc citreum KM20 were expressed in E. coli and purified. The native structure of domain III was confirmed by ELISA antibody kit and sera of patients with tick-borne encephalitis. Immunogenic and protective properties of the preparation comprising these recombinant proteins immobilized on a dextran carrier with CpG oligonucleotides as an adjuvant were investigated on the mice model.ResultsAll 3 variants of recombinant proteins immobilized on dextran demonstrate specific interaction with antibodies from the sera of TBE patients. Thus, constructed recombinant proteins seem to be promising for TBE diagnostics. The formulation comprising the 3 variants of recombinant antigens immobilized on dextran and CpG oligonucleotides, induces the production of neutralizing antibodies against TBEV of different subtypes and demonstrates partial protectivity against TBEV infection.ConclusionsStudied proteins interact with the sera of TBE patients, and, in combination with dextran and CPGs, demonstrate immunogenicity and limited protectivity on mice compared with reference “Tick-E-Vac” vaccine.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-016-1884-5) contains supplementary material, which is available to authorized users.

Full genome sequences and molecular characterization of tick-borne encephalitis virus strains isolated from human patients

Tick-borne encephalitis virus (TBEV) causes tick-borne encephalitis (TBE), one of the most important human neuroinfections across Eurasia. Up to date, only three full genome sequences of human European TBEV isolates are available, mostly due to difficulties with isolation of the virus from human patients. Here we present full genome characterization of an additional five low-passage TBEV strains isolated from human patients with severe forms of TBE. These strains were isolated in 1953 within Central Bohemia in the former Czechoslovakia, and belong to the historically oldest human TBEV isolates in Europe. We demonstrate here that all analyzed isolates are distantly phylogenetically related, indicating that the emergence of TBE in Central Europe was not caused by one predominant strain, but rather a pool of distantly related TBEV strains. Nucleotide identity between individual sequenced TBEV strains ranged from 97.5% to 99.6% and all strains shared large deletions in the 3′ non-coding region, which has been recently suggested to be an important determinant of virulence. The number of unique amino acid substitutions varied from 3 to 9 in individual isolates, but no characteristic amino acid substitution typical exclusively for all human TBEV isolates was identified when compared to the isolates from ticks. We did, however, correlate that the exploration of the TBEV envelope glycoprotein by specific antibodies were in close proximity to these unique amino acid substitutions. Taken together, we report here the largest number of patient-derived European TBEV full genome sequences to date and provide a platform for further studies on evolution of TBEV since the first emergence of human TBE in Europe.

Cloning and Expression of Recombinant Tick-Borne Encephalitis Virus-like Particles in Pichia pastoris

ObjectivesThe purpose of this study was to verify the feasibility of using the glyceraldehyde-3-phosphate dehydrogenase (GAP) promotor based Pichia pastoris expression system to produce tick-borne encephalitis virus (TBEV) virus-like particles (VLPs). MethodsThe complementary DNA encoding the TBEV prM signal peptide, prM, and E proteins of TBEV Korean strain (KrM 93) was cloned into the plasmid vector pGAPZɑA, then integrated into the genome of P. pastoris, under the control of the GAP promoter. Expression of TBEV VLPs was determined by Western blotting using monoclonal antibody against TBEV envelope (E) protein. ResultsRecombinant TBEV VLPs consisting of prM and E protein were successfully expressed using the GAP promoter-based P. pastoris expression system. The results of Western blotting showed that the recombinant proteins were secreted into the culture supernatant from the P. pastoris and glycosylated. ConclusionThis study suggests that recombinant TBEV VLPs from P. pastoris offer a promising approach to the production of VLPs for use as vaccines and diagnostic antigens.

N-linked glycan in tick-borne encephalitis virus envelope protein affects viral secretion in mammalian cells, but not in tick cells

Tick-borne encephalitis virus (TBEV) is a zoonotic disease agent that causes severe encephalitis in humans. The envelope protein E of TBEV has one N-linked glycosylation consensus sequence, but little is known about the biological function of the N-linked glycan. In this study, the function of protein E glycosylation was investigated using recombinant TBEV with or without the protein E N-linked glycan. Virion infectivity was not affected after removing the N-linked glycans using N-glycosidase F. In mammalian cells, loss of glycosylation affected the conformation of protein E during secretion, reducing the infectivity of secreted virions. Mice subcutaneously infected with TBEV lacking protein E glycosylation showed no signs of disease, and viral multiplication in peripheral organs was reduced relative to that with the parental virus. In contrast, loss of glycosylation did not affect the secretory process of infectious virions in tick cells. Furthermore, inhibition of transport to the Golgi apparatus affected TBEV secretion in mammalian cells, but not in tick cells, indicating that TBEV was secreted through an unidentified pathway after synthesis in endoplasmic reticulum in tick cells. These results increase our understanding of the molecular mechanisms of TBEV maturation.

