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Filovirus family consists of Ebola virus and Marburg virus which can cause severe hemorrhagic fever in human and non-human primates with high mortality. Ebola viruses are classified into five species named after the location of their first outbreaks: Zaire, Sudan, Ivory Coast, Bundibugyo, and Reston [46]. Three species of fruit bats, Hypsignathus monstrosus, Epomops franqueti, Myonycteris torquata, captured near the sites of 2001 and 2005 outbreaks of Ebola, are implicated as the natural reservoir of Ebola virus[20, 21]. Ebola virus infects humans and non-human primates through direct contact with body fluid, while aerosol transmission has also been suggested[19]. Since there are no vaccines or therapeutics available for humans, Ebola virus has become one of the major concerns on bio-terrorism.
Ebola viruses are enveloped viruses with an ~19 kb single-stranded RNA genome containing 7 genes. The GP gene encodes two glycoproteins, a secreted form called sGP and a transmembrane form called GP which is generated by RNA editing[34, 35, 44]. GP is responsible for viral entry, including attachment of viruses to the target cells and fusion of the virus-cell membranes[43, 48, 49]. GP is modified by N-linked glycosylation in the Endoplasmic Reticulum to form PreGP, and further undergoes O-linked glycosylation in Golgi to become mature GP[10].
Ebola GP processing by proteases has been well documented. First, the N-terminal signal peptide (32 residues) of GP is cleaved cotranslationally. The GP precursor is then cleaved into GP1 and GP2 in trans-Golgi by a furin-like cellular protease, and GP1 and GP2 are linked by disulfide bonding[14, 36, 45]. Although the cleavage site between GP1 and GP2 is conserved among the Ebola species, this cleavage event appears not required for Ebola entry[13, 28, 29, 49]. Lastly, GP was shown to be cleaved at D637 by the tumor necrosis factor a-converting enzyme (TACE) to shed the truncated GP from the surface of infected cells[7]. In addition to these cleavage events during GP maturation, two intracellular proteases, Cathepsin B and L, residing in the endosomes, have been shown to process GP1 into an 18-19 kDa fragment during viral entry[5, 16, 37]. The endosomal cleavage appears to be critical on viral entry. It is interesting to note that these endosomal proteases play a role in entry of other viruses[9, 12, 30, 31, 39]
Many cellular proteins have been implicated in facilitating Ebola entry, including asialoglycoprotein receptor, folate receptor a, dendritic cell-specific ICAM3 grabbing non-integrin, liver/lymph node-specific ICAM3 grabbing non-integrin, human macrophage galactose and N-acetylgalactosamine-specific C-type lectin, and Tyro3 receptor kinase family [1, 4, 8, 22, 32, 33, 38, 42]. However, none of them seems to be absolutely required for the entry. Interestingly, expression of Ebola GP on the target cells can specifically enhance Ebola GP-mediated viral entry [23].
The N-terminal 200 residues of GP1 are relatively conserved compared to the highly variable C-terminal mucin-like domain (MUC, amino acids 309~476). The MUC region is heavily modified by O-and N-linked glycosylation. It has been shown that MUC is responsible for the cytopathic effects induced by GP expression, but it is dispensable for GP-mediated viral entry, at least in tissue culture using pseudotyping systems[14, 24, 40, 50]. Previous studies have shown that the N-terminal region of GP1 (approximately 150 residues in length after cleavage of the signal peptide) is critical for receptor binding, which is referred to as the receptor-binding domain, or RBD[3, 24]. Extensive mutational analyses have been performed to charac-terize the roles of RBD residues in protein folding and function in viral entry. In this report, we examined the roles of the RBD residues by substitutions in protein expression, virion incorporation, and viral entry[3, 14, 24, 26]. By entry interference assay and binding assay, we identified several key residues involved in receptor binding and the post-binding steps in viral entry. In addition, the results further substantiated the notion that Ebola and Marburg viruses use the same or similar receptor.
Characterization of the Receptor-binding Domain of Ebola Glycoprotein in Viral Entry
- Received Date: 31 March 2011
- Accepted Date: 25 April 2011
Abstract: Ebola virus infection causes severe hemorrhagic fever in human and non-human primates with high mortality. Viral entry/infection is initiated by binding of glycoprotein GP protein on Ebola virion to host cells, followed by fusion of virus-cell membrane also mediated by GP. Using an human immunodeficiency virus (HIV)-based pseudotyping system, the roles of 41 Ebola GP1 residues in the receptor-binding domain in viral entry were studied by alanine scanning substitutions. We identified that four residues appear to be involved in protein folding/structure and four residues are important for viral entry. An improved entry interference assay was developed and used to study the role of these residues that are important for viral entry. It was found that R64 and K95 are involved in receptor binding. In contrast, some residues such as I170 are important for viral entry, but do not play a major role in receptor binding as indicated by entry interference assay and/or protein binding data, suggesting that these residues are involved in post-binding steps of viral entry. Furthermore, our results also suggested that Ebola and Marburg viruses share a common cellular molecule for entry.