Journal of General Virology

Biosynthesis and role of filoviral glycoproteins

Heinz Feldmann,¹ Viktor E. Volchkov,² Valentina A. Volchkova,² Ute Ströher³ and Hans-Dieter Klenk³

¹ Canadian Science Centre for Human and Animal Health, 1015 Arlington Street, Winnipeg, Manitoba, Canada R3E 3R2
² Biologie des Filovirus, Claude Bernard University Lyon-1, 46 Allée d'Italie, 69007 Lyon, France
³ Institut für Virologie, Philipps-Universität, Robert-Koch-Str. 17, D-35037 Marburg, Germany

Marburg and Ebola viruses cause systemic infections leading to severe haemorrhagic fever in human and non-human primates. There is evidence that the filovirus glycoproteins play an important role in cell tropism, the spread of infection and pathogenicity. Biosynthesis of the transmembrane glycoprotein involves a series of co- and post-translational events, including proteolytic cleavage by a host cell protease. Cleavage leads to two disulphide-linked subunits, GP₁ and GP₂, of which GP₂ anchors the molecule into the membrane. Although cleavage is not a requirement for Ebola virus infectivity in cell culture, the role of cleavage in vivo is unknown. Different strains of Ebola virus show variations in the cleavability of the glycoprotein that may account for differences in pathogenicity. Expression of the transmembrane glycoprotein of Ebola virus, but not of Marburg virus, requires transcriptional editing. Unedited transcripts yield the non-structural glycoprotein sGP, which is extensively secreted from infected cells. The role of the different soluble glycoproteins produced during filovirus infections is unknown, but they may interfere with the host immune response and other defence mechanisms.

Infections with filoviruses cause a fulminant haemorrhagic disease in human and non-human primates. Among all of the virus haemorrhagic fevers, Marburg and Ebola virus infections are characterized as the most severe forms, with case-fatality rates ranging from 22 to 90 %. The pathophysiological changes that make filovirus infections so devastating are not well understood. The viruses are pantropic, but there is not a single organ that shows sufficient damage to account for either the onset of severe shock syndrome or the tendency to bleed. As in some other virus haemorrhagic fevers, such as haemorrhagic fever with renal syndrome, dengue haemorrhagic fever and Lassa fever, filovirus infections are associated with generalized fluid distribution problems, hypotension and coagulation disorders. Thus, Marburg and Ebola virus haemorrhagic fevers may be compared to a syndrome provoked by systemic treatment with cytokines, which is also seen in endotoxin-induced shock (Feldmann & Klenk, 1996; Peters et al., 1996; Schnittler & Feldmann, 1999).

Filoviruses are enveloped, non-segmented, negative-stranded RNA viruses that constitute a separate family within the order Mononegavirales. The family comprise the genera Marburg virus (MBGV) and Ebola virus (EBOV). The genus Ebola virus is further subdivided into three distinct African species, Cote d'Ivoire, Sudan and Zaire, and the Asian species Reston (van Regenmortel et al., 2000). The history and epidemiology of filoviruses have been reviewed elsewhere (Feldmann & Klenk, 1996; Peters et al., 1996; Feldmann et al., 1998; Peters & LeDuc, 1999).

Filoviruses comprise a single, negative-stranded, linear RNA genome that is non-infectious and does not contain a poly(A) tail. Upon entry into the host cell cytoplasm, the RNA is transcribed to generate a polyadenylated, subgenomic mRNA species. The genome shows the following characteristic gene order: 3´ leader, nucleoprotein (NP), virion protein (VP) 35, VP40, glycoprotein (GP), VP30, VP24, polymerase protein (L) and 5´ trailer. Transcription and translation lead to the synthesis of seven structural polypeptides, with presumed identical functions for each of the different filoviruses. Four proteins, NP, VP30, VP35 and L, are associated with the viral genomic RNA in the ribonucleoprotein complex. The three remaining structural proteins are membrane-associated; GP_1,2 is a type I transmembrane protein, while VP24 and VP40 are probably located on the inner side of the membrane. A non-structural, secreted glycoprotein (sGP) is expressed by EBOV, but not MBGV (Feldmann & Kiley, 1999).

