Integrative structure of norovirus NS3 suggests a role in RNA transport

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.