title: Mechanistic understanding ofN-glycosylation in Ebola virus glycoprotein maturation and function.
abstract: The Ebola virus (EBOV) trimeric envelope glycoprotein (GP) precursors are cleaved into the receptor-binding GP1and the fusion-mediating GP2subunits and incorporated into virions to initiate infection. GP1and GP2form heterodimers that have 15 or twoN-glycosylation sites (NGSs), respectively. Here we investigated the mechanism of howN-glycosylation contributes to GP expression, maturation, and function. As reported before, we found that, although GP1NGSs are not critical, the two GP2NGSs, Asn563and Asn618, are essential for GP function. Further analysis uncovered that Asn563and Asn618regulate GP processing, demannosylation, oligomerization, and conformation. Consequently, these two NGSs are required for GP incorporation into EBOV-like particles and HIV type 1 (HIV-1) pseudovirions and determine viral transduction efficiency. Using CRISPR/Cas9 technology, we knocked out the two classical endoplasmic reticulum chaperones calnexin (CNX) and/or calreticulin (CRT) and found that both CNX and CRT increase GP expression. Nevertheless, NGSs are not required for the GP interaction with CNX or CRT. Together, we conclude that, although Asn563and Asn618are not required for EBOV GP expression, they synergistically regulate its maturation, which determines its functionality.
authors.affiliation: From the Harbin Veterinary Research Institute, CAAS-Michigan State University Joint Laboratory of Innate Immunity, State Key Laboratory of Veterinary Biotechnology, Chinese Academy of Agricultural Sciences, Harbin 150059, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Wang
authors.affiliation: From the Harbin Veterinary Research Institute, CAAS-Michigan State University Joint Laboratory of Innate Immunity, State Key Laboratory of Veterinary Biotechnology, Chinese Academy of Agricultural Sciences, Harbin 150059, China.
authors.forename: Yujie
authors.initials: Y
authors.lastname: Wang
authors.affiliation: the Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan 48824.
authors.forename: Dylan A
authors.initials: DA
authors.lastname: Frabutt
authors.affiliation: the Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan 48824.
authors.forename: Xihe
authors.initials: X
authors.lastname: Zhang
authors.affiliation: From the Harbin Veterinary Research Institute, CAAS-Michigan State University Joint Laboratory of Innate Immunity, State Key Laboratory of Veterinary Biotechnology, Chinese Academy of Agricultural Sciences, Harbin 150059, China.
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Yao
authors.affiliation: From the Harbin Veterinary Research Institute, CAAS-Michigan State University Joint Laboratory of Innate Immunity, State Key Laboratory of Veterinary Biotechnology, Chinese Academy of Agricultural Sciences, Harbin 150059, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Hu
authors.affiliation: From the Harbin Veterinary Research Institute, CAAS-Michigan State University Joint Laboratory of Innate Immunity, State Key Laboratory of Veterinary Biotechnology, Chinese Academy of Agricultural Sciences, Harbin 150059, China.
authors.forename: Zhuo
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: the College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China.
authors.forename: Chaonan
authors.initials: C
authors.lastname: Liu
authors.affiliation: the College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China.
authors.forename: Shimin
authors.initials: S
authors.lastname: Zheng
authors.affiliation: the Nebraska Center for Virology and School of Veterinary Medicine and Biomedical Sciences, University of Nebraska, Lincoln, Nebraska 68583, and.
authors.forename: Shi-Hua
authors.initials: SH
authors.lastname: Xiang
authors.affiliation: From the Harbin Veterinary Research Institute, CAAS-Michigan State University Joint Laboratory of Innate Immunity, State Key Laboratory of Veterinary Biotechnology, Chinese Academy of Agricultural Sciences, Harbin 150059, China, zhengyo@msu.edu.|the Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan 48824.
authors.forename: Yong-Hui
authors.identifier: 0000-0002-1098-7385
authors.initials: YH
authors.lastname: Zheng
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D029043:Ebolavirus / Q000235:genetics / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral; D006031:Glycosylation; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D011499:Protein Processing, Post-Translational; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D014759:Viral Envelope Proteins; C113189:envelope glycoprotein, Ebola virus
keywords: Ebola virus; calnexin; chaperone; glycoprotein; glycosylation; virus entry