# Photodynamic Inactivation of Human Coronaviruses
## 4 Discussion  
The COVID-19 pandemic has provided new motivation to discover and develop antivirals against coronaviruses. We demonstrated that PhytoQuin, a photosensitizing botanical extract, inactivated distantly related human coronaviruses HCoV-229E and HCoV-OC43, as well as pseudotyped lentiviruses bearing SARS-CoV-2 S protein. PhytoQuin acts in a light-dependent manner to damage nearby macromolecules, which is consistent with a ROS-mediated mechanism of action as observed for other photosensizers. Our study  
Figure 6: Light-activated emodin is sufficient to inactivate HCoV-OC43. (**A**) HPLC trace identifying the constituents of the current lot of PhytoQuin. AU, absorbance units. (**B**) HCoV-OC43 was combined with 0.5% DMSO, 4 ug/mL PhytoQuin, or 117 ng/mL emodin prior to exposure to LED light in black tubes (“Dark”, black dots/lines) or clear tubes (“Light”, blue dots/lines) at 49 J/ cm\({}^{2}\). The treated inocula were serially diluted and used to infect cells in 96-well plates to assay for infectivity using TCID\({}_{50}\) assays. The resulting TCID\({}_{50}\)/ mL values are plotted showing the individual data points with each bar graph (DMSO, grey; PhytoQuin, white) representing the average \(\pm\) SEM from seven independent experiments. The LOD for the TCID\({}_{50}\) assays is indicated by the horizontal dotted line. * \(p\) = 0.01–0.05, ** \(p\) = 0.001–0.01 using unpaired \(t\)-tests with the indicated comparisons. Abbreviations: AU, absorbance units; DMSO, dimethyl sulfoxide; LED, light-emitting diode; LOD, limit of detection; SEM, standard error of the mean; TCID\({}_{50}\), median tissue culture infectious dose. provides strong support for the addition of PhytoQuin and the PhytoQuin constituent emodin to the growing list of PSs with antiviral activity against CoVs [30; 31; 32; 33; 34; 35; 36; 37]. We observed that while PhytoQuin PDI did not cause gross structural changes in viral particles (Figure 3), it did disrupt the viral lipid envelope to expose the viral genome to ribonucleases (Figure 4) and altered the electrophoretic mobility of S proteins (Figure 5). These changes strongly correlated with diminished infectivity of CoVs and S-pseudotyped lentiviruses following PhytoQuin PDI (Figures 2 and 5A). Unlike within the cell where quenching by antioxidants can occur, free radicals generated in the extracellular milieu have unfettered access to damage viral structural proteins and inactivate viruses. Certain surface-exposed amino acid side chains are sensitive to oxidative damage, which can result in peptide bond cleavage, amino acid modifications, and intra- and inter-protein crosslinking [38]. Our observation of reduced electrophoretic mobility of SARS-CoV-2 S from purified lentiviruses following exposure to photoactivated PhytoQuin is suggestive of aggregation or cross-linking of these viral glycoproteins (Figure 5B,C). CoV S proteins contain numerous cysteine residues in their ectodomains, where the majority form disulfide bonds to support protein structure and function [39; 40; 41; 42; 43; 44]. Cysteines of S proteins may be oxidized via photoactivation to form inter-protein linkages similar to heterodimeric protein crosslinking observed in vitro with the PS rose bengal [45]. Furthermore, multi-step oxidation reactions particularly of Tyr, Trp, Met, and His residues can generate reactive peroxyl or carbonyl derivatives that react with nucleophilic protein side chains to crosslink proteins [46]. Spurious crosslinking of proximal S proteins may reduce protein flexibility and interfere with conformational changes required for receptor interactions and membrane fusion [47]. The appearance of new S protein species with reduced electrophoretic mobility under reducing conditions suggests that changes in S due to PhytoQuin treatment are likely linked to irreversible protein cross-linking.