Mutational Analysis of Human Norovirus VP2 Elucidates Critical Molecular Interactions for Virus Assembly.

UNLABELLED

Human noroviruses ( s) 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 (VLPs), 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 VP2 is the molecular bridge for assembly of particles containing VP1, VP2 and VPg. We used deletion constructs and mutational analyses, guided by bioinformatic analyses, 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 AA residues 40-43 on VP2 are required for interaction with VP1; mutation of VP2 AA 40-43 abrogates VP2 encapsidation. Computational analyses predicted VP2 has a highly conserved N-terminal α-helical domain and an intrinsically disordered C-terminal domain that exhibits significant sequence diversity. We identified VP2, not VP1, uniquely binds 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.

IMPORTANCE

Human noroviruses (HuNoVs) are the leading cause of epidemic and sporadic gastroenteritis in all age groups worldwide. Yet, we currently lack vaccines or therapeutics for these pathogens. Knowledge about HuNoV biology is limited, including the fundamental mechanisms governing particle assembly. Modern structural techniques have not resolved the complete structure of pandemic GII.4 norovirus that includes the localization of the interior capsid proteins VP2 and VPg. Furthermore, VP2's functional role(s) during infection remains obscure. Studies of feline and murine caliciviruses show VP2 may be involved in delivering the viral genome into cells, suggesting it synergizes with VP1 and VPg. We identify a motif on 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 binds 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.