Strain-Specific Functional Diversity in GII.4 Norovirus Entry: HBGA-Dependent Capsid Clustering and Membrane Remodeling

B.Vijayalakshmi Ayyar, Baylor College of Medicine

B. Vijayalakshmi Ayyar1, Carmen V. Apostol2, Janam Dave1, Soni Kaundal2, Frederick H. Neill1, Khalil Ettayebi1, Sarah Maher1, Ramakrishnan Anish2, Göran Larson3, Robert L. Atmar1,4, Sue E. Crawford1, B. V. Venkataram Prasad2, Mary K. Estes1,4* 1Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA, 2Verna and Marrs McLean Department of Biochemistry and Molecular Pharmacology, 3 The Glycobiology Group, Department of Laboratory Medicine, Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden, and 4Department of Medicine, Baylor College of Medicine, Houston, TX, USA

Entry and Replication

Human noroviruses (HuNoVs), especially GII.4 strains, are the leading cause of acute viral gastroenteritis globally, yet no vaccines or antivirals exist. Although the pandemic GII.4 Sydney 2012 (GII.4 Syd) hijacks a clathrin-independent carrier (CLIC) pathway, initiated by membrane wounding, it is unclear whether other GII.4 variants employ the same entry route. To address this, we systematically compared the early attachment and entry of multiple GII.4 variants using wild-type and mutant virus-like particles (VLPs) and modified human intestinal enteroid (HIE) cultures. Only a subset of GII.4 variant VLPs, including GII.4 Syd, form distinct, HBGA-dependent capsid clusters on the cell surface. These clustering strains display significantly enhanced membrane wounding and endocytosis compared to non-clustering strains and outcompete them in replication assays. Mutagenesis of the VP1 protruding domain (including a HBGA–binding–deficient mutant R345A), identified residues V333 and R339 as essential for clustering and endocytosis. Mutating either of these residues abolishes clustering and uptake without affecting HBGA binding, indicating an important role of these residues in mediating a post-attachment reorganization of the virion–cell interface. Although HBGA engagement is required to trigger clustering and entry, inhibitor studies reveal these processes proceed independently of host protein glycosylation and instead depend on cholesterol- and ceramide-driven lipid raft remodeling. Quantitative analyses across multiple GII.4 variants reveal a dichotomy between clustering and non-clustering phenotypes, with the former exhibiting higher entry competence. This mechanistic distinction offers insight into strain-specific cell entry mechanisms and may aid in identifying the elusive proteinaceous HuNoV cellular receptor(s) supporting targeted therapeutic development.