Defining The Fusion Peptide On The Glycoprotein Of Hantavirus GM04-38 Strain

Objective Prior computational methods showed that the glycoprotein G2 of Hantavirus was its fusion protein,which was classⅡfusion protein,and that the internal highly conserved domain(aa763～785) maybe was critical for membrane fusion and was named fusion peptide.In addition,there were experimental results demonstrating the fusion peptide was located on glycoprotein G2 but not G1.Finding out that there was a similar conserved domain in Hantavirus GM04-38 strain,we investigated the role of this domain to estimate whether it was its fusion peptide preliminarily,so as to provide elements for elucidating the fusion mechanism of Hantavirus and developing effective vaccines and drugs for Hantavirus.Methods Primers were designed according to the sequence of M segment cDNA by Primer 5.Recombinant PCR was used for constructing ten clone mutants, in which there was only one amino acid mutated.Then we amplified their glycoprotein G2 coding region by PCR,digested the products with KpnⅠand XhoⅠ. The same digestion was made for the vector pCAGGS/MCS.Consequently,the G2 coding region and the vector were ligated and transformed into E.coli DH5α.After being screened by ampicillin resistant,the expressing mutants were constructed.After being confirmed by enzyme digestion and sequence analysis,the wild G1Bgl and GmG2/or one of the expressing mutants were co-transfected into Vero E6 cells.IFA was performed to observe expression efficiencies.After treated with acidic MEM,the cells were fixed and stained by Giemsa and observed under microscope to examine cell fusion activities.Results 1.Ten clone mutants were constructed,in which there was only one amino acid mutated.According to the amino acid substituted,they were named C765A,P767D,P768D,C770A,P771D,G772D,G774D,G776D,C777A and C780A.They were digested with KpnⅠand HindⅢ,the fragments were 1.6 kb and 2.7 kb respectively.The results were confirmed by sequence analysis.2.Ten expressing mutants were constructed,in which there was only one amino acid mutated.They were named GmG2-C765A,GmG2-P767D,GmG2-P768D, GmG2-C770A,GmG2-P771D,GmG2-G772D,GmG2-G774D,GmG2-G776D, GmG2-C777A and GmG2-C780A accordingly.They were digested with KpnⅠand XhoⅠ,the fragments were 1.6kb and 4.7kb respectively.The results were confirmed by sequence analysis.3.IFA showed there were robust fluorescent signals in the cells co-transfected with the wild G1Bgl and GmG2/or one of the expressing mutants.They were concentrated in the perinuclear region of the transfected cells.4.Giemsa staining showed cell-cell fusion could be induced under acidic conditions in the cells co-transfected with the wild G1Bgl and GmG2.But there were not cell fusion activities in the cells co-transfected with the wild G1Bgl and one of the expressing mutants.Conclusion The mutation of ten amino acids in the putative fusion domain (aa763～785) can block cell fusion activities completely but can not influence the expressions of glycoproteins.This is the most compelling evidence for the direct involvement of the conserved domain in fusion.Here we present evidence that this domain is likely to be the fusion peptide of the virus.