The Role of the Charged Residues of the GP2 Helical Regions in Ebola Entry

The glycoprotein （GP） of Ebola is the sole structural protein that forms the spikes on the viral envelope. The GP contains two subunits, GPI 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.

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.