Pharmacological Induction of Heme Oxygenase-1 Impairs Nuclear Accumulation of Herpes Simplex Virus Capsids upon Infection

Abstract

Heme oxygenase-1 (HO-1) is an inducible enzyme that is expressed in response to physical and chemical stresses, such as ultraviolet radiation, hyperthermia, hypoxia, reactive oxygen species (ROS), as well as cytokines, among others. Its activity can be positively modulated by cobalt protoporphyrin (CoPP) and negatively by tin protoporphirin (SnPP). Once induced, HO-1 degrades iron-containing heme into ferrous iron (Fe2+), carbon monoxide (CO) and biliverdin. Importantly, numerous products of HO-1 are cytoprotective with anti-apoptotic, anti-oxidant, anti-inflammatory, and anti-cancer effects. The products of HO-1 also display antiviral properties against several viruses, such as the human immunodeficiency virus (HIV), influenza, hepatitis B, hepatitis C, and Ebola virus. Here, we sought to assess the effect of modulating HO-1 activity over herpes simplex virus type 2 (HSV-2) infection in epithelial cells and neurons. There are no vaccines against HSV-2 and treatment options are scarce in the immunosuppressed, in which drug-resistant variants emerge. By using HSV strains that encode structural and non-structural forms of the green fluorescent protein (GFP), we found that pharmacological induction of HO-1 activity with CoPP significantly decreases virus plaque formation and the expression of virus-encoded genes in epithelial cells as determined by flow cytometry and western blot assays. CoPP treatment did not affect virus binding to the cell surface or entry into the cytoplasm, but rather downstream events in the virus infection cycle. Furthermore, we observed that treating cells with a CO-releasing molecule (CORM-2) recapitulated some of the anti-HSV effects elicited by CoPP. Taken together, these findings indicate that HO-1 activity interferes with the replication cycle of HSV and that its antiviral effects can be recapitulated by CO.

Keywords: antiviral drug; capsid; carbon monoxide; heme oxygenase-1; herpes simplex virus; pharmacological induction.

Figures

Figure 1

HO-1 expression in epithelial cells treated with CoPP or SnPP and infected with HSV. (A,B) Western blot analyses of HO-1 expression in Vero (left) and HeLa cells (right) after treatment with HO-1-modulating drugs and/or infection with HSV-2 at an MOI 1 for 16 h. (C,D) Flow cytometry analyses of HO-1 expression in Vero (left) and HeLa cells (right) after treatment with HO-1-modulating drugs and/or infection with HSV-2 at an MOI 1 (MFI, mean fluorescence intensity). (E,F) Virus plaque formation (PFU, plaque forming units) in Vero (left) and HeLa cells (right) treated with HO-1-modulating drugs and then infected with 150 PFU of HSV-2. Plaque forming units were determined at 18 and 36 h post-infection for Vero and HeLa cells, respectively. Data are means ± SEM of three independent experiments. Representative images are shown for western blots. Two-way ANOVA, and Tukey's multiple comparison test were used for statistical analyses (**p < 0.01, ***p < 0.001).

Figure 2

Pharmacological induction of HO-1 restricts the expression of HSV-encoded genes in epithelial cells. (A,B) Flow cytometry analyses of GFP-derived fluorescence in Vero (left) and HeLa cells (right), respectively infected at MOI 1 with HSV-2 that encodes a non-structural GFP reporter gene under the control of a constitutive promotor, in response to varying concentrations of HO-1-modulating drugs. (C,D) Cell viability was assessed for Vero (left) and HeLa cells (right) for treatment with varying concentrations of HO-1-modulating drugs. (E,F) Western blot analyses for viral proteins gD and VP16 in Vero (left) and HeLa (right) cells at 16 h post-infection with HSV-2 at an MOI 1 after treatment with 60 and 10 μM, respectively of HO-1-modulating drugs. (G,H) Quantification of viral genome copies in Vero (left) and HeLa cells (right) by qPCR at 18 h post-infection at MOI 10. Data are means ± SEM of three independent experiments for experiments (A–F) and two independent experiments for (G,H). Representative images are shown for Western blots. Two-way ANOVA, and Tukey's multiple comparison test were used for statistical analyses (A): statistics is shown for the CoPP treated group vs. SnPP-treated and untreated (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 3

Pharmacological induction of HO-1 activity does not interfere with virus binding to the cell surface nor capsid entry into the cytoplasm. Western blot analyses for determining virus binding to the surface of (A). Vero cells and (B). HeLa cells. Cells were incubated with varying doses of virus for 5 h at 4°C, washed with saline buffer and then immediately prepared for protein extraction. HSV gD protein expression was assessed to determine the amount of virus bound to the surface of cells. (C,D) Flow cytometry analyses measuring virus-derived GFP fluorescence of capsids internalized into the cytoplasm 2 h post-infection with a MOI of 100 after treatment with CoPP, SnPP, or vehicle in Vero cells (left) and HeLa cells (right). Cells were thoroughly washed with saline buffer and treated with trypsin to remove any surface-bound virus before analysis. Data are means ± SEM of three independent experiments. Representative images are shown for Western blots. Two-way ANOVA, and Tukey's multiple comparison test were used for statistical analyses (n.s., non-significant differences).

Figure 4

Pharmacological induction of HO-1 with CoPP modulates capsid distribution in infected cells and elicits differential cell morphology after treatment after HSV-infection. Representative confocal microscopy images of (A). Vero and (B). HeLa cells treated or not with CoPP and infected with an HSV virus encoding a structural GFP fluorescent capsid (green: GFP-capsid fusion protein). Cell nuclei were stained with Hoescht (blue), and membrane were stained with WGA (red) (C,D). Quantification of the distribution of capsid GFP-fluorescence in treated and infected Vero (left) and HeLa cells (right), respectively relative to the position of the nucleus. An average of fifteen fields and 150–200 cells were analyzed per experiment in a blind manner (E,F). Quantification of total green fluorescence (virus encoded structural GFP fluorescent capsid) in confocal microscopy images of HSV-infected Vero (left) and HeLa cells (right), respectively treated or not with CoPP. TCCF is the integral density of fluorescence of the region of interest minus the equivalent of the cell area*mean fluorescence intensity of the background in the complete z-axis of the analyzed cells. Representative confocal microscopy images at 63X magnification are shown. Data are means ± SEM of two independent experiments. One way ANOVA, Two-way ANOVA, and Tukey's multiple comparison test were used for statistical analyses (**p < 0.01, ***p < 0.001).

Figure 5

Treatment with a carbon monoxide-releasing molecule recapitulates the antiviral effects of CoPP after HSV infection. Quantification of plaque forming units (PFU) after HSV-2 titration over (A). Vero and (B). HeLa cells treated with CORM-2 and inactivated CORM-2 (iCORM2). Virus-encoded GFP fluorescence (non-structural GFP reporter) determined by flow cytometry in (C). Vero and (D). HeLa cells infected with HSV at different MOIs at 20 h post-infection. Data are means ± SEM of three independent experiments. One-way ANOVA, and Tukey's multiple comparison test were used for statistical analyses (*p < 0.05, **p < 0.01, ***p < 0.001).