A human-derived Pseudorabies virus isolate induces Cav2.2-sensitive Ca ²⁺ influx and JNK-associated retinal epithelial injury

Figures

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  Fig 1. ARPE-19 cells support hSD-1/2019 infection under the experimental conditions. (A) Growth kinetics of the classical PRV strain Ea and the human-derived PRV strain hSD-1/2019 in ARPE-19 cells infected at MOI = 0.5. Total viral genome copies were quantified by qPCR at the indicated time points post-infection. (B) Growth kinetics of parental hSD-1/2019 and the VP26-mCherry reporter virus derived from hSD-1/2019 in ARPE-19 cells infected at MOI = 0.5. Viral genome copies were quantified by qPCR. (C) Representative fluorescence microscopy images showing VP26-mCherry signal in ARPE-19 cells infected with hSD-1/2019-VP26-mCherry at the indicated time points. Scale bar, 200 μm. (D) Quantification of VP26-mCherry fluorescence intensity in infected ARPE-19 cells over time. Data are presented as mean ± SD from three independent experiments.

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  Fig 2. hSD-1/2019 infection induces cytopathic retinal epithelial injury with increased membrane permeability. ARPE-19 cells were infected with hSD-1/2019 at MOI = 0.5. (A) Phase-contrast microscopy showing CPE in infected ARPE-19 cells at 16 hpi. Arrows indicate swollen, detached, or aggregated cells. Scale bar, 200 μm. (B) Cytotoxicity in infected ARPE-19 cells at 16 hpi, assessed by LDH release assay (OD490 nm). (C) Representative fluorescence microscopy images of infected cells co-stained with PI and Hoechst 33342 at 16 hpi. Scale bar, 200 μm. (D) Quantification of PI-positive area following infection. (E) Flow cytometry analysis of Annexin V-FITC/PI-stained ARPE-19 cells at the indicated time points post-infection. (F) Transmission electron microscopy (TEM) of control and hSD-1/2019-infected ARPE-19 cells. Scale bar, 2 μm. Data are presented as mean ± SD from three independent experiments. Statistical significance was determined using a two-tailed Student’s t-test (**P < 0.01; ***P < 0.001).

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  Fig 3. hSD-1/2019-induced injury depends on extracellular Ca2+ influx. ARPE-19 cells were infected with hSD-1/2019 at MOI = 0.5, and intracellular Ca2+ concentration ([Ca2+]ᵢ) was measured using Fluo-3/AM. (A) Time-course analysis of [Ca2+]ᵢ in infected ARPE-19 cells, measured by flow cytometry. (B, C) [Ca2+]ᵢ in infected cells at 16 hpi following pretreatment with the intracellular Ca2+ chelator BAPTA-AM or the extracellular Ca2+ chelator EGTA at the indicated concentrations. (D) Quantification of [Ca²+]ᵢ in infected cells treated with EGTA (2 mM) or cultured in Ca2+-free medium, measured by Fluo-3/AM fluorescence intensity. (E) PI/Hoechst staining of infected cells treated with EGTA (2 mM) or cultured in Ca2+-free medium. Scale bar, 400 μm. (F) Quantification of PI-positive area. (G) LDH release in infected cells treated with EGTA (2 mM) or cultured in Ca2+-free medium. (H) Phase-contrast microscopy showing CPE in infected cells treated as indicated. Scale bar, 400 μm. Data are presented as mean ± SD from three independent experiments. Statistical significance was determined using a two-tailed Student’s t-test (**P < 0.01; ***P < 0.001).

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  Fig 4. JNK1/2 activation is associated with hSD-1/2019-induced retinal epithelial injury. (A) Western blot analysis of the phosphorylation levels of ERK1/2, JNK1/2, and p38 in ARPE-19 cells infected with hSD-1/2019 (MOI = 0.5) at the indicated times post-infection. Total ERK1/2, JNK1/2, and p38 were used as corresponding controls, and β-actin served as the loading control. (B) CPE (scale bar, 400 μm), (C) PI/Hoechst staining (scale bar, 400 μm), (D) quantification of PI-positive area, and (E) LDH release in infected cells at 16 hpi following pretreatment with the JNK1/2 inhibitor SP600125 (10 μM) or the p38 inhibitor SB203580 (10 μM). Data are presented as mean ± SD from three independent experiments. Statistical significance was determined using a two-tailed Student’s t-test (***P < 0.001; ns, not significant).

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  Fig 5. CaMKII inhibition attenuates hSD-1/2019-induced retinal epithelial cell injury. (A) Western blot analysis of phospho-CaMKII levels in ARPE-19 cells infected with hSD-1/2019 (MOI = 0.5) for the indicated times. β-actin served as the loading control. (B) Western blot analysis of phospho-CaMKII levels in infected cells treated with the CaMKII inhibitor KN-93 at the indicated concentrations. β-actin served as the loading control. (C) PI/Hoechst staining (scale bar, 400 μm), (D) quantification of PI-positive area, (E) CPE (scale bar, 400 μm), and (F) LDH release in infected cells pretreated with KN-93 (10 μM). Data are presented as mean ± SD from three independent experiments. Statistical significance was determined using a two-tailed Student’s t-test (***P < 0.001).

