Viruses 2012, 4, 117-139; doi:10.3390/v4010117
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`OPEN ACCESS
`
`viruses
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`ISSN 1999-4915
`www.mdpi.com/journal/viruses
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`Review
`Molecular and Cellular Aspects of Rhabdovirus Entry
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`Aurélie A. V. Albertini, Eduard Baquero, Anna Ferlin and Yves Gaudin *
`
`Laboratoire de Virologie Moléculaire et Structurale, Centre de Recherche de Gif, CNRS (UPR 3296),
`Avenue de la Terrasse, 91198, Gif sur Yvette Cedex, France;
`E-Mails: alberti@vms.cnrs-gif.fr (A.A.V.A.); baquero@vms.cnrs-gif.fr (E.B.);
`ferlin@vms.cnrs-gif.fr (A.F.)
`
`* Author to whom correspondence should be addressed; E-Mail: gaudin@vms.cnrs-gif.fr;
`Tel.: +33-1-6982-3836; Fax: +33-1-6982-4308.
`
`Received: 25 November 2011; in revised form: 5 January 2012 / Accepted: 10 January 2012 /
`Published: 18 January 2012
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`Abstract: Rhabdoviruses enter the cell via the endocytic pathway and subsequently fuse
`with a cellular membrane within the acidic environment of the endosome. Both receptor
`recognition and membrane fusion are mediated by a single transmembrane viral
`glycoprotein (G). Fusion is triggered via a low-pH induced structural rearrangement. G is
`an atypical fusion protein as there is a pH-dependent equilibrium between its pre- and
`post-fusion conformations. The elucidation of the atomic structures of these two
`conformations for the vesicular stomatitis virus (VSV) G has revealed that it is different
`from the previously characterized class I and class II fusion proteins. In this review, the
`pre- and post-fusion VSV G structures are presented in detail demonstrating that G
`combines the features of the class I and class II fusion proteins. In addition to these
`similarities, these G structures also reveal some particularities that expand our understanding
`of the working of fusion machineries. Combined with data from recent studies that
`revealed the cellular aspects of the initial stages of rhabdovirus infection, all these data give
`an integrated view of the entry pathway of rhabdoviruses into their host cell.
`
`Keywords: rhabdovirus; rabies virus; vesicular stomatitis virus; endocytosis; membrane
`fusion; glycoprotein
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`1. Introduction
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`The Rhabdoviridae family is grouped in the order Mononegavirales together with the Filoviridae
`(e.g., Ebola Virus), the Paramyxoviridae (e.g., measles and respiratory syncytial viruses) and the
`Bornaviridae (e.g., Borna disease virus). All of these viruses are enveloped and have a non-segmented
`genome made of a single stranded negative-sense RNA molecule.
`Among Mononegavirales, Rhabdoviruses have the most diverse hosts. They are widespread among
`a great diversity of organisms such as plants, insects, fishes, mammals, reptiles and crustaceans [1].
`This family has a long history and it has been recently shown that the genomes of several arthropods
`contain numerous integrated elements from Rhabdoviruses with some integration events that are at
`least 11 million years old [2].
`Based on their structural properties, antigenicity and phylogenetic analyses, Rhabdoviruses have
`been grouped into six genera. Lyssavirus (prototype: rabies virus—RABV) and Vesiculovirus
`(prototype: vesicular stomatitis virus—VSV) are the two best studied genera. Other genera include the
`Ephemerovirus genus (prototype: bovine ephemeral fever virus), the Novirhabdovirus genus (which
`contains many fish viruses such as the infectious hematopoietic necrosis virus) and two genera that are
`arthropod-borne and infect plants: Cytorhabdoviruses (prototype: Lettuce necrotic yellows virus) and
`Nucleorhabdoviruses (prototype: Potato yellow dwarf virus). In addition, numerous identified
`rhabdoviruses are still unclassified.
`All rhabdoviruses have a rigid bullet shape with a flat base and a round tip. The genome of
`rhabdoviruses comprises up to ten genes among which only five are common to all members of the
`family. These genes encode the nucleoprotein (N), the phosphoprotein (P), the matrix protein (M), the
`glycoprotein (G) and the viral polymerase (L). The genome is associated with N, L and P to form the
`nucleocapsid, which is condensed by the matrix protein into a tightly coiled helical structure. The
`condensed nucleocapsid is surrounded by a lipid bilayer containing the viral glycoprotein G that
`constitutes the spikes that protrude from the viral surface.
