Ebola virus is an enveloped, nonsegmented, negative-stranded RNA virus. Together with Marburg virus, they are classified in the order of Mononegavirales and the family of Filoviridae (29). Ebola virus is further grouped into four species: Zaire, Sudan, Ivory Coast and Reston. Infection by Ebola viruses can cause severe hemorrhagic fever characterized by widespread tissue infection and damage in multiple organ and tissue in human and other primates, and result in high mortality, up to 90% by Zaire species (29). Currently there is no effective vaccine or treatment. against Ebola infection in human.
The viral genome of Ebola is approximately 19Kb in length, and encodes seven genes: NP, VP35, VP40, GP, VP30, VP24, and L (30). The GP gene encodes two products through transcriptional editing (31, 36). The non-edited ORF encodes a smaller, secreted glycoprotein (sGP) which can be detected in large amount in the serum of the early phase of infected patients (31, 36). However the role of sGP in viral replication and pathogenesis is still not clear. The -1 ORF of the GP gene encodes a membrane-anchored glycoprotein (GP) which is the sole structural protein that forms the spikes on the virion surface (32).
GP is synthesized as the pre-GP in the ER, fully glycosylated in Golgi (GP0), and transported to the plasma membrane where virus budding occurs (13, 25, 32). GP0 is cleaved in the Golgi apparatus by furin-like proteases to generate two subunits, GP1 and GP2, which are linked by a disulfide bond (17, 24, 25). The native form of GP is a homotrimer of GP1/GP2 heterodimer on virions (32). GP1 is highly glycosylated containing both N-and O-linked carbohydrates, especially in the mucin-like region (13). GP1 is believed to be responsible for receptor binding, while GP2 for mediating membrane fusion and viral entry during Ebola infection. An X-ray structure of GP1/GP2 complex, believed to be at the native conformation, was recently reported. Together with the biochemical and extensive functional analysis of GP1, the receptorbinding domain (RBD) and some critical residues in RBD have been elucidated (4, 15, 16, 20, 21, 23).
In contrast, characterization of the Ebola GP2 subunit has been limited in scope. Nevertheless, the available information reveals that the Ebola GP2 subunit shares several characteristic features with other viruses. It was proposed that GP2 is structurally similar to the transmembrane subunits (TM) of retroviruses, particularly that of Rous sarcoma viruses (11, 37). The putative fusion peptide is located near the N-terminus. Following the putative fusion peptide is a region of heptad repeats (N-helical region) which are implicated in formation of coiled-coil structures. Another predicted amphiphathic helical region (C-helical region) is located to the C-terminal end of the GP2 ectodomain. The X-ray structures of the core GP2 ectodomain show that the core GP2 ectodomain forms a trimer in which a long central three-stranded coiled-coil is surrounded by shorter C-terminal helices that are packed in an antiparallel orientation into hydrophobic grooves formed by the inner coiled-coil (19, 39). This structural feature is shared by several viral fusion proteins such as HA2 of influenza virus, gp41 of human immunodeficiency virus, suggesting a common membrane fusion mechanism mediated by different viral glycoproteins. These proteins have been classified as the class Ⅰ fusion proteins (8). Mutational analysis of the hydrophobic residues in the N-and C-helices of GP2 indicated that some of these residues are indeed important in mediating Ebola entry (34), consistent with the structural features of the core GP2 ectodomain.
In this study, we examined the role of the charged residues in the GP2 helical regions in Ebola entry using a HIV pseudotyping system as a surrogate functional assay. We found that substitution of many charged residues in these regions greatly impaired the ability of Ebola GP to mediate efficient viral entry, indicating that these charged residues play an important role in viral entry. Elucidating the role(s) of these residues in protein folding and function of GP may provide insightful information on the molecular mechanism of Ebola entry and on the development of potential entry inhibitors against Ebola infection.
The Role of the Charged Residues of the GP2 Helical Regions in Ebola Entry
- Received Date: 05 December 2008
- Accepted Date: 12 December 2008
Abstract: The glycoprotein (GP) of Ebola is the sole structural protein that forms the spikes on the viral envelope. The GP contains two subunits, GP1 and GP2, linked by a disulfide bond, which are responsible for receptor binding and membrane fusion, respectively. In this study, the full length of GP gene of Ebola Zaire species, 2028 base pairs in length, was synthesized using 38 overlapping oligonucleotides by multiple rounds of polymerase chain reaction (PCR). The synthesized GP gene was shown to be efficiently expressed in mammalian cells. Furthermore, an efficient HIV-based pseudotyping system was developed using the synthetic GP gene, providing a safe approach to dissecting the entry mechanism of Ebola viruses. Using this pseudotyping system and mutational analysis, the role of the charged residues in the GP2 helical regions was examined. It was found that substitutions of the most charged residues in the regions did not adversely affect GP expression, processing, or viral incorporation, however, most of the mutations greatly impaired the ability of GP to mediate efficient viral infection. These results demonstrate that these charged residues of GP2 play an important role in GP-mediated Ebola entry into its host cells. We propose that these charged residues are involved in forming the intermediate conformation(s) of GP in membrane fusion and Ebola entry.