Porcine Circovirus 2 Induction of ROS Is Responsible for Mitophagy in PK-15 Cells Via Activation of Drp1 Phosphorylation

Article Porcine Circovirus 2 Induction of ROS Is Responsible for Mitophagy in PK-15 Cells via Activation of Drp1 Phosphorylation

Yikai Zhang, Renjie Sun, Xiaoliang Li * and Weihuan Fang *

Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, Institute of Preventive Veterinary Medicine, Zhejiang University, Hangzhou 310058, China; [email protected] (Y.Z.); [email protected] (R.S.) * Correspondence: [email protected] (X.L.); [email protected] (W.F.); Tel.: +86-571-88982291 (X.L.); +86-571-88982242 (W.F.) Received: 2 February 2020; Accepted: 4 March 2020; Published: 6 March 2020

Abstract: Mitochondrial dynamics is essential for the maintenance of cell homeostasis. Previous studies have shown that porcine circovirus 2 (PCV2) infection decreases the mitochondrial membrane potential and causes the elevation of reactive oxygen species (ROS), which may ultimately lead to mitochondrial apoptosis. However, whether PCV2 induce mitophagy remains unknown. Here we show that PCV2-induced mitophagy in PK-15 cells via Drp1 phosphorylation and PINK1/Parkin activation. PCV2 infection enhanced the phosphorylation of Drp1 and its subsequent translocation to mitochondria. PCV2-induced Drp1 phosphorylation could be suppressed by speciﬁc CDK1 inhibitor RO-3306, suggesting CDK1 as its possible upstream molecule. PCV2 infection increased the amount of ROS, up-regulated PINK1 expression, and stimulated recruitment of Parkin to mitochondria. N-acetyl-L-cysteine (NAC) markedly decreased PCV2-induced ROS, down-regulated Drp1 phosphorylation, and lessened PINK1 expression and mitochondrial accumulation of Parkin. Inhibition of Drp1 by mitochondrial division inhibitor-1 Mdivi-1 or RNA silencing not only resulted in the reduction of ROS and PINK1, improved mitochondrial mass and mitochondrial membrane potential, and decreased mitochondrial translocation of Parkin, but also led to reduced apoptotic responses. Together, our study shows that ROS induction due to PCV2 infection is responsible for the activation of Drp1 and the subsequent mitophagic and mitochondrial apoptotic responses.

Keywords: porcine circovirus 2; ROS; mitophagy; Drp1

1. Introduction Porcine circovirus 2 (PCV2) is the major pathogen of several diseases, which are collectively referred to as Porcine-Circovirus-Related Diseases (PCVAD) [1]. As a single-stranded circular DNA virus of only approximately 1.7 kb, PCV2 contains three major open reading frames (ORFs): ORF1 coding for the replicase protein (Rep), ORF2 for the capsid protein (Cap), and ORF3 for a putative protein with pro-apoptotic activity [2,3]. Our recent work has revealed that PCV2 infection induces mitochondrial apoptosis independent of ORF3 [4]. We have also found that PCV2 infection elicits autophagy via activation of CaMKKβ/AMPK/mTOR [5,6]. Autophagy is an intracellular event that degrades surplus cytoplasmic components [7,8]. There are three diﬀerent patterns of autophagy: microautophagy [9], chaperone-mediated autophagy [10], and macroautophagy [11]. Organelle-speciﬁc selective autophagy of the mitochondria (mitophagy) is a part of the macroautophagy that plays an important role in the quality control of mitochondria and the maintenance of cell homeostasis via selective engulfment of damaged mitochondria by autophagosomes and their subsequent degradation by lysosomes [12]. Mitochondria are highly dynamic organelles that

Viruses 2020, 12, 289; doi:10.3390/v12030289 www.mdpi.com/journal/viruses Viruses 2020, 12, 289 2 of 19 constantly divide and fuse. Mitochondrial fission is regulated by cytoplasmic dynamic relative protein 1 (Drp1), which is recruited to the surface of mitochondria after its phosphorylation at Ser616 [13] under the control of cyclin B/cyclin-dependent kinase 1 (CDK1) [14]. The PTEN-induced putative kinase 1 (PINK1)-Parkin (E3 ubiquitin ligase) pathway is one of the well-studied multiple molecular regulatory mechanisms in mitophagy [15]. Through the rapid degradation of mitochondrial proteases, PINK1 is maintained at a low level [16]. However, during mitochondrial stress, PINK1 stabilizes on the outer membrane of mitochondria and recruits cytosolic Parkin to the mitochondria [16,17]. Once transferred to the mitochondrial surface, Parkin mediates the engulfment and subsequent destruction of mitochondria through autophagosomes [18]. As mitochondria function as the central executioner in the mitochondrial apoptotic pathway, it is possible that mitochondrial dynamics related proteins might participate in the modulation of mitophagy and mitochondrial apoptosis. It has been proved that the Hepatitis B virus (HBV) [19] and the Hepatitis C virus (HCV) [20] could stimulate mitophagy. Much evidence suggests that ROS promotes mitochondrial dysfunction and apoptosis [21,22]. PCV2 infection is known to induce oxidative stress in PK-15 cells [23], autophagy [24], and even mitochondrial apoptosis via the elevation of cellular ROS and cytosolic Ca2+ [4,6]. However, whether PCV2 induces mitophagy and whether mitophagy and mitochondrial apoptosis are associated with mitochondrial fission remains to be studied. We attempted to examine the effects and mechanisms of PCV2 infection on the mitophagic and mitochondrial apoptotic responses in PK-15 cells. Our data reveal that ROS induced by PCV2 infection elicits Drp1 phosphorylation and activation of the PINK1/Parkin pathway with subsequent mitophagy and mitochondrial apoptosis.

2. Materials and Methods

2.1. Cell Culture and Virus

The porcine kidney cell line (PK-15) free of PCV1 was used. The cells were cultured at 37 ◦C with 5% CO2 in Dulbecco’s Modified Eagle’s Medium (DMEM) (HyClone, South Logan, UT, USA) containing 4% heat-inactivated fetal bovine serum (GIBCO, Grand Island, NY, USA) with 1% L-glutamine, 1% non-essential amino acids, 100 U/mL penicillin G, and 100 µg/mL streptomycin (100 , GIBCO). × The PK-15 cells that stably expressed EGFP-LC3 (PK-15/EGFP-LC3) were generated and stored in our laboratory. The PCV2 strain YW (GenBank accession no. MG245866) belonged to PCV2b and was originally isolated from the lung of a pig with naturally occurring PCVAD in Zhejiang Province, China. The virus was propagated in PK-15 cells and was used to generate the isogenic mutant with inactivation of the ORF3 protein (∆ORF3) [4]. The PCV2 parent strain YW and its ∆ORF3 mutant contained 1 105.25 and × 1 105.1 infectious virus particles per mL, respectively. As PCV2b has been the predominant genotype × in the swine population and had a more significant effect than the PCV2d in inducing mitochondrial apoptosis [4], this genotype was chosen in this study to investigate its role in mitophagy.

2.2. Chemicals and Antibodies N-acetyl-L-cysteine (NAC) (Beyotime, Hangzhou, China); Mdivi-1 (MedChemExpress, Monmouth Junction, NJ, USA), a selective cell-permeable inhibitor of mitochondrial division; and RO-3306 (Selleck Chemicals LLC, Houston, TX, USA), a selective CDK1 inhibitor were used in their respective experiments. Antibodies against β-actin, caspase 3A/B, PINK1, LC3I/II, GADPH, CDK1 (cdc2), and Drp1 and its phosphorylated form (p-Drp1) were purchased from Cell Signaling Technology (CST, Boston, USA). Anti-VDAC1 antibody was from Abcam and the Anti-Parkin antibody was from Proteintech (Chicago, USA). Mouse anti-Cap monoclonal antibody was produced in our laboratory. Goat anti-rabbit or goat anti-mouse antibodies conjugated to horseradish peroxidase (HRP) were purchased from KPL (Gaithersburg, MD, USA). Viruses 2020, 12, 289 3 of 19

2.3. Virus Infection and Chemical Treatments PK-15 cells at approximately 50–60% confluence were infected with PCV2 or its ∆ORF3 mutant strain at a multiplicity of infection (MOI) of 1 for 48 h to examine mitophagy, oxidative stress, or apoptosis. To investigate the effects of the oxidative stress and mitochondrial fission inhibitors on the changes of ROS and relevant molecules, PK-15 cells were first infected with PCV2 and then treated with NAC (10 mM) or Mdivi-1 (4 µM) at 2 h post infection (hpi). The cells were subjected to further incubation for specified periods according to the experimental goals. Mock-infected cells were included as controls. All infected and/or treated cells were collected for relevant assays described below.

