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.Enveloped viruses enter cells by fusing their membranes with a host cell membrane, either at the cell surface or at an internal site after uptake by endocytosis. This is mediated by metastable surface proteins that undergo a triggered conformational change upon binding to a receptor or exposure to the acidic environment of the endosome, allowing a previously buried portion of the protein, the fusion peptide, to insert into the target membrane (22). Exposure and insertion of the fusion peptide is believed to be the crucial step in the initiation of the fusion process, although further conformational changes may be required for achieving the complete merger of the lipid bilayers.One structurally related group of well-characterized viral fusion proteins includes the spike proteins of influenza A and C viruses, human immunodeficiency virus and other retroviruses, paramyxoviruses, and filoviruses such as Ebola virus (for reviews, see references 44, 48, and 5). These fusion proteins require proteolytic cleavage for activity (27), and their fusion peptides reside at or near the N-terminal end of the membrane-anchored subunit. When activated by the appropriate trigger, they all adopt a characteristic six-helix rod-like structure with a long coiled coil at the trimer interface. During the formation of the core, the fusion peptide is tr...

Flaviviruses are assembled intracellularly in an immature form containing heterodimers of two envelope proteins, E and prM. Shortly before the virion exits the cell, prM is cleaved by a cellular enzyme, and this processing step can be blocked by treatment with agents that raise the pH of exocytic compartments. We carried out in vivo and in vitro studies with tick-borne encephalitis (TBE) virus to investigate the possible role of furin in this process as well as the functional consequences of prM cleavage. We found that prM in immature virions can be correctly cleaved in vitro by recombinant bovine furin but that efficient cleavage occurs only after exposure of the virion to mildly acidic pH. The data suggest that exposure to an acidic environment induces an irreversible structural change that renders the cleavage site accessible to the enzyme. Cleavage by furin in vitro resulted in biological activation, as shown by a 100-fold increase in specific infectivity, the acquisition of membrane fusion and hemagglutination activity, and the ability of the envelope proteins to undergo low-pHinduced structural rearrangements characteristic of mature virions. In vivo, prM cleavage was blocked by a furin inhibitor, and infection of the furin-deficient cell line LoVo yielded only immature virions, suggesting that furin is essential for cleavage activation of flaviviruses.

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.

We present a kinetic analysis of the membrane fusion activity of tick-borne encephalitis (TBE) virus and TBE-derived recombinant subviral particles (RSPs) in a liposomal model system. Fusion was monitored using a fluorescence assay involving pyrene-labeled phospholipids. Fusion was strictly dependent on low pH, with the optimum being at pH 5.3-5.5 and the threshold at pH 6.8. Fusion did not require a protein or carbohydrate receptor in the target liposomes. Preexposure to low pH of the virus alone resulted in inactivation of its fusion activity. At the optimum pH for fusion and 37 degrees C, the rate and extent of fusion were very high, with more than 50% of the virus fusing within 2 s and the final extent of fusion being 70%. Lowering of the temperature did not result in a significant decrease in the rate and extent of fusion, suggesting that TBE virus fusion is a facile process with a low activation energy, possibly due to the flat orientation of the E glycoprotein on the viral surface facilitating the establishment of direct intermembrane contact. The fusion characteristics of TBE virus and RSPs were similar, indicating that RSPs provide a reliable and convenient model for further study of the membrane fusion properties of TBE virus.

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.

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.Enveloped viruses have evolved different but conceptually related mechanisms to fuse their membranes with cellular membranes during entry into cells. The process is controlled by viral surface glycoproteins that undergo triggered conformational changes required for mediating fusion (16,30).At least two different classes of viral fusion proteins can be distinguished (21). Class I is represented by orthomyxo-, retro-, paramyxo-, and filoviruses. Their fusion proteins mature by proteolytic cleavage of a precursor protein, yielding a membrane-anchored subunit with an amino-terminal or aminoproximal fusion peptide. Application of the fusion trigger (receptor binding or low pH) results in the formation of a characteristic trimeric postfusion structure with a triplestranded coiled coil at its core (6,17,27,30). Class II fusion proteins are not proteolytically cleaved and have internal rather than amino-terminal fusion peptides. They are synthesized as a complex with a second membrane glycoprotein, and the activation of the fusogenic potential involves the cleavage of this accessory protein (11,12,18). X-ray crystallography of two class II fusion proteins, the E protein of the flavivirus tick-borne encephalitis (TBE) virus (24) and the E1 protein of the alphavirus Semliki Forest virus...

69:5816-5820, 1995) were extensively characterized and shown to be ordered structures containing envelope glycoproteins with structural and functional properties very similar to those in the virion envelope. The particles were spherical, with a diameter of about 30 nm and a buoyant density of 1.14 g/cm 3 in sucrose gradients. They contained mature E proteins with endoglycosidase H-resistant glycans as well as fully cleaved mature M proteins. Cleavage of prM, which requires an acidic pH in exocytic compartments, could be inhibited by treatment of transfected cells with ammonium chloride, implying a common maturation pathway for RSPs and virions. RSPs incorporated [ 14 C]choline but not [ 3 H]uridine, demonstrating that they contain lipid but probably lack nucleic acid. The envelope proteins of RSPs exhibited a native antigenic and oligomeric structure compared with virions, and incubation at an acidic pH (pH <6.5) induced identical conformational changes and structural rearrangements, including an irreversible quantitative conversion of dimers to trimers.The RSPs were also shown to be functionally active, inducing membrane fusion in a low-pH-dependent manner and demonstrating the same specific hemagglutination activity as whole virions. Tick-borne encephalitis virus RSPs thus represent an excellent model system for investigating the structural basis of viral envelope glycoprotein functions.

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