`G plays a critical role during the initial steps of the infectious cycle. First, it recognizes receptors at
`the viral surface and after virion endocytosis, it mediates the fusion between the viral and the
`endosomal membranes.
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`2. Basic Biochemical Properties of the Rhabdovirus Glycoprotein
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`G is a type I membrane glycoprotein. After cleavage of the amino-terminal signal peptide, the
`complete mature glycoprotein is approximately 500 amino acids long (495 for VSV and 505 for
`RABV). The bulk of the mass of G is located outside the viral membrane and constitutes the
`amino-terminal ectodomain (452 for VSV and 440 for RABV). G is anchored in the membrane by a
`single -helical transmembrane (TM) segment. The small intraviral domain is likely involved in
`interactions with internal proteins and there is evidence for RABV G interaction with M protein [3].
`For both VSV and RABV, the glycoproteins form trimers [4–8]. This oligomeric organization
`is not stable at high pH and is sensitive to detergent solubilization [5]. In the case of VSV, there
`exists a dynamic equilibrium between monomers and trimers of G, both in vitro after detergent
`solubilization [9,10] and in vivo in the endoplasmic reticulum [11].
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`The rhabdovirus glycoprotein is N-glycosylated. The number of glycosylation sites may vary from
`one virus to another. For both VSV G and RABV G, it has been shown that the oligosaccharide chains
`are required for proper folding of the protein at two different levels: first, they increase the solubility of
`the folding intermediates and second, they allow the interaction of these folding intermediates with
`calnexin and calreticulin, which are chaperones with lectin properties [12–14].
`The G ectodomain is the target for neutralizing antibodies [15–19]. The major antigenic sites of
`both VSV G and RABV G have been characterized. In the case of RABV, several hundred monoclonal
`antibodies (Mabs) have been used to characterize the antigenic structure of G. RABV G has two major
`antigenic sites: antigenic site II is located between amino-acids 34 and 42 and amino-acids 198 and
`200 [17], and antigenic site III extends from amino acid 330 to 338 [18]. This latter site is associated
`with virulence and the replacement of arginine 333 by any other amino acid (except lysine) leads to an
`attenuated phenotype [18,20,21]. In addition to these major antigenic sites, one minor antigenic site
`and a few isolated epitopes have been described [22–26].
`
`3. Rhabdovirus Receptors
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`VSV has a wide host spectrum: It infects both vertebrates and insects cells. Therefore, its receptor
`is a rather ubiquitous molecule. Phosphatidylserine (PS) has long been considered to be a viral
`receptor [27] despite the fact that it is only present at the surface of apoptotic cells. Recent results
`indicate that PS is not a receptor for VSV [28]. Other studies have suggested that gangliosides might
`play the role of the receptors in CER (chicken embryo related) cells [29]. Moreover, recent work has
`demonstrated that the endoplasmic reticulum chaperone gp96 is essential for infection with VSV [30].
`Cells without gp96 or with catalytically inactive gp96 do not bind VSV-G. From these data, it has been
`proposed that gp96 is essential for the occurrence of functional VSV-G receptors at the cell surface,
`most likely because it is required for correct folding of either a proteinaceous receptor or an enzyme
`required for the synthesis of a glycolipid receptor [30].
`In the case of RABV, apart from the very beginning and end of the infectious process, non-adapted
`isolates exclusively multiply and propagate in neurons, and in vitro, they can only infect established
`cell lines of neuronal origin. Several passages are required to select a fixed strain that is adapted to the
`multiplication in non-neuronal cell lines (such as BHK21 and Vero cells) [31–33]. Most of the fixed
`RABV laboratory strains have resulted from such an adaptation process. Although they have kept their
`neurotropism and propagate in the nervous system like street viruses, they have also acquired the
`ability to use ubiquitous receptors that are present at the surface of non-neuronal cell types [34]. As a
`consequence, among the many molecules that have been proposed to be RABV receptors [35], it is not
`clear which are actually used by natural isolates during animal infection.
