Analysis of gene mutation in the interaction domain of head and stalk of Newcastle disease virus hemagglutinin-neuraminidase glycoprotein

Zhankui Cao; Ying Liu; Miaomiao Chi; Jingjing Jiang; Jingxue Liu; Hongling Wen; Li Zhao; Lianli Chi; Zhiyu Wang

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Analysis of gene mutation in the interaction domain of head and stalk of Newcastle disease virus hemagglutinin-neuraminidase glycoprotein

VernacularTitle: 新城疫病毒HN糖蛋白头颈部相互作用区基因突变分析
Author: Zhankui CAO ¹ ; Ying LIU ¹ ; Miaomiao CHI ¹ ; Jingjing JIANG ¹ ; Jingxue LIU ¹ ; Hongling WEN ¹ ; Li ZHAO ¹ ; Lianli CHI ² ; Zhiyu WANG ¹
Author Information

1. Department of Virology, School of Public Health, Shandong University, Jinan 250012, China
2. National Glycoengineering Research Center, State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China
Publication Type:Journal Article
Keywords: Newcastle disease virus; Hemagglutinin-neuraminidase; Fusion protein; Homologous recombination
From: Chinese Journal of Experimental and Clinical Virology 2018;32(5):463-468
CountryChina
Language:Chinese
Abstract: Objective:To identify the function of 91-112 amino acids (aa) fragment, the interaction domain of head and stalk of Newcastle disease virus(NDV) HN glycoprotein, and clarify the role of the fragment in promoting cell specific membrane fusion.
Methods:The specific gene sequences were identified by aligning 91-112 amino acids of NDV HN protein with amino acids of MeV H, RSV G, hPIV3 HN protein. The fragment deletion, fragment substitution and intermolecular homologous recombination method were combined to construct the deletion mutant, De(HN), and three chimeras, Ch(MeV), Ch(RSV), Ch(hPIV3). Cationic transfection reagent was used to transfect the plasmids into baby hamster kidney cells (BHK-21), in which vaccinia virus-T7 RNA polymerase expression system was expressed. Indirect immunofluorescence assay (IIFA) and flow cytometry (FCM) were executed to analyze the cell surface expression level. Cell fusion promotion activity, receptor recognition activity and neuraminidase activity of each mutant were also detected.
Results:Cell surface expression efficiency of De(HN) and Ch(MeV), Ch(RSV), Ch(hPIV3) proteins were 9.04%, 82.20%, 70.16%, 75.65% of that of wild-type (wt) HN. Fusion promotion activity of De(HN), Ch(MeV), Ch(RSV), Ch(hPIV3) were 3.83%, 24.76%, 29.42%, 57.84% of that of wt HN. The fusion promotion activity of De(HN) almost disappeared and syncytium couldn’t be found under the microscope. Hemadsorption activity was 13.48%, 36.25%, 34.93%, 65.22%, respectively (P<0.05), which was consistent with the fusion promotion activity of mutant proteins. Neuraminidase activity was 10.81%, 54.42%, 50.13%, 60.35% of that of wt HN, respectively (P<0.05).
Conclusions:The amino acids fragment (91-112) of NDV HN protein plays an important role in promotion fusion. The loss of fusion promotion activity of De(HN) protein was related to the failure of effective cell surface expression of the mutant.