2.4. Protein Extraction and SDS-PAGE/Western Blotting PK-15 cells cultured in 24-well plates were washed with phosphate-buffered saline (PBS at pH 7.2, Beyotime) and collected in a cell lysis buffer (Beyotime) containing protease inhibitors (Roche Molecular Biochemicals, Mannheim, Germany) on ice. The cell lysates were centrifuged at 14,000 g × for 10 min at 4 ◦C and the supernatant samples were collected. To isolate the mitochondrial fraction, PK-15 cells were infected with PCV2. At 48 hpi, the cytosolic and mitochondrial fractions were isolated using a Mitochondria Isolation Kit (Beyotime) according to the manufacturer’s recommendations. The protein concentration was determined using a Bradford assay kit (Beyotime). Equal amounts of protein samples were loaded and separated on 8%, 10%, or 15% SDS–PAGE gels. The samples were transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Darmstadt, Germany). After blocking for 1 h with PBS with 0.05% Tween-20 (PBST) containing 5% nonfat milk power at 37 ◦C, the membranes were incubated with primary antibodies at 4 ◦C overnight. The membranes were then washed with PBST and incubated with secondary antibodies at 37 ◦C for 1 h. Images of the immunoblots were captured using a Gel 3100 chemiluminescent imaging system (Sagecreation, Beijing, China). The target protein blots were quantified using the NIH ImageJ software (National Institutes of Health, Germany).

2.5. Detection of Mitophagy Mitophagy Detection Kit® (Dojindo, Kumamoto, Japan) containing Mtphagy Dye® and Lyso Dye® was used to detect the mitophagy induced by the PCV2 infection. PK-15 cells were incubated in 24-well plates and stained with 100 nM Mtphagy Dye for 15 min in the dark before washing with DMEM (HyClone). The cells were then infected with PCV2 (MOI = 1). At 48 hpi, the infected cells were washed with DMEM and Lyso Dye (1 µM) was added into each well. The plate was incubated for another 10 min, followed by washing with Hanks’ balanced salt solution (HBSS) (Beyotime). The cells were imaged on a confocal fluorescence microscope (IX81-FV1000, Olympus, Markham, ON, Canada). The fluorescence intensity was quantified using the NIH ImageJ software. The colocalization of mitochondria autophagosomes was detected using confocal microscopy (IX81-FV1000). PK-15/EGFP-LC3 cells and MitoTracker™ Red CMXRos (ThermoFisher, Waltham, USA) labeling were used to examine the colocalization of mitochondria with GFP-LC3-positive autophagosomal structures.

2.6. Transmission Electron Microscopy Transmission electron microscopy (TEM) was used to assess the mitochondrial morphology. PK-15 cells were incubated in a six-well plate. Mock- and PCV2-infected cells (48 hpi) were prepared for the TEM. The specimens were first fixed with 2.5% glutaraldehyde in a phosphate buffer (PB) (0.1 M, pH 7.0) for 4–5 h, and then washed three times with PB for 15 min at each step. The specimens were postfixed with 1% OsO4 in PB for 1–2 h and washed three times with PB as per above. Dehydration of the specimens was conducted using a graded series of ethanol (30%, 50%, 70%, 80%, 90%, 95%, and 100%) for about 15 to 20 min at each step, and then using absolute acetone for 20 min. For embedding and ultrathin sectioning, specimens were placed in Eppendorf tubes containing Spurr resin and heated Viruses 2020, 12, 289 4 of 19

at 70 ◦C for at least 9 h. The LEICA EM UC7 ultratome (Wetzlar, Germany) was used to prepare the sections, which were stained with uranyl acetate and alkaline lead citrate for 5 to 10 min, and then observed using the Hitachi Model H-7650 TEM (Tokyo, Japan).

2.7. Immunofluorescence PK-15 cells were cultured in Petri dishes (10 mm in diameter) (Xinyou, Hangzhou, China) and infected with PCV2 at MOI = 1. Mock-infected cells were included as a control. At 48 hpi, MitoTracker™ Red CMXRos (ThermoFisher) was used to stain the mitochondria for 15 min at 37 ◦C. The cells were fixed with 4% paraformaldehyde for 30 min, washed twice with HBSS, and then permeabilized with HBSS containing 0.25% TritonX-100 for 15 min. The cells were then probed with the Anti-p-Drp1 antibody. Images were visualized under a 60 oil objective on a confocal microscope (IX81-FV1000, Olympus). × The quantification of the cell fluorescence intensity was conducted with the NIH ImageJ software.

2.8. Measurement of the Mitochondrial Mass, ROS, and Mitochondrial Membrane Potential For the determination of mitochondrial mass, ROS, and mitochondrial membrane potential (MMP), PK-15 cells were cultured in 24-well plates. Briefly, after removing the culture medium, cells were washed with HBSS. The mitochondrial mass was probed using 0.1 µM MitoTracker Green (excitation—490 nm; emission—516 nm) (Dojindo), ROS levels were probed using 10 µM of 20,70-dichlorofluorescein diacetate (DCFH-DA) (excitation—480 nm; emission—525 nm) (Sigma-Aldrich, St. Louis, MO, USA), and MMP was probed using 0.1 µM JC-1 (excitation—490 nm; the monomeric form: emission—527 nm; J-aggregates: emission—590 nm.) (BD Biosciences, San Diego, CA, USA) for 30 min at 37 ◦C in the dark following the manufacturer’s instructions. After staining, the cells were trypsinized, collected using centrifugation (3 min, 500 g), and re-suspended in HBSS for flow × cytometric analysis. All flow cytometric experiments were performed on the BD FACSCalibur™ Flow Cytometer (BD Biosciences, San Diego, CA, USA). Data were analyzed using FlowJo software, version 10 (Treestar, San Carlos, CA, USA).

2.9. Measurement of Apoptosis For the determination of the apoptotic rate, an Annexin V-FITC Apoptosis Detection Kit (Vazyme, Nanjing, China) was used for the flow cytometry. After 48 hpi, PK-15 cells were trypsinized and collected in HBSS, where 400 µL 1 binding buffer was added to re-suspended cells for each sample. × The cells were stained with 5 µL Annexin V-FITC and 5 µL propidium iodide (PI) for 10 min in the dark at room temperature. At least 1 104 cells in each sample were measured to identify the apoptotic × cell populations. To detect whether virus infection activated caspase 3 and caspase 9, the Caspase 3/9 Activity Assay Kit (Beyotime) was used. The kit contains Ac-DEHD-pNA and Ac-LEHD-pNA as caspase 3 and 9 specific substrates, respectively. The active caspases could cleave substrates to become fluorescent (excitation at 485 nm and emission at 530 nm).

2.10. Statistical Analysis The results are expressed as mean SEM from at least three independent experiments. Statistical ± comparisons were made using one-way analysis of variance (ANOVA) followed by a least significant difference (LSD) post hoc test. Differences between groups were considered significant if * p < 0.05 and highly significant if ** p < 0.01.

3. Results

3.1. PCV2 Induced Mitophagy with the Accumulation of Mitophagosomes A previous work in our lab has shown that PCV2 could induce autophagy in PK-15 cells [24]. To examine whether PCV2 could induce mitophagy, PK-15/EGFP-LC3 cells were infected with PCV2 Viruses 2020, 12, 289 5 of 19

