Targeting Picornavirus Nonstructural 2C Protein: Structure, Function, and Antiviral Discovery

1. A schematic representation of the viral particle of picornavirus, and the genome organization of the virion with proteolytic processing of the polyprotein, using EV-A71 as a representative. (A) Viral particle structure of EV-A71 (PDB ID: 4AED). (B) The diagram of the genome organization and the polyprotein cleavage of EV-A71 during replication.

2. Structures of monomeric 2C proteins and modeled hexameric assemblies. (A) Diagram of major functional motifs in 2C. The protein contains an N-terminal membrane-binding region (AH domain), a conserved superfamily 3 (SF3) helicase-like AAA+ ATPase core, a zinc-binding domain in selected picornaviruses, and a C-terminal helical domain (α6) that includes the RNA-binding segment and PBD. (B) Alignment of reported picornaviral 2C crystal structures. (C) Overview of 2C hexameric models generated based on the JC polyomavirus large T antigen structure (PDB ID: 5J4Y), colored by chain. ATP-γ-S is shown as cyan sticks, and ATP is shown as blue sticks.

3. Schematic overview of the picornaviral life cycle and the roles of 2C in replication and morphogenesis. After receptor binding and entry, the viral RNA genome is released into the cytoplasm, where translation is initiated through the IRES in the 5′UTR, and the viral polyprotein is synthesized and processed. During the middle stage of infection, 2C contributes to oligomer formation, replication-organelle biogenesis, membrane remodeling, and RNA replication. During the late stage, 2C also participates in encapsidation and virion morphogenesis through functional interactions with structural proteins. In parallel, 2C engages host factors that promote replication (not all interactions shown). Figure created with BioRender.

4. Identification of 2C inhibitors through lead optimization. (A) HBB analogs and other benzimidazole derivatives as 2C inhibitors. (B) SAR study of 2C-Compound 1 and its derivatives as 2C inhibitors. (C) Optimization of pyrazolopyridine derivatives as 2C inhibitors. (D) SAR study of dibucaine and its derivatives as 2C inhibitors.

5. Identification of 2C inhibitors through virtual screening. (A) Comparison of the chemical structures of adenosine triphosphate (ATP) and screening hits, the pharmacophore model of the selected analogs of the hits, and the structures of the lead candidates as 2C inhibitors. (B) Discovery scheme of “Y”-shaped virtual screening hits as 2C inhibitors.

6. Identification of 2C inhibitors through rational peptide design and summary of reported binding pocket and resistant residues. (A) Design of an EV-A71 2C-targeting peptide (PDB ID: 5GRB). The amino acid sequence of the C-terminal domain, 2CL, and B-2CL. Chemical structural formulas of the three peptidomimetics, 2CA-1, 2CA-2, 2CA-3 obtained after further optimization. (B) The pockets of reported 2C inhibitors shown on EV-A71 2C (PDB ID: 5GRB) and CV-B3 2C (PDB ID: 6S3A). (C) Resistance mapping of the 2C proteins of CVB3, EV-A71, and EV-D68 with representative residues shown mutation.

7. Assays for evaluating inhibitors targeting the Picornavirus 2C protein. (A) Biophysical assays (DSF, ITC) provide direct evidence of ligand–2C binding. (B) 2C ATPase assays measure phosphate release using engineered hexameric 2C constructs. (C) 2C FP assays enable direct, quantitative measurement of binding. Figure created with BioRender.