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  Fig 6. Extracellular Ca2+ influx and CaMKII act upstream of JNK1/2 activation. (A) Western blot analysis of phospho-JNK1/2 levels in ARPE-19 cells infected with hSD-1/2019 (MOI = 0.5) for 16 h, cultured in Ca2+-free medium or treated with EGTA at the indicated concentrations. Total JNK1/2 and β-actin were used as controls. (B) Western blot analysis of phospho-JNK1/2 levels in infected cells treated with KN-93 at the indicated concentrations. Total JNK1/2 and β-actin were used as controls. (C) [Ca2+]ᵢ in infected cells pretreated with the JNK1/2 inhibitor SP600125 (10 μM) was measured at 12 or 24 hpi by Fluo-3/AM staining and flow cytometry.

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  Fig 7. The Ca2+/CaMKII/JNK axis contributes to mitochondrial dysfunction in hSD-1/2019-infected cells. ARPE-19 cells were pretreated with EGTA (2 mM), KN-93 (10 μM), or SP600125 (10 μM) before infection with hSD-1/2019 (MOI = 0.5) for 16 h. (A) ΔΨm was assessed by JC-1 staining and confocal microscopy. A decrease in the red/green fluorescence ratio indicates ΔΨm loss. Scale bar, 20 μm. (B) Intracellular ROS levels were measured using DCFH-DA staining and flow cytometry. (C) Cellular ATP levels were quantified using an enhanced ATP assay kit. Data are presented as mean ± SD from three independent experiments. Statistical significance was determined using a two-tailed Student’s t-test (***P < 0.001).

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  Fig 8. Pharmacological screening identifies a Cav2.2-sensitive component in hSD-1/2019-induced Ca2+ influx. ARPE-19 cells were infected with hSD-1/2019 (MOI = 0.5) for 16 h, and [Ca2+]ᵢ was measured by Fluo-3/AM staining and flow cytometry. (A) Screening of Ca2+ influx inhibitors affecting virus-induced [Ca2+]ᵢ elevation, including EGTA (extracellular Ca2+ chelator, 2 mM), 2-APB (SOCE inhibitor, 50 μM), Synta66 (Orai1 inhibitor, 10 μM), Dizocilpine (NMDA receptor antagonist, 10 μM), Pyr3 (TRPC3/ROC inhibitor, 10 μM), Ethosuximide (T-type voltage-gated Ca2+ channel inhibitor, 2 mM), Cilnidipine (L/N-type Ca2+ channel blocker, 20 μM), Nifedipine (L-type voltage-gated Ca2+ channel blocker, 10 μM), and NP118809 (N-type/Cav2.2 channel blocker, 20 μM). (B) Representative flow cytometry histograms showing dose-dependent effects of Cilnidipine or NP118809 on hSD-1/2019-induced [Ca2+]ᵢ elevation. EGTA (2 mM) was used as a positive control. (C) Quantification of [Ca2+]ᵢ in infected cells treated with Cilnidipine (20 μM) or NP118809 (20 μM). Data are presented as mean ± SD from three independent experiments. Statistical significance was determined using a two-tailed Student’s t-test (**P < 0.01;…

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  Fig 9. Cav2.2-sensitive channel blockade attenuates hSD-1/2019-induced cell injury and JNK1/2 activation. ARPE-19 cells were pretreated with Cilnidipine or NP118809 for 2 h before infection with hSD-1/2019 (MOI = 0.5) for 16 h. (A) PI/Hoechst staining of infected cells treated with Cilnidipine (20 μM) or NP118809 (20 μM). Scale bar, 400 μm. (B) Quantification of PI-positive area. (C) LDH release in infected cells treated with the indicated concentrations of Cilnidipine or NP118809. (D) CPE in infected cells treated with Cilnidipine (20 μM) or NP118809 (20 μM). Scale bar, 400 μm. (E) Western blot analysis of phospho-JNK1/2 levels in infected cells treated with the indicated concentrations of Cilnidipine or NP118809. EGTA (2 mM) was used as a positive control for extracellular Ca2+ restriction. Total JNK1/2 and β-actin were used as controls. Data are presented as mean ± SD from three independent experiments. Statistical significance was determined using a two-tailed Student’s t-test (***P < 0.001).

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  Fig 10. gK expression promotes Cav2.2-sensitive Ca2+ elevation and JNK1/2 activation. (A) [Ca2+]ᵢ in HEK-293T cells transfected with increasing amounts of gK-expressing plasmid or empty vector for 24 h, measured by Fluo-3/AM staining and flow cytometry. hSD-1/2019-infected HEK-293T cells were included as a reference. (B) [Ca2+]ᵢ in gK-transfected HEK-293T cells pretreated with Cilnidipine (20 μM) or NP118809 (20 μM). (C) [Ca2+]ᵢ in ARPE-19 cells transfected with increasing amounts of gK-expressing plasmid, with or without Cilnidipine (20 μM) or NP118809 (20 μM) treatment. (D) Western blot analysis of JNK1/2 phosphorylation in ARPE-19 cells transfected with increasing amounts of gK-expressing plasmid. (E) Western blot analysis of JNK1/2 phosphorylation in gK-transfected ARPE-19 cells treated with Cilnidipine (20 μM) or NP118809 (20 μM). Total JNK1/2 and β-actin were used as controls.

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  Fig 11. Proposed model of hSD-1/2019-induced retinal epithelial cell injury. hSD-1/2019 infection induces extracellular Ca2+ influx through a Cav2.2-sensitive pathway in ARPE-19 cells. The resulting increase in [Ca2+]ᵢ is associated with CaMKII activation and subsequent JNK1/2 phosphorylation. Activation of this signaling axis is linked to mitochondrial dysfunction, including increased ROS production, decreased ΔΨm, and ATP depletion, thereby contributing to retinal epithelial cell injury. PRV gK is proposed as a candidate viral contributor to the upstream Cav2.2-sensitive Ca2+ signal.