The Effect Of The N-Linked Glycosylation Site Of Hantavirus GM04-38 On Cell Fusion

Objective N-Linked oligosaccharide chain can affect protein function in many aspects.There were experimental results have demonstrated the missing of oligosaccharide chain of viral fusion protein in some virus can lead to defects or loss of cell fusion. In view of Hantavirus GM04-38 Zhu envelope glycoprotein G1 and G2 are also associated by the N-glycosylation, we investigated the role of N-Linked glycosylation site in cell fusion, 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 five clone mutants, in which there was only one amino acid mutated. Then we amplified their glycoprotein G1 and G2 coding region by PCR, digested the products with Kpn I and Xho I. The same digestion was made for the vector pcaggs/mcs.Consequently, the G1 and G2 coding region and the vector were ligated and transformed into E.coli DH5a. After being screened by ampicillin resistant, the expressing mutants were constructed. After being confirmed by enzyme digestion and sequence analysis, the wild G1Bgl, GmG2,four mutants of G1 and one mutant of G2 were co-transfected into Vero E6 cells. Western Blot and IFA were performed to test the expression of GPs genes. After treated with acidic MEM, the cells were fixed and stained by Giemsa and observed under microscope to examine cell fusion activities.Results 1.Five clone mutants were constructed, in which there was only one amino acid mutated. According to the amino acid substituted, they were named N134A, N235A, N347A, N399A and N928A.They were digested with Kpn I and Hind III, the vector was 2.7kb; the N134A, N235A, N347A and N399A mutants were 2.1kb, and the N928A mutant 1.6kb. The results were confirmed by sequence analysis.2.Five expressing mutants were constructed, in which there was only one amino acid mutated. They were named GlBgl-N134A, GlBgl-N235A, GlBgl-N347A, GlBgl-N399A and GmG2-N928A. They were digested with Kpn I and Xho I, the vector was 4.7kb;the N134A, N235A, N347A and N399A mutants were 2.1kb, and the N928A mutant 1.6kb. 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.Western Blot showed all the mutants expressed G1 and G2 gene except the N134A mutant only expressed G2 gene. The fragments of G1 and G2 are 68KDa and 55Kda respectively.5.Giemsa staining showed no cell-cell fusion could be induced under acidic conditions in the cells co-transfected with the mutants N134A or N928A; co-transfected with other mutants and wild G1Bgl and GmG2 showed cell-cell fusion.Conclusion It is likely that N-linked glycosylation at 134 site is crucial in directing correctfolding of G1,which made the mutant N134A was retained in the ER and could no longer be recognized by anti-G1 MAbs. The mutation of the single site on G2 (N928) resulted in a loss of cell fusion, which suggests the 928 site on G2 is crucial for cell fusion and the fusion peptide is likely on G2.