# Photodynamic Inactivation of Human Coronaviruses
## 3 Results
### PhytoQuin PDI of HCoV Virions Damages the Viral Lipid Envelope
The remaining RNA was combined with _Luc_ RNA and was extracted, reverse transcribed, and quantitated using qPCR to measure the viral sequences, _ORF1_ (red) and \(N\) (yellow), and the control _GFP_ (green) or _Luc_ (grey) cDNAs. The averaged amplification curves from one representative experiment performed in triplicate from three independent replicates are shown with the difference in Cq values between untreated and Triton + RNase treatment indicated above the curves. (**B**) HCOV-229E virions were combined with 0.5% DMSO or 4 ug/mL PhytoQuin 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 viral particles were then left untreated (–), treated with RNase A/T1 (+R), or treated with both Triton X-100 and RNase A/T1 (+T/R) prior to RNA extraction, reverse transcription, and qPCR analysis. The fold-changes in cDNA levels were plotted relative to the DMSO/Dark-treated samples showing the individual data points from three independent experiments with each bar graph (DMSO, grey; PhytoQuin, white) representing averages \(\pm\) SEM. * \(p\) = 0.01–0.05, ** \(p\) = 0.001–0.01, *** \(p\) = 0.001–0.0001 using unpaired _t_-tests compared to the respective DMSO control. Abbreviations: cDNA, complementary DNA; Cq, quantification cycle; DMSO, dimethyl sulfoxide; _GFP_, green fluorescent protein; LED, light-emitting diode; _Luc_, luciferase; N, nucleocapsid; ORF1, open-reading frame; Phyto., PhytoQuin; SEM, standard error of the mean; T, Triton X-100; R/RNase, ribonuclease; vRNA, viral RNA. PhytoQuin PDI Prevents Infection of ACE2/TMPRSS2-Expressing Cells by SARS-CoV-2 S-Pseudotyped Lentiviruses by Altering S Glycoprotein  
Having established that PhytoQuin PDI damages the viral lipid envelope, we next sought to determine whether viral glycoproteins could also be affected by PDI. As we lacked antibodies to visualize HCoV-229E or OC43 Spike (S), we created lentivirusises pseudotyped with SARS-CoV-2 S for which Western blotting reagents were readily available. These lentivirusises also carried a firefly luciferase gene to measure infection. Lentiviruses were incubated with 2-4 \(\mu\)g/mL of PhytoQuin (or equivalent DMSO) in the presence or absence of LED light at 49 J/cm\({}^{2}\). Following treatment, the lentivirusises were used to infect human HEK 293A cells co-expressing angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2), which act as a key receptor and accessory factor, respectively, for SARS-CoV-2 S-mediated entry [25; 26; 27]. Light treatment in the presence of DMSO led to <1.5-fold decrease in luciferase expression, demonstrating that lentivirusises also have a small degree of light sensitivity (Figure 5A), similar to HCoVs (Figures 2A and 3B). Importantly, we observed that PhytoQuin PDI strongly inhibited luciferase gene expression in a light-dependent and dose-dependent manner (Figure 5A). This suggests that either PDI interferes with S interactions with the cell surface or that the lentiviral lipid envelope is disrupted in such a way as to prevent proper delivery of the luciferase reporter gene to the cell nucleus. Regardless, these data provide further evidence for the antiviral potency of PhytoQuin PDI and support our use of this model to study the effects of PhytoQuin on viral glycoproteins. Since viral glycoproteins stud the surface of enveloped viruses like HCoVs, they are particularly susceptible to damage initiated from the extracellular environment. This would include any oxidative damage to viral glycoproteins caused by PhytoQuin PDI. We used our pseudotyped lentivirusises to investigate any changes in protein integrity potentially caused by PhytoQuin using Western blotting. Using wild-type, S (D614)-pseudotyped lentivirusises, we demonstrated that treating samples with PhytoQuin and light caused alterations in S protein electrophoretic mobility, with the emergence of slow-migrating forms of S with abnormally high predicted molecular weights (Figure 5B).