Human herpesvirus type 1 and type 2 disrupt mitochondrial dynamics in human keratinocytes

Human herpesvirus type 1 and type 2 disrupt mitochondrial dynamics in human keratinocytes Mitochondrial movement and distribution throughout the cytoplasm is crucial for maintaining cell homeostasis. Mitochon- dria are dynamic organelles but can be functionally disrupted during infection. Here, we show that the ubiquitous human pathogens HHV-1 and HHV-2 induce changes in the mitochondrial morphology and distribution in the early and late phases of productive infection in human keratinocytes (HaCaT cells). We observed a decrease in the mitochondrial potential at 2 h postinfection and a decrease in cell vitality at 24 h postinfection. Moreover, we found that mitochondria migrated to the perinuclear area, where HHV-1 and HHV-2 antigens were also observed, mainly in the early stages of infection. Positive results of real-time PCR showed a high level of HHV-1 and HHV-2 DNA in HaCaT cells and culture medium. Our data demonstrate that HHV-1 and HHV-2 cause mitochondrial dysfunction in human keratinocytes. Introduction simplex labialis, genital herpes, keratitis and encephalitis [3, 12]. Keratinocytes serve as the r fi st line of defense during Human herpesviruses types 1 and 2 (HHV-1 and HHV-2) skin infections. Their role includes recognition of infectious belong to the subfamily Alphaherpesvirinae of the family agents and initiation of innate immune response, which leads Herpesviridae. Infection with these viruses is widespread to production of cytokines and recruitment of neutrophils. in human populations all over the world. To enter its host, However, viruses have evolved multiple strategies that allow a virus must overcome a barrier of mucosal surfaces, skin them to escape from the immune response and complete or cornea. HHV-1 and HHV-2 target keratinocytes during their replication. One of these strategies includes utiliza- initial entry and establish a primary infection in the epithe- tion of host cell mitochondria. Mitochondria are organelles lium, which is followed by latent infection in neurons. Infec- that are involved in a variety of metabolic and cellular func- 2+ tions are usually mild but may spread to the central nervous tions, including Ca homeostasis, ATP production, and pro- system, causing serious neurological disorders. HHV-1 and grammed cell death. They also participate in the synthesis of HHV-2 have been identified as causative agents of various, key metabolites and are the primary source of endogenous mild and even life-threatening diseases, namely, herpes reactive oxygen species [6, 16]. Mitochondria form a network distributed throughout the cell [17]. They are dynamic organelles that constantly Handling Editor: Zhongjie Shi. change their shape, length and movement along cytoskel- * Joanna Cymerys etal tracks. There are two main processes responsible for jcymerys@op.pl mitochondrial homeostasis: fission and fusion. These are crucial for maintenance of a proper number of functional Division of Microbiology, Department of Preclinical mitochondria. Fusion allows the exchange of DNA, con- Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences, Ciszewskiego 8, tents and metabolites between neighboring mitochondria. 02-786 Warsaw, Poland Fission of the mitochondrial network enables the distribution Department of Physiological Sciences, Faculty of Veterinary and transport of mitochondria within and outside the cell Medicine, Warsaw University of Life Sciences, to a place where energy demand is high. Moreover, fission Nowoursynowska 159, 02-776 Warsaw, Poland adjusts apoptosis to eliminate damaged mitochondria [1, 13]. Military Institute of Hygiene and Epidemiology, Kozielska 4, The main proteins responsible for fusion are Mfn-1, Mfn-2 01-163 Warsaw, Poland Vol.:(0123456789) 1 3 2664 M. Chodkowski et al. and Opa-1. In mice, knockouts of the genes encoding these no. CRL1587). To produce the virus for the experiments, proteins results in lethality to embryos and mitochondrial the Vero cells were infected with HHV-1 or HHV-2 at 0.001 dysfunction. Cells with defective Mfn1 and Mfn2 have a plaque-forming units (PFU)/cell. One hour after infection disrupted mitochondrial network and many small, punctate at 37 °C, the inoculum was removed by aspiration, fresh mitochondria. The most important protein participating in culture