We now have information regarding the processing, structure and function of some of the filoviral proteins. Studies using reconstituted replication systems demonstrated that NP, VP35 and L were essential and sufficient for transcription, as well as for the replication of MBGV monocistronic minigenomes, while EBOV-specific transcription was also dependent on the presence of VP30 (Mühlberger et al., 1998, 1999). Of the two membrane-associated non-glycosylated proteins, VP40 functions as the matrix protein. Recently, its crystal structure has been elucidated (Dessen et al., 2000; Ruigrok et al., 2000). The structure and function of VP24, the other membrane-associated protein, has not yet been studied. Reverse genetics systems for EBOV were developed based on the artificial replication systems mentioned above (G. Neumann et al., unpublished data; Volchkov et al., 2001). These newly developed systems will help to determine protein functions.

Despite the fact that all virus components may contribute to the development of disease, the glycoproteins of filoviruses are considered to be major determinants in virus pathogenesis. Therefore, this review focuses on the biosynthesis and role of filoviral glycoproteins.

Expression strategies of the glycoprotein genes

Filoviral glycoproteins are encoded by gene 4 (GP gene) of the non-segmented, negative-stranded RNA genome (Fig. 1). MBGV gene 4 encodes a single open reading frame (ORF) of 2043 nucleotides, which translates into the transmembrane glycoprotein (Feldmann et al., 1992; Will et al., 1993; Bukreyev et al., 1995; Sanchez et al., 1993, 1998 a). In contrast, the expression strategy of gene 4 of all EBOV involves transcriptional editing and gives rise to different glycosylated proteins (Volchkov et al., 1995, 1998 b; Sanchez et al., 1996). The primary structure of the editing site is a run of seven uridine residues in the genomic sequence. Transcriptional editing is performed by the viral RNA-dependent RNA polymerase (L protein). Unedited viral mRNA species (about 80 % of GP-specific mRNAs) encode the primary product of gene 4, the secreted, non-structural glycoprotein (sGP). The transmembrane glycoprotein, GP, is translated from edited GP gene-specific mRNA species and is the result of the addition of a single adenosine residue at the editing site (Fig. 1). This event, which shifts the ORF to –1, seems to occur in about 20 % of the GP gene-specific transcripts (Volchkov et al., 1995; Sanchez et al., 1996).

Fig. 1. Transcription and expression strategies of the glycoprotein genes of filoviruses. Unlike MBGV, the EBOV transmembrane glycoprotein GP_1,2 can only be expressed through transcriptional editing. The primary product of the EBOV glycoprotein gene (gene 4) is the precursor of the secreted glycoprotein (pre-sGP) and is expressed from unedited transcripts and post-translationally cleaved into the soluble products sGP and -peptide. GP_1,2 comprises the disulphide-linked (S–S) subunits GP₁ and GP₂; it mediates binding to target cells. Significant amounts of GP₁ are released from expressing HeLa cells. The binding specificities of GP₁, sGP and -peptide are not yet defined clearly.

EBOV Zaire variants, which have been selected from both guinea pigs (Volchkov et al., 2000 a) and tissue culture (Sanchez et al., 1993), have incorporated an additional uridine residue at the editing site. This insertion leads to an inversion of the sGP:GP ratio, with about 80 % of the mRNAs encoding the transmembrane glycoprotein GP and 20 % encoding sGP. Here, the deletion of one or the insertion of two adenosine residues is also observed and these changes allow a switch into a third ORF (–2). This ORF terminates two amino acids downstream of the editing site and generates a third non-structural small secreted protein (ssGP) that has not been demonstrated following infection with wild-type EBOV in cell culture (Volchkova et al., 1998; Volchkov et al., 2000 a).

