# Photodynamic Inactivation of Human Coronaviruses
## 3 Results
### PhytoQuin PDI of HCoV Virions Damages the Viral Lipid Envelope  
Even though PhytoQuin PDI did not cause gross morphological changes in HCoV particles, we reasoned that PDI could nevertheless breach the lipid envelope and render the genome susceptible to degradation by ribonucleases (RNases). We developed an RNase protection assay for measuring vRNA levels in virions to monitor for PDI-mediated disruption of the CoV lipid envelope. To establish the parameters for the assay, HCoV-229E virions in infected cell supernatants were incubated in the presence or absence of RNase at 37 \({}^{\circ}\)C for 20 min or incubated with RNase in the presence of Triton X-100, a non-ionic detergent that disrupts lipid bilayers like the viral envelope. After a 20 min incubation, RNase activity was inhibited using TRIzol, and viral RNA was isolated and reverse transcribed. As a positive control, a synthetic _GFP_ RNA was included prior to the RNase treatments as an unprotected RNA species susceptible to RNase activity. A synthetic _Luc_ RNA was added post-TRIzol extraction to confirm RNase inhibition by monitoring for undesired RNA degradation following TRIzol disassociation of the virions/viral ribonucleoprotein (vRNP) complexes. qPCR was performed using oligonucleotide primers that amplify portions of the HCoV-229E \(N\), HCoV-229E _ORF1_, _GFP_, or _Luc_ genes. Our pilot experiments validated that vRNA was protected from exogenous RNase activity by the viral lipid envelope and was only susceptible to RNase activity after the viral lipid envelope was disrupted (Figure 4A); as indicated by the increase in Cq values after Triton X-100 addition. _GFP_ RNA was immediately susceptible to degradation by RNase whereas little effect was observed on _Luc_ RNA which was added after RNase inhibition (Figure 4A). The higher abundance (earlier amplification) of HCoV-229E \(N\) relative to _ORF1_ may be due to the presence of viral subgenomic RNA (sgRNA) in exosomes, as this has also been observed in SARS-CoV-2 qPCR experiments [24]. This assay was subsequently used to determine whether damage induced by PhytoQuin PDI breaches the HCoV lipid envelope. HCoV-229E stocks were incubated with 4 ug/mL of PhytoQuin in the presence of LED light at 49 J/cm\({}^{2}\), which we previously demonstrated was sufficient to inactivate HCoV-229E (Figure 2A). In parallel, virion preparations were treated with PhytoQuin in the dark or treated with DMSO vehicle control with and without light exposure. All samples were then treated with RNase +/ - Triton X-100 for 20 min at 37 \({}^{\circ}\)C or left untreated. This analysis revealed that PhytoQuin treatment alone, in the absence of light treatment, did not render the viral genome more susceptible to RNase digestion (Figure 4B). However, when light treatment was coupled with PhytoQuin treatment, significant reductions in \(N\) and _ORF1_ RNA copies were observed (Figure 4B). Strikingly, the loss of \(N\) or _ORF1_ RNA occurred irrespective of the addition of exogenous RNase (Figure 4B), suggesting that either there was a low level of RNase contamination in the PhytoQuin extract or that PhytoQuin, or the ROS generated from it, can directly damage the vRNA. As we observed an equivalent decrease in _GFP_ levels following PhytoQuin addition with or without photoactivation (Figure 4B), this suggests that some component of the extract can damage RNA directly in a light-independent manner. Overall, it is clear from these data that PhytoQuin PDI damages enveloped particles in a manner that renders HCoV RNA susceptible to degradation. Figure 4: Light-activated PhytoQuin damages HCOV-229E lipid envelopes and renders vRNA susceptible to degradation by RNases. (**A**) Development of an RNase protection assay to quantitate vRNA levels in HCOV-229E inocula. Virions in serum-free medium were combined with in vitro transcribed _GFP_ RNA and left untreated (—), treated with RNase A/T1 (- - -), or lysed with Triton X-100 prior to RNase A/T1 treatment (- -).