`Host cell treatment with different phospholipases has been shown to reduce the binding of fixed
`RABV strains suggesting that some phospholipids can play the role of viral receptors [36]. Similarly,
`cells pretreated with neuraminidase were shown to be non-susceptible to viral infection [37]. After
`incorporation of exogenous gangliosides such as GT1b and GQ1b, the cells recovered their
`susceptibility to RABV infection [38]. These results indicate that highly sialylated gangliosides are
`part of the cellular membrane receptor structure for the attachment of fixed RABV strains.
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`In addition to phospholipids and gangliosides, three proteins have been proposed to play the role of
`viral receptors. Some evidence indicates that the nicotinic acetylcholine receptor (nAChR) acts as a
`RABV receptor [39]. First, a segment of RABV G has a sequence similarity to the snake venom
`curaremimetic neurotoxins which are potent ligands of nAChR [40]. Second, an interaction between
`RABV and purified Torpedo nAChR was demonstrated [41]. Finally, purified RABV was shown to
`bind the  subunit of nAChR in an overlay assay [42]. However, there is no direct evidence in animal
`models that this molecule is a RABV receptor. Furthermore, RABV can infect neurons that do not
`express nAChR [43], and nAChR is located mainly on muscle cells. Thus, although nAChR could
`account for the ability of street RABV to multiply locally in myotubes at the site of inoculation [44],
`which would facilitate the subsequent penetration into neurons, other molecules are needed to mediate
`viral entry into neurons.
`The second protein that has been proposed to play the role of a RABV receptor is the neural cell
`adhesion molecule (NCAM) [45]. Preincubation of RABV with soluble NCAM inhibited its ability to
`infect susceptible cells. Moreover, the transfection of resistant L fibroblasts with the NCAM-encoding
`gene induces RABV susceptibility. Additionally, the infection of NCAM-deficient mice by RABV
`resulted in slightly delayed mortality and restricted brain invasion. This suggests that NCAM is a
`bona fide receptor in vivo.
`The low-affinity nerve-growth factor receptor, p75NTR was identified as a ligand of a soluble form
`of RABV G [46]. The ability of RABV G to bind p75NTR was dependent on the presence of a lysine
`and an arginine in positions 330 and 333, respectively, which were known to control virus penetration
`into the motor and sensory neurons of adult mice [18,20,21]. Replacement of amino acids 318 and 352
`were also shown to abolish interaction between p75NTR and RABV G [47]. Furthermore,
`p75NTR-expressing BSR cells were permissive for a non-adapted fox RABV isolate (street virus). The
`glycoprotein from another genotype of lyssavirus (GT 6, European bat lyssavirus type 2) was also
`shown to bind p75NTR [48]. Nevertheless, mice lacking all the extracellular receptor domains were
`still susceptible to infection (of which the rate and specificity were unchanged), indicating that the
`RABVG-p75(NTR) interaction is not necessary for RABV infection of primary neurons [49].
`Very little is known concerning the receptor of other rhabdoviruses, the only exception is the viral
`hemorrhagic septicemia virus (VHSV), a salmonid rhabdovirus, for which it has been shown that
`monoclonal antibodies (MAbs) directed against a fibronectin containing complex protect cells from the
`infection. Because the purified rainbow trout fibronectin was able to bind specifically to VHSV,
`fibronectin was proposed to be a receptor for VHSV and other fish rhabdoviruses [50].
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`4. Fusion Properties of Rhabdoviruses
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`After receptor binding, both RABV and VSV enter the cell by the endocytic pathway. The acidic
`environment of the endosomal compartment triggers a series of conformational changes of the viral
`glycoproteins that catalyze fusion between the viral and the endosomal membranes [51–53].
`The pH dependence of fusion has been characterized for several rhabdoviruses [54–56]. Fusion is
`optimal around pH 6 and the threshold for fusion activity is approximately pH 6.5.
`As for other viruses that fuse at low pH, the exposure of the virion to low pH in the absence of a
`target membrane leads to the inactivation of the fusion properties of G [57–59]. Remarkably, unlike
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`other viruses for which the low-pH induced fusion inactivation is irreversible [57], reincubation of the
`virus above pH 7 leads to the recovery of the initial fusion activity of rhabdoviruses [58,59].