Chimeric Tick-Borne Encephalitis/Dengue Virus Is Attenuated inIxodes scapularisTicks andAedes aegyptiMosquitoes

In an effort to derive an efficacious live attenuated vaccine against tick-borne encephalitis, we generated a chimeric virus bearing the structural protein genes of a Far Eastern subtype of tick-borne encephalitis virus (TBEV) on the genetic background of recombinant dengue 4 (DEN4) virus. Introduction of attenuating mutations into the TBEV envelope protein gene, as well as the DEN4 NS5 protein gene and 3' noncoding region in the chimeric genome, results in decreased neurovirulence and neuroinvasiveness in mice, and restricted replication in mouse brain. Since TBEV and DEN4 viruses are transmitted in nature by ticks and mosquitoes, respectively, it was of interest to investigate the infectivity of the chimeric virus for both arthropod vectors. Therefore, parental and chimeric viruses were tested for growth in mosquito and tick cells and for oral infection in vivo. Although all chimeric viruses demonstrated moderate levels of replication in C6/36 mosquito cells, they were unable to replicate in ISE6 tick cells. Further, the chimeric viruses were unable to infect or replicate in Aedes aegypti mosquitoes and Ixodes scapularis tick larvae. The poor infectivity for both potential vectors reinforces the safety of chimeric virus-based vaccine candidates for the environment and for use in humans.

P1182 Differences of immunoreactivity of selected antigenic epitope of tick-borne encephalitis virus envelope protein gE modelled by different variants of recombinant fusion proteins

P1182 Differences of immunoreactivity of selected antigenic epitope of tick-borne encephalitis virus envelope protein gE modelled by different variants of recombinant fusion proteins

Folding and dimerization of tick-borne encephalitis virus envelope proteins prM and E in the endoplasmic reticulum.

Flavivirus envelope proteins are synthesized as part of large polyproteins that are co- and posttranslationally cleaved into their individual chains. To investigate whether the interaction of neighboring proteins within the precursor protein is required to ensure proper maturation of the individual components, we have analyzed the folding of the flavivirus tick-borne encephalitis (TBE) virus envelope glycoproteins prM and E by using a recombinant plasmid expression system and virus-infected cells. When expressed in their polyprotein context, prM and E achieved their native folded structures with half-times of approximately 4 min for prM and about 15 min for E. They formed heterodimeric complexes within a few minutes after synthesis that were required for the final folding of E but not for that of prM. Heterodimers could also be formed in trans when these proteins were coexpressed from separate constructs. When expressed without prM, E could form disulfide bonds but did not express a specific conformational epitope and remained sensitive to reduction by dithiothreitol. This is consistent with a chaperone-like role for prM in the folding of E. PrM was able to achieve its native folded structure without coexpression of E, but signal sequence cleavage at the N terminus was delayed. Our results show that prM is an especially rapidly folding viral glycoprotein, that polyprotein cleavage and folding of the TBE virus envelope proteins occurs in a coordinated sequence of processing steps, and that proper and efficient maturation of prM and E can only be achieved by cosynthesis of these two proteins.

A DNA Immunization Model Study with Constructs Expressing the Tick-Borne Encephalitis Virus Envelope Protein E in Different Physical Forms

We have conducted a DNA immunization study to evaluate how the immune response is influenced by the physical structure and secretion of the expressed Ag. For this purpose, we used a series of plasmid constructs encoding different forms of the envelope glycoprotein E of the flavivirus tick-borne encephalitis virus. These included a secreted recombinant subviral particle, a secreted carboxyl-terminally truncated soluble homodimer, a nonsecreted full-length form, and an inefficiently secreted truncated form. Mice were immunized using both i.m. injection and Gene Gun-mediated application of plasmids. The functional immune response was evaluated by determining specific neutralizing and hemagglutination-inhibiting Ab activities and by challenging the mice with a lethal dose of the virus. As a measure for the induction of a Th1 and/or Th2 response, we determined specific IgG subclasses and examined IFN-gamma, Il-4, and Il-5 induction. The plasmid construct encoding a secreted subviral particle, which carries multiple copies of the protective Ag on its surface, was superior to the other constructs in terms of extent and functionality of the Ab response as well as protection against virus challenge. As expected, the type of Th response was largely dependent on the mode of application (i.m. vs Gene Gun), but our data show that it was also strongly influenced by the properties of the Ag. Most significantly, the plasmid encoding the particulate form was able to partially overcome the Th2 bias imposed by the Gene Gun, resulting in a balanced Th1/Th2 response.