Biosynthesis, processing and maturation of glycoproteins

Transmembrane glycoprotein (GP). The open reading frames for the transmembrane glycoproteins (pre-GP) of MBGV, strains Musoke and Popp, and EBOV, strain Mayinga, encode polypeptides of 681 and 676 amino acids in length, respectively. These are type I transmembrane proteins and can be subdivided into a large ectodomain, a lipid membrane-spanning domain of approximately 30 amino acids and a short cytoplasmic tail of four (EBOV) and eight amino acids (MBGV) (Fig. 2 A). pre-GP undergoes a complex sequence of processing events in the endoplasmic reticulum (ER). This includes the removal of the signal peptide (Will et al., 1993; Sanchez et al., 1998 b), N-glycosylation (Feldmann et al., 1991, 1994; Volchkov et al., 1995; Becker et al., 1996; Sanchez et al., 1998 b) and oligomerization (Feldmann et al., 1991; Sanchez et al., 1998 b). ER processing is followed by acylation in a pre-Golgi compartment (Funke et al., 1995; Ito et al., 2001) and by O-glycosylation and maturation of N-glycans in the Golgi apparatus (Feldmann et al., 1991, 1994; Geyer et al., 1992; Will et al., 1993; Volchkov et al., 1995; Becker et al., 1996). Finally, pre-GP gets cleaved proteolytically into a large amino-terminal (GP₁) and a small carboxy-terminal (GP₂) subunit in the trans-Golgi network by the subtilisin-like proprotein convertase furin (Figs 1 and 2 A, B) (Volchkov et al., 1998 a, 2000 b).

Fig. 2. The transmembrane glycoprotein of filoviruses. (A) Schematic illustration of the primary structure. The type I transmembrane glycoprotein GP_1,2 carries three hydrophobic domains (grey boxes): a signal peptide (SP) at the amino-terminal end, a fusion domain (FD) and a transmembrane domain (TD) at the carboxy-terminal end. The glycoprotein precursor pre-GP is cleaved proteolytically into the subunits GP₁ and GP₂. Arrows indicate cleavage sites. Both subunits are disulphide-linked in the mature molecule. x, potential N-linked carbohydrate site chain; C, cysteine residue; C*, acylated cysteine residue, RRKR, MBGV cleavage site; RTRR, EBOV cleavage site. (B) Proposed structure of GP₂. The ectodomain of GP₂ contains the fusion peptide followed by an amino-terminal helix, a peptide loop and a carboxy-terminal helix. Helices were proposed by the GARNIER program of PC/GENE (IntelliGenetics) (Weissenhorn et al., 1998 a, b). (C) Proposed structure of the transmembrane glycoprotein molecule. The transmembrane glycoprotein GP_1,2 is anchored into the lipid membrane by a hydrophobic domain (FD in part A) at the carboxy terminus of GP₂. GP₁ is attached to GP₂ by a intramolecular disulphide bond probably involving cysteine residue 53.

The mature envelope glycoprotein GP_1,2 is anchored in the membrane by a carboxy-terminal hydrophobic domain of GP₂ (Fig. 2) (Volchkov et al., 1998 b). The middle region of GP_1,2 is variable, extremely hydrophilic and carries the bulk of N- and O-glycans, which account for more than one-third of the molecular mass of the mature protein (Geyer et al., 1992; Will et al., 1993; Feldmann et al., 1991; Volchkov et al., 1995; Becker et al., 1996). Oligosaccharide side chains differ in their terminal sialylation patterns. These patterns seem to be isolate as well as cell line-dependent (Feldmann et al., 1994). Detailed structural analyses of filoviral carbohydrates are only available for MBGV (Geyer et al., 1992). Comparison of the pre-GP sequences of MBGV and EBOV shows conservation at both the amino-terminal and the carboxy-terminal ends. Two carboxy-terminal cysteine residues are acylated (Funke et al., 1995; Ito et al., 2001). GP₂ contains a sequence of several uncharged, hydrophobic amino acids at a distance of 22 (EBOV) or 91 (MBGV) amino acids from the cleavage site and which bears some structural similarity to the fusion peptides of retroviruses (Fig. 2) (Volchkov et al., 1992, 2000 b; Gallaher, 1996).