for theViruses confocal 2020, 12 microscopy, x FOR PEER REVIEW analysis of mitophagosome formation by merging GFP-LC3 puncta5 of 19 with MitoTracker Red. A significantly higher number of EGFP-LC3 puncta were seen in the mitochondria in PCV2-infectedMitoTracker Red. cells A compared significantly to higher the uninfected number of EGFP cells‐ (**LC3p puncta< 0.01) were (Figure seen in1A,B). the mitochondria Mtphagy Dye exhibitedin PCV2 a higher‐infected fluorescence cells compared intensity to the upon uninfected fusion cells between (** p < the0.01) mitochondria (Figure 1A,B). and Mtphagy lysosomes Dye as an indicationexhibited ofa higher mitophagy. fluorescence Figure intensity1C shows upon thatfusion the between mitophagic the mitochondria vacuoles and and lysosomes lysosomes as an were colocalizedindication in PCV2-infectedof mitophagy. Figure PK-15 1C cells. shows The that fluorescent the mitophagic intensity vacuoles was higher and lysosomes in PCV2-infected were colocalized in PCV2‐infected PK‐15 cells. The fluorescent intensity was higher in PCV2‐infected cells cells than mock-infected ones (** p < 0.01) (Figure1D). TEM images revealed mitochondrial swelling than mock‐infected ones (** p < 0.01) (Figure 1D). TEM images revealed mitochondrial swelling and and mitochondriamitochondria lackinglacking cristae in in PCV2 PCV2-infected‐infected cells cells (Figure (Figure 1Ea–c).1Ea–c). PCV2 PCV2 infection infection induced induced the the appearanceappearance of autophagic of autophagic vacuoles vacuoles (Figure (Figure1Ed), 1Ed), suggesting suggesting the the occurrence of of autophagy autophagy of damaged of damaged mitochondria.mitochondria. In the In mock-infectedthe mock‐infected cells, cells, mitochondria mitochondria with intact intact cristae cristae could could be bereadily readily observed observed (Figure(Figure1Ee–h). 1Ee–h). These These data data suggest suggest that that the the PCV2 PCV2 infection infection inducedinduced mitochondrial mitochondrial damage damage and and might might induceinduce the selected the selected elimination elimination of damagedof damaged mitochondria mitochondria via autophagy.

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Figure 1. The PCV2‐induced formation of mitophagosomes. PK‐15 cells stably expressing EGFP‐LC3 withFigure PCV2 1. The infection PCV2-induced (MOI = formation 1) or without of mitophagosomes. (Mock) at 48 hpi PK-15 were cells examined stably expressing for the formation EGFP-LC3 of withmitophagosomes. PCV2 infection (A) (MOIThe cells= 1) were or without stained (Mock)with MitoTracker at 48 hpi were Red. examinedImages on for the the right formation depict the of magnifiedmitophagosomes. fields of (theA) Theboxed cells areas were in the stained merge with images, MitoTracker showing Red. colocalization Images on of the GFP right‐LC3 depict punctae the (green)magnified with fields mitochondria of the boxed (Red). areas Images in the in merge the white images, boxes showing were enlarged colocalization 2.5‐fold. of GFP-LC3 Scale bars: punctae 5 μm. (green) with mitochondria (Red). Images in the white boxes were enlarged 2.5-fold. Scale bars: 5 µm. The black arrows indicate the colocalization of mitochondria and LC3 dots. (B) PCV2 infection The black arrows indicate the colocalization of mitochondria and LC3 dots. (B) PCV2 infection induced induced a higher number of mitophagosomes in mitochondria compared to the mock control. (C) The a higher number of mitophagosomes in mitochondria compared to the mock control. (C) The cells cells were stained with Mtphagy Dye and Lyso Dye. Images on the right depict the magnified fields were stained with Mtphagy Dye and Lyso Dye. Images on the right depict the magnified fields of the of the boxed areas in the merge images. Images in the white boxes were enlarged 2.5‐fold. Scale bars: boxed areas in the merge images. Images in the white boxes were enlarged 2.5-fold. Scale bars: 5 µm. 5 μm. The Mtphagy punctae (red) and Lyso puncta (green) were colocalized (red arrows), indicating The Mtphagy punctae (red) and Lyso puncta (green) were colocalized (red arrows), indicating the the occurrence of mitochondrial autophagy (mitophagy). (D) PCV2 infection induced a higher occurrence of mitochondrial autophagy (mitophagy). (D) PCV2 infection induced a higher intensity intensity of Mtphagy Dye than the mock control. (E) Transmission electron microscopy images of Mtphagy Dye than the mock control. (E) Transmission electron microscopy images revealed the revealed the mitochondrial ultrastructure in PCV2‐infected or mock‐infected cells. In the magnified mitochondrial ultrastructure in PCV2-infected or mock-infected cells. In the magnified images, the red images, the red dotted boxes indicate swollen mitochondria (a,b) and an autophagic vacuole that dotted boxes indicate swollen mitochondria (a,b) and an autophagic vacuole that contains mitochondria contains mitochondria (c,d). In the mock‐infected cells, the normal mitochondria were seen as clear (c,d). In the mock-infected cells, the normal mitochondria were seen as clear cristae and filling matrix cristae and filling matrix (e–h). The magnified images were enlarged twofold. Scale bars: 200 nm. Bar (e–h). The magnified images were enlarged twofold. Scale bars: 200 nm. Bar charts (B,D) show the charts (B,D) show the mean ± SEM of three independent experiments. * p < 0.05 and ** p < 0.01. mean SEM of three independent experiments. ** p < 0.01. ± 3.2. PCV2 PCV2 Enhanced Enhanced Drp1 Drp1 Ser616 Ser616 Phosphorylation Phosphorylation and and Its Mitochondrial Translocation Viruses, such as HBV [[19],19], HCV [[20],20], and the classical swine fever virus (CSFV) [[25],25], could perturb mitochondrial dynamics by causing mitochondrial fission fission and mitophagy. However, However, it is unclear whether abnormal mitochondrial dynamics occur in cellscells infectedinfected withwith PCV2.PCV2. Confocal immunofluorescenceimmunofluorescence waswas performed toto observe the changes in mitochondrial dynamics in PK PK-15‐15 cells infected by PCV2. Figure 22A,BA,B showsshows thatthat PCV2PCV2 infectioninfection shiftedshifted thethe morphologymorphology ofof mitochondrial mitochondrial networks towardtoward a a significantly significantly higher higher proportion proportion of fragmentedof fragmented structures structures (higher (higher proportion proportion of >70% of >70%fragmented fragmented than the than proportion the proportion of <30% of fragmented<30% fragmented structures structures in the PCV2 in the infected PCV2 infected cells, ** cells,p < 0.01). ** p

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<30% Fragmented 80 ** ** 30-70% Fragmented >70% Fragmented 60

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FigureFigure 2. 2.Defects Defects in in the the mitochondrial mitochondrial tubular tubular networks networks and and mitochondrial mitochondrial translocation translocation of of the the mitochondrial-fission-relatedmitochondrial‐fission‐related protein protein Drp1 Drp1 induced induced by PCV2. by PCV2. PK-15 PK cells‐15 cells were were infected infected with PCV2with PCV2 for 48for h in 48 Petri h in dishes Petri fordishes confocal for confocal microscopy. microscopy. Mock-infected Mock‐ cellsinfected were cells used were as a control.used as ( Aa )control. Confocal (A) microscopyConfocal microscopy images showing images mitochondrial showing mitochondrial fragmentation fragmentation in PCV2-infected in PCV2‐infected cells. At cells. 48 hpi, At 48 the hpi, cellsthe were cells were stained stained with with MitoTracker MitoTracker (red) (red) (top (top panels) panels) or immunostained or immunostained with with anti-PCV2 anti‐PCV2 capsid capsid proteinprotein antibody antibody (green) (green) (bottom (bottom panel). panel). In In the the magnified magnified images, images, typical typical tubular tubular mitochondria mitochondria in in mock-infectedmock‐infected cells cells and and fragmented fragmented mitochondria mitochondria in in infected infected cells cells are are shown. shown. (B ()B Quantification) Quantification of of thethe mitochondrial mitochondrial morphology morphology in in (A). (A). Cells Cells were were scored scored as as one one of of the the three three morphological morphological categories categories asas depicted depicted in in the the inset. inset. In In eacheach group, at least least 50 50 cells cells were were scored. scored. (C ()C The) The cells cells were were prestained prestained with withMitoTracker MitoTracker Red Red (100 (100 nM) nM) and and then then immune immune-stained‐stained with with anti anti-phospho-Drp1‐phospho‐Drp1 (Ser616) (Ser616) (green) (green) at at 48 48hpi. hpi. ( (DD)) The The quantification quantification of thethe p-Drp1p‐Drp1 (Ser616)(Ser616) fluorescence fluorescence intensity intensity of of (C (C) was) was analyzed analyzed using using NIHNIH ImageJ ImageJ software software in in at leastat least 50 50 cells cells of of each each group. group. Bar Bar charts charts (B ,(DB,)D show) show the the mean mean SEM± SEM of of three three ± independentindependent experiments. experiments. * p *

