Photodynamic Inactivation of Herpes Simplex Viruses

Abstract

Herpes simplex virus (HSV) infections can be treated with direct acting antivirals like acyclovir and foscarnet, but long-term use can lead to drug resistance, which motivates research into broadly-acting antivirals that can provide a greater genetic barrier to resistance. Photodynamic inactivation (PDI) employs a photosensitizer, light, and oxygen to create a local burst of reactive oxygen species that inactivate microorganisms. The botanical plant extract Orthoquin^TM is a powerful photosensitizer with antimicrobial properties. Here we report that Orthoquin also has antiviral properties. Photoactivated Orthoquin inhibited herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) infection of target cells in a dose-dependent manner across a broad range of sub-cytotoxic concentrations. HSV inactivation required direct contact between Orthoquin and the inoculum, whereas pre-treatment of target cells had no effect. Orthoquin did not cause appreciable damage to viral capsids or premature release of viral genomes, as measured by qPCR for the HSV-1 genome. By contrast, immunoblotting for HSV-1 antigens in purified virion preparations suggested that higher doses of Orthoquin had a physical impact on certain HSV-1 proteins that altered protein mobility or antigen detection. Orthoquin PDI also inhibited the non-enveloped adenovirus (AdV) in a dose-dependent manner, whereas Orthoquin-mediated inhibition of the enveloped vesicular stomatitis virus (VSV) was light-independent. Together, these findings suggest that the broad antiviral effects of Orthoquin-mediated PDI may stem from damage to viral attachment proteins.

Keywords: HSV-1; HSV-2; antiviral; natural product; photodynamic inactivation; plaque assay.

Figure 1

Overview of the preparation and characterization steps for producing Orthoquin^TM.

Figure 2

Establishment of phototoxic threshold of Orthoquin on HeLa (A) and hTert-immortalized human BJ cells (B). Cells were seeded in a 96-well plate and treated the following day with Orthoquin or DMSO vehicle control. At 16 h post-treatment, plates were exposed to a visible LED light for 30 min, with the dark plate wrapped in aluminum foil. alamarBlue^® cell viability assay was performed 48 h post-treatment. n = 3 ± SEM independent biological replicates. CC₅₀ values, denoted with a vertical line, were calculated using Prism 7.

Figure 3

Differential sensitivity of HSV-1 and HSV-2 to white light. Viral inoculum was diluted and exposed to a 65 W LED light in clear (light) or black (dark) tubes for the indicated times, and used to infect HeLa cell monolayers as described in the methods. (A) Representative image of HSV-1 plaque assay, quantified in (B). (C) Representative HSV-2 plaque assay, quantified in (D). Values in (B,D) are normalized the number of PFUs detected after 5 min of light exposure. n = 3 ± SEM from three independent biological replicates. * = p < 0.05 by two-way ANOVA.

Figure 4

Light-dependent reduction of HSV-1 and HSV-2 plaque formation with Orthoquin treatment. HSV-1 and HSV-2 inocula were mixed with Orthoquin at concentrations ranging from 0.01 to 1 μg/mL and exposed to a 65 W LED light in clear (light) or black (dark) tubes for 10 min (HSV-1) or 5 min (HSV-2). Treated inocula were used to infect HeLa cell monolayers, as described in the methods. (A) Representative image of HSV-1 plaque assay, quantified in (B). (C) Representative HSV-2 plaque assay, quantified in (D). Values in (B,D) are normalized to the number of PFUs detected in DMSO, light- or dark-treated wells. n = 3 ± SEM from three independent biological replicates. * = p < 0.05 by two-way ANOVA. nd = none detected.

Figure 5

Orthoquin PDI inactivates HSV-1 across a 100-fold range of inoculum titer. Four dilutions of HSV-1 inocula (10⁻³, 10⁻⁴, 10⁻⁵, and 10⁻⁶) were mixed with 0.1 µg/mL Orthoquin and exposed to a 65 W LED light in clear (light) or black (dark) tubes for 10 min. Following treatment, each inoculum was diluted in multiple 10-fold dilutions to a final dilution of 10⁻⁶ and used to infect HeLa cell monolayers. Cells were fixed and stained as described in the methods. n = 3 independent biological replicates. Representative plaque assays are shown.

Figure 6

Orthoquin anti-HSV-1 activity depends on close proximity with virions during the photoactivation step. (A) Experimental schema for Orthoquin treatments: (i) Orthoquin was exposed to light in clear or dark tubes for 10 min and was then combined with HSV-1 virions and used to infect HeLa cells; (ii) Orthoquin was exposed to light in clear or dark tubes then used to treat a monolayer of HeLa cells for 1 h. Afterwards, the cells were infected with HSV-1; (iii) Orthoquin PDI of virions prior to infection as in Figure 3 and Figure 4. (B) Representative plaque assays are shown for the three experimental schema. (C) Plaque assay data from three independent experiments were quantified. Values were normalized to the number of plaques detected to the combined Orthoquin/HSV-1 treatment in the dark tubes (schema iii). n = 3 ± SEM from three independent biological replicates. * = p < 0.05 by two-way ANOVA. Oq = Orthoquin.

Figure 7

Orthoquin does not damage HSV-1 capsids. Four 10-fold dilutions of HSV-1 inocula were mixed with 0.1 μg/mL Orthoquin and exposed to a 65 W LED light in clear (light) or black (dark) tubes for 10 min. Following treatment, samples were treated with DNase I for 30 min at 37 °C. Viral DNA was then extracted in lysis buffer and qPCR was performed using primers amplifying UL54 (viral DNA) and luciferase (recovery control). The data shown are means ± standard deviation of three independent experiments. n = 3 ± SD from three independent biological replicates; ns = non-significant by two-way ANOVA.

Figure 8

High doses of Orthoquin inhibit detection of HSV-1 structural proteins. Culture supernatants from mock-infected HeLa cells (A) or HSV-1-infected HeLa cells (B) were processed by differential centrifugation and exposed to 0.1 μg/mL or 0.1 mg/mL Orthoquin, or DMSO vehicle control, or mock-treated for 10 min and exposed to a 65 W LED light in clear (light) or black (dark) tubes. Following treatment, HSV-1 particle preparations were subjected to SDS-PAGE and immunoblotting with a rabbit polyclonal pan-anti-HSV1 antibody that detects viral structural proteins. Uninfected and infected HeLa cell lysates were included as controls for HSV-1 antigen detection.

Figure 9

Orthoquin inhibits vesicular stomatitis virus (VSV) and human adenovirus type 5 (AdV) plaque formation. (A) VSV inocula were mixed with Orthoquin at concentrations ranging from 0.01 to 1 μg/mL and exposed to a 65 W LED light in clear (light) or black (dark) tubes for 5 min. Treated inocula were then plaqued on a monolayer of Vero cells, as described in the methods. A representative image of VSV plaques is shown. (B) Quantification of plaques in (A). Values were normalized to the number of PFUs detected in DMSO, light or dark treated wells. n = 3 ± SEM from three independent biological replicates. (C) AdV inocula were mixed with Orthoquin at concentrations ranging from 0.01 to 1 μg/mL and exposed to a 65 W LED light in clear (light) or black (dark) tubes for 10 min. Treated-inocula were then plaqued on monolayers of HEK293A cells, as described in the methods. Plaques were counted using a fluorescent microscope. n = 4 ± SEM from four independent biological replicates. * = p < 0.05 by two-way ANOVA. ns = non-significant by two-way ANOVA. nd = none detected.