Canadian Science Centre for Human and Animal Health, 1015 Arlington Street, Winnipeg, Manitoba, CanadaR3E 3R21
Biologie des Filovirus, Claude Bernard University Lyon-1, 46 Allée dItalie, 69007 Lyon, France2
Institut für Virologie, Philipps-Universität, Robert-Koch-Str. 17, D-35037 Marburg, Germany3
Author for correspondence: Heinz Feldmann. Fax +1 204 789 2140. e-mail Heinz_Feldmann{at}hc-sc.gc.ca
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Introduction |
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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 dIvoire, 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; GP1,2 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.
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Filoviral glycoproteins |
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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. 2A). 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., 1998b
), N-glycosylation (Feldmann et al., 1991
, 1994
; Volchkov et al., 1995
; Becker et al., 1996
; Sanchez et al., 1998b
) and oligomerization (Feldmann et al., 1991
; Sanchez et al., 1998b
). 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 2A
, B
) (Volchkov et al., 1998a
, 2000b
).
|
The special arrangement of the cysteine residues in the GP1,2 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. 2C). It can be assumed that cysteine residue 53 is also critical for maintaining the structure of GP1,2, as discussed below for sGP (Volchkova et al., 1998
). The mature transmembrane glycoprotein is a trimer comprising disulphide-bonded GP1,2 molecules (Feldmann et al., 1991
; Sanchez et al., 1998b
). As demonstrated for GP1,2 expression using a recombinant vaccinia virus, the mature transmembrane glycoprotein forms spikes, without the need for other viral proteins (Volchkov et al., 1998b
). 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. 2B
) (Weissenhorn et al., 1998a
, 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. 3B
) (Volchkov et al., 1998b
).
|
-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 1014 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., 2000b
). 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., 2000b
). 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 524539). 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., 2000b
).
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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 data). MBGV may be distinct due to obvious differences in the position of its fusion domain compared with EBOV (Fig. 2A
, 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, 1994a
). 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 RXK/RR, 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., 1998a
). MBGV and all highly pathogenic EBOV strains display the canonical furin motif RXK/RR 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 KQKR (Table 1
and Fig. 5
) (Volchkov et al., 1998a
). 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., 1998a
). 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.
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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. 3B
) 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., 1998b
). 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 bloodtissue 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., 2000a
). 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 GP1,2 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.
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Acknowledgments |
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