The special arrangement of the cysteine residues in the GP_1,2 molecules allows the formation of an intramolecular disulphide bridge between the two cleavage products. This suggests a stem region consisting of GP₁ and GP₂ and a crown-like domain on the top formed by GP₁ that carries the mass of the carbohydrate side chains (Fig. 2 C). It can be assumed that cysteine residue 53 is also critical for maintaining the structure of GP_1,2, as discussed below for sGP (Volchkova et al., 1998). The mature transmembrane glycoprotein is a trimer comprising disulphide-bonded GP_1,2 molecules (Feldmann et al., 1991; Sanchez et al., 1998 b). As demonstrated for GP_1,2 expression using a recombinant vaccinia virus, the mature transmembrane glycoprotein forms spikes, without the need for other viral proteins (Volchkov et al., 1998 b). X-ray crystallography demonstrated that the central structural feature of the GP₂ ectodomain is a long, triple-stranded, coiled-coil, followed by a disulphide-bonded loop that reverses the chain direction and connects to an -helix packed antiparallel to the core helices (Fig. 2 B) (Weissenhorn et al., 1998 a, b; Malashkevich et al., 1999). During maturation, GP₁ is partly shed in monomeric form after the release of its disulphide linkage to the transmembrane subunit GP₂ (Fig. 3 B) (Volchkov et al., 1998 b).

Fig. 3. The soluble glycoproteins of EBOV. (A) Schematic structure of secreted glycoprotein precursor (pre-sGP). Numbers indicate the positions of cysteine residues. The proteolytic cleavage site at amino acid position 324 (RxRR) is indicated. Cleavage will produce the soluble products sGP and -peptide. (B) Soluble glycoproteins. Vero E6 cells were infected with EBOV wild-type at 0.01 p.f.u. per cell. The culture medium was harvested at 5 days post-infection, clarified by low-speed centrifugation and subjected to sucrose equi../LIBrium gradient analysis (10–40 % sucrose gradient). Following centrifuging at 36000 r.p.m. at 4 °C for 20 h in an SW41 rotor (Beckman), 12 fractions (1 ml) were collected from the bottom to the top. Proteins were analysed by SDS–PAGE (10 % gel) followed by immunoblotting using goat anti-EBOV immunoglobulins (1:3000). (C) Schematic structure of an sGP dimer, which comprise antiparallel-orientated monomers stabilized by intermolecular disulphide bonds between cysteine residues at positions 53 and 306.

Secreted glycoprotein (sGP). The secreted glycoprotein precursor (pre-sGP) of EBOV, species Zaire, is 364 amino acids in length and shares the amino-terminal 295 amino acids with the transmembrane glycoprotein GP (Fig. 3 A). The different carboxy terminus (69 amino acids) contains several charged residues as well as cysteine residues. As with pre-GP, pre-sGP undergoes several co- and post-translational processing events, such as signal peptide cleavage, glycosylation, oligomerization and proteolytic cleavage (Volchkova et al., 1998, 1999). The limiting step during maturation and transport seems to be oligomerization in the ER (Volchkova et al., 1998). After oligomerization, pre-sGP is transported into the Golgi compartments where glycosylation is completed and post-translational cleavage into sGP and a small peptide, designated -peptide, occurs (Volchkova et al., 1999). Cleavage is mediated by furin, which is also responsible for the cleavage of pre-GP, as discussed above (Figs 1, 2 and 3) (Volchkov et al., 1998 a). Due to a lack of a transmembrane anchor, sGP is secreted efficiently from infected cells (Fig. 3 B). Secretion also occurs if cleavage is abolished (Volchkova et al., 1999). sGP and uncleaved pre-sGP appear as a disulphide-linked homodimer that shows an antiparallel orientation (Fig. 3 C) (Sanchez et al., 1998 b; Volchkova et al., 1998). Dimerization is due to an intermolecular disulphide linkage between the amino- and carboxy-terminal cysteine residues at positions 53 and 306, respectively. The remaining four highly conserved cysteine residues at the amino terminus seem to be involved in intramolecular folding of monomers (Volchkova et al., 1998).