medium was added, and the cells were cultured for the defragmentation of the mitochondrial network in the 3 days. Culture supernatants were harvested at 72 h after cell is dynamin-related protein 1 (Drp1), which has GTPase virus challenge, and after three cycles of freezing (-80 °C) activity. This protein migrates between the cytosol and the and thawing at RT, they were clarified by centrifugation at mitochondrial network and binds to the mitochondrial outer 800 g for 10 min and stored in small volumes at -80 °C. A membrane during fission. Dysregulation of mitochondrial virus stock suspension containing approximately 10 PFU/ motility has been observed in many human diseases, mainly ml was used in all the experiments. neurodegenerative disorders, cancer, diabetes, and arrhyth- HaCaT cells (10 cells per well) were infected with mias, and also during ageing [9]. HHV-1 or HHV-2 for 60 min at 37 °C. After adsorption, It is believed that mitochondria play a major role in viral the inoculum was removed by aspiration and fresh culture infections. Murata et al. [10] have shown that, in Vero cells, medium was added. The cells were then incubated for 2, 24 mitochondria are recruited to the site of viral replication and or 48 hours at 37 °C with 5% CO . morphogenesis. They have also shown that mitochondria migrate to the perinuclear area where HHV-1 tegument was Real‑time cell growth analysis present. It is possible that mitochondria, as energy centers of the cell, provide the energy necessary for replication of the The growth kinetics, behaviour and morphology of HaCaT virus. It has also been shown that the mitochondrial potential cells infected with HHV-1 or HHV-2 were analysed using a is stable up to 6 hours postinfection (h p.i.) but decreases JuLI™ Br Live Cell Analyser system for a bright-field image during the late phase of infection [10]. In addition, many analysis (NanoEnTek, Korea) [4]. HaCaT cells (10 cells per types of viral proteins have been identified as responsible well) were seeded in a 6-well plate and infected with HHV-1 for the modulation of apoptosis. Alphaherpesviruses, like or HHV-2 as described above. Cell-growth images were cap- other large DNA viruses, encode proteins that interfere with tured for 48 h at 7-min intervals. Cell confluence analysis mitochondrial function and localization to block the apop- was done and a real-time cell growth curve was generated totic pathway. However, little is known about the role of using JuLI Br PC software. All images were captured at mitochondria in skin cells, which function as the primary objective magnification of ×4. barrier and, on the other hand, constitute a very important replication site. Real‑time PCR To the best of our knowledge, there are no data available concerning the effects of productive viral infection on mito- To determine the number of viral DNA copies per reac- chondria and the mitochondrial network in keratinocytes, tion, a standard curve was prepared as described previously which are crucial in the first steps of herpesviral infection. [7]. Briefly, fragments of glycoprotein B gene sequence of Therefore, in the present study we investigated the effect HHV-1 and HHV-2 were amplified using appropriate prim- of HHV-1 and HHV-2 infection on cell vitality, apoptosis, ers: HSV -1F ext (G T G A T G TT G AGG T CG A T G AAGG T) mitochondrial network rearrangement, and mitochondrial and HS V -1R e xt (A C A A CG CG A CGC A C A T C A A GG T) potential in human keratinocytes in vitro. a n d H SV - 2 F ex t ( C G T AC G A T G AG T T T G T G T T G G C G A ) and HSV -2R ext (T CA GCT GG T GAG AG T ACG CG T A). The products were cloned in pGEM-T Easy Vector. Serial Materials and methods dilutions of recombinant plasmids were prepared ranging from 10 to 10 copies per reaction. Real-time PCR was Cell culture and virus strains performed in 96-well plates using a 7500 Real Time PCR System thermocycler (Applied Biosystems) with TaqMan Human keratinocytes (HaCaT cells) were cultured as a mon- Universal Master Mix II (Applied Biosystems) and probes olayer using Dulbecco’s modified Eagle’s medium (DMEM; labelled with JOE as described previously [11]. Gibco) supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, 100 U of penicillin per ml and 100 mg of Immunofluorescent staining procedures streptomycin per ml, at 37 °C in a humidified atmosphere of 5% CO . Cells were