Mapping of Functional Elements in the Stem-Anchor Region of Tick-Borne Encephalitis Virus Envelope Protein E

ABSTRACT 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 α-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 α-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.

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

Journal of General Virology ◽

10.1099/vir.0.055269-0 ◽

2013 ◽

Vol 94 (10) ◽

pp. 2249-2258 ◽

Cited By ~ 22

Author(s):

Kentaro Yoshii ◽

Natsumi Yanagihara ◽

Mariko Ishizuka ◽

Mizuki Sakai ◽

Hiroaki Kariwa

Keyword(s):

Envelope Protein ◽

Mammalian Cells ◽

Molecular Mechanisms ◽

Consensus Sequence ◽

Encephalitis Virus ◽

Secretory Process ◽

Virus Envelope ◽

Tick Borne Encephalitis ◽

Virus Envelope Protein ◽

Tick Borne Encephalitis Virus

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.

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

Archives of Virology ◽

10.1007/bf01309857 ◽

1995 ◽

Vol 140 (2) ◽

pp. 213-221 ◽

Cited By ~ 21

Author(s):

H. Holzmann ◽

K. Stiasny ◽

H. York ◽

F. Dorner ◽

C. Kunz ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Conformational Changes ◽

Envelope Protein ◽

Low Ph ◽

Encephalitis Virus ◽

Virus Envelope ◽

Tick Borne Encephalitis ◽

Virus Envelope Protein ◽

Tick Borne Encephalitis Virus

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Effect of Membrane Curvature-Modifying Lipids on Membrane Fusion by Tick-Borne Encephalitis Virus

Journal of Virology ◽

10.1128/jvi.78.16.8536-8542.2004 ◽

2004 ◽

Vol 78 (16) ◽

pp. 8536-8542 ◽

Cited By ~ 40

Author(s):

Karin Stiasny ◽

Franz X. Heinz

Keyword(s):

Fusion Protein ◽

Membrane Fusion ◽

Low Ph ◽

Encephalitis Virus ◽

Soluble Form ◽

Inverted Cone ◽

Outer Leaflet ◽

Tick Borne Encephalitis ◽

Target Membrane ◽

Tick Borne Encephalitis Virus

ABSTRACT Enveloped viruses enter cells by fusion of their own membrane with a cellular membrane. Incorporation of inverted-cone-shaped lipids such as lysophosphatidylcholine (LPC) into the outer leaflet of target membranes has been shown previously to impair fusion mediated by class I viral fusion proteins, e.g., the influenza virus hemagglutinin. It has been suggested that these results provide evidence for the stalk-pore model of fusion, which involves a hemifusion intermediate (stalk) with highly bent outer membrane leaflets. Here, we investigated the effect of inverted-cone-shaped LPCs and the cone-shaped oleic acid (OA) on the membrane fusion activity of a virus with a class II fusion protein, the flavivirus tick-borne encephalitis virus (TBEV). This study included an analysis of lipid mixing, as well as of the steps preceding or accompanying fusion, i.e., binding to the target membrane and lipid-induced conformational changes in the fusion protein E. We show that the presence of LPC in the outer leaflet of target liposomes strongly inhibited TBEV-mediated fusion, whereas OA caused a very slight enhancement, consistent with a fusion mechanism involving a lipid stalk. However, LPC also impaired the low-pH-induced binding of a soluble form of the E protein to liposomes and its conversion into a trimeric postfusion structure that requires membrane binding at low pH. Because inhibition is already observed before the lipid-mixing step, it cannot be determined whether impairment of stalk formation is a contributing factor in the inhibition of fusion by LPC. These data emphasize, however, the importance of the composition of the target membrane in its interactions with the fusion peptide that are crucial for the initiation of fusion.

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Membrane Interactions of the Tick-Borne Encephalitis Virus Fusion Protein E at Low pH

Journal of Virology ◽

10.1128/jvi.76.8.3784-3790.2002 ◽

2002 ◽

Vol 76 (8) ◽

pp. 3784-3790 ◽

Cited By ~ 89

Author(s):

Karin Stiasny ◽

Steven L. Allison ◽

Juliane Schalich ◽

Franz X. Heinz

Keyword(s):