`Along with the fusion properties, it has been demonstrated that G can assume at least three different
`conformational states that have different biochemical properties [54,58]. The native pre-fusion state is
`observed at the viral surface above pH 7. In this conformation, G is thought to bind the viral receptors.
`Upon acidification, the virions are initially more hydrophobic [58], a feature that, in the absence of a
`target membrane, results in viral aggregation [59]. Using hydrophobic photolabeling, it has been
`demonstrated for both RABV and VSV, that G is then in an activated state that is able to interact with
`the target membrane as the first step of the fusion reaction [60]. After a longer incubation at low pH,
`the G post-fusion conformation is reached. In this conformation, the structure of G is antigenically
`distinct from both the pre-fusion and the activated conformation and, in electron microscopy, its
`ectodomain appears much more elongated than in the pre-fusion state (11 nm versus 8 nm) [58].
`The structural transition is reversible and the pre-fusion state can be recovered from the post-fusion
`state by readjusting the pH above 7 [58]. In fact, there is a pH-dependent thermodynamic equilibrium
`between different states of G that is shifted toward the post-fusion state at low pH [61]. The transition
`toward the post-fusion state is highly cooperative upon proton binding: approximately 2.8 protons bind
`simultaneously to trimeric G to induce the conformational change [61].
`This equilibrium explains why the low-pH induced inactivation is reversible. The reversibility of
`the conformational change is required to allow G to be transported through the acidic compartments of
`the Golgi apparatus and to recover its native pre-fusion state when incorporated in newly synthesized
`virions [62]. Other viruses, for which the fusogenic conformational change is irreversible (such as
`influenza virus, tick borne encephalitis virus or Semliki forest virus), have evolved different
`mechanisms to protect their fusion proteins from undergoing irreversible conformational changes in
`the Golgi apparatus [63]. For these viruses, it has been proposed that the high amount of energy
`released during the conformational change from the metastable pre-fusion state to the final stable
`post-fusion state is used to form the high energy lipidic intermediates during the fusion reaction [64].
`In the case of rhabdoviruses, the existence of an equilibrium between the different states implies that
`the energy released during the structural transition of a single trimer is small compared with the
`energetic barrier of the fusion reaction (the activation energy of the fusion process has been estimated
`to be in the range of 40 kcal/mol [54,65,66]). This indicates that a concerted action of several trimers is
`required. Indeed, for RABV, the minimal number of spikes involved in a fusion complex has been
`estimated to be approximately 15 [61].
`The fusion pathway of rhabdoviruses with liposomes has been studied in detail. Neither RABV nor
`VSV require a specific lipid for fusion [59,67,68]. However, recent studies have indicated that the
`target membrane composition has an influence on the efficiency of the process. Particularly, it has
`been demonstrated that lipid bis(monoacylglycero)phosphate (BMP, also called lysobisphosphatidic
`acid), present in the internal vesicles of the endocytic pathway, favors VSV fusion when it is present in
`the target membrane [69].
`An investigation of the effects of lipids with various dynamic molecular shapes on RABV-induced
`fusion has suggested that, similar to other enveloped viruses [70,71], RABV-induced fusion proceeds
`via the formation of an intermediate stalk that is a local lipidic connection between the outer leaflets of
`the fusing membranes [57]. A radial expansion of the stalk would induce the formation of a transient
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`hemi-fusion diaphragm (i.e., a local bilayer made by two initial inner leaflets) in which the formation
`of a pore and its enlargement would lead to complete fusion. It has been possible to trap and
`characterize an RABV fusion intermediate under suboptimal fusion conditions (pH slightly above the
`pH threshold for fusion and cold temperatures) [57]. This intermediate is apparently at a well-advanced
`stage of the fusion process when the hemi-fusion diaphragm is destabilized, but while lipid mixing is
`still restricted.
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`5. Structural Studies on VSV G
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`5.1. Crystallographic Structure of the Pre- and Post-Fusion States of the VSV G Ectodomain
`
`The atomic structures of the pre- and post-fusion conformations of a soluble form of the VSV G
`ectodomain (Gth, aa residues 1–422) have been recently determined [72,73] (Figure 1a,b). The soluble
`ectodomain had been generated by limited proteolysis of the viral particle with thermolysin. The
`structural organization of the two conformations of G appears to be very different from other viral
`fusion proteins described so far. However, amino acid sequence alignments revealed that all
`rhabdovirus glycoproteins share the same fold.
