GII.23/24/25 noroviruses recognize glycans via a conventional glycan-binding site

Hanbo Li^1,2Xin Cong^2,3*Xiaoman Sun²Jianxun Qi⁴Xinyu Li⁵Miao Jin²Zhaojun Duan^2*

• ¹South China Agricultural University College of Animal Science, Guangzhou, China
• ²Chinese Center For Disease Control and Prevention, Beijing, China
• ³Zhejiang University School of Medicine Children's Hospital National Clinical Research Center for Child Health, Hangzhou, China
• ⁴Institute of Microbiology Chinese Academy of Sciences, Beijing, China
• ⁵Chengde Medical University, Chengde, China

Human noroviruses (HuNoVs) are genetically diverse RNA viruses that cause acute gastroenteritis, with genogroup II (GII) accounting for over 90% of global infections. Glycans, particularly histo-blood group antigens (HBGAs), have been identified as attachment factors or receptors for HuNoVs infection. However, the glycan-binding receptors of the later-identified GII genotypes GII.23/24/25 remain elusive. Here, we identified the binding spectra of GII.23/24/25 strains through saliva-and glycan-based ELISA assays and determined their crystal structures. We found that the P domains of GII.24 and GII.25 can recognize multiple types of saliva samples, including both A/B/O secretor and non-secretor individuals. In contrast, GII.23 primarily binds to B secretor saliva samples. Furthermore, GII.23/24 P domains are able to interact with the H disaccharide, whereas GII.25 exhibits binding affinity for both H disaccharide and B triaccharide. Crystal structures of GII.23/24/25 P domains revealed high structural similarity, and the complex of GII.25 P domains with H disaccharide was resolved. Single-point mutagenesis identified N352, R353, D382, G443, G444, and H445 as critical residues for H disaccharide binding in GII.25 P domain, while A351 determines glycan-binding specificity. These findings demonstrate that GII.23/24/25 exhibit glycan-binding patterns similar to most GII HuNoV genotypes. Our results provide structural insights into virus-host evolution and inform therapeutic strategies against HuNoVs.