Structure of the Norovirus NTPase

Daniel Hurdiss, Utrecht University

Meryl Haas (1), Jake Mills (2), Cynthia Kelley (3), Henrietta Hóf (1), Coco Niemel (1), Tim Donselaar (1), Ieva Drulyte (5), Frank J.M. van Kuppeveld (1), Joost Snijder (3), Morgan Herod (2), and Daniel L. 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. 2) Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom. 3) Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research, Department of Chemistry, Faculty of Science, Utrecht University, 3584 CH Utrecht, The Netherlands. 4) Institute for Life Sciences and Chemistry, Utrecht University of Applied Sciences, The Netherlands. 5) Materials and Structural Analysis, Thermo Fisher Scientific, 5651 GG Eindhoven, The Netherlands.

Structure & Pathogenesis

Human Noroviruses (huNoVs) are the leading cause of non-bacterial gastroenteritis, responsible for an estimated 700 million infections and 200,000 deaths annually. Currently, no vaccines or small-molecule drugs are available. Here, we investigated the viral non-structural protein NS3, also known as NTPase. NS3 is a membrane-bound AAA+ protein, belonging to the helicase superfamily III (SF3), with several proposed functions in the norovirus lifecycle. We rationally engineered a soluble, hexameric, and enzymatically active NS3 protein and determined a 2.8 Å resolution cryo-EM structure of the nucleotide analogue-bound complex. This revealed a split lock-washer conformation consistent with AAA+ proteins that employ a coordinated hand-over-hand substrate translocation mechanism. We resolved the nucleotide-binding site at 2.2 Å resolution, revealing the molecular basis for nucleotide binding and hydrolysis. Collectively, our structure provides the first view of a hexameric SF3 ATPase from an RNA virus and serves as a roadmap for the development of anti-noroviral therapies.