-Peptide. The small cleavage product of pre-sGP, -peptide, varies in length between 40 and 48 amino acids for the different EBOV (Fig. 3) (Volchkova et al., 1999). Its molecular mass of about 10–14 kDa is significantly larger than the one predicted from the amino acid sequence (about 4.7 kDa). This difference in size is due to the attachment of several O-glycans that carry terminal sialic acids. In this respect, it differs from sGP, which seems mainly to carry N-linked carbohydrates. -peptide is secreted from cells but this process seems not to be as efficient as the secretion of sGP (Volchkova et al., 1999).

Small secreted glycoprotein (ssGP). The small secreted glycoprotein (ssGP) of EBOV resembles a natural carboxy-terminal truncated variant of sGP. Due to the lack of three carboxy-terminal cysteine residues, including the cysteine residue at position 306 involved in the dimerization of sGP, ssGP is secreted in a monomeric form (Volchkov et al., 1995; Volchkova et al., 1998). ssGP has not yet been demonstrated after infection of cell culture with wild-type EBOV.

Potential role of glycoproteins in filovirus pathogenesis

Cell tropism and entry. Using vesicular stomatitis virus and retrovirus pseudotypes, several groups demonstrated independently that the transmembrane glycoprotein of filoviruses mediates receptor binding and subsequent fusion with susceptible cells (Fig. 1) (Takada et al., 1997; Wool-Levis & Bates, 1998; Yang et al., 1998; Chan et al., 2000 b). There is evidence that MBGV uses the asialoglycoprotein receptor to infect hepatocytes (Becker et al., 1995). For EBOV, it was suggested that integrins, especially the 1 group, might interact with the glycoprotein and perhaps be involved in entry into the cells (Takada et al., 2000). More recent studies indicate that the folate receptor- serves as a cofactor for cellular entry by MBGV and EBOV (Chan et al., 2001). Using standard methodology, fusion activity has never been demonstrated experimentally. Early post-infection filovirus particles are associated with coated pits along the plasma membrane, indicating endocytosis as a possible mechanism for entry (Geisbert & Jahrling, 1995). This is supported by studies that employ lysosomotropic agents (Mariyankova et al., 1993; Chan et al., 2000 b). Based on the structural similarity to the fusion peptides of retroviruses, Gallaher (1996) postulated a fusion peptide for EBOV at a distance of 22 amino acids from the cleavage site (amino acids 524–539). Recently, it was demonstrated that the same peptide induces fusion with liposomes (Ruiz-Argüello et al., 1998). This observation, together with mutational analysis of the putative fusion domain (Ito et al., 1999), offers compelling support for a role for this conserved hydrophobic region in the EBOV transmembrane glycoprotein GP as a fusion peptide (Figs 2 and 4). Recently, it was shown that the coiled-coil motif of GP₂ (see below) plays an important role in facilitating the entry of EBOV (Watanabe et al., 2000). For MBGV, a similar putative fusion domain can be found at a distance of 91 amino acids from the cleavage site (Volchkov et al., 2000 b).

Fig. 4. Structural features of fusogenic transmembrane glycoprotein domains. (A) Structural similarities between EBOV GP₂ and the transmembrane subunits HA2 of influenza virus haemagglutinin, gp41 of HIV env protein and F1 of simian virus type 5 fusion protein are shown. Four domains can be distinguished in the fusion active state: the fusion peptide (a), an amino-terminal helix (b), a carboxy-terminal helix (c) and the membrane anchor (d). The transmembrane proteins assemble into trimers in which the large amino-terminal helices form an interior, parallel coiled-coil with the smaller carboxy-terminal helices packing in an antiparallel fashion at the surface. The fusion peptide and the membrane anchor are therefore located at one end of the rod-like trimers. (B) Fusion model. The close proximity of the fusion peptide and the membrane anchor brings both membranes together and thereby promotes fusion (taken from Feldmann et al., 1999; reproduced with permission from Springer–Verlag).

