N-Linked Glycosylation on Anthrax Toxin Receptor 1 Is Essential for Seneca Valley Virus Infection.

Nadishka Jayawardena,Matthias Wolf,Charles Rudin,John T Poirier,Laura N Burga,Linde A Miles,Mihnea Bostina

doi:10.3390/v13050769

Abstract

Seneca Valley virus (SVV) is a picornavirus with potency in selectively infecting and lysing cancerous cells. The cellular receptor for SVV mediating the selective tropism for tumors is anthrax toxin receptor 1 (ANTXR1), a type I transmembrane protein expressed in tumors. Similar to other mammalian receptors, ANTXR1 has been shown to harbor N-linked glycosylation sites in its extracellular vWA domain. However, the exact role of ANTXR1 glycosylation on SVV attachment and cellular entry was unknown. Here we show that N-linked glycosylation in the ANTXR1 vWA domain is necessary for SVV attachment and entry. In our study, tandem mass spectrometry analysis of recombinant ANTXR1-Fc revealed the presence of complex glycans at N166, N184 in the vWA domain, and N81 in the Fc domain. Symmetry-expanded cryo-EM reconstruction of SVV-ANTXR1-Fc further validated the presence of N166 and N184 in the vWA domain. Cell blocking, co-immunoprecipitation, and plaque formation assays confirmed that deglycosylation of ANTXR1 prevents SVV attachment and subsequent entry. Overall, our results identified N-glycosylation in ANTXR1 as a necessary post-translational modification for establishing stable interactions with SVV. We anticipate our findings will aid in selecting patients for future cancer therapeutics, where screening for both ANTXR1 and its glycosylation could lead to an improved outcome from SVV therapy.

Highlights

Seneca Valley virus (SVV) is a small, non-enveloped RNA virus belonging to the genus Senecavirus in the family Picornaviridae [1]
As a first step toward recognizing potential N-linked glysosylation sites in anthrax toxin receptor 1 (ANTXR1)-Fc recombinant preparations used in this study, we combined different proteolytic degradation protocols followed by deglycosylation with PNGase F in the presence of 18O water to obtain glycopeptides suited for LC-nSI-MS/MS analysis
Viral protein or receptor glycosylation can generally be broadly categorized into two main groups: N-linked and O-linked

Summary

Introduction

Seneca Valley virus (SVV) is a small, non-enveloped RNA virus belonging to the genus Senecavirus in the family Picornaviridae [1]. The SVV ORF encodes a single 2181 amino acid long polyprotein, which is later cleaved into a characteristic picornaviral protein layout, L-4-3-4 [1] In this layout, L stands for the leader protein and 4-3-4 represents the four structural proteins VP4, VP2, VP3, and VP1 and seven non-structural proteins 2A–2C and 3A–3D. In the SVV life cycle, two forms of particles exist: mature capsid with a packaged genome, and native empty capsid without RNA [2]. In both capsids, VP1 is arranged around the 5-fold axis, while VP2 and VP3 alternate around the 3-fold axis. VP4 is located in the capsid interior, forming contacts with the RNA near the 5-fold axis [2,3]

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