Fusion Protein ◽

Membrane Binding ◽

Low Ph ◽

Encephalitis Virus ◽

Soluble Form ◽

Viral Fusion ◽

Sequence Element ◽

Viral Fusion Protein ◽

Tick Borne Encephalitis ◽

Tick Borne Encephalitis Virus

ABSTRACT Membrane fusion of the flavivirus tick-borne encephalitis virus is triggered by the mildly acidic pH of the endosome and is mediated by envelope protein E, a class II viral fusion protein. The low-pH trigger induces an oligomeric rearrangement in which the subunits of the native E homodimers dissociate and the monomeric subunits then reassociate into homotrimers. Here we provide evidence that membrane binding is mediated by the intermediate monomeric form of E, generated by low-pH-induced dissociation of the dimer. Liposome coflotation experiments revealed that association with target membranes occurred only when liposomes were present at the time of acidification, whereas pretreating virions at low pH in the absence of membranes resulted in the loss of their ability to stably attach to liposomes. With the cleavable cross-linker ethylene glycolbis(succinimidylsuccinate), it was shown that a truncated soluble form of the E protein (sE) could bind to membranes only when the dimers were free to dissociate at low pH, and binding could be blocked by a monoclonal antibody that recognizes the fusion peptide, which is at the distal tip of the E monomer but is buried in the native dimer. Surprisingly, analysis of the membrane-associated sE proteins revealed that they had formed trimers. This was unexpected because this protein lacks a sequence element in the C-terminal stem-anchor region, which was shown to be essential for trimerization in the absence of a target membrane. It can therefore be concluded that the formation of a trimeric form of sE is facilitated by membrane binding. Its stability is apparently maintained by contacts between the ectodomains only and is not dependent on sequence elements in the stem-anchor region as previously assumed.

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Expression of cloned envelope protein genes from the flavivirus tick-borne encephalitis virus in mammalian cells and random mutagenesis by PCR

Virus Genes ◽

10.1007/bf01703077 ◽

1994 ◽

Vol 8 (2) ◽

pp. 187-198 ◽

Cited By ~ 17

Author(s):

Steven L. Allison ◽

Christian W. Mandl ◽

Christian Kunz ◽

Franz X. Heinz

Keyword(s):

Envelope Protein ◽

Mammalian Cells ◽

Random Mutagenesis ◽

Encephalitis Virus ◽

Tick Borne Encephalitis ◽

Tick Borne Encephalitis Virus

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

Journal of Virology ◽

10.1128/jvi.02132-18 ◽

2019 ◽

Vol 93 (8) ◽

Cited By ~ 2

Author(s):

Xu Yang ◽

Jianxun Qi ◽

Ruchao Peng ◽

Lianpan Dai ◽

Ernest A. Gould ◽

...

Keyword(s):

Structural Analysis ◽

Encephalitis Virus ◽

Infection Rates ◽

E Proteins ◽

E Protein ◽

Tick Borne Encephalitis ◽

The Family ◽

Lateral Ridge ◽

Louping Ill ◽

Tick Borne Encephalitis Virus

ABSTRACT 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.

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Structure-Based Mutational Analysis of Several Sites in the E Protein: Implications for Understanding the Entry Mechanism of Japanese Encephalitis Virus

Journal of Virology ◽

10.1128/jvi.00293-15 ◽

2015 ◽

Vol 89 (10) ◽

pp. 5668-5686 ◽

Cited By ~ 22

Author(s):

Haibin Liu ◽

Yi Liu ◽

Shaobo Wang ◽

Yanjun Zhang ◽

Xiangyang Zu ◽

...

Keyword(s):

Amino Acids ◽

Japanese Encephalitis Virus ◽

Membrane Fusion ◽

Japanese Encephalitis ◽

Receptor Binding ◽

Viral Entry ◽

Envelope Protein ◽

Encephalitis Virus ◽

Entry Pathway ◽

Entry Mechanism

ABSTRACTJapanese encephalitis virus (JEV), which causes viral encephalitis in humans, is a serious risk to global public health. The JEV envelope protein mediates the viral entry pathway, including receptor-binding and low-pH-triggered membrane fusion. Utilizing mutagenesis of a JEV infectious cDNA clone, mutations were introduced into the potential receptor-binding motif or into residues critical for membrane fusion in the envelope protein to systematically investigate the JEV entry mechanism. We conducted experiments evaluating infectious particle, recombinant viral particle, and virus-like particle production and found that most mutations impaired virus production. Subcellular fractionation confirmed that five mutations—in I0, ij, BC, and FG and the R9A substitution—impaired virus assembly, and the assembled virus particles of another five mutations—in kl and the E373A, F407A, L221S, and W217A substitutions—were not released into the secretory pathway. Next, we examined the entry activity of six mutations yielding infectious virus. The results showed N154 and the DE loop are not the only or major receptor-binding motifs for JEV entry into BHK-21 cells; four residues, H144, H319, T410, and Q258, participating in the domain I (DI)-DIII interaction or zippering reaction are important to maintain the efficiency of viral membrane fusion. By continuous passaging of mutants, adaptive mutations from negatively charged amino acids to positively charged or neutral amino acids, such as E138K and D389G, were selected and could restore the viral entry activity.IMPORTANCERecently, there has been much interest in the entry mechanism of flaviviruses into host cells, including the viral entry pathway and membrane fusion mechanism. Our study provides strong evidence for the critical role of several residues in the envelope protein in the assembly, release, and entry of JEV, which also contributes to our understanding of the flaviviral entry mechanism. Furthermore, we demonstrate that the H144A, H319A, T410A, and Q258A mutants exhibit attenuated fusion competence, which may be used to develop novel vaccine candidates for flaviviruses.

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