Mechanism of membrane fusion

Electronics Repair Manuals

Schematic Diagrams and Service Manuals

Get Instant Access

The fusion of flaviviruses with membranes is strictly dependent on low pH, consistent with the cell entry by receptor-mediated endocytosis. The pH threshold of fusion has been shown to be around pH 6.4, suggesting that this process already occurs in early endosomes. In vitro studies on the fusion of TBE virus with liposomes have revealed that this process is extremely efficient, does not require an interaction with specific receptors and proceeds significantly faster than that mediated by class I viral fusion proteins, with t./a of a few seconds as compared to several minutes, respectively (Corver et al 2000, Melikyan et al 2000, Gallo et al 2001). In contrast to what is known from alphaviruses (Kielian et al 2000), which also possess class II fusion proteins, flavivirus fusion does not have an absolute requirement for the presence of cholesterol or sphingomyelin in the target membrane (Corver et al 2000, Stiasny et al 2003). However, using liposome preparations of different lipid compositions, Stiansy et al (2003) showed that specific interactions involving the 3'-OH group of cholesterol facilitates the binding of the E protein to target membranes as well as the structural transitions necessary for fusion.

Like class I fusion proteins, the class II flavivirus fusion protein exists in a metastable conformation at the surface of mature virions and undergoes dramatic structural changes when it encounters the low pH fusion trigger. As revealed by studies with TBE virus, acidic pH leads to the dissociation of the E dimers (Stiasny et al 1996), the exposure of the internal fusion peptide loop at the tip of domain II (Allison et al 2001, Stiasny et al 2002), and an oligomeric transition into E trimers (the post-fusion structure) (Allison et al 1995) that are energetically more stable than the E dimers (Stiasny et al 2001). The energy released during these transitions is believed to be used for driving the merger of the two opposing membranes. So far it has not been possible to crystallize the full-length form of the E trimer. The soluble forms of TBE and dengue E dimers, however, could be converted into a crystallizable trimeric post-fusion form through the interaction with liposomal membranes at acidic pH (Stiasny et al 2004, Modis et al 2004) and their structures were determined by X-ray crystallography (Bressanelli et al 2004, Modis et al 2004). Apart from the reorientation of the molecule relative to the membrane from horizontal in the virion to perpendicular in the fused membrane, the most prominent difference is a change in the position of domain III relative to the other domains through its movement from the end of the monomer to the side next to domain II (Fig. 2). Although these structures lack the 'stem' element and the two trans-membrane anchors, the arrangement of the three domains and modelling experiments (Bressanelli et al 2004) suggest that the post-fusion structure of E has a hairpin-like organization (similar to that of class I fusion proteins) in which the fusion peptide loop and the membrane anchors are juxtaposed in the fused membranes (Fig. 2C). Based on the atomic structures of E in its dimeric pre- and trimeric post-fusion forms (Rey et al 1995, Modis et al 2003, 2004, 2005, Zhang et al 2004, Bressanelli et al 2004) as well as a number of biochemical and functional studies (Allison et al 1995, 2001, Stiasny et al 1996, 2001, 2002), models of the flavivirus fusion mechanism were proposed consisting of several steps

FIG. 2. Schematic diagrams (compare with Fig. 1 C, D) showing the structural changes in the E protein during the fusion process. (A) The dimeric E protein in its native state at the surface of the mature virion. (B) Monomeric E interacting with a target membrane via its fusion peptide loop. Possible hinge movements at the junction between domains II and I are indicated by dotted lines. (C) Formation of the final post-fusion conformation trough relocation of domain III and interactions of the stem-anchor region with domain II, leading to the juxtaposition of the fusion peptide loops and the membrane anchors in the fused membrane.

FIG. 2. Schematic diagrams (compare with Fig. 1 C, D) showing the structural changes in the E protein during the fusion process. (A) The dimeric E protein in its native state at the surface of the mature virion. (B) Monomeric E interacting with a target membrane via its fusion peptide loop. Possible hinge movements at the junction between domains II and I are indicated by dotted lines. (C) Formation of the final post-fusion conformation trough relocation of domain III and interactions of the stem-anchor region with domain II, leading to the juxtaposition of the fusion peptide loops and the membrane anchors in the fused membrane.

including (i) the dissociation of the E dimer resulting in the exposure of the previously buried fusion peptide loop, (ii) the attachment of the E protein to a target membrane via the fusion peptide loop, and (iii) the trimerization of E and the formation of a 'hairpin' structure resulting in the merger of the two membranes