An important mechanism for controlling fusion activity of viral surface proteins is the processing by protein convertases (Klenk & Garten 1994 a, b). Proteolytic cleavage, which often occurs next to a protein domain involved in fusion, is the first step in the activation of these fusion proteins and is followed by a conformational change resulting in the exposure of the fusion domain (Bullough et al., 1994; Chan et al., 1997; Weissenhorn et al., 1997). The conformational change may be triggered by low pH, such as in endosomes (Skehel et al., 1982), or by the interaction with a secondary receptor protein at the cell surface (Feng et al., 1996). The central structural features of the EBOV GP₂ ectodomain (Weissenhorn et al., 1998 a, b), as described previously, suggest that the fusion peptide and membrane anchor domain are located at one end of the rod-like trimer. Such structures have been observed with the transmembrane subunits HA2 of influenza virus haemagglutinin (Bullough et al., 1994), gp41 of human immunodeficiency virus (HIV) env protein (Chan et al., 1997; Weissenhorn et al., 1997) and F1 of paramyxovirus fusion protein (Joshi et al., 1998) (Fig. 4). All of these proteins require cleavage and conformational changes to activate their fusogenic potency. Therefore, glycoprotein cleavage by furin and other host cell proteases is absolutely necessary for the infectivity of these viruses. The structural similarities and the difference in the folding of uncleaved and cleaved GP, as judged by mobility during SDS–PAGE (Volchkov et al., 1998 b), strongly suggest that the fusion process of filoviruses occurs in a similar fashion.

However, studies using pseudotype viruses have demonstrated that proteolytic cleavage of the transmembrane glycoprotein is dispensable for the replication of EBOV, at least in cell culture (Wool-Levis & Bates, 1999; Ito et al., 2001). This theory was supported recently by a rescued cleavage site mutant using an EBOV reverse genetics system (G. Neumann et al., unpublished). MBGV may be distinct due to obvious differences in the position of its fusion domain compared with EBOV (Fig. 2 A, B).

Virus spread. To date, the furin cleavage motif is highly conserved among all filovirus transmembrane glycoprotein sequences (Table 1) and its conservation suggests a role in the virus life cycle. Hence, cleavage may be required for virus replication in the host or natural reservoir. Animal studies will have to verify whether proteolytic cleavage has a role in establishing filovirus infection in vivo. Cleavage by furin may be an important factor for pantropism (Klenk et al., 1998). Furin is a processing enzyme of the constitutive secretory pathway and is expressed in most mammalian cells. Furin, which is localized predominantly in the trans-Golgi network (Molloy et al., 1994; Schäfer et al., 1995) but also secreted from cells in a truncated form (Wise et al., 1990; Vey et al., 1995), appears to be the key enzyme in virus activation (Klenk & Garten, 1994 a). The enzyme belongs to the proprotein convertases, a family of subtilisin-like eukaryotic endoproteases that also includes PC1/PC3, PC2, PC4, PACE4, PC5/PC6 and LPC/PC7 (Seidah et al., 1996). These enzymes are expressed differentially in cells and tissues and display similar, but not identical, specificity for basic motifs, such as R–X–K/R–R, at the cleavage sites of their substrates. Variation in the cleavage site of the glycoprotein GP may account for differences in the pathogenicity of EBOV (Table 1 and Fig. 5) (Volchkov et al., 1998 a). MBGV and all highly pathogenic EBOV strains display the canonical furin motif R–X–K/R–R at the cleavage site and are highly susceptible to cleavage. Only the glycoproteins of the EBOV Reston strains, which appear to be less pathogenic for humans and only moderately pathogenic for at least some monkey species (Fisher-Hoch et al., 1992), show a reduced cleavability because of the suboptimal cleavage site sequence K–Q–K–R (Table 1 and Fig. 5) (Volchkov et al., 1998 a). Expression of the Reston virus glycoprotein in transfected mammalian cells demonstrated a low cleavability of this protein, which could be increased by a single amino acid change (Fig. 5) (Volchkov et al., 1998 a). Thus, highly pathogenic variants may emerge from Reston-like strains by mutations restricted to the cleavage site (Klenk et al., 1998). On the other hand, inhibition of furin cleavage, which can be achieved with peptidyl chloromethylketones or other compounds (Stieneke-Grober et al., 1992; Anderson et al., 1993), may be a valuable concept for treatment strategies of acute infections with filoviruses.

