Franz X. Heinz

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.

Mutational Evidence for an Internal Fusion Peptide in Flavivirus Envelope Protein E

ABSTRACT The envelope protein E of the flavivirus tick-borne encephalitis (TBE) virus promotes cell entry by inducing fusion of the viral membrane with an intracellular membrane after uptake by endocytosis. This protein differs from other well-studied viral and cellular fusion proteins because of its distinct molecular architecture and apparent lack of involvement of coiled coils in the low-pH-induced structural transitions that lead to fusion. A highly conserved loop (the cd loop), which resides at the distal tip of each subunit and is mostly buried in the subunit interface of the native E homodimer at neutral pH, has been hypothesized to function as an internal fusion peptide at low pH, but this has not yet been shown experimentally. It was predicted by examination of the X-ray crystal structure of the TBE virus E protein (F. A. Rey et al., Nature 375:291–298, 1995) that mutations at a specific residue within this loop (Leu 107) would not cause the native structure to be disrupted. We therefore introduced amino acid substitutions at this position and, using recombinant subviral particles, investigated the effects of these changes on fusion and related properties. Replacement of Leu with hydrophilic amino acids strongly impaired (Thr) or abolished (Asp) fusion activity, whereas a Phe mutant still retained a significant degree of fusion activity. Liposome coflotation experiments showed that the fusion-negative Asp mutant did not form a stable interaction with membranes at low pH, although it was still capable of undergoing the structural rearrangements required for fusion. These data support the hypothesis that the cd loop may be directly involved in interactions with target membranes during fusion.

Molecular Organization of a Recombinant Subviral Particle from Tick-Borne Encephalitis Virus

The tick-borne encephalitis (TBE) flavivirus contains two transmembrane proteins, E and M. Coexpression of E and the M precursor (prM) leads to secretion of recombinant subviral particles (RSPs). In the most common form of these RSPs, analyzed at a 19 A resolution by cryo-electron microscopy (cryo-EM), 60 copies of E pack as dimers in a T = 1 icosahedral surface lattice (outer diameter, 315 A). Fitting the high-resolution structure of a soluble E fragment into the RSP density defines interaction sites between E dimers, positions M relative to E, and allows assignment of transmembrane regions of E and M. Lateral interactions among the glycoproteins stabilize this capsidless particle; similar interactions probably contribute to assembly of virions. The structure suggests a picture for trimer association under fusion-inducing conditions.

Adaptation 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 Vivo

ABSTRACT Propagation of the flavivirus tick-borne encephalitis virus in BHK-21 cells selected for mutations within the large surface glycoprotein E that increased the net positive charge of the protein. In the course of 16 independent experiments, 12 different protein E mutation patterns were identified. These were located in all three of the structural domains and distributed over almost the entire upper and lateral surface of protein E. The mutations resulted in the formation of local patches of predominantly positive surface charge. Recombinant viruses carrying some of these mutations in a defined genetic backbone showed heparan sulfate (HS)-dependent phenotypes, resulting in an increased specific infectivity and binding affinity for BHK-21 cells, small plaque formation in porcine kidney cells, and significant attenuation of neuroinvasiveness in adult mice. Our results corroborate the notion that the selection of attenuated HS binding mutants is a common and frequent phenomenon during the propagation of viruses in cell culture and suggest a major role for HS dependence in flavivirus attenuation. Recognition of this principle may be of practical value for designing attenuated flavivirus strains in the future.

Structural basis of potent Zika–dengue virus antibody cross-neutralization

Zika virus is a member of the Flavivirus genus that had not been associated with severe disease in humans until the recent outbreaks, when it was linked to microcephaly in newborns in Brazil and to Guillain–Barré syndrome in adults in French Polynesia. Zika virus is related to dengue virus, and here we report that a subset of antibodies targeting a conformational epitope isolated from patients with dengue virus also potently neutralize Zika virus. The crystal structure of two of these antibodies in complex with the envelope protein of Zika virus reveals the details of a conserved epitope, which is also the site of interaction of the envelope protein dimer with the precursor membrane (prM) protein during virus maturation. Comparison of the Zika and dengue virus immunocomplexes provides a lead for rational, epitope-focused design of a universal vaccine capable of eliciting potent cross-neutralizing antibodies to protect simultaneously against both Zika and dengue virus infections.Zika virus is a member of the Flavivirus genus that had not been associated with severe disease in humans until the recent outbreaks, when it was linked to microcephaly in newborns in Brazil and to Guillain-Barré syndrome in adults in French Polynesia. Zika virus is related to dengue virus, and here we report that a subset of antibodies targeting a conformational epitope isolated from patients with dengue virus also potently neutralize Zika virus. The crystal structure of two of these antibodies in complex with the envelope protein of Zika virus reveals the details of a conserved epitope, which is also the site of interaction of the envelope protein dimer with the precursor membrane (prM) protein during virus maturation. Comparison of the Zika and dengue virus immunocomplexes provides a lead for rational, epitope-focused design of a universal vaccine capable of eliciting potent cross-neutralizing antibodies to protect simultaneously against both Zika and dengue virus infections.