TheThe ORF3 ORF3 protein protein was was found found to to causecause apoptosis in in PCV2 PCV2-infected‐infected cells cells [2]. [2 ].We We hypothesized hypothesized that thatPCV2 PCV2-induced‐induced mitochondrial mitochondrial fission fission might might occur occur independently ofof ORF3. ORF3. An An ORF3-inactivated ORF3‐inactivated PCV2PCV2 mutant mutant (∆ (ORF3)ORF3) was was used used to to infect infect the the PK-15 PK‐15 cells. cells. In In the the whole-cell whole‐cell lysate, lysate, there there was was increased increased p-Drp1p‐Drp1 in in PCV2-infected PCV2‐infected cells cells compared compared with with the the control control cells cells (** (**p < p 0.01).< 0.01). The The ORF3-deficient ORF3‐deficient virus virus couldcould still still induce induce Drp1 Drp1 phosphorylation phosphorylation (* p <(* 0.05),p < 0.05), although although the responses the responses were generallywere generally lower thanlower thosethan observed those observed with the with parental the parental strain (Figurestrain (Figure3A,B). To3A,B). further To further substantiate substantiate the observation the observation that PCV2that inducedPCV2 induced Drp1 translocation Drp1 translocation to mitochondria, to mitochondria, we extracted we mitochondria extracted mitochondria from virus-infected from virus and ‐ mock-infectedinfected and PK-15mock‐ cellsinfected for Western PK‐15 cells blotting. for Western We found blotting. that p-Drp1 We found was that significantly p‐Drp1 was enriched significantly in the mitochondrialenriched in the fraction mitochondrial of PCV2-infected fraction of cells PCV2 (* p‐infected< 0.05) (Figure cells (*3 pC,D). < 0.05) These (Figure results 3C,D). indicated These results that PCV2indicated promoted that PCV2 mitochondrial promoted fission mitochondrial via Drp1 fission phosphorylation via Drp1 phosphorylation and its mitochondrial and its translocation. mitochondrial translocation.

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FigureFigure 3. 3. PCV2PCV2 stimulated stimulated Drp1 Drp1 phosphorylation phosphorylation and and its its translocation translocation to to mitochondria mitochondria independent independent ofof ORF3. ORF3. The The cells cells were were infected infected with with PCV2 PCV2 or its or mutant its mutant ΔORF3∆ORF3 (MOI (MOI = 1) in= 241)‐ inwell 24-well plates plates for 48 forh. Mock48 h.‐ Mock-infectedinfected cells were cells used were as used a control. as a control. (A) Whole (A) Whole-cell‐cell lysates lysates (WCL) (WCL) extracted extracted from PK from‐15 PK-15 cells infectedcells infected with withPCV2 PCV2 and Δ andORF3∆ORF3 infection infection were were analyzed analyzed using using Western Western blotting blotting with with antibodies antibodies againstagainst Drp1, Drp1, p p-Drp1‐Drp1 (Ser616), (Ser616), Cap Cap (PCV2 (PCV2 capsid capsid protein), protein), and and β‐βactin-actin (used (used as as a a protein protein loading loading control).control). (B ()B The) The ratio ratio of ofp‐Drp1/Drp1 p-Drp1/Drp1 in whole in whole-cell‐cell lysates lysates (WCL) (WCL) is shown. is shown. (C) Cytosolic (C) Cytosolic (Cyto) (Cyto) and mitochondrialand mitochondrial (Mito) (Mito) fractions fractions isolated isolated from from PK‐ PK-1515 cells cells with with or orwithout without PCV2 PCV2 infection infection were were analyzedanalyzed using using Western Western blotting withwith p-Drp1,p‐Drp1, VDAC1, VDAC1, and and GAPDH GAPDH antibodies. antibodies. GADPH GADPH was was used used as asthe the internal internal control control of of cytoplasmic cytoplasmic proteins proteins and and VDAC1 VDAC1 asas thatthat ofof mitochondrial proteins. ( (DD)) The The ratioratio of of p p-Drp1‐Drp1/VDAC1/VDAC1 inin the the mitochondrial mitochondrial fraction fraction is shown.is shown. Bar Bar charts charts (B, D(B), showD) show the meanthe meanSEM ± ± SEMof three of three independent independent experiments. experiments. * p < *0.05 p < 0.05 and and ** p <** 0.01.p < 0.01.

3.3. PCV2-Induced ROS was Involved in Drp1 Phosphorylation 3.3. PCV2‐Induced ROS was Involved in Drp1 Phosphorylation WeWe previously foundfound that that the the PCV2 PCV2 infection infection of PK-15 of PK cells‐15 cells caused caused a significant a significant elevation elevation of cellular of cellularROS [4 ].ROS To investigate [4]. To investigate the relationship the relationship between increased between ROS increased levels and ROS Drp1 levels phosphorylation and Drp1 phosphorylationinduced by PCV2, induced we collected by PCV2, cells we at di collectedfferent time cells points at different from 24 time hpi points to 48 hpi from for 24 Western hpi to blotting 48 hpi forand Western flow cytometry. blotting and Figure flow4 cytometry.shows that Figure there was4 shows a significant that there increase was a significant of both p-Drp1 increase and of both ROS pwith‐Drp1 the and progression ROS with ofthe the progression PCV2 infection, of the PCV2 particularly infection, after particularly 36 hpi (Figure after4 A–C).36 hpi NAC (Figure treatment 4A–C). NACwas e treatmentffective at was suppressing effective the at suppressing ROS generation the ROS in PCV2-infected generation in cells PCV2 (Figure‐infected4D) cells and at(Figure inhibiting 4D) andDrp1 at phosphorylation, inhibiting Drp1 suggesting phosphorylation, that ROS wassuggesting involved inthat the ROS Drp1 phosphorylationwas involved in (Figure the 4Drp1E,G). phosphorylationThe cyclin B/CDK1 (Figure complex 4E,G). is oneThe of cyclin the mitotic B/CDK1 kinases complex that is knownone of tothe cooperate mitotic kinases with the that small is knownG protein to cooperate RALA and with its etheffector small RALBP1 G protein to promoteRALA and Drp1 its phosphorylationeffector RALBP1 andto promote mitochondrial Drp1 phosphorylationfission [26,27]. A and specific mitochondrial CDK1 inhibitor fission RO-3306 [26,27]. wasA specific used to CDK1 treat inhibitor PCV2-infected RO‐3306 cells was to examineused to treatwhether PCV2 CDK1‐infected regulated cells to the examine phosphorylation whether CDK1 of Drp1 regulated during thethe PCV2 phosphorylation infection. We of found Drp1 thatduring the thesuppression PCV2 infection. of CDK1 We by RO-3306found that inhibited the suppression the Drp1 phosphorylation of CDK1 by RO induced‐3306 inhibited by PCV2 (*thep < Drp10.05), phosphorylationwhile the NAC treatmentinduced by decreased PCV2 (* the p < CDK1 0.05), expression while the (*NACp < 0.05)treatment (Figure decreased4F). These the findings CDK1 expressionsuggest that (* CDK1p < 0.05) might (Figure be the4F). upstream These findings signal suggest of Drp1 that phosphorylation CDK1 might be in responsethe upstream to ROS. signal of Drp1 phosphorylation in response to ROS.

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0.0 p-Drp1/Drp1 ratio (WCL) 06 ck 0.0 o PCV2 NAC M CDK1 Cap 2+ V Targeted protein/βTargeted ratio -actin C +RO-33 P 2 PCV (F) (G)

Figure 4. PCV2 infection promoted the Drp1 phosphorylation and reactive oxygen species (ROS) generation, and Drp1 activation could be reversed via treatment with N‐acetyl‐L‐cysteine (NAC) or via the inhibition of CDK1 with RO‐3306. PK‐15 cells were infected with PCV2 (MOI = 1) for 24, 36, or 48 h. Mock‐infected cells were used as a control. (A) Target proteins of the PK‐15 cell extracts were separated using SDS‐PAGE and analyzed using Western blotting with antibodies to p‐Drp1 (Ser616), Drp1, and PCV2‐Cap. β‐actin was used as a protein loading control. The gels shown are representative of three individual experiments. (B) The p‐Drp1/Drp1 ratio in WCL. (C) After 48 hpi, cells were harvested to assess cytosolic ROS using flow cytometry. Cells were treated with 10 μM DCFH‐DA for 30 min at 37 °C in the dark. The cytosolic ROS levels are shown as the mean value of DCF fluorescence intensity. (D) The PK‐15 cells were infected with PCV2, treated with 10 mM NAC at 2 hpi, and then incubated for an additional 46 h. The cells were collected for flow cytometry to examine the changes in the cytosolic ROS level. (E) The cells were infected with PCV2, treated with 10 mM NAC or 200 nM

