Integrative structure of norovirus NS3 suggests a role in RNA transport.
6 affiliations
- 1Section of Virology, Division of Infectious Diseases and Immunology, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
- 2Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK.
- 3Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research, Department of Chemistry, Faculty of Science, Utrecht University, Utrecht, The Netherlands.
- 4Department of Physics and Astronomy, and LaserLaB Amsterdam, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands.
- 5Materials and Structural Analysis, Thermo Fisher Scientific, Eindhoven, The Netherlands.
- 6Section of Virology, Division of Infectious Diseases and Immunology, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands. [email protected].
Abstract
Human noroviruses (HuNoVs) are the leading global cause of acute gastroenteritis, yet no vaccines or antiviral therapies are currently approved. The non-structural protein NS3 is a membrane-bound AAA+ ATPase of superfamily 3 (SF3) with multiple proposed roles in the norovirus replication cycle. However, the structure of NS3, and the mechanisms by which it contributes to genome replication and membrane remodeling, have remained unknown. We engineered a soluble, hexameric, and catalytically active form of NS3 and determined its cryo-EM structure in the presence of a nucleotide analogue at 2.9 Å resolution. The structure adopts a split lock-washer architecture characteristic of AAA+ motors that operate via a hand-over-hand translocation mechanism. Complementary biochemical, single-molecule, and virological assays support oligomerization-dependent ATPase activity, ssRNA engagement, and the functional importance of conserved structural elements. Using integrative modeling with AlphaFold3, supported by targeted mutagenesis, we generated a full-length, membrane-associated model in which NS3 forms a continuous conduit across the membrane. This model supports a role for NS3 as a candidate membrane-spanning RNA translocase that may couple ATP hydrolysis to genome movement. This structural and functional framework helps address long-standing gaps in our understanding of norovirus replication and establishes a basis for mechanistic studies and structure-guided antiviral design.
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BibTeX
@article{haas2026integrative,
title = {{Integrative structure of norovirus NS3 suggests a role in RNA transport.}},
author = {Meryl Haas and Tino Hoeksma and Jake T Mills and Cynthia Kelley and Arnab Das and Kankipati Teja Shyam and Robin Veenstra and Owen Byford and Henrietta Hóf and Coco Niemel and Tim Donselaar and Ieva Drulyte and Frank J M van Kuppeveld and David Dulin and Joost Snijder and Morgan R Herod and Daniel L Hurdiss},
journal = {Nature communications},
year = {2026},
month = sep,
doi = {10.1038/s41467-026-77946-7},
url = {https://europepmc.org/article/MED/42901927},
note = {CC BY},
}
RIS
TY - JOUR
TI - Integrative structure of norovirus NS3 suggests a role in RNA transport.
AU - Meryl Haas
AU - Tino Hoeksma
AU - Jake T Mills
AU - Cynthia Kelley
AU - Arnab Das
AU - Kankipati Teja Shyam
AU - Robin Veenstra
AU - Owen Byford
AU - Henrietta Hóf
AU - Coco Niemel
AU - Tim Donselaar
AU - Ieva Drulyte
AU - Frank J M van Kuppeveld
AU - David Dulin
AU - Joost Snijder
AU - Morgan R Herod
AU - Daniel L Hurdiss
PY - 2026
DA - 2026/09/21
JO - Nature communications
DO - 10.1038/s41467-026-77946-7
UR - https://europepmc.org/article/MED/42901927
AB - Human noroviruses (HuNoVs) are the leading global cause of acute gastroenteritis, yet no vaccines or antiviral therapies are currently approved. The non-structural protein NS3 is a membrane-bound AAA+ ATPase of superfamily 3 (SF3) with multiple proposed roles in the norovirus replication cycle. However, the structure of NS3, and the mechanisms by which it contributes to genome replication and membrane remodeling, have remained unknown. We engineered a soluble, hexameric, and catalytically active form of NS3 and determined its cryo-EM structure in the presence of a nucleotide analogue at 2.9 Å resolution. The structure adopts a split lock-washer architecture characteristic of AAA+ motors that operate via a hand-over-hand translocation mechanism. Complementary biochemical, single-molecule, and virological assays support oligomerization-dependent ATPase activity, ssRNA engagement, and the functional importance of conserved structural elements. Using integrative modeling with AlphaFold3, supported by targeted mutagenesis, we generated a full-length, membrane-associated model in which NS3 forms a continuous conduit across the membrane. This model supports a role for NS3 as a candidate membrane-spanning RNA translocase that may couple ATP hydrolysis to genome movement. This structural and functional framework helps address long-standing gaps in our understanding of norovirus replication and establishes a basis for mechanistic studies and structure-guided antiviral design.
SN - 2041-1723
ER -