Because the flavivirus fusion machinery is extremely fast and efficient it has not yet been possible to define structural intermediates of the E protein during its transition into the post-fusion conformation by the use of specifically designed peptide inhibitors, a technology that has been applied successfully to viruses with class I fusion proteins (Earp et al 2005, Matthews et al 2004). In experiments with TBE virus, however, new experimental information about structural intermediates of the flavivirus fusion process was obtained through the exposure of virions to alkaline pH (Stiasny et al 2006a). It was shown that under these conditions the icosahedral envelope organization is opened and the E dimers dissociate into their monomeric constituents (Fig. 2). Similar to what occurs under physiological (acid pH) conditions, the exposure of the fusion peptide and the apparent outward projection of E monomers at alkaline pH lead to their stable attachment to target membranes. Under these conditions, however, the process is arrested at this intermediate stage, and neither fusion activity nor E trimer formation can be observed, suggesting that the domain relocation necessary for hairpin-formation does not occur spontaneously upon dimer dissociation but requires specific protonation events in the monomers.

Molecular antigenic structure

Sites involved in virus neutralization

Since the flavivirus E protein has the dual function of receptor binding and membrane fusion it is the principle target of neutralizing antibodies. A significant degree of information on the binding sites for such antibodies has become available, primarily by the mapping of mutations that lead to escape from neutralizing mouse monoclonal antibodies (Mabs) and, more recently, by the structure determination of protein E-Fab complexes (Oliphant et al 2005) and the specific mutagenesis of E in recombinant forms (Kiermayr et al, in preparation). Figure 3 shows a summary of the positions of single amino acid substitutions found in Mab escape mutants of different flaviviruses. Although there are only limited sets of data for individual flaviviruses, this compilation suggests that the binding of antibodies to sites at the exposed outer surface of each of the three domains can lead to neutralization. Such binding sites can be restricted to individual domains only (e.g. a Fab interacting with domain III of the West Nile virus E protein has been defined in its atomic details), but site-specific mutagenesis experiments with

Mechanism Fusion

FIG. 3. Surface representation of the TBE virus E dimer. Highlighted in black are the positions of Mab escape mutants identified with different flaviviruses that were assigned to the homologous positions in the TBE virus E protein. Abbreviations are as follows: TBE (tickborne encephalitis), Den (dengue), JE (Japanese encephalitis), Li (Louping ill), MVE (Murray Valley encephalitis), WN (West Nile), YF (yellow fever).

FIG. 3. Surface representation of the TBE virus E dimer. Highlighted in black are the positions of Mab escape mutants identified with different flaviviruses that were assigned to the homologous positions in the TBE virus E protein. Abbreviations are as follows: TBE (tickborne encephalitis), Den (dengue), JE (Japanese encephalitis), Li (Louping ill), MVE (Murray Valley encephalitis), WN (West Nile), YF (yellow fever).

recombinant subviral particles also provide evidence for subunit-overlapping sites involving amino acid residues from each of the monomeric subunits in the E dimer (Kiermayr et al, in preparation).

Flavivirus cross-reactive sites

Originally, flaviviviruses were grouped together on the basis of antigenic relationships observed in certain assays with polyclonal animal and human immune sera (Calisher et al 1989). These broad cross-reactivities between all of the flaviviruses are characteristic of haemagglutination-inhibition and enzyme immunoassays, whereas neutralization assays are significantly more specific and have allowed the definition of so-called serocomplexes. These comprise more closely related flavivi-ruses, and cross-neutralization is only observed within but not between such serocomplexes. Molecular details of the sites in E that induce and bind broadly flavivirus cross-reactive antibodies have recently been elucidated in the TBE virus system by the use of cross-reactive Mabs raised against several different flavivi-ruses as well as by dissecting the antibody populations present in post-infection polyclonal human immune sera (Stiasny et al 2006b). It was shown with low affinity only that the cross-reactive antibodies bind to the surface of native infectious virions and recognize a cryptic site involving conserved amino acids present in the fusion peptide loop at the tip of domain II. Experimentally this site can be made more accessible by dissociating the tightly packed viral envelope into its dimeric E protein constituents, but it also becomes exposed in the course of antigen coating to solid phases (as in certain enzyme immunoassay formats) as well as during the incubation steps in haemagglutination inhibition assays. The information gained so far with monoclonal and polyclonal antibodies suggests that flavi-viruses have a single dominant site in their E protein that induces broadly cross-reactive antibodies. This site, however, appears to be only partially accessible at the surface of native and infectious virions and its cryptic nature can explain the lack of cross-neutralization despite extensive cross-reactivities observed in haemagglutination-inhibition and enzyme immunoassays.

Was this article helpful?

0 0
Swine Influenza

Swine Influenza

SWINE INFLUENZA frightening you? CONCERNED about the health implications? Coughs and Sneezes Spread Diseases! Stop The Swine Flu from Spreading. Follow the advice to keep your family and friends safe from this virus and not become another victim. These simple cost free guidelines will help you to protect yourself from the swine flu.

Get My Free Ebook


Post a comment