Microbiology
Mechanistic understanding of N-glycosylation in Ebola virus glycoprotein maturation and function

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The Ebola virus (EBOV) trimeric envelope glycoprotein (GP) precursors are cleaved into the receptor-binding GP1 and the fusion-mediating GP2 subunits and incorporated into virions to initiate infection. GP1 and GP2 form heterodimers that have 15 or two N-glycosylation sites (NGSs), respectively. Here we investigated the mechanism of how N-glycosylation contributes to GP expression, maturation, and function. As reported before, we found that, although GP1 NGSs are not critical, the two GP2 NGSs, Asn563 and Asn618, are essential for GP function. Further analysis uncovered that Asn563 and Asn618 regulate 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 Asn563 and Asn618 are not required for EBOV GP expression, they synergistically regulate its maturation, which determines its functionality.

chaperone
Ebola virus
glycoprotein
glycosylation
virus entry
calnexin

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The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

This article contains supplemental Table 1.

1

Both authors contributed equally to this work.

2

Supported by National Natural Science Foundation of China Grant 31502089 and Harbin Veterinary Research Institute Grant SKLVBP2015012. The authors declare that they have no conflicts of interest with the contents of this article.