Requirements within the Ebola Viral Glycoprotein for Tetherin Antagonism.

Nathan Vande Burgt,Paul Bates,Rachel Kaletsky

doi:10.3390/v7102888

Abstract

Tetherin is an interferon-induced, intrinsic cellular response factor that blocks release of numerous viruses, including Ebola virus, from infected cells. As with many viruses targeted by host factors, Ebola virus employs a tetherin antagonist, the viral glycoprotein (EboGP), to counteract restriction and promote virus release. Unlike other tetherin antagonists such as HIV-1 Vpu or KSHV K5, the features within EboGP needed to overcome tetherin are not well characterized. Here, we describe sequences within the EboGP ectodomain and membrane spanning domain (msd) as necessary to relieve tetherin restriction of viral particle budding. Fusing the EboGP msd to a normally secreted form of the glycoprotein effectively promotes Ebola virus particle release. Cellular protein or lipid anchors could not substitute for the EboGP msd. The requirement for the EboGP msd was not specific for filovirus budding, as similar results were seen with HIV particles. Furthermore trafficking of chimeric proteins to budding sites did not correlate with an ability to counter tetherin. Additionally, we find that a glycoprotein construct, which mimics the cathepsin-activated species by proteolytic removal of the EboGP glycan cap and mucin domains, is unable to counteract tetherin. Combining these results suggests an important role for the EboGP glycan cap and msd in tetherin antagonism.

Highlights

The innate immune system is the first line of defense against viral pathogens
We found that neither soluble glycoprotein (sGP) nor secGP, a soluble version of EboGP cleaved at the extracellular base by tumor necrosis factor-converting enzyme (TACE) protease [31], could effectively counteract tetherin [2]
Overall these experiments define a minimal 320 residue portion of the Ebola glycoprotein ectodomain, containing the receptor binding domain and glycan cap regions of EboGP, that when anchored to the cell surface is sufficient to antagonize tetherin activity. These data indicate that the mucin domain and the extracellular region of the GP2 subunit of EboGP are dispensable for anti-tetherin activity

Summary

Introduction

The innate immune system is the first line of defense against viral pathogens. mammalian cells employ numerous innate cellular mechanisms to inhibit viral replication and spread. Intrinsic antiviral factors comprise a form of innate immunity that directly limit viral entry, replication or assembly. Polymerase stuttering incorporates an additional nucleotide in a small percentage of the GP transcripts causing a frameshift and production of the full-length, virion associated glycoprotein (EboGP) [20,21] Due to this method of production, sGP and EboGP share 295 N-terminal residues, including regions within EboGP needed for receptor recognition and cell binding as well as a domain called the glycan cap. We define a minimal 320 residue portion of the Ebola glycoprotein ectodomain, containing the receptor binding domain and glycan cap regions of EboGP, that when anchored to the cell surface is sufficient to antagonize tetherin activity. Cell lysates and purified VLPs were analyzed by immunoblot

Immunoprecipitaion Assay

Immunoblot Analysis

Flow Cytometry Analysis

Requirements within EboGP for Tetherin Antagonism

Discussion