Engineering customized receptors to culture human norovirus

Robin Veenstra, Utrecht University

Robin Veenstra(1) and Daniel Hurdiss(1) (1) Section of Virology, Division of Infectious Disease and Immunology, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, 3584 CL Utrecht, The Netherlands

Entry and Replication

Human noroviruses are the most common cause of acute gastroenteritis worldwide with an estimated 685 million infections and 200,000 deaths annually. Despite advancements in culturing human noroviruses using enteroid models, the study of these viruses remains challenging due to a lack of reverse genetics system and a robust monolayer cell culture model. Furthermore, designing such a cell culture model is complicated by the lack of a known, proteinaceous receptor for human norovirus. However, breakthroughs in synthetic biology and AI-driven protein design have recently made it possible to design customized coronavirus receptors that enable viral entry without the need to express the native receptor. In this study, we aim to design such engineered customized receptors for human norovirus. Our receptors consist of four modular domains: a cytosolic domain, a transmembrane domain, a spacer domain and a viral binding domain. The cytosolic and transmembrane domain will be derived from known viral receptors, such as the murine norovirus receptor (murine CD300lf) or the SARS-COV-2 receptor (human ACE2). The spacer domain is generated from Ig or Ig-like domains or 23-aa tandem repeats from the CLEC4M protein. The viral binding domain is based on published, broad-spectrum nanobodies or created de novo using AI programs such as AlphaFold3, RFdiffusion and ProteinMPNN. The engineered receptors are expressed in 293T cells to verify permissiveness for human norovirus. Our research could pave the way for the development of a robust human norovirus cell culture model that can be used for antiviral drug screening and fundamental research.