# Structural and Functional Analysis of Murine Polyomavirus Capsid Proteins Establish the Determinants of Ligand Recognition and Pathogenicity

## TL;DR

This study explores how small changes in a virus's protein affect its ability to bind to cell receptors and cause disease in mice.

## Contribution

The study identifies a new ganglioside receptor and reveals how subtle amino acid changes in the virus's protein influence receptor binding and pathogenicity.

## Key findings

- A new infectious ganglioside receptor with an [α-2,8]-linked sialic acid was identified.
- Crystal structures show how different MuPyV strains bind to sialylated glycans with varying affinities.
- Amino acid changes in VP1 affect receptor affinity but not binding mode, influencing viral pathogenicity.

## Abstract

Murine polyomavirus (MuPyV) causes tumors of various origins in newborn mice and hamsters. Infection is initiated by attachment of the virus to ganglioside receptors at the cell surface. Single amino acid exchanges in the receptor-binding pocket of the major capsid protein VP1 are known to drastically alter tumorigenicity and spread in closely related MuPyV strains. The virus represents a rare example of differential receptor recognition directly influencing viral pathogenicity, although the factors underlying these differences remain unclear. We performed structural and functional analyses of three MuPyV strains with strikingly different pathogenicities: the low-tumorigenicity strain RA, the high-pathogenicity strain PTA, and the rapidly growing, lethal laboratory isolate strain LID. Using ganglioside deficient mouse embryo fibroblasts, we show that addition of specific gangliosides restores infectability for all strains, and we uncover a complex relationship between virus attachment and infection. We identify a new infectious ganglioside receptor that carries an additional linear [α-2,8]-linked sialic acid. Crystal structures of all three strains complexed with representative oligosaccharides from the three main pathways of ganglioside biosynthesis provide the molecular basis of receptor recognition. All strains bind to a range of sialylated glycans featuring the central [α-2,3]-linked sialic acid present in the established receptors GD1a and GT1b, but the presence of additional sialic acids modulates binding. An extra [α-2,8]-linked sialic acid engages a protein pocket that is conserved among the three strains, while another, [α-2,6]-linked branching sialic acid lies near the strain-defining amino acids but can be accommodated by all strains. By comparing electron density of the oligosaccharides within the binding pockets at various concentrations, we show that the [α-2,8]-linked sialic acid increases the strength of binding. Moreover, the amino acid exchanges have subtle effects on their affinity for the validated receptor GD1a. Our results indicate that both receptor specificity and affinity influence MuPyV pathogenesis.

Viruses are obligate intracellular pathogens, and all of them share one crucial step in their life cycle—the attachment to their host cell via cellular receptors, which are usually proteins or carbohydrates. This step is decisive for the selection of target cells and virus entry. In this study, we investigated murine polyomavirus (MuPyV), which attaches to host gangliosides with its major capsid protein, VP1. We have solved the crystal structures of VP1 in complex with previously known interaction partners as well as with the ganglioside GT1a, which we have identified as a novel functional receptor for MuPyV. Earlier studies have shown that different strains with singular amino acid exchanges in the receptor binding pocket of VP1 display altered pathogenicity and viral spread. Our investigations show that, while these exchanges do not abolish binding or significantly alter interaction modes to our investigated carbohydrates, they have subtle effects on glycan affinity. The combination of receptor specificity, abundance, and affinity reveals a much more intricate regulation of pathogenicity than previously believed. Our results exemplify how delicate changes to the receptor binding pocket of MuPyV VP1 are able to drastically alter virus behavior. This system provides a unique example to study how the first step in the life cycle of a virus can dictate its biological properties.