grown to full confluence with a medium HaCaT cells seeded on glass coverslips in a 12-well plate change every 2 days. The McIntyre strain of HHV-1 and were infected with HHV-1 or HHV-2. At 2, 24 and 48 h p.i. HHV-2 strain 333 were grown in Vero cell cultures (ATCC 1 3 Disruption of mitochondrial dynamics by human herpesviruses 2665 (hour postinfection) coverslips were incubated with 100 nM images, obtained by confocal microscopy were used accord- MitoRed (Sigma-Aldrich) for 30 min at 37 °C, then washed ing to the protocol established by Valente et al. [18]. Each three times with culture medium and fixed in 3.7% para- analysis was performed on ten cells. formaldehyde/PBS (Sigma-Aldrich). The presence of viral antigens was detected by means of direct immunofluores- Image cytometry cence, using FITC-conjugated polyclonal rabbit anti-herpes simplex virus 1/2 serum (Dako, dilution 1:200). Cellular fluorescence was quantified using a NucleoCoun- Additionally, at 2 and 24 h p.i., HaCaT cells were stained for dynamin-related protein 1 (Drp1) detection. At the begin- ter NC-3000 image cytometer (ChemoMetec). The Nucle- oCounter system was used for evaluation of mitochondrial ning, cells were washed twice in PBS (Sigma-Aldrich) and fixed in 3.7% paraformaldehyde/PBS (Sigma-Aldrich) for transmembrane potential (Δψ) and cell vitality at 24 h p.i. In the mitochondrial potential assay, HaCaT cells were stained 30 min at room temperature (RT), then permeabilized with 0.5% Triton X-100 (Sigma-Aldrich) solution in PBS. Before with JC-1 (cationic dye 5,5,6,6-tetrachloro-1,1,3,3-tetraeth- ylbenzimidazol-carbocyanine iodide; ChemoMetec A/S). staining, fixed HaCaT cells on coverslips were blocked with PBS containing 1% bovine serum albumin (BSA) (Sigma First, the suspended HaCaT cells were diluted with PBS to a final concentration of 1.5 ×10 cells/mL. The samples Chemicals) for 30 min at room temperature. The presence of Drp1 was detected by using DNM1L polyclonal antibody were then incubated with 12.5 mL of a 200 mg/mL solution of JC-1 for 10 min at 37 °C. After incubation, samples were (Invitrogen, dilution 1:500) and Alexa Fluor 488 goat anti- rabbit (Invitrogen; dilution 1:250). washed twice in PBS and resuspended in 250 mL of a 1 mg/ mL solution of 4′,6-diamidino-2-phenylindole in PBS. In Cell nuclei were stained with Bisbenzimidine/Hoechst 33258 according to manufacturer’s recommendations. the vitality assay (detection of changes in the intracellular level of thiols), HaCaT cells were stained with VitaBright-48 Afterwards, coverslips were mounted on microscope slides using anti-fade mounting medium (Sigma-Aldrich). Unin- (ChemoMetec A/S), acridine orange (ChemoMetec A/S), and propidium iodide (PI; ChemoMetec A/S). Suspended fected HaCaT cells served as a negative control. For Drp1 staining, HaCaT cells pre-incubated with Dynasore (GTPase HaCaT cells were diluted with PBS to a final concentra- tion of 2.0 × 10 cells/mL and were mixed with 5 mL of inhibitor; 80 µM/ml) for 60 min before infection served as a positive control. Results were evaluated using a confocal VitaBright-48·PI·acridine orange. Subsequently, the samples were examined using a Nucleo-Counter NC-3000 according microscope (Leica TCS SP8-WWL). to manufacturer’s instructions. The results were analyzed using the NucleoView NC-3000 software (details of the Confocal microscopy NucleoCounter NC-3000 design and capabilities are avail- able at www.chemo metec .com) [2]. Confocal images were acquired using a Leica white light A positive control for mitochondrial potential analysis laser scanning confocal microscope (Leica TCS SP8-WWL, was prepared by adding CCCP (carbonyl cyanide m-chlo- KAWA.SKA Sp. z o.o., Poland) with a 63x oil-immersion rophenyl hydrazone; 5 µl per ml of cell culture medium). lens, using excitation at 405 nm, 499 nm, 569 nm for Hoe- In parallel, uninfected HaCaT cells served as a negative chst, FITC and MitoRed, respectively. Images were captured control. and converted to 24-bit tiff files for visualization using the Leica Application Suite X (LAS X) software platform (Leica Microsystems). Statistical evaluation Analysis of mitochondrial morphology The results were statistically evaluated by one-way analysis of variance (ANOVA) using the Student–Newman–Keuls For mitochondrial morphology analysis, MiNa Single Image multiple comparisons test and the