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

Bin Wang,Yujie Wang,Dylan A Frabutt,Xihe Zhang,Xiaoyu Yao,Dan Hu,Zhuo Zhang,Chaonan Liu,Shimin Zheng,Shi-Hua Xiang,Yong-Hui Zheng

doi:10.1074/jbc.m116.768168

Abstract

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.

Highlights

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
These two N-glycosylation sites (NGSs) are required for GP incorporation into EBOV-like particles and HIV type 1 (HIV-1) pseudovirions and determine viral transduction efficiency
GP1 is divided into core domain and mucin-like domain (MLD), and the core domain is further divided into the receptor-binding domain (RBD) and glycan cap (Fig. 1A) [13]

Summary

Results

We used MLD-deleted (⌬MLD) GP in most of our experiments to study GP activity. Each of the nine NGSs on ⌬MLD-GP were deglycosylated by replacing Asn with Gln, creating nine mutants: N40Q, N204Q, N228Q, N238Q, N257Q, N268Q, N296Q, N563Q, and N618Q When their expression in cells and HIV-1 pseudovirions was determined, it was found that none of these single mutations reduced GP expression and processing, except for N563Q and N618Q, which reduced processing (Fig. 2A, lanes 1–10). KIF reduced HIV-EBOV GP transduction in a dose-dependent manner without reducing the levels of the GP incorporation (Fig. 3, C and D) These results demonstrate that demannosylation is required for EBOV GP function. More CRT proteins were pulled down from WT and ⌬CNX than ⌬CRT and ⌬⌬ cells These results demonstrate that GPs interact with CRT in a CNXindependent manner

CRT Actin

Experimental procedures

Virus production

EBOV transduction assay

Western blotting and immunoprecipitation

Gene knockout