Table 1. Proteolytic cleavage sites of filovirus glycoproteins

Cleavage site sequences of all known glycoproteins. The amino acid sequences of the cleavage sites are presented in positions –1 to –4. Proteolytic cleavage occurs at the carboxy-terminal of the arginine residue at position –1. The relative pathogenicity in human and non-human primates is indicated.

Fig. 5. Processing of wild-type and cleavage site mutants of EBOV, species Reston. HeLa cells were infected with a vaccinia virus expressing the T7 polymerase (vTF7-3) and then transfected with the plasmids pGEM-PR8 (wild-type Reston GP, WT) and pGEM-R/R (mutant R/R). At 6 h post-infection, cells were pulse-labelled for 20 min with [³⁵S]cysteine and then chased for 240 min. Immunoprecipitated proteins were separated under reducing conditions on an 8 % polyacrylamide gel. The positions of the non-cleaved precursors (pre-GP_ER and pre-GP) and the cleavage subunit GP₁ are indicated.

Target cell destruction. Filovirus infections lead to a moderate cytopathogenic effect in target cells. However, the mechanism that causes cell destruction is unknown. It is possible that either the massive production and accumulation of viral proteins or the maturation of virus particles at the plasma membrane are involved in this process. Alternatively, a viral protein may have specific cytotoxic potential. Studies published recently have demonstrated cell destruction upon the expression of EBOV transmembrane glycoproteins (Chan et al., 2000 a; Takada et al., 2000; Yang et al., 2000). In one study, it was reported that a serine/threonine-rich mucin-like domain located on GP₁ mediated cytotoxicity in 293 T and endothelial cells. This was confirmed in vessel explants in infections with recombinant adenovirus vectors expressing EBOV GP (Yang et al., 2000). A second study demonstrated cell detachment of 293 T cells following expression of EBOV, but not MBGV, GP. Cell detachment in this case occurred without cell death. It was attributed largely to a domain within the extracellular region of GP₂ and seemed to involve a phosphorylation-dependent signal cascade (Chan et al., 2000 a). The ectodomain of the glycoprotein and its anchorage to the membrane are required for GP-induced morphological changes (Takada et al., 2000). Using a reverse genetics system, it was demonstrated recently that the cytotoxicity of EBOV depends on the level of GP expression. Overexpression of GP leads to an early detachment and cytotoxicity of infected cells (Volchkov et al., 2001). These data show that GP expression is controlled by RNA editing, which allows expression from approximately 20 % of the GP-specific transcripts and therefore down-regulates its synthesis. It appears that the editing of the GP gene of EBOV, while not being required for virus replication, is linked with the need to control cytotoxicity.