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Figure 4. PCV2 infection promoted the Drp1 phosphorylation and reactive oxygen species (ROS) generation, and Drp1 activation could be reversed via treatment with N-acetyl-L-cysteine (NAC) or via the inhibition of CDK1 with RO-3306. PK-15 cells were infected with PCV2 (MOI = 1) for 24, 36, or 48 h. Mock-infected cells were used as a control. (A) Target proteins of the PK-15 cell extracts were separated using SDS-PAGE and analyzed using Western blotting with antibodies to p-Drp1 (Ser616), Drp1, and PCV2-Cap. β-actin was used as a protein loading control. The gels shown are representative of three individual experiments. (B) The p-Drp1/Drp1 ratio in WCL. (C) After 48 hpi, cells were harvested to assess cytosolic ROS using ﬂow cytometry. Cells were treated with 10 µM DCFH-DA for 30 min at

37 ◦C in the dark. The cytosolic ROS levels are shown as the mean value of DCF fluorescence intensity. (D) The PK-15 cells were infected with PCV2, treated with 10 mM NAC at 2 hpi, and then incubated for an additional 46 h. The cells were collected for flow cytometry to examine the changes in the cytosolic Viruses 2020, 12, x FOR PEER REVIEW 10 of 19 ROS level. (E) The cells were infected with PCV2, treated with 10 mM NAC or 200 nM RO-3306 at 2 hpi,RO and‐3306 then at 2 incubated hpi, and then for incubated an additional for an 46 additional h before 46 measurement h before measurement of the target of the protein target protein expression usingexpression Western blottingusing Western for CDK1, blotting Drp1, for CDK1, p-Drp1 Drp1, (Ser616), p‐Drp1 and (Ser616), PCV2-Cap. and PCV2β-actin‐Cap. was β‐actin used was as used a protein loadingas a control.protein loading The gels control. shown The are gels representative shown are representative of three individual of three experiments. individual experiments. (F) Ratios of(F) CDK1 and PCV2-CapRatios of CDK1 to β and-actin. PCV2 (G‐Cap) The to p-Drp1 β‐actin./ Drp1(G) The ratio p‐Drp1/Drp1 in WCL. Dataratio in in WCL. bar charts Data in (B bar–D ,chartsF,G) show(B– the meanD,F,SEMG) show of three the mean independent ± SEM of three experiments. independent * p experiments.< 0.05 and ** * pp << 0.050.01. and ** p < 0.01. ± 3.4. PCV2-Induced3.4. PCV2‐Induced Drp1 Drp1 Phosphorylation Phosphorylation andand Mitophagy Could Could be be Reversed Reversed Using Using ROS ROS Scavenging Scavenging N‐ N-acetyl-L-cysteineacetyl‐L‐cysteine MitochondrialMitochondrial function function is is accompanied accompanied by ROS generation. generation. Excessive Excessive ROS ROS generated generated by by mitochondrialmitochondrial dysfunction dysfunction might might induce induce mitophagy, a a mechanism mechanism used used to eliminate to eliminate the damaged the damaged organellesorganelles [28 ].[28]. PCV2 PCV2 infection infection is known known to to elevate elevate the theROS ROS load [4,23,29]. load [4,23 ROS,29‐].scavenging ROS-scavenging NAC was NAC wasemployed employed to to treat treat the the PCV2 PCV2-infected‐infected cells cells to toinvestigate investigate the theeffect eff ofect PCV2 of PCV2-induced‐induced ROS ROSon on mitophagosome formation shown as colocalization of the GFP‐LC3 puncta with mitochondria. Figure mitophagosome formation shown as colocalization of the GFP-LC3 puncta with mitochondria. Figure5 5 reveals that NAC treatment significantly reduced mitophagosome formation in PCV2‐infected cells reveals(** p that < 0.01). NAC treatment significantly reduced mitophagosome formation in PCV2-infected cells (** p < 0.01).

50 ** 40

20 per cell per

0 Number Number of GFP-LC3 puncta ck o PCV2 M

CV2+NAC P Mock+NAC

(A) (B)

FigureFigure 5. Colocalization 5. Colocalization of of the the EGFP-LC3 EGFP‐LC3 punctae and and MitoTracker MitoTracker induced induced by PCV2 by PCV2 was inhibitable was inhibitable usingusing a ROS-scavenger a ROS‐scavenger N-acetyl-L-cysteine N‐acetyl‐L‐cysteine (NAC). The The PK PK-15‐15 cells cells stably stably expressing expressing EGFP EGFP-LC3‐LC3 in in PetriPetri dishes dishes were were infected infected with with PCV2, PCV2, treated treated with 10 10 mM mM NAC NAC at at2 hpi 2 hpi or untreated, or untreated, and subjected and subjected to furtherto further incubation incubation for for 46 46 h. h. ( A(A)) Cells Cells werewere treated treated with with 100 100 nM nM MitoTracker MitoTracker Red Red for 15 for min 15 in min the in the dark. Representative confocal images showing the colocalization of MitoTracker Red and EGFP‐LC3 dark. Representative confocal images showing the colocalization of MitoTracker Red and EGFP-LC3 punctae in the cells with or without (Mock) PCV2 infection. Scale bars: 10 μm. (B) Average numbers punctae in the cells with or without (Mock) PCV2 infection. Scale bars: 10 µm. (B) Average numbers of colocalization of EGFP‐LC3 and MitoTracker Red punctae per cell from at least 50 cells. Bar chart of colocalization(B) shows the ofmean EGFP-LC3 ± SEM of andthree MitoTracker independent experiments. Red punctae ** per p < cell0.01. from at least 50 cells. Bar chart (B) shows the mean SEM of three independent experiments. ** p < 0.01. ± To further address how ROS were involved in the Drp1 phosphorylation and PCV2‐induced mitophagy, we examined the changes of PINK1, Parkin, LC3II, Drp1, and p‐Drp1 in whole lysates of cells infected with PCV2 with or without NAC treatment. We observed that PCV2 infection led not only to autophagy, shown as a marked increase of the LC3II/LC3I ratio, but also to the elevated expression of PINK1 and Parkin in PK‐15 cells, as well as increased Drp1 phosphorylation, which are indicative of mitophagic responses. All these changes due to PCV2 infection were reversed using an NAC treatment (Figure 6A–D) (* p < 0.05 or ** p < 0.01). PCV2 replication was also inhibited using an NAC treatment, which is shown as a decreased PCV2 Cap expression and a reduced number of PCV2 infected cells (Figure 6D,E). Further analysis revealed that increased Parkin accumulation in the mitochondrial fraction of PCV2 infected cells could be blocked using an NAC treatment (* p < 0.05)

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To further address how ROS were involved in the Drp1 phosphorylation and PCV2-induced mitophagy, we examined the changes of PINK1, Parkin, LC3II, Drp1, and p-Drp1 in whole lysates of cells infected with PCV2 with or without NAC treatment. We observed that PCV2 infection led not only to autophagy, shown as a marked increase of the LC3II/LC3I ratio, but also to the elevated expression of PINK1 and Parkin in PK-15 cells, as well as increased Drp1 phosphorylation, which are indicative of mitophagic responses. All these changes due to PCV2 infection were reversed using an NAC treatment (Figure6A–D) (* p < 0.05 or ** p < 0.01). PCV2 replication was also inhibited using an NAC treatment, which is shown as a decreased PCV2 Cap expression and a reduced number of PCV2Viruses infected 2020, 12, cells x FOR (Figure PEER REVIEW6D,E). Further analysis revealed that increased Parkin accumulation11 of 19 in the mitochondrial fraction of PCV2 infected cells could be blocked using an NAC treatment (* p < 0.05) (Figure(Figure6F,G). 6F,G). Thus, Thus, we we provide provide clear clear evidence that that there there was was a causal a causal link link between between mitophagy mitophagy and and excessive ROS production during PCV2 infection, and that ROS might function as an upstream signal excessive ROS production during PCV2 infection, and that ROS might function as an upstream signal for the activation of Drp1 phosphorylation with subsequent mitochondrial fission and mitophagy. for the activation of Drp1 phosphorylation with subsequent mitochondrial ﬁssion and mitophagy.

