Karin Stiasny

Structure of a flavivirus envelope glycoprotein in its low-pH-induced membrane fusion conformation

Enveloped viruses enter cells via a membrane fusion reaction driven by conformational changes of specific viral envelope proteins. We report here the structure of the ectodomain of the tick‐borne encephalitis virus envelope glycoprotein, E, a prototypical class II fusion protein, in its trimeric low‐pH‐induced conformation. We show that, in the conformational transition, the three domains of the neutral‐pH form are maintained but their relative orientation is altered. Similar to the postfusion class I proteins, the subunits rearrange such that the fusion peptide loops cluster at one end of an elongated molecule and the C‐terminal segments, connecting to the viral transmembrane region, run along the sides of the trimer pointing toward the fusion peptide loops. Comparison with the low‐pH‐induced form of the alphavirus class II fusion protein reveals striking differences at the end of the molecule bearing the fusion peptides, suggesting an important conformational effect of the missing membrane connecting segment.

Correlation between ELISA, hemagglutination inhibition, and neutralization tests after vaccination against tick-borne encephalitis.

The significance of IgG antibody levels determined by a binding assay (ELISA) was investigated as a surrogate marker for the presence of neutralizing and hemagglutination inhibiting antibodies in sera from individuals vaccinated against tick‐borne encephalitis (TBE). To assess the extent of interference by flavivirus cross‐reactive antibodies, sera from persons with a proven or suspected history of other flavivirus infections and/or vaccinations were also examined. An excellent and highly significant correlation was found between ELISA IgG units and the antibody titers obtained by the hemagglutination inhibition (HI) as well as by the neutralization test (NT), provided that there was no other exposure to flavivirus antigens except TBE vaccination. Yellow fever vaccination and/or dengue virus infections induced significant levels of antibodies reactive in the TBE ELISA and HI test, which did not exhibit, however, neutralizing activity against TBE virus. The phenomenon and problem of “original antigenic sin” was demonstrated in a TBE vaccinee with a history of previous flavivirus infections. TBE vaccination first induced a booster reaction resulting in a rise in the level of cross‐reactive antibodies only, whereas TBE virus‐neutralizing antibodies became detectable only after the third vaccination. It is concluded that the level of IgG antibodies determined by ELISA is a good marker for predicting the presence of neutralizing antibodies after TBE vaccination, but only in the absence of flavivirus cross‐reactive antibodies. Otherwise, a neutralization assay is necessary for assessing immunity.

Recombinant and virion-derived soluble and particulate immunogens for vaccination against tick-borne encephalitis

Using different forms of the envelope glycoprotein E from tick-borne encephalitis virus we investigated the influence of physical and antigenic structure on the efficacy of vaccination. Different protein E-containing preparations were either derived from purified virions or were produced as recombinant proteins in COS cells. These included soluble dimeric forms (virion-derived protein E dimers with and without membrane anchor; recombinant protein E dimers without membrane anchor), micellar aggregates of protein E (rosettes), and recombinant subviral particles (RSPs). The structural differences between these immunogens were verified by sedimentation analysis, immunoblotting and epitope mapping with a panel of monoclonal antibodies. Specific immunogenicities were determined in mice in comparison to formalin-inactivated whole virus. Rosettes and RSPs were excellent immunogens and exhibited similar efficacies as inactivated virus in terms of antibody induction and protection against challenge, whereas all of the soluble forms were much less immunogenic. These data emphasize the importance of the immunogens antigenic and physical structure for an effective stimulation of the immune system and indicate that RSPs represent an excellent candidate for a recombinant vaccine against tick-borne encephalitis.

Characterization of monoclonal antibody-escape mutants of tick-borne encephalitis virus with reduced neuroinvasiveness in mice.

Escape mutants of tick-borne encephalitis (TBE) virus were selected using neutralizing monoclonal antibodies (MAbs) that react with three different and previously unrecognized epitopes in the envelope protein E of TBE virus. Two of these variants (V-IC3 and V-IE3) exhibited a significantly reduced reactivity with their selecting MAbs, as determined by ELISA, whereas with one variant (V-IO3), reactivity was completely unchanged. Comparative sequence analyses demonstrated that each of the variants differed from the wild-type virus by a single amino acid substitution located at exposed positions within domains I, II and III of protein E. In the mouse model, all three mutants were still neuro-virulent but exhibited a significantly reduced neuro-invasiveness after subcutaneous inoculation. Virus replication, however, was sufficient to induce a specific antibody response. The observed alterations in virulence properties were not associated with reduced growth rates in vertebrate cell cultures, but one variant (V-IE3) exhibited a small plaque phenotype. The mutation of variant V-IO3 resulted in a temperature-sensitive phenotype and a significant elevation of the pH-threshold of the conformational change necessary for fusion activity.

The interactions of the flavivirus envelope proteins: implications for virus entry and release

Viral membrane proteins play an important role in the assembly and disassembly of enveloped viruses. Oligomerization and proteolytic cleavage events are involved in controlling the functions of these proteins during virus entry and release. Using tick-borne encephalitis virus as a model we have studied the role of the flavivirus envelope proteins E and prM/M in these processes. Experiments with acidotropic agents provide evidence that the virus is taken up by receptor-mediated endocytosis and that the acidic pH in endosomes plays an important role for virus entry. The envelope glycoprotein E undergoes irreversible conformational changes at acidic pH, as indicated by the loss of several monoclonal antibody-defined epitopes, which coincide with the viral fusion activity in vitro. Sedimentation analysis reveals that these conformational changes lead to aggregation of virus particles, apparently by the exposure of hydrophobic sequence elements. None of these features are exhibited by immature virions containing E and prM rather than E and M. Detergent solubilization, sedimentation, and crosslinking experiments provide evidence that prM forms a complex with protein E which prevents the conformational changes necessary for fusion activity. The functional role of prM before its endoproteolytic cleavage by a cellular protease thus seems to be the protection of protein E from acid-inactivation during its passage through acidic trans Golgi vesicles in the course of virus release.

The entry machinery of flaviviruses

We have been using the flavivirus tick-borne encephalitis virus (TBEV) as a model system for investigating the molecular mechanisms underlying the membrane fusion process mediated by a class II viral fusion protein, the flavivirus envelope protein E. In the mature virion this protein exists as a metastable dimer that dissociates at the acidic pH in endosomes and is converted into a more stable trimeric conformation. The dimer dissociation step liberates an internal fusion peptide that interacts with the target endosomal membrane, and then further conformational changes are believed to drive membrane fusion. Although flavivirus fusion appears to be a more facile and efficient process than that of alphaviruses, which also possess a class II viral fusion protein, the fusion mechanism in both viral systems involves structurally related interactions with lipids, specifically the 3beta-hydroxyl group at C3 of cholesterol. The class II viral fusion machineries are structurally different from those involving class I viral fusion proteins, such as those found in orthomyxoviruses, paramyxoviruses, retroviruses, and filoviruses, but have certain similarities in common with bacterial pore-forming proteins.

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

SummaryA 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.