`Remarkably, the fold of Gth in its post-fusion state was the same as that of the HSV1 glycoprotein
`gB whose structure was published at the same time [74]. Together with the Epstein Barr virus
`glycoprotein gB [75] and the baculovirus glycoprotein gp64 [76] a new class of fusion proteins
`(class III) was defined (Figure 1c). VSV G is the only class III fusion protein for which the structures
`of both the pre- and post-fusion states have been determined by X-ray crystallography. The structures
`determined for other class III fusion glycoproteins are presumptive post-fusion conformations, based
`on their structural similarity with the post-fusion conformation of VSV G (Figure 1).
`Four distinct domains of Gth have been identified: a -sheet rich lateral domain (domain I in red), a
`central domain involved in the trimerization of the molecule (domain II, in blue), a pleckstrin
`homology domain (domain III, in orange) and a fusion domain (domain IV, in yellow). The
`organization of the VSV G fusion domain is similar (although not homologous) to that of the class II
`fusion proteins. It contains an extended -sheet structure made of three -strands that have two loops
`located at the tip that constitute the membrane-interacting motif of the G ectodomain. After the end of
`the trimerization domain (amino acid residue 405), 40 amino acids remain so that the polypeptide
`chain can reach the G TM segment but the structural organization is unknown after amino-acid
`residue 413 (resp. 410) for the pre-fusion domain (resp. post-fusion domain) (Figure 1a,b).
`The major antigenic sites of RABV G are located in domain I (antigenic site III) and domain III
`(antigenic site II). Additionally, for RABV G, the p75NTR binding domain is located in lateral domain
`I and the putative binding site of nAChR is located in domain III.
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`Figure 1. (a) Ribbon diagrams of the vesicular stomatitis virus (VSV) glycoprotein (G)
`pre-fusion trimer (top) and protomer (bottom) (PDB code = 2J6J [73]). (b) Ribbon diagram
`of the VSV G post-fusion trimer (top) and protomer (bottom) (PDB code = 2CMZ [72]). G
`is depicted by domains; the lateral domain (domain I) is in red, the pleckstrin homology
`domain (domain III) is in orange, and the fusion domain (domain IV) is in yellow. The
`trimerization domain is colored with two shades of blue (the part of G that retains its
`structure during refolding is in cyan, and the part of G that is refolded during the structural
`transition is in deep blue). The fusion loops are green and the C-terminus is pink. (c) Ribbon
`diagram of the HSV-1 gB trimer (top) and protomer (bottom) (PUB: 2GUM [74]). gB is
`colored by domains with the same color code as VSV G. The orientation of the VSV G and
`HSV-1 gB trimers toward the viral membrane is indicated. The VSV G protomers are
`aligned on the rigid block made of the lateral domain (in red) and the cyan part of the
`trimerization domain. The orientation of the post-fusion protomer is thus different in the
`top and the bottom of the figure.
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`5.2. The Structural Transition
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`The Gth structures revealed that the conformational change from the pre- to post-fusion state
`involves a dramatic reorganization of the glycoprotein [51,73]. During the structural transition,
`domains I, III and IV keep their tertiary structure. However, they undergo rearrangements in their
`relative orientation due to secondary structure changes in the hinge region between the fusion
`(domain IV) and the pleckstrin homology domain (domain III) and due to a major refolding of the
`trimerization domain (domain II).