Mapping of functional elements in the stem-anchor region of tick-borne encephalitis virus envelope protein E.

Envelope protein E of the flavivirus tick-borne encephalitis virus mediates membrane fusion, and the structure of the N-terminal 80% of this 496-amino-acid-long protein has been shown to differ significantly from that of other viral fusion proteins. The structure of the carboxy-terminal 20%, the stem-anchor region, is not known. It contains sequences that are important for membrane anchoring, interactions with prM (the precursor of membrane protein M) during virion assembly, and low-pH-induced structural changes associated with the fusion process. To identify specific functional elements in this region, a series of C-terminal deletion mutants were constructed and the properties of the resulting truncated recombinant E proteins were examined. Full-length E proteins and proteins lacking the second of two predicted transmembrane segments were secreted in a particulate form when coexpressed with prM, whereas deletion of both segments resulted in the secretion of soluble homodimeric E proteins. Sites located within a predicted alpha-helical region of the stem (amino acids 431 to 449) and the first membrane-spanning region (amino acids 450 to 472) were found to be important for the stability of the prM-E heterodimer but not essential for prM-mediated intracellular transport and secretion of soluble E proteins. A separate site in the stem, also corresponding to a predicted alpha-helix (amino acids 401 to 413), was essential for the conversion of soluble protein E dimers to a homotrimeric form upon low-pH treatment, a process resembling the transition to the fusogenic state in whole virions. This functional mapping will aid in the understanding of the molecular mechanisms of membrane fusion and virus assembly.

Tick-borne encephalitis virus envelope protein E-specific monoclonal antibodies for the study of low pH-induced conformational changes and immature virions.

A set of ten monoconal antibodies (mabs) specific for the tick-borne encephalitis (TBE) virus envelope protein E were prepared and characterized with respect to their functional activities, the location of their binding sites on protein E and the involvement of their epitopes in acid pH-induced conformational changes and interactions with the precursor to the membrane protein (prM) in immature virions. The majority of these mabs mapped to the previously defined antigenic domain A. All of the mabs recognize parts of the E protein which undergo low pH-induced structural rearrangements believed to be necessary for the fusion activity of the virus, and six of the mabs define epitopes which are affected by the prM-E interaction in immature virions. They are therefore of potential value as specific reagents for studying the structure and function of protein E, as well as the function of the prM-E association. Five of the mabs exhibited neutralizing activity, and can therefore be used for the selection of escape mutants.

Oligomeric rearrangement of tick-borne encephalitis virus envelope proteins induced by an acidic pH.

The flavivirus envelope protein E undergoes irreversible conformational changes at a mildly acidic pH which are believed to be necessary for membrane fusion in endosomes. In this study we used a combination of chemical cross-linking and sedimentation analysis to show that the envelope proteins of the flavivirus tick-borne encephalitis virus also change their oligomeric structure when exposed to a mildly acidic environment. Under neutral or slightly alkaline conditions, protein E on the surface of native virions exists as a homodimer which can be isolated by solubilization with the nonionic detergent Triton X-100. Solubilization with the same detergent after pretreatment at an acidic pH, however, yielded homotrimers rather than homodimers, suggesting that exposure to an acidic pH had induced a simultaneous weakening of dimeric contacts and a strengthening of trimeric ones. The pH threshold for the dimer-to-trimer transition was found to be 6.5. Because the pH dependence of this transition parallels that of previously observed changes in the conformation and hydrophobicity of protein E and that of virus-induced membrane fusion, it appears likely that the mechanism of fusion with endosomal membranes involves a specific rearrangement of the proteins in the viral envelope. Immature virions in which protein E is associated with the uncleaved precursor (prM) of the membrane protein M did not undergo a low-pH-induced rearrangement. This is consistent with a protective role of protein prM for protein E during intracellular transport of immature virions through acidic compartments of the trans-Golgi network.