# An Evolutionary Distinct Nipah Virus N-Glycosylation Site Provides Stability for Receptor Engagement

## TL;DR

This study explores how glycosylation affects the Nipah virus's ability to bind to host cells, revealing key residues important for stability and receptor engagement.

## Contribution

The first site-specific glycan characterization of the Nipah virus G glycoprotein and identification of critical residues for receptor binding.

## Key findings

- The N481 N-glycan site is not evolutionarily conserved across Nipah species.
- The Ser/Thr residue in the N481 sequon is crucial for maintaining G glycoprotein stability and ephrin B2 binding.
- Hydrogen bonding networks contribute to G stability and host engagement.

## Abstract

Nipah virus is a deadly paramyxovirus with 40 to 75% mortality and >750 cases since 1998. Currently there are no clinically approved vaccines or therapeutics to treat infection. Nipah is an enveloped virus with two surface glycoproteins, the trimeric fusion glycoprotein (F), and the tetrameric attachment glycoprotein (G), which is responsible for cellular attachment via binding to the host ephrin B2/B3 receptor. Glycosylation can substantially affect immunogenicity, receptor binding, and structural conformations for virus glycoproteins, but its effects on Nipah G receptor engagement have not been studied. Here, phylogenetic and mass spectrometry analysis of the Nipah G Malaysia strain reveal how N-glycosylation has evolved since the appearance of the virus in 1998. We discovered that the N481 sequon is not conserved and the threonine/serine in the glycosylation site is critical for maintaining long-range stability of G subunits that facilitates ephrin B2 binding affinity. Together, these data reveal plasticity of N-glycosylation sites across Nipah species and the presence of hydrogen bonding networks that contribute to G stability and host engagement—results that are valuable for understanding virus attachment/entry mechanisms and the rational design of structure-based vaccines.

•First site-specific glycan characterization of the Nipah virus glycoprotein (G) tetramer.•Phylogenetic analysis reveals the N481 N-glycan site is not evolutionary conserved.•The absence of the N481 glycan does not contribute to ephrin B2 receptor binding.•However, the Ser/Thr in the sequon is conserved and maintains Nipah G stability.

First site-specific glycan characterization of the Nipah virus glycoprotein (G) tetramer.

Phylogenetic analysis reveals the N481 N-glycan site is not evolutionary conserved.

The absence of the N481 glycan does not contribute to ephrin B2 receptor binding.

However, the Ser/Thr in the sequon is conserved and maintains Nipah G stability.

Nipah virus is a deadly pathogen and the roles of glycosylation in its infection are poorly understood. Here, we investigated the evolution of the Nipah virus attachment glycoprotein (G) and characterized its site-specific glycosylation. We determined key allosteric residues in a nonconserved N-glycan site that are critical for receptor binding. Our data provide structural and dynamic evidence for Nipah glycoprotein stabilization within the N481 sequon, offering insights for therapeutic development.