2.0 * * 1.5

1.0

0.5 p-Drp1/Drp1 ratio 0.0

2 C k C V A C P NA Moc 2+ V C P Mock+N

(A) (B)

* 2.0 ** * 2.5 ** PCV2 1.5 * PCV2+NAC 2.0 * Mock Mock+NAC 1.0 1.5 ** 0.5 1.0 LC3Ⅱ /LC3Ⅰ ratio

Relative ratio of ratio Relative 0.5 0.0

targeted protein/β -actin0.0 PCV2 Mock PINK1 Parkin Cap V2+NAC PC Mock+NAC (C) (D)

(F)

2.0 * *

1.5

1.0 of Parkin of 0.5

0.0 Mitochondrial accumulation Mitochondrial ck o NAC PCV2 + M V2 C ock+NAC P M (G) (E)

Figure 6. PCV2 increased the Drp1 phosphorylation, LC3II conversion, and PINK1 and Parkin expression, which could be down‐regulated using an N‐acetyl‐L‐cysteine (NAC) treatment. The PK‐ 15 cells were infected with PCV2, treated with 10 mM NAC at 2 hpi, and then incubated for an additional 46 h before measuring the target protein expression using Western blotting or immunofluorescence. (A) Whole‐cell extracts were separated using SDS‐PAGE and analyzed using Western blotting with antibodies against Parkin, PINK1, Drp1, p‐Drp1 (Ser616), LC3Ⅰ/Ⅱ, Cap, and β‐actin. β‐actin was used as a protein loading control. The gels shown here are representative of three individual experiments. (B–D) Ratio of p‐Drp1 to Drp1 (B); LC3Ⅱ/LC3Ⅰ(C); and PINK1, Parkin, and Cap to β‐actin (D). (E) The PCV2‐infected cells with or without NAC treatment were immunostained

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Figure 6. PCV2 increased the Drp1 phosphorylation, LC3II conversion, and PINK1 and Parkin expression, which could be down-regulated using an N-acetyl-L-cysteine (NAC) treatment. The PK-15 cells were infected with PCV2, treated with 10 mM NAC at 2 hpi, and then incubated for an additional 46 h before measuring the target protein expression using Western blotting or immunoﬂuorescence. (A) Whole-cell extracts were separated using SDS-PAGE and analyzed using Western blotting with antibodies against Parkin, PINK1, Drp1, p-Drp1 (Ser616), LC3I/II, Cap, and β-actin. β-actin was used as a protein loading control. The gels shown here are representative of three individual experiments. (B–D) Ratio of p-Drp1 to Drp1 (B); LC3II/LC3I(C); and PINK1, Parkin, and Cap to β-actin (D). (E) The PCV2-infected cells with or without NAC treatment were immunostained with an anti-PCV2-Cap

monoclonalViruses 2020 antibody., 12, x FOR PEER (F) ParkinREVIEW in cytoplasmic (Cyto) and mitochondrial (Mito) fractions12 wasof 19 visualized using Western blotting. GAPDH was used as the internal control for the cytoplasmic proteins and VDAC1with for an that anti of‐PCV2 the‐ mitochondrialCap monoclonal proteins. antibody. ((GF)) Parkin Ratio ofin Parkincytoplasmic to VDAC1 (Cyto) and in the mitochondrial mitochondrial fraction. Bar(Mito) charts fractions (B–D was,G) visualized show the using mean WesternSEM blotting. of three GAPDH independent was used experiments. as the internal control * p < 0.05 for and the cytoplasmic proteins and VDAC1 for± that of the mitochondrial proteins. (G) Ratio of Parkin to ** p < 0.01. VDAC1 in the mitochondrial fraction. Bar charts (B–D,G) show the mean ± SEM of three independent 3.5. Suppressionexperiments. of the Mitochondrial * p < 0.05 and ** Fission p < 0.01. Protein Drp1 Inhibited PCV2-Induced Mitophagy and Mitochondrial Apoptosis 3.5. Suppression of the Mitochondrial Fission Protein Drp1 Inhibited PCV2‐Induced Mitophagy and MitochondrialMitochondrial dynamics Apoptosis is regulated by Drp1, a key protein involved in mitochondrial fission [30], and integrallyMitochondrial linked to apoptosis dynamics [31 is]. regulated We showed by Drp1, that PCV2a key protein could induceinvolved mitochondrial in mitochondrial apoptosis fission [4]. To gain insight[30], and into integrally the role linked of Drp1 to apoptosis in PCV2-induced [31]. We showed mitophagy that PCV2 and could mitochondrial induce mitochondrial apoptosis, we tested whetherapoptosis the specific[4]. To gain suppression insight into of the Drp1 role of with Drp1 lentivirus-mediated in PCV2‐induced mitophagy shRNA and (shDrp1) mitochondrial or chemical treatmentapoptosis, with Mdivi-1 we tested would whether affect thethe mitophagicspecific suppression and apoptotic of Drp1 responses.with lentivirus The‐mediated chemical shRNA inhibition (shDrp1) or chemical treatment with Mdivi‐1 would affect the mitophagic and apoptotic responses. of Drp1 or RNA silencing with shDrp1 could e ectively alleviate mitochondrial fragmentation caused The chemical inhibition of Drp1 or RNAff silencing with shDrp1 could effectively alleviate by PCV2 infectionmitochondrial (Figure fragmentation7A). To confirm caused by the PCV2 relationship infection (Figure between 7A). Drp1 To confirm and mitophagy, the relationship Western blotting wasbetween used Drp1 to examine and mitophagy, the effects Western of Drp1 blotting inhibition was used on theto examine expression the effects of key of molecules Drp1 inhibition involved in mitophagyon the and expression apoptosis. of key We molecules found that involved the inhibition in mitophagy of Drp1 and expression apoptosis. We resulted found inthat decreased the PINK1 andinhibition caspase of 3 cleavageDrp1 expression (Figure resulted7B,C), in as decreased well as a PINK1 diminished and caspase Parkin 3 cleavage accumulation (Figure and7B,C), reduced as well as a diminished Parkin accumulation and reduced LC3II/I ratio in the mitochondrial fraction LC3II/I ratio in the mitochondrial fraction (Figure7D,E)(* p < 0.05 compared with those of the cells (Figure 7D,E)(* p < 0.05 compared with those of the cells infected with PCV2 only). infected with PCV2 only).

<30% Fragmented 30-70% Fragmented 80 >70% Fragmented * ns 60 ns **

20 Percent of cells of Percent

2 -1 1 V vi rp PC di M +shD 2+ 2 V V C C PCV2+shNC P P (A) (B)

3 ** Mock Mock+Mdivi-1 * * PCV2+Mdivi-1 2 * * PCV2 ** PCV2+shDrp1 PCV2+shNC 1 Relative ratio of ratio Relative

targeted protein/β -actin 0 Drp1 p-Drp1 PINK1 C-cas 3 Cap

(C)

Figure 7. Cont.

VirusesViruses2020 2020, 12,, 289 12, x FOR PEER REVIEW 13 of13 19 of 19

(D)

(E) (F)

FigureFigure 7. Inhibition 7. Inhibition of Drp1 of Drp1 by shDrp1by shDrp1 and and Mdivi-1 Mdivi suppressed‐1 suppressed mitochondrial mitochondrial fragmentation, fragmentation, Drp1 Drp1 phosphorylation,phosphorylation, and and PINK1 PINK1 expression, expression, as well as well as reduced as reduced the the accumulation accumulation of Parkin of Parkin and and LC3II LC3II in in mitochondrialmitochondrial fraction fraction and caspaseand caspase 3 cleavage. 3 cleavage. PK-15 PK cells‐15 were cells incubated were incubated in 24-well in plates.24‐well Cells plates. were Cells eitherwere infected either with infected the lentivirus with the containinglentivirus containing shDrp1 (shDrp1) shDrp1 for (shDrp1) 12 h and for then 12 infectedh and then with infected PCV2 or with thePCV2 cells were or the infected cells were with PCV2infected and with then PCV2 treated and with then 4 µ treatedM Mdivi-1 with at 4 2 μ hpi,M Mdivi and then‐1 at incubated 2 hpi, and for then an additionalincubated 46for h an before additional confocal 46 microscopyh before confocal analysis microscopy or measuring analysis the targetor measuring proteins the using target Western proteins blotting.using (WesternA) Quantiﬁcation blotting. (A of) Quantification mitochondrial of fragmentation mitochondrial in fragmentation PCV2-infected in cells. PCV2 (‐Binfected) Whole-cell cells. (B) extractsWhole were‐cell separated extracts usingwere SDS-PAGEseparated using and analyzed SDS‐PAGE using and Western analyzed blotting using with Western antibodies blotting against with Drp1,antibodies p-Drp1 (Ser616), against Drp1, PINK1, p‐ PCV2Drp1 (Ser616), Cap, cleaved PINK1, caspase PCV2 3 (C-casCap, cleaved 3), and caspaseβ-actin. 3β (C-actin‐cas was3), and used β‐ asactin. a proteinβ‐actin loading was used control. as a Theprotein results loading shown control. here are The representative results shown of threehere individualare representative experiments. of three (C)individual The ratios ofexperiments. Drp1, p-Drp1 (C) (Ser616), The ratios PINK1, of Drp1, C-cas p‐Drp1 3, and (Ser616), Cap to βPINK1,-actin. C (D‐cas) The 3, and levels Cap of Parkinto β‐actin. and(D LC3II) The in levels the cytoplasmic of Parkin and (Cyto) LC3II and in mitochondrialthe cytoplasmic (Mito) (Cyto) fractions and mitochondrial were analyzed (Mito) using fractions Western were blotting.analyzed GAPDH using wasWestern used blotting. as the internal GAPDH control was used of cytoplasmic as the internal proteins control and of VDAC1 cytoplasmic as that proteins of theand mitochondrial VDAC1 as proteins.that of the ( Emitochondrial) Ratio of Parkin proteins. to VDAC1 (E) Ratio in the of mitochondrialParkin to VDAC1 fraction. in the ( mitochondrialF) Ratio of LC3IIfraction./I in the (F mitochondrial) Ratio of LC3 fraction.Ⅱ/Ⅰ in Bar the charts mitochondrial (A,C,E,F) fraction. show the Bar mean chartsSEM (A,C of,E three,F) show independent the mean ± ± experiments.SEM of three * p

