The antinoroviral effect of copper ions is well known, yet most of this work has previously been conducted in copper and copper alloy surfaces, not copper ions in solution. In this work, we characterized the effects that Cu ions have on human norovirus capsids’ and surrogates’ integrity to explain empirical data, indicating virus inactivation by copper alloy surfaces, and as means of developing novel metal ion-based virucides. Comparatively high concentrations of Cu(II) ions (>10 mM) had little effect on the infectivity of human norovirus surrogates, so we used sodium ascorbate as a reducing agent to generate unstable Cu(I) ions from solutions of copper bromide. We found that significantly lower concentrations of monovalent copper ions (∼0.1 mM) compared to divalent copper ions cause capsid protein damage that prevents human norovirus capsids from binding to cell receptors in vitro and induce a greater than 4-log reduction in infectivity of Tulane virus, a human norovirus surrogate. Further, these Cu(I) solutions caused reduction of GII.4 norovirus from stool in suspension, producing about a 2-log reduction of virus as measured by a reverse transcriptase-quantitative polymerase chain reaction. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) data indicate substantial major capsid protein cleavage of both GI.7 and GII.4 norovirus capsids, and TEM images show the complete loss of capsid integrity of GI.7 norovirus. GII.4 virus-like particles (VLPs) were less susceptible to inactivation by copper ion treatments than GI.7 VLPs based upon receptor binding and SDS-PAGE analysis of viral capsids. The combined data demonstrate that stabilized Cu(I) ion solutions show promise as highly effective noroviral disinfectants in solution that can potentially be utilized at low concentrations for inactivation of human noroviruses. Published by American Chemical Society 2022 The Authors https://creativecommons.org/licenses/by-nc-nd/4.0/ In this work, we characterized the effects that Cu ions have on human norovirus capsids’ and surrogates’ integrity to explain empirical data, indicating virus inactivation by copper alloy surfaces, and as means of developing novel metal ion-based virucides. Comparatively high concentrations of Cu(II) ions (>10 mM) had little effect on the infectivity of human norovirus surrogates, so we used sodium ascorbate as a reducing agent to generate unstable Cu(I) ions from solutions of copper bromide. We found that significantly lower concentrations of monovalent copper ions (∼0.1 mM) compared to divalent copper ions cause capsid protein damage that prevents human norovirus capsids from binding to cell receptors in vitro and induce a greater than 4-log reduction in infectivity of Tulane virus, a human norovirus surrogate. Further, these Cu(I) solutions caused reduction of GII.4 norovirus from stool in suspension, producing about a 2-log reduction of virus as measured by a reverse transcriptase-quantitative polymerase chain reaction. 17 Many bacterial pathogens have developed resistance to the toxic effects of copper ions using ion-specific pumps to remove elevated ion levels, 18 , 19 but viruses do not have the ability to acquire this kind of defense mechanism. Copper iodide nanoparticles that release Cu(I) ions have demonstrated high efficacy against a number of human norovirus surrogates. 8 , 9 , 20 The biocidal effects of Cu(I) ions appear to be a result of the oxidative effects from copper’s redox activity. 21 Copper is a redox-active transition metal that can be present in its monovalent or divalent form in solution. Results and Discussion We used plaque assays of a human norovirus surrogate, Tulane virus (TV), in combination with HBGA binding assays of human norovirus VLPs to assess the effects of different copper ion formulations on human norovirus infectivity. We selected copper bromide as a source of divalent copper ions and added sodium ascorbate in at least 10-fold excess to reduce Cu(II) and create a redox active environment. For plaque assay experiments, we treated TV stocks with CuBr 2 solutions with concentrations ranging from 0.01 to 1 mM for 30 min to evaluate the effects of Cu(II) ions alone. Complete reduction in binding occurred after treatment with CuBr 2 at 10 5 -fold lower concentration in combination with ascorbate. At 100 mM Cu(II) + ascorbate, a higher VLP-HBGA binding was observed than with Cu(II) alone. We believe that this may be due to the fact that ascorbate is only in excess of Cu(II) ions below 1 mM CuBr 2 , and it is possible that the mixture of Cu(I) and Cu(II) ions actually had a counteractive effect compared to the predominantly one type of ion alone. Thus, we used only copper concentrations below 1 mM to evaluate the efficacy of CuBr 2 /ascorbate mixtures to reduce VLP-HBGA binding at shorter treatment times to ensure excess ascorbate. Further work investigating this possibility and the potential influence of mixtures of copper ions on antiviral efficacy should be conducted in the future. As seen in Figure 2 b, we observed very strong antiviral activity at low Cu ion concentrations. Only 1 min was required to damage capsid integrity with as low as 0.01 mM CuBr 2 combined with ascorbate. Even 0.001 mM CuBr 2 with ascorbate was found to eliminate VLP-HBGA binding in 15 min. Figure 2 Copper(I) solutions dramatically reduce the ability of norovirus capsids to bind HBGAs. In the presence of ascorbate as a reducing agent, copper ions cycle between the +2 and +1 oxidation state. Ascorbate oxidizes to dehydroascorbate as it reduces Cu(II) to Cu(I), and Cu(I) oxidizes back to Cu(II) by either dissolved oxygen and its reduction products or by reacting directly with the protein capsid. Cu(I) may reduce disulfide bonds within the major capsid protein and cause protein unfolding that inhibits VLP binding to HBGAs, which has been reported in binding assays to rely heavily on maintenance of a higher order capsid protein structure.

embrane transport of copper ion [ 14 ] STEAP Metalloreductase Reduction of divalent copper ions to monovalent copper ions [ 143 ] DCYTB Metalloreductase Promote the conversion of copper ions to the reduced state [ 144 ] ATOX1 Antioxidant 1 copper chaperone Transports cytoplasmic copper to organelles [ 145 ] ATP7A/B Copper‐transporting ATPases Regulation of copper ion transport [ 146 ] CP Cuproenzymes Regulates copper distribution [ 15 ] MT1/2 Metallothionein Chelated copper reduces it’s metal toxicity [ 17 ] GSH Copper chelator Chelated copper reduces it’s metal toxicity [ 23 ] COX17 Copper chaperone Regulates mitochondrial copper [ 22 ] CCO Cuproenzymes Regulates cellular biochemical reactions [ 31 ] CCS Copper chaperone Regulates transport of copper [ 34 ] SOD1 Cuproenzymes Antioxidant damage [ 145 ] Open in a new tab STEAP six-transmembrane epithelial antigen of the prostate, DCYTB duodenal cytochrome b, CP ceruloplasmin, GSH glutathione, COX17 cytochrome c oxidase copper chaperone, CCO cytochrome C oxidase, CCS Cu chaperone for superoxide dismutase, SOD1 superoxide dismutase 1. Importance of copper homeostasis at the cellular level Overall, copper homeostasis plays an important role at the cellular level. Extracellular Cu 2+ participates in the regulation of the physiological functions of various cytokines, Cu 2+ that arrives at the cell membrane is reduced to Cu + , which alters the structure of membrane proteins to change their active state. Cytoplasmic Cu + contributes to the maintenance of normal physiological functions of various organelles and avoids cytotoxicity produced by peroxidation [ 43 ]. The binding of Cu + to transcription factors in the nucleus can regulate gene expression and protein synthesis [ 44 ]. The maintenance of intracellular copper homeostasis is mainly dependent on the above copper chaperone proteins and copper transporter proteins, the imbalance of copper homeostasis will lead to cellular metabolic disorganization and even death [ 45