# Mutational analysis of human norovirus VP2 elucidates critical molecular interactions for virus assembly

## TL;DR

This study identifies how the VP2 protein in human noroviruses interacts with other proteins to assemble the virus, offering insights into its structure and function.

## Contribution

The study reveals VP2 as a molecular bridge for capsid assembly and identifies specific interaction sites with VP1 and VPg.

## Key findings

- VP2 is essential for interactions with VP1 and VPg during capsid assembly.
- AAs 40–43 on VP2 are critical for binding to VP1.
- The C-terminal domain of VP2 is sufficient to interact with VPg.

## Abstract

Human noroviruses (HuNoVs) are the leading cause of viral gastroenteritis, with ≥80% of infections caused by the GII genogroup. HuNoVs are non-enveloped, with an icosahedral capsid composed of 90 dimers of the major capsid protein VP1, which encloses a minor structural protein, VP2, and a VPg-linked, positive-sense ssRNA genome. Although the atomic structure of the icosahedral capsid formed by VP1 is well characterized using crystallography and cryo-electron microscopy analyses of HuNoV virus-like particles, the structures and the localization of VP2 and VPg inside the capsid, how they are incorporated into the capsid, and whether this process requires interactions between them remain unresolved. Herein, we show that VP2 is the molecular bridge for assembly of particles containing VP1, VP2, and VPg. We used deletion constructs and mutational analyses in transfected HEK293FT cells, guided by bioinformatics, to determine the interaction site on VP2 for VP1 of the pandemic-causing GII.4 Sydney HuNoV. GII.4 HuNoV VP2 contains a unique insertion site at amino acids (AAs) 43–53, relative to VP2s of other GII HuNoV genotypes. We identified that AAs 40–43 on VP2 are required for interaction with VP1, and mutation of VP2 AAs 40–43 abrogates VP2-VP1 interactions. Computational analyses predicted that VP2 has a highly conserved N-terminal α-helical domain and an intrinsically disordered C-terminal domain that exhibits significant sequence diversity. We identified that VP2, not VP1, pulls down VPg; the VP2 C-terminal domain is sufficient to interact with VPg. These findings reveal domain-specific functions of VP2 that are essential for coordinating capsid protein interactions for HuNoV assembly.

Human noroviruses (HuNoVs) are the leading cause of epidemic and sporadic gastroenteritis in all age groups worldwide. Yet, we lack vaccines or therapeutics for HuNoVs. Knowledge of the fundamental mechanisms governing HuNoV particle assembly is limited. Modern structural techniques have not resolved the complete structure of pandemic GII.4 norovirus or the localization of interior capsid proteins VP2 and VPg. Furthermore, VP2’s functional roles during infection remain obscure. Studies of feline and murine caliciviruses show that VP2 may help deliver the viral genome into cells, suggesting that VP2 synergizes with VP1 and VPg. We identified a motif within the N-terminal α-helical domain of VP2, adjacent to a unique insertion site, that is essential for interaction with the major capsid protein VP1. We show VP2 uniquely interacts with the translation initiation protein VPg via its disordered C-terminus. These findings reveal principles of HuNoV capsid protein interactions and highlight VP2 as a bridge facilitating capsid assembly.