Photodynamic Inactivation of Human Coronaviruses

Abstract

Photodynamic inactivation (PDI) employs a photosensitizer, light, and oxygen to create a local burst of reactive oxygen species (ROS) that can inactivate microorganisms. The botanical extract PhytoQuin^TM is a powerful photosensitizer with antimicrobial properties. We previously demonstrated that photoactivated PhytoQuin also has antiviral properties against herpes simplex viruses and adenoviruses in a dose-dependent manner across a broad range of sub-cytotoxic concentrations. Here, we report that human coronaviruses (HCoVs) are also susceptible to photodynamic inactivation. Photoactivated-PhytoQuin inhibited the replication of the alphacoronavirus HCoV-229E and the betacoronavirus HCoV-OC43 in cultured cells across a range of sub-cytotoxic doses. This antiviral effect was light-dependent, as we observed minimal antiviral effect of PhytoQuin in the absence of photoactivation. Using RNase protection assays, we observed that PDI disrupted HCoV particle integrity allowing for the digestion of viral RNA by exogenous ribonucleases. Using lentiviruses pseudotyped with the SARS-CoV-2 Spike (S) protein, we once again observed a strong, light-dependent antiviral effect of PhytoQuin, which prevented S-mediated entry into human cells. We also observed that PhytoQuin PDI altered S protein electrophoretic mobility. The PhytoQuin constituent emodin displayed equivalent light-dependent antiviral activity to PhytoQuin in matched-dose experiments, indicating that it plays a central role in PhytoQuin PDI against CoVs. Together, these findings demonstrate that HCoV lipid envelopes and proteins are damaged by PhytoQuin PDI and expands the list of susceptible viruses.

Keywords: HCoV-229E; HCoV-OC43; PhytoQuin; ROS; SARS-CoV-2; antiviral; coronavirus; emodin; lentivirus; natural product; photodynamic inactivation; photosensitizer; spike.

Figure 1

Cell viability following DMSO and PhytoQuin treatment of Huh7.5 and BHK-21 cells. Huh7.5 cells (A) or BHK-21 cells (B) in low-serum infection media were exposed to increasing concentrations of DMSO (solvent, left panels) or PhytoQuin (right panels) with (49 J/cm², blue points; 98 J/cm², light blue points) or without (black points) LED light treatment. Cell viability was assayed at 48 h post-treatment using alamarBlue cell viability reagent. The vertical dotted lines indicate the highest concentrations of DMSO and PhytoQuin used in subsequent experiments. Data are plotted as averages ± SEM from three independent experiments. The 50% cytotoxic concentration (CC₅₀) values were calculated using Prism v9.2.0. Abbreviations: DMSO, dimethyl sulfoxide; LED, light-emitting diode; SEM, standard error of the mean.

Figure 2

PhytoQuin potently inactivates human coronaviruses 229E and OC43. (A) HCoV-229E was combined with 0.5% DMSO or PhytoQuin (0.5, 1, 2, or 4 µg/mL) prior to exposure to LED light in black tubes (“Dark”, black dots/lines) or clear tubes (“Light”, blue dots/lines) at 49 J/cm². The treated inocula were serially diluted and used to infect Huh7.5 cells and titered using TCID₅₀ assays. (B) HCoV-OC43 was combined with 0.5% DMSO or PhytoQuin (0.5, 1, 2, or 4 µg/mL) prior to exposure to light as indicated for panel A. The treated inocula were serially diluted and used to infect BHK-21 cells and titered using TCID₅₀ assays. The resulting TCID₅₀/mL values are plotted showing the individual data points with each bar graph (DMSO, grey; PhytoQuin, white) representing the average ± SEM from four or six independent experiments. The LOD for the TCID50 assays is indicated by the horizontal dotted line. * p = 0.01–0.05, ** p = 0.001–0.01, using unpaired t-tests comparing to DMSO/+Light. Abbreviations: DMSO, dimethyl sulfoxide; LED, light-emitting diode; LOD, limit of detection; SEM, standard error of the mean; TCID₅₀, median tissue culture infectious dose.

Figure 3

PhytoQuin treatment does not cause overt damage to HCoV-OC43 particles. HCoV-OC43 particles treated with 0.5% DMSO (+/−LED light) or 4 µg/mL PhytoQuin (+/−LED light) were fixed, negative stained with uranyl acetate, and imaged using a transmission electron microscope. Five to seven viral particles are shown per condition. Scale bar = 100 nm. Abbreviations: DMSO, dimethyl sulfoxide; LED, light-emitting diode.

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 (···). 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 µg/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/cm². 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 ± 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.

Figure 5

Light-activated PhytoQuin inhibits SARS-CoV-2 S-pseudotyped lentivirus entry and alters S protein electrophoretic mobility. (A) SARS-CoV-2 S (D614)-pseudotyped lentiviruses were combined with 0.5% DMSO or PhytoQuin (2 or 4 µg/mL) prior to exposure to LED light in black tubes (Dark, black dots/lines) or clear tubes (Light, blue dots/lines) at 49 J/cm². The treated lentiviruses were then used to transduce ACE2/TMPRSS2-expressing 293A cells, and luciferase transgene expression was measured 48 h post-transduction. The resulting relative luminescence units (RLU) are plotted showing the individual data points with each bar graph (DMSO, grey; PhytoQuin, white) representing the average ± SEM from four independent experiments. * p = 0.01–0.05, *** p = 0.0001–0.001 using unpaired t-tests comparing to DMSO/+Light. Two independent batches of SARS-CoV-2 S (D614)-pseudotyped lentiviruses (B) or SARS-CoV-2 S (Δfurin)-pseudotyped lentiviruses (C) were purified via ultracentrifugation and then combined with 0.5% DMSO or 4 µg/mL PhytoQuin prior to exposure to light in black tubes (Light: −/black font) or clear tubes (Light: +/blue font) at 49 J/cm². After treatment, lentiviral particles were solubilized in 2X Laemmli buffer and subjected to SDS-PAGE followed by immunoblotting to visualize SARS-CoV-2 S protein (α-S-RBD) and HIV-1 p24 (α-p24). Abbreviations: DMSO, dimethyl sulfoxide; kDa, kilodalton; LED, light-emitting diode; Phyto., PhytoQuin; RBD, receptor binding domain; Rep, replicate; SEM, standard error of the mean; S, spike.

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 µg/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/cm². The treated inocula were serially diluted and used to infect cells in 96-well plates to assay for infectivity using TCID₅₀ assays. The resulting TCID₅₀/mL values are plotted showing the individual data points with each bar graph (DMSO, grey; PhytoQuin, white) representing the average ± SEM from seven independent experiments. The LOD for the TCID₅₀ 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; TCID₅₀, median tissue culture infectious dose.