Tukey–Kramer multi- macro was used. This tool allows the number of individuals, ple comparisons test. This analysis was performed using number of networks, mean length of branches/rod, mean net- TM GraphPad Prism version 4.03 software (GraphPad Soft- work size, mean network size per branch, and mitochondrial ware Inc., San Diego, CA, USA). Statistical differences were footprint to be computed. In order to perform this analysis 1 3 2666 M. Chodkowski et al. Fig. 1 A and B. Morphological changes of HaCaT cells infected with Real-time PCR analysis of viral DNA copy number in HaCaT cells HHV-1. Cells were observed for 48 h using a JuLI™ Br Live Cell and cell medium during HHV-1 and HHV-2 infection. Statistical dif- Analyzer. CPE was manifested as cell destruction and fusion of cells, ferences were interpreted as significant at P < 0.05 (*) and P < 0.01 as confirmed by a growth curve. Objective magnification, x4. C. (**) interpreted as significant at P < 0.05 (*) and highly signifi- Fig. 2 The mitochondrial network in uninfected control HaCaT cells. ► A-F. Immunofluorescence staining of mitochondria (red fluorescence) cant at P < 0.01 (**). and the nucleus (blue fluorescence). A yellow arrow indicates tubular mitochondria. G. The original image was processed using “unsharp mask”, “CLAHE”, “median”, “binarize” and “skeletonize” for detec- Results tion different mitochondrial shapes and structures. H. Tubular mito- chondria. I. Punctate mitochondria. J. Branched mitochondrial net- work HHV‑1 and HHV‑2 replication in HaCaT cells During HHV-1 infection, a cytopathic effect (CPE) was medium at 24 h p.i. and 48 h p.i., which was most probably observed as morphology changes in HaCaT cells. After 24 the result of the release of progeny virions from the cell h p.i. and 48 h p.i., we observed a diffuse cytopathic effect, (Fig. 1C). manifesting as cell rounding, shrinking and lysis of indi- vidual cells. Interestingly, we did not observe large changes Changes in the mitochondrial network in the confluence of monolayer during the study. At 48 h p.i., during HHV‑1 and HHV‑2 infection the confluence has decreased to around 90% (Fig.  1A and B). Similar results were observed with cells infected with In uninfected HaCaT cells, the mitochondrial network was HHV-2 (data not shown). dense, branched and spread evenly throughout the cell The quantitative PCR analysis showed a statistically (Fig. 2). We observed many tubular, long and highly inter- significant increase in the DNA copy number of analyzed connected mitochondria localized in the subcellular region viruses in comparison to the uninfected control (Fig. 1C). and a small number of punctate mitochondria. Moreover, The highest, statistically significant, increase in the copy we observed fusion of mitochondria of dividing cells, number of viral DNA was observed at 48 h p.i. with HHV-1 which accumulated in close proximity to the chromosomes 7 7 and HHV-2 (2.8 ± 1.01 × 10 and 1.9 ± 1.31 × 10 cop- (Fig. 2A and D). In uninfected cells, we distinguished three ies/ml, respectively; P <0.01). We also found a signifi- types of mitochondrial shape: tubular (Fig. 2H), punctate cant increase in the viral DNA copy number in the culture (Fig. 2I) and branched mitochondrial network (Fig. 2 J). 1 3 Disruption of mitochondrial dynamics by human herpesviruses 2667 1 3 2668 M. Chodkowski et al. HHV-1 and HHV-2 infection caused changes in the mor- plaques and multinucleated cells was also observed (Fig. 3B phology of the mitochondrial network. At 2 h p.i., with I and II). both HHV-1 and HHV-2, we observed the interaction of Examples of cells selected for mitochondrial morphology viral particles with the mitochondrial network. The viral analysis are shown in Fig. 4A-G’. At 2 h p.i., with HHV-1, antigens were located near the cell nucleus (Fig. 3A), and we observed a decrease in the number of mitochondrial net- interestingly, they partially colocalized with mitochondria works, together with an increase in the number of individual (Fig. 3A XIII and XIV). At 24 h p.i. with HHV-1 (Fig. 3B), mitochondrial objects. In addition, both the percentage of we observed changes in the shape of the mitochondrial net- cross-linked mitochondria and the length of the network work and its distribution within the cell in comparison to branches decreased. Moreover, the total area of mitochon- uninfected HaCaT cells. The mitochondrial