TheThe down-regulation down‐regulation of Drp1 of Drp1 by Mdivi-1by Mdivi treatment‐1 treatment or shDrp1or shDrp1 signiﬁcantly significantly decreased decreased ROS ROS and and rescuedrescued MMP MMP in PCV2-infectedin PCV2‐infected cells cells compared compared with with PCV2-infected PCV2‐infected but but untreated untreated cells cells (* p (*< p0.05) < 0.05) (Figure(Figure8A,B). 8A,B). Drp1 Drp1 suppression suppression inhibited inhibited the the loss loss of mitochondrialof mitochondrial mass mass in in cells cells infected infected by by PCV2 PCV2 (* (* pp< <0.05, 0.05, ** **p p< <0.01 0.01 vs. vs. control control cells) cells) (Figure (Figure8C). 8C). Treatment Treatment with with either either Mdivi-1 Mdivi or‐1 shDrp1or shDrp1 could could alleviatealleviate apoptotic apoptotic responses responses in PK-15 in PK cells,‐15 cells, which which was shown was shown as a decreased as a decreased apoptotic apoptotic rate (* p rate< 0.05) (* p < (Figure0.05)8 D)(Figure and 8D) reduced and reduced activities activities of caspases of caspases 3 and 93 and that 9 were that were closely closely related related to mitochondrial to mitochondrial apoptosisapoptosis (* p (*< p0.05) < 0.05) (Figure (Figure8E,F). 8E,F). These These ﬁndings findings indicate indicate that that Drp1 Drp1 might might function function as an as upstream an upstream moleculemolecule and and cooperate cooperate with with ROS ROS to regulate to regulate the the PINK1 PINK1/Parkin/Parkin pathway, pathway, as well as well as mitophagic as mitophagic and and mitochondrialmitochondrial apoptotic apoptotic responses. responses.

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(A) (B)

(E) (F)

FigureFigure 8. 8.Inhibition Inhibition of Drp1 of Drp1 by shDrp1 by shDrp1 and Mdivi-1 and decreasedMdivi‐1 thedecreased cytosolic the ROS, cytosolic rescued mitochondrialROS, rescued massmitochondrial and MMP, mass and reducedand MMP, apoptosis and reduced due to apoptosis the infection due byto the PCV2. infection PK-15 by cells PCV2. in 24-well PK‐15 platescells in were24‐well first plates infected were with first the infected lentivirus with containing the lentivirus specific containing shDrp1 specific (shDrp1) shDrp1 for 12 (shDrp1) h and then for infected 12 h and withthen PCV2 infected for 48with h. PCV2 Alternatively, for 48 h. PK-15 Alternatively, cells were PK infected‐15 cells with were PCV2, infected treated with with PCV2, 4 µ Mtreated Mdivi-1 with at 4 2μ hpi,M Mdivi and then‐1 at incubated 2 hpi, and for then an additionalincubated for 46 h.an ( Aadditional) Cytosolic 46 ROS h. (A in) Cytosolic the cells were ROS evaluated in the cells using were flowevaluated cytometry, using shown flow cytometry, as the mean shown value ofas thethe DCFmean fluorescence value of the intensity. DCF fluorescence (B) JC-1 fluorescence intensity. (B was) JC‐ depicted1 fluorescence as the ratio was betweendepicted redas the and ratio green between fluorescent red and signals green to fluorescent indicate changes signals in to the indicate mitochondrial changes membranein the mitochondrial potential. ( membraneC) Mitochondrial potential. mass (C was) Mitochondrial measured with mass MitoTracker was measured green with staining MitoTracker using flowgreen cytometry. staining ( Dusing) Apoptosis flow cytometry. rates of PK-15 (D) cells Apoptosis examined rates using of flow PK‐ cytometry.15 cells examined (E,F) The using bar charts flow showcytometry. fold changes (E,F) The of caspase bar charts 3 (E show) and fold caspase changes 9 (F) of enzyme caspase activities 3 (E) and in caspase the cells. 9 Bar(F) enzyme charts (A activities,C,E,F) showin the the cells. mean Bar chartsSEM of (A three,C,E, independentF) show the mean experiments. ± SEM of * pthree< 0.05 independent and ** p < 0.01. experiments. * p < 0.05 ± and ** p < 0.01.

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4.4. Discussion Discussion VirusesViruses induce induce host host cell cell responses, responses, such such as as the the unfoldedunfolded proteinprotein responseresponse (UPR)(UPR) andand autophagy, andand can can hijackhijack UPRUPR and autophagy to promote promote their their replication replication and and infectivity infectivity [32–34]. [32–34]. Induction Induction of ofautophagy autophagy and and UPR UPR via viaviral viral infection infection could could lead to lead the toactivation the activation of persistent of persistent cellular cellularstresses, stresses,including including oxidative oxidative stress, which stress, is whichinvolved is involved in the activation in the activation or modulation or modulation of apoptotic of apoptoticpathways pathways[34–36]. PCV2, [34–36 ].thoughPCV2, the though smallest the smallest one known one known to infect to infect mammals, mammals, is a is virus a virus that that is is versatile inin activatingactivating host host cell cell responses, responses, such such as as ER ER stress stress [ [4,37,38],4,37,38], oxidative oxidative stress stress [ [4,23,29],4,23,29], autophagyautophagy [[5,6,24],5,6,24], ORF3-dependentORF3‐dependent apoptosisapoptosis [3[3],], andand ORF3-independent ORF3‐independent mitochondrial mitochondrial apoptosis apoptosis [ 4[4].]. TheThe majormajor questionsquestions here here are are where where these these cellular cellular responses responses are are intertwined intertwined during during PCV2 PCV2 infection infection and and what what areare the the mechanisms mechanisms of of such such a a crosstalk. crosstalk. Because Because PCV2 PCV2 infection infection induces induces increased increased ROS ROS generation generation andand oxidative oxidative stressstress [ 23[23,29],,29], we we were were particularly particularly interested interested in in understanding understanding whatwhat rolesroles the the ROS ROS playplay inin mitochondrial mitochondrial dynamicsdynamics towardtoward autophagy autophagy and and apoptosis. apoptosis. Here,Here, forfor thethe ﬁrstfirst time,time, wewe demonstrateddemonstrated that that the the generation generation of of ROS ROS during during PCV2 PCV2 infection infection induced induced mitophagy mitophagy in PK-15 in PK cells‐15 cells via thevia ROS-mediated the ROS‐mediated activation activation of Drp1 of Drp1 (Figure (Figure9). 9).

