Defining the N-linked glycosylation site of Hantaan virus envelope glycoproteins essential for cell fusion.
The Hantaan virus (HTNV) is an enveloped virus that is capable of inducing low pH-dependent cell fusion. We molecularly cloned the viral glycoprotein (GP) and nucleocapsid (NP) cDNA of HTNV and expressed them in Vero E6 cells under the control of a CMV promoter. The viral gene expression was assessed using an indirect immunofluorescence assay and immunoprecipitation. The transfected Vero E6 cells expressing GPs, but not those expressing NP, fused and formed a syncytium following exposure to a low pH. Monoclonal antibodies (MAbs) against envelope GPs inhibited cell fusion, whereas MAbs against NP did not. We also investigated the N-linked glycosylation of HTNV GPs and its role in cell fusion. The envelope GPs of HTNV are modified by N-linked glycosylation at five sites: four sites on G1 (N134, N235, N347, and N399) and one site on G2 (N928). Site-directed mutagenesis was used to construct eight GP gene mutants, including five single N-glycosylation site mutants and three double-site mutants, which were then expressed in Vero E6 cells. The oligosaccharide chain on residue N928 of G2 was found to be crucial for cell fusion after exposure to a low pH. These results suggest that G2 is likely to be the fusion protein of HTNV.
Zheng
Feng
F
Qilu Hospital of Shandong University, Shandong Province, Jinan 250012, China.
Ma
Lixian
L
Shao
Lihua
L
Wang
Gang
G
Chen
Fengzhe
F
Zhang
Ying
Y
Yang
Song
S
D000818:Animals; D002459:Cell Fusion; D002522:Chlorocebus aethiops; D015726:Giant Cells / Q000821:virology; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D018063:Hantaan virus / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D019251:Nucleocapsid / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000378:metabolism*; D014760:Viral Fusion Proteins / Q000737:chemistry / Q000378:metabolism*
D006023:Glycoproteins; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; D014760:Viral Fusion Proteins