Folding and Dimerization of Tick-Borne Encephalitis Virus Envelope Proteins prM and E in the Endoplasmic Reticulum

ABSTRACT 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.

Cryptic Properties of a Cluster of Dominant Flavivirus Cross-Reactive Antigenic Sites

ABSTRACT A number of flaviviruses are important human pathogens, including yellow fever, dengue, West Nile, Japanese encephalitis, and tick-borne encephalitis (TBE) viruses. Infection with or immunization against any of these viruses induces a subset of antibodies that are broadly flavivirus cross-reactive but do not exhibit significant cross-neutralization. Nevertheless, these antibodies can efficiently bind to the major envelope protein (E), which is the main target of neutralizing and protective antibodies because of its receptor-binding and membrane fusion functions. The structural basis for this phenomenon is still unclear. In our studies with TBE virus, we have provided evidence that such cross-reactive antibodies are specific for a cluster of epitopes that are partially occluded in the cage-like assembly of E proteins at the surfaces of infectious virions and involve—but are not restricted to—amino acids of the highly conserved internal fusion peptide loop. Virus disintegration leads to increased accessibility of these epitopes, allowing the cross-reactive antibodies to bind with strongly increased avidity. The cryptic properties of these sites in the context of infectious virions can thus provide an explanation for the observed lack of efficient neutralizing activity of broadly cross-reactive antibodies, despite their specificity for a functionally important structural element in the E protein.

Epitope mapping of flavivirus glycoproteins

Publisher Summary The vast majority of monoclonal antibodies to flaviviruses produced so far are specific for the E glycoprotein; therefore, this chapter deals with the characteristics of epitopes on this immunologically dominant protein. In addition, recent studies with NV3-specific monoclonal antibodies provide evidence that this nonstructural glycoprotein may play an important role in mounting a protective immune response. These new aspects have been emphasized. The analysis of flavivirus structural glycoproteins with monoclonal antibodies revealed a serological complexity that goes far beyond that established by polyclonal sera. At the epitope level, antigenic relationships are strongly determined by the assay system used, and differing patterns of cross-reactivities are obtained when different functional tests are applied. For the tick-borne encephalitis (TBE) virus system, it has been shown that increased binding is due to up to six-fold enhancement of antibody avidity. The structural characteristics of monoclonal antibody-defined epitopes have been determined for TBE virus. Epitopes in both major domains appear to be strongly conformational dependent, with the difference that domain A is very labile and easily denatured, whereas domain B is strongly stabilized by disulfide bridges. For all flaviviruses analyzed with monoclonal antibodies, the dissociation of different functional activities is apparent at the epitope level. All types of different qualitative and quantitative combinations can be observed.

A topological and functional model of epitopes on the structural glycoprotein of tick-borne encephalitis virus defined by monoclonal antibodies.

A topological and functional model of eight distinct epitopes on the structural glycoprotein of tick-borne encephalitis (TBE) virus was established by the use of monoclonal antibodies. The unique specificities and spatial relationships of these antibodies were determined by variant analysis, haemagglutination inhibition (HI), neutralization, passive mouse protection, and antibody blocking assays. Seven out of the eight distinct epitopes were shown to be partially linked and to cluster in two antigenically reactive domains (A, B). Each of these domains is inhomogeneous and contains constituents with different serological specificities and functions. Domain A is defined by three HA-inhibiting antibodies, two of which are flavivirus group-reactive, whereas the third is TBE virus subtype specific. Within this domain only the subtype-specific antibody is involved in virus neutralization, thus explaining the observation that neutralization tests with flaviviruses show higher serological specificities than HI tests and that HI tests can be made type and subtype specific by antibody absorption. Domain B is composed of three TBE-complex reactive epitopes, and the corresponding antibodies inhibit HA and neutralize the virus. A fourth epitope linked to this domain is neither involved in HA nor in neutralization and the same holds true for a subtype-specific epitope which is topologically independent of domains A and B. Each of two different nonneutralizing antibodies was capable of blocking the binding of distinct neutralizing antibodies. All eight epitopes are indistinguishably present on strains of the western subtype of TBE virus isolated all over Europe in different years from different hosts, thus again confirming the great stability of this virus.

Structures and mechanisms in flavivirus fusion.

Publisher Summary This chapter focuses on the work carried out with tick-borne encephalitis (TBE) virus, the structurally best characterized of the flaviviruses. The data is related to those obtained with other flaviviruses, which are assumed to have a conserved structural organization, and compare the characteristics of flavivirus fusion to those of other enveloped viruses. Fusion proteins from several different virus families, including Orthomyxoviridae, Paramyxoviridae, Retroviridae, and Filoviridae have been shown to exhibit striking structural similarities; they all use a common mechanism for inducing membrane fusion, and the same general model applies to all of these cases. The flavivirus genome is a positive-stranded RNA molecule consisting of a single, long open reading frame of more than 10,000 nucleotides flanked by noncoding regions at the 5′ and 3′ ends. The fusion properties of flaviviruses have been investigated using several different assay systems, including virus-induced cell–cell fusion and virus–liposome fusion. All of these studies indicate that flaviviruses require an acidic pH for fusion, consistent with their proposed mode of entry.