Figure 9. A proposed model of mitophagy and mitochondrial apoptosis induced by PCV2 infection. Figure 9. A proposed model of mitophagy and mitochondrial apoptosis induced by PCV2 infection. PCV2 infection induces oxidative stress, loss of mitochondrial mass, and mitochondrial membrane PCV2 infection induces oxidative stress, loss of mitochondrial mass, and mitochondrial membrane potential, probably through its capsid protein [4], leading to mitochondrial fission, mitophagy, and potential, probably through its capsid protein [4], leading to mitochondrial fission, mitophagy, and mitochondrial apoptosis. ROS induced by PCV2 infection stimulates Drp1 phosphorylation, probably mitochondrial apoptosis. ROS induced by PCV2 infection stimulates Drp1 phosphorylation, probably by activating its upstream molecule CDK1. N-acetyl-L-cysteine (NAC) treatment could reverse these by activating its upstream molecule CDK1. N‐acetyl‐L‐cysteine (NAC) treatment could reverse these PCV2-induced phenotypes, and inhibited Drp1 phosphorylation, PINK1 expression, and Parkin PCV2‐induced phenotypes, and inhibited Drp1 phosphorylation, PINK1 expression, and Parkin translocation to mitochondria. Inhibition of Drp1 via Mdivi-1 treatment or specific RNA silencing translocation to mitochondria. Inhibition of Drp1 via Mdivi‐1 treatment or specific RNA silencing could reduce the amount of ROS and alleviate PCV2-induced mitophagy and mitochondrial apoptosis. could reduce the amount of ROS and alleviate PCV2‐induced mitophagy and mitochondrial Theapoptosis. recruitment of Parkin to mitochondria is considered to be a hallmark of mitophagy [12]. PINK1 is another essential component of mitophagy, which is involved in Parkin’s translocation to The recruitment of Parkin to mitochondria is considered to be a hallmark of mitophagy [12]. damaged mitochondria and its subsequent activation [17]. Our study revealed that PCV2-induced PINK1 is another essential component of mitophagy, which is involved in Parkin’s translocation to mitophagy was shown as a significant translocation of Parkin to mitochondria and increased levels of damaged mitochondria and its subsequent activation [17]. Our study revealed that PCV2‐induced PINK1 and LC3II in infected cells, as well as perinuclear accumulation of damaged mitochondria and mitophagy was shown as a significant translocation of Parkin to mitochondria and increased levels the subsequent formation of mitophagosomes and mitophagolysosomes. Drp1 is known to mediate the of PINK1 and LC3II in infected cells, as well as perinuclear accumulation of damaged mitochondria scission of mitochondrial membranes, regulate mitochondrial dynamics, and influence mitochondrial and the subsequent formation of mitophagosomes and mitophagolysosomes. Drp1 is known to apoptosis and mitophagy [39,40]. We found that PCV2 infection caused a time-dependent increase in mediate the scission of mitochondrial membranes, regulate mitochondrial dynamics, and influence p-Drp1 and ROS. The fact that PCV2-induced Drp1 phosphorylation and mitophagic responses could mitochondrial apoptosis and mitophagy [39,40]. We found that PCV2 infection caused a time‐ be markedly reduced by the ROS scavenger NAC (Figure6A,B) indicates that ROS generated during dependent increase in p‐Drp1 and ROS. The fact that PCV2‐induced Drp1 phosphorylation and PCV2 infection might be the upstream molecule of Drp1 activation with subsequent mitochondrial mitophagic responses could be markedly reduced by the ROS scavenger NAC (Figure 6A,B) indicates fission and mitophagy. that ROS generated during PCV2 infection might be the upstream molecule of Drp1 activation with subsequent mitochondrial fission and mitophagy.

Viruses 2020, 12, 289 16 of 19

The link between PINK1 and Parkin is well established. PINK1 stabilizes dysfunctional mitochondria and recruits Parkin from the cytosol to damaged mitochondria [41]. However, the relationship between Drp1 and PINK1/Parkin remains elusive. Drp1 and Parkin were co-recruited to mitochondria in the proximity of PINK1 following mitochondrial depolarization, indicating a spatial coordination between these molecules [42]. Our results suggest that Drp1 might have positive effects on the PINK1/Parkin pathway in PCV2-infected cells because the inhibition of Drp1 by Mdivi-1 or RNA silencing decreased the PINK1 expression and reduced the Parkin recruitment to mitochondria (Figure7). This is similar to the work of Park et al. who showed that the knockdown of Drp1 significantly blocked the increase of PINK1 and Parkin expression in the mitochondrial fraction of cells treated with carbonyl cyanide m-chlorophenylhydrazone (CCCP) [43]. Xiao et al. showed that ROS was able to trigger PINK1/Parkin-dependent mitophagy in CCCP-treated cells [44]. These findings imply that there is a close relationship between Drp1 and PINK1/Parkin in CCCP-induced mitophagy. CDK1 is known to mediate mitochondrial activities in the cell cycle progression and stress response [45]. Kashatus et al. showed that the small Ras-like GTPase RALA could be relocalized upon phosphorylation to the mitochondria, where it concentrated RALBP1 and Drp1. RALBP1 was associated with CDK1, which led to phosphorylation of Drp1 on Ser616 [46]. Another report also revealed the involvement of CDK1 in Drp1 phosphorylation and mitochondrial fission [27]. Here, we showed that the specific inhibition of CDK1 by RO-3306 suppressed Drp1 phosphorylation in PCV2 infected cells, and NAC treatment reduced the level of both CDK1 and p-Drp1 (Figure4). Thus, we speculate that CDK1 might be involved in Drp1 phosphorylation by ROS during PCV2 infection. However, the specific mechanism regarding how CDK1 is activated remains to be explored. Mitochondria are known to be the main source of cellular ROS. Mitochondrial dysfunction may lead to the overproduction of ROS [47]. We found that the silencing of Drp1 by shRNA reduced the cellular ROS level. Similar results have been reported in other cell models. In T lymphocytes, ROS generation is dependent on mitochondrial fission. The inhibition of Drp1 resulted in reduced ROS levels [41]. In a high-glucose-induced cell death model, mitochondrial fragmentation was found as an upstream factor for ROS overproduction, and the inhibition of mitochondrial fission normalized the cellular ROS level [48]. Therefore, the role of Drp1 in PCV2-induced mitophagy seems to be two-sided: one as a receiver of the ROS signal, probably via its upstream molecules, and the other as a stimulator of ROS generation via its regulated mitochondrial fission (Figure9). Mitophagy, as a contrast to apoptosis, could be pro-survival to the host cells, favoring viral propagation and persistence. We found that the inhibition of Drp1 by Mdivi-1 or shDrp1 decreased both the mitophagic and mitochondrial apoptotic responses of PCV2-infected cells. This is different from what was seen in other viruses, such as HBV, HCV, CSFV, porcine reproductive and respiratory syndrome virus (PRRSV), and transmissible gastroenteritis virus (TGEV). In cells infected with these viruses, the induction of mitophagy was found to prevent apoptosis, while the inhibition of mitophagy promoted apoptosis [19,20,26,49–51]. This may result from different mechanisms of induction of either mitophagy or apoptosis by different viruses. Zhu et al. reported that TGEV infection induced mitophagy and increased the expression of DJ-1 [51], a molecule that is involved in several signaling pathways, including the control of mitochondrial quality and reaction to oxidative stress [52,53], while the silencing of DJ-1 inhibited mitophagy with reduced ROS, but increased apoptosis in the virus-infected cells [51]. They found marked localization of the nucleocapsid protein N of TGEV in the mitochondrial fraction, while the role of the N protein either in mitophagy or apoptosis was unexplored. Results from this study and our recent work have revealed that an ORF3-inactivated PCV2 mutant could still induce mitophagy and mitochondrial apoptosis [4]. PCV2 Cap was sufficient to induce ROS [4], in addition to its ability to cause an unfolded protein response and apoptosis [38]. Therefore, we tend to believe that it is the general oxidative stress due to excessive generation of ROS during PCV2 infection that targets both mitophagy and mitochondrial apoptosis via Drp1 activation, with mitochondrial fission being the initial step of these cellular events [41]. Viruses 2020, 12, 289 17 of 19

In conclusion, our study shows that PCV2-induced ROS was responsible for mitophagic and/or mitochondrial apoptotic responses through the activation of Drp1. Oxidative stress is one of the major factors in PCV2 pathogenesis. This warrants the use of antioxidant treatments in PCVAD as an adjunctive therapy.

Author Contributions: Y.Z., W.F., and X.L. designed the experiments. Y.Z. and R.S. performed the experiments. Y.Z. and R.S. analyzed the data. Y.Z. wrote the original manuscript. W.F. and X.L. reviewed the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This work is part of the project sponsored by the National Natural Science Foundation of China (31272534). Conﬂicts of Interest: The authors declare no conﬂicts of interest, and all the authors approved this manuscript for publication.

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