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.
Meryl Haas, T. Hoeksma, J. Mills et al.· Nature Communications· 0 citations
Viral proteases represent validated targets for direct-acting antivirals and the treatment of associated infections. In co-crystal structures of Mpro of SARS-CoV-2 with peptidomimetic inhibitors, we noticed a spatial proximity of sidechains filling the S1’ and S2 pockets, as well as those filling S3 and S1 pockets. To enhance molecular rigidity, the proximal residues were conformationally fixed by macrocyclization. We report the synthesis of two macrocyclic series, i.e. exocyclic nitriles with linked P3 and P1 residues and endocyclic α-ketoamides with linked P1’ and P2 residues, and characterize their binding modes and bioactivities. The 17-membered macrocyclic α-ketoamide 20 f inhibited Mpro (IC₅₀ = 370 nM) and exerted anti-SARS-CoV-2 effects (EC₅₀ = 1.9 μM). Leveraging structural similarities between Mpro and the 3Cpro of enterovirus D68, we describe with two co-crystal structures how α-ketoamide macrocycles bound to and inhibited the enteroviral protease. Notably, 20 f exhibited very potent antiviral activities with EC₅₀‘s of 33, 133, and 146 nM against EV-D68, EV-A71, and CVB3, respectively. The study demonstrates how broad-spectrum activity can be achieved with direct-acting antivirals. Viral proteases are key targets for developing direct-acting antivirals to combat infections like SARS-CoV-2. Here, the authors synthesize macrocyclic α-ketoamide inhibitors, demonstrating potent inhibition of SARS-CoV-2 and enteroviruses, highlighting macrocyclization’s potential to enhance antiviral efficacy and achieve broad-spectrum activity.
R. Akula, Haifa El Kilani, Alina Metzen et al.· Communications Chemistry· 0 citations
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