`The refolding of G from pre- to post-fusion conformation is reminiscent of that of class I fusion
`protein such as the paramyxovirus fusion protein F [77] and the influenza hemagglutinin subunit 2
`(HA2) [78]. As for class I, the pre- and post-fusion states are related by flipping both the fusion
`domain (domain IV) and the C-terminal segment relative to a rigid block composed of the lateral
`domain (domain I) and part of the trimerization domain (domain II) that retains its structure during
`molecule refolding. The reversal of the molecule around the rigid block involves the lengthening of the
`central helices (that form the trimeric central core of the post-fusion conformation) and the refolding of
`the three carboxy-terminal segments into helices that position themselves in the grooves of the central
`core in an anti-parallel manner to form a six helix bundle (Figure 1a,b). This organization is obviously
`very similar to that of the post-fusion hairpin structure of class I proteins. The Gth post-fusion trimer
`displays a six-helix bundle with the fusion domain at the amino-terminus of the central helices and the
`TM segments at the carboxy-terminus of the anti-parallel outer helices. The result is an elongated
`structure with the fusion domain and the TM segment at the same extremity of the molecule
`(Figure 2a).
`Both structures also allow the identification of amino acid residues playing the role of pH-dependent
`molecular switches. In the pre-fusion state [73], there is a cluster of 3 conserved histidines (H60 and
`H162 in the fusion domain and H407 in the carboxy-terminal segment of the protein) (Figure 2b). At
`low pH, the protonation of these residues, by creating a cluster of positive charges, is likely to
`destabilize the interaction between the fusion domain and the C-terminal segment and might trigger the
`transition from the pre- to the post-fusion conformation. Indeed, it has been shown that chemical
`modification of histidines with diethylpyrocarbonate inhibits G fusion properties [79]. Reciprocally, in
`the post-fusion state, there are several acidic amino acids in the trimerization domain (domain II) that
`are sequestered close together [72] (Figure 2c). Sequence alignments reveal that this clustering is a
`conserved feature for all rhabdovirus glycoproteins [72]. In the VSV G post-fusion conformation, these
`residues are protonated and form hydrogen bonds. Their deprotonation induces strong repulsive forces
`that destabilize the post-fusion trimer and triggers the conformational change toward the pre-fusion
`state. These residues are buried in the post-fusion conformation and are exposed to solvent in the
`pre-fusion state. As a consequence, their pKa is much higher in the post-fusion state than in the
`pre-fusion state. This stronger affinity of the post-fusion state for protons explains the cooperativity of
`the rhabdovirus glycoprotein structural transition [61].
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`5.3. Interaction between G and Membranes
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`As mentioned above, the organization of the fusion domain resembles that of class II fusion
`proteins [80]. The main difference is that the membrane-interacting domain, located at the tip of the
`three-stranded -sheet, is bipartite, made of two loops (Figure 2d). An alignment of the amino-acid
`sequences of several rhabdovirus glycoproteins reveals that these loops are only slightly hydrophobic
`but always contain polar aromatic residues (tryptophans and tyrosines). That these loops are indeed an
`essential part of the membrane interacting motif is consistent with mutagenesis experiments [81–84].
`
`Figure 2. (a) A comparison of the central six-helix bundle of the class I viral fusion
`proteins (HIV gp41 and hPIV3 F) and class III viral fusion proteins (VSV G) in their
`post-fusion conformation. The amino-terminal trimeric coiled-coil is in blue and the
`antiparallel lateral helices are in pink. (b) A close up view of the histidine cluster located at
`the fusion domain/C-terminal domain interface that has to be disrupted during the
`structural transition from pre- to post fusion structure. (c) Top view and close up of the
`VSV G trimerization domain showing the clusters of acidic residues in the post-fusion
`state. (d) The structural organization of the Dengue virus E fusion domain (left) and the
`VSV G fusion domain (right). The hydrophobic residues in the tips of the fusion domains
`are depicted in green and, for VSV G, charged residues that impede deep penetration of the
`fusion domain inside the membrane are in blue.
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`The presence of charged residues in the vicinity of the loops precludes any deep penetration of the
`fusion domain inside the target membrane. It is likely that the tryptophans and the tyrosines position
`themselves at the interface between the fatty acid chains and the head group layers of lipids [85]
`(Figure 2d). This interfacial interaction involving only a few residues cannot create a strong point of
`anchoring that can be used to pull the target membrane toward the viral one. Rather, this interaction
`likely facilitates the formation of point-like protrusions that have been proposed to be stalk
`precursors [86]. In addition, membrane deformation may be facilitated by the large number of spikes at
`the viral surface and their trimeric status, which allows multiple fusion loops to interact with the
`external leaflet of the target bilayer.