Interference with the host defence system. Immunosuppression seems to be an important factor in the pathogenesis of filovirus haemorrhagic fever. However, the mechanisms leading to the immunosuppressed status of the hosts are unknown and are currently being investigated. For EBOV, it has been reported that sGP interacts with the host immune response by binding to neutrophils through CD16b, the neutrophil-specific form of the Fc receptor III. Subsequently, sGP binding appears to inhibit early activation of these cells (Yang et al., 1998; Kindzelskii et al., 2000). However, this concept has been challenged by a report from Maruyama et al. (1998). Relatively high amounts of glycoprotein are released into the medium of filovirus-infected cells (Fig. 3 B) and it has been discussed that this soluble portion, as well as sGP (Fig. 3), may effectively bind antibodies that might otherwise be protective (Sanchez et al., 1996; Volchkov et al., 1998 b). In addition, filovirus transmembrane glycoprotein molecules possess a sequence close to the carboxy terminus resembling a presumptive immunosuppressive domain found in retrovirus glycoproteins (Volchkov et al., 1992; Will et al., 1993; Bukreyev et al., 1995). Peptides synthesized according to this 26 amino acid long region inhibit the blastogenesis of lymphocytes in response to mitogens, inhibit the production of cytokines and decrease the proliferation of mononuclear cells in vitro (Ignatyev, 1999). It is not yet known if the immunosuppressive domain on the GP is functional on mature molecules.

Recently, neutralizing anti-GP antibodies have been generated from several species, including humans, which were immunized or infected with Ebola virus. These neutralizing antibodies showed protective and therapeutic properties in animal models (Maruyama et al., 1999; Wilson et al., 2000). Protective properties, most likely due to neutralizing antibodies, were also associated with convalescent sera (Mupapa et al., 1999). The successful use of the transmembrane glycoprotein in different immunization approaches has demonstrated clearly the immunogenic and protective properties of this protein in small animal models and in non-human primates (Hevey et al., 1998; Vanderzanden et al., 1998; Xu et al., 1998; Pushko et al., 2000; Sullivan et al., 2000). Progress has been made towards the development of a human vaccine, but there is still a lot of work to be done (Burton & Parren, 2000; Klenk, 2000).

Soluble glycoproteins in pathogenesis. The disturbance of the blood–tissue barrier, which is controlled primarily by endothelial cells, is another important factor in pathogenesis. The endothelium seems to be affected in two ways: directly by virus infection leading to activation and eventual cytopathogenic replication and indirectly by a mediator-induced inflammatory response. These mediators originate from virus-activated cells of the mononuclear phagocytic system, especially macrophages, which are the primary target cells (Schnittler & Feldmann, 1999). Current data indicate that the activation of endothelial and mononuclear phagocytotic cells could be triggered by either virus infection or the binding of soluble viral or cellular factors produced during virus infection.

Several soluble glycoproteins are being expressed and secreted or released during infections with filoviruses. The comparison of the different filoviruses and virus variants raises questions concerning the relative roles of these proteins in pathogenesis. MBGV, which causes a comparable disease in primates, releases glycoproteins in amounts similar to EBOV, but does not express sGP due to a different organization of its glycoprotein gene (Fig. 1). The EBOV Zaire strains (discussed above) that express only small amounts of sGP display high pathogenicity in animal models (Ryabchikova et al., 1999; Volchkov et al., 2000 a). The less pathogenic or apathogenic subtype EBOV Reston has been shown to produce high levels of sGP (Sanchez et al., 1996). All of this argues against a more general role of sGP in the pathogenesis of filoviruses and may point towards a potential role of the soluble ectodomain of GP_1,2 in pathogenesis. This may highlight the importance of cleavage in pathogenesis, since the production of soluble glycoprotein GP₁ depends on the efficacy of furin cleavage. However, this does not exclude a role of sGP as a biologically active protein in infection.

H.F., V. E. V. and H. D. K. hold several grants on filoviruses provided by the Deutsche Forschungsgemeinschaft (SFB 286, Fe 286/4-1), the Kempkes-Stiftung (21/95), INSERM, Fondation Pour La Recherche Medicale, DGA (P01008SH), Canadian Institutes of Health Research (MOP-43921) and the European Community (INCO grant ERBIC 18 CT9803832). The authors are thankful for the contributions of several graduate students supported by these grants.

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