Novel fold of rotavirus glycan-binding domain predicted by AlphaFold2 and determined by X-ray crystallography

Abstract

The VP8* domain of spike protein VP4 in group A and C rotaviruses, which cause epidemic gastroenteritis in children, exhibits a conserved galectin-like fold for recognizing glycans during cell entry. In group B rotavirus, which causes significant diarrheal outbreaks in adults, the VP8* domain (VP8*B) surprisingly lacks sequence similarity with VP8* of group A or group C rotavirus. Here, by using the recently developed AlphaFold2 for ab initio structure prediction and validating the predicted model by determining a 1.3-Å crystal structure, we show that VP8*B exhibits a novel fold distinct from the galectin fold. This fold with a β-sheet clasping an α-helix represents a new fold for glycan recognition based on glycan array screening, which shows that VP8*B recognizes glycans containing N-acetyllactosamine moiety. Although uncommon, our study illustrates how evolution can incorporate structurally distinct folds with similar functionality in a homologous protein within the same virus genus.

Introduction

Rotaviruses are non-enveloped icosahedral double-stranded RNA (dsRNA) viruses belonging to the Reoviridae family¹. These viruses exhibit enormous genetic and serological diversity. Based on the sequence and antigenic differences of the capsid protein VP6, they are classified into ten different species or groups (A–J)^2,3. Group A, B, C, and H rotaviruses infect both humans and animals^4,5. Epidemiologically, groups A, B, and C are the best characterized. While group A rotaviruses (RVA) and to a lesser extent group C rotaviruses (RVC) are the causative agents of most gastroenteric infections worldwide, the group B rotaviruses (RVB) have been associated with large epidemic outbreaks of severe gastroenteritis in China^6,7 and sporadic infection in several countries^8,9,10. Unlike RVA, which infects mainly young children, RVB causes cholera-like severe diarrhea predominantly in adults, although children can be infected as well⁸. Antibodies to the RVB have been detected in people in developed countries such as the USA, Canada, and the UK^11,12, indicating a broader prevalence of RVB. RVB also infects animals and caused recent hemorrhagic diarrheal outbreaks in piglets and foals, resulting in significant economic impact and posing a threat of potential zoonotic transmission^13,14. Considering that these rotaviruses have segmented dsRNA genomes, with a propensity for evolving by gene reassortment from co-infections and mutations, the potential for the emergence of new variants that can cause severe epidemics cannot be discounted. A case in point is the current ongoing global COVID-19 pandemic caused by SARS-CoV-2¹⁵.

Like RVA, which remains the best characterized thus far, and RVC, the genome of RVB consists of 11 dsRNA segments that encode 11 proteins¹⁶. Cryo-EM reconstructions of RVA have shown that trimers of VP4 form 60 protruding spikes attached to the outer VP7 and middle VP6 layers of the triple-layered capsid^17,18,19. The proteolytic treatment of VP4, which significantly enhances infectivity, results in two fragments, VP8* and VP5*, that remain associated with the virion. Sequence comparison of the structural proteins encoded by different rotavirus groups shows that the VP8* domain of the spike protein VP4 is the most variable²⁰. Extensive structural studies have shown that the galectin-like VP8* of human RVA and RVC (VP8*A and VP8*C) recognize various cellular glycans in a genotype-dependent manner (Fig. 1a, b)^{21,22,23,24,25,26,27,28,29}. The VP8* of human RVA exhibits genotype-dependent glycan specificity by recognizing different histo-blood group antigens (HBGA)³⁰. Differential recognition of HBGA has provided a possible rationale for why some RVAs specifically infect neonates, and some cause sporadic outbreaks while others infect a wider population^22,23,24,31. Unlike the VP8*A and VP8*C, the structure of VP8*B and its glycan specificity have not been characterized. The VP8*B shares no sequence identity with either VP8*A or VP8*C (Supplementary Fig. 1a and Supplementary Table 1), which could differentially impact not only the structure but also the glycan-binding properties. Here we show by determining the crystal structure of VP8*B, using a model predicted using the recently developed AlphaFold2, that the VP8*B exhibits a fold with a twisted β-sheet clasping an α-helix that is entirely different from VP8*A or VP8*C. Our glycan array screening and in silico docking analysis show VP8*B recognizes glycans containing N-acetyllactosamine exemplifying how viruses can evolve by incorporating structurally distinct modules with similar functionality….