`In the pre-fusion structure, unlike class I and class II fusion proteins, the fusion loops are not buried
`at an oligomeric interface and instead point toward the viral membrane [73]. It is therefore possible
`that in the pre-fusion conformation, the fusion loops also interact with the membrane.
`In addition to the fusion loops, two other domains have been demonstrated to play a major role in
`the fusion process. The first one is the TM segment. As for other viruses [87], replacement of VSV G
`TM segment by a glycophosphatidylinositol anchor results in the loss of the fusion properties of the
`protein [88]. Furthermore, mutagenesis studies have revealed a crucial role for the glycine residues of
`the TM segment at a late stage of the fusion process as mutations of these residues block fusion at the
`hemi-fusion stage [89].
`The second domain is the membrane proximal domain [90]. A deletion of the 13 membrane-proximal
`amino acids reduces both the cell-cell fusion and the virus infectivity. The structure of this segment is
`not known but in the case of RABV G, it has been proposed to adopt an α-helical conformation with a
`strong amphipathic signature [91].
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`5.4. Cooperativity between Glycoproteins during the Fusion Process
`
`As mentioned above, for rhabdoviruses, the pH-dependent equilibrium between pre- and post-fusion
`conformations of G implies that a large number of spikes cooperate to achieve the fusion reaction [61].
`Some recent data have provided new insights into the organization of the fusion machinery and suggest
`that distinct assemblies of fusion glycoproteins are involved at distinct stages of the fusion process [92]
`(Figure 3).
`For RABV, a local hexagonal lattice of G was observed at the surface of some RABV mutants with
`a mutation in G affecting the kinetics of the structural transition when incubated under suboptimal
`fusion conditions [93]. Each hexagon was made up of 6 G trimers. Remarkably, a similar organization
`of Gth was found in the crystals of the pre-fusion form (that had p622 crystalline symmetry) [73].
`Furthermore, a recent EM study performed on VSV revealed that the flat base of the virion is a
`privileged site for fusion [92] (Figure 3). The planar nature of this part of the virion could be propitious
`for the protein to assemble into a hexagonal p6 lattice. This local organization could favor the
`formation of the initial lipid intermediates (stalk or initial fusion pore) but also favor a concerted
`transition for the proteins making up the local network.
`A second network of glycoproteins in their post-fusion conformation, in this case helical, can be
`observed on the cylindrical part of the VSV particles incubated below pH 6 [92]. When formed in the
`absence of a target membrane, this helical array disrupts the viral membrane suggesting that this
`
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`network formation induces tension in the viral membrane. It has thus been proposed that these
`interactions between G glycoproteins, in their post-fusion conformation and located outside the contact
`zone, drive the enlargement of the initial fusion pore, which would have been formed in the contact
`zone between the flat base of the virion and the target membrane (Figure 3).
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`Figure 3. A model for VSV fusion [88]. In the first step (a), the flat base of VSV interacts
`with the target membrane most likely via activated glycoproteins that expose their fusion
`domain at their top. The local organization of G in this region may facilitate the formation
`of stalks and initial fusion pores (b). Then, a helical network of G in their post-fusion
`conformations is formed on the lateral part of the viral particle (c). The formation of this
`regular array likely induces tension in the membrane (red arrows) that drives pore
`enlargement and leads to complete fusion (d).
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`5.5. Intermediates during the Conformational Change
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`During the conformational change, an intermediate conformation is formed with the fusion loops
`and the TM segment at the opposite ends of the molecule. In this conformation, the fusion loops are
`distal to the viral membrane and can easily interact with the target membrane. For RABV, this
`intermediate activated state is stabilized at low temperature and is formed early during the fusion
`process as demonstrated by hydrophobic photolabeling [60]. This type of intermediate is common for
`fusion proteins and is often referred to as the extended intermediate conformation [94].
`Complex topological issues exist for the structural transition pathway [51]. The initial steps leading
`to exposure of the fusion loops may maintain strict three-fold symmetry; however, this symmetry is
`disrupted by the refolding of the C-terminal portion of the molecule [95]. Furthermore, in the
`pre-fusion state as well as in a putative extended trimeric intermediate conformation, the fusion
`domains are located outside the pyramidal volume defined by the viral membrane and t