network was dria decreased. At 24 h p.i., the number of mitochondrial organized near the nucleus. At the same time, we observed objects increased, and we observed a decrease in the number colocalization of some viral particles with mitochondria. of mitochondrial networks. We also observed a reduction in Similar observations were made in the case of infection with the total mitochondrial area. At 48 h p.i., we observed a sig- HHV-2. After 48 h p.i. we observed an increase in viral rep- nificant increase in the number of mitochondrial objects. The lication. During HHV-1 infection, the mitochondrial network mean number of branches per network was reduced at 2, 24, was completely fragmented. In the case of HHV-2 infec- and 48 h p.i., but the results were not statistically significant. tion, after 48 h we observed accumulation of viral antigens HHV-1 infection caused various changes in the morphology within the cells. Moreover, a cytopathic effect in the form of of the mitochondrial network in HaCaT cells. This analysis Fig. 3 Mitochondrial network organization in HaCaT cells infected effect in HaCaT cells during infection with HHV-2 at 48 h p.i. is vis- with HHV-1 or HHV-2 at 2 (A), 24 and 48 h p.i. (B). A yellow arrow ible. The red arrow in panel B (II) indicates the distribution of viral indicates colocalization of mitochondria with viral antigen (A, VIII). antigen along the edges of the cell, and the white arrow indicates A profile plot of fluorescence signal intensities along the yellow line interruption of the mitochondrial network between infected cells (B, visible in panel A XIV indicates the colocalization of mitochondria II) and viral antigen (A, XIII). In panel B (I), the syncytial cytopathic 1 3 Disruption of mitochondrial dynamics by human herpesviruses 2669 Fig. 4 A-G. Mitochondrial network analysis performed on 10 HaCaT at 24 h p.i; G’, a single cell infected with HHV-1 at 48 h p.i. H and I. cells. A’, a single uninfected control cell; B’, a single cell infected Summary statistics for all infected and control HaCaT cells. The box with HHV-1 at 2 h p.i; C’, a single cell infected with HHV-1 at 24 h plot shows median (horizontal lines), first-to-third quartile (box), and p.i; D’, a single cell infected with HHV-1 at 48 h p.i; E’, a single cell extreme values (%) (**, P < 0.01; *, P < 0.05). Each analysis was infected with HHV-1 at 2 h p.i.; F’, a single cell infected with HHV-1 performed on 10 cells shows that the mitochondrial network underwent s fi sion and mitochondria (Fig. 5B). During infection, we observed that was fragmented after infection (Fig 4H). Drp1 was partially translocated from the cytoplasm to the HHV-2 infection caused changes similar to those caused outer membrane of the mitochondria. After 2 h p.i., with by HHV-1. We observed a gradual increase in the number of both HHV-1 and HHV-2, we observed a colocalization of mitochondria, which reached a maximum at 48 h p.i. We also Drp1 with mitochondria localized in perinuclear area. After observed changes in mitochondrial cross-linking, as well as 24 h p.i. we observed a decrease in Drp1 protein expression a decrease in the length of the mitochondrial branches, but in infected cells together with progressive disintegration of these differences were statistically insignificant. At 24 and the mitochondrial network (Fig. 5C-F). 48 h p.i., a statistically significant decrease in the overall mitochondrial surface area was observed (Fig. 4I). The mitochondrial potential and vitality of HaCaT We then investigated the distribution of Drp1 in HaCaT cells during HHV‑1 and HHV‑2 infection cells. In uninfected control cells, Drp1, as well as the mito- chondrial network, was distributed evenly in the cytoplasm In this assay, changes in the mitochondrial potential that (Fig. 5A). After 24 h of treatment with Dynasore, which occurred during HHV-1 or HHV-2 infection were evaluated is an inhibitor of GTPases, we observed a decrease in the (Fig. 6A, C). Considering the pivotal role of mitochondria in Drp1 level in treated cells and the presence of tubular 1 3 2670 M. Chodkowski et al. Fig. 5 Localization of Drp1 in HaCaT cells. Immunofluo- rescence staining was used to examine mitochondrial translo- cation of Drp1 fission protein (white arrows indicate localiza- tion of Drp1). A. Uninfected HaCaT cells. B. Cells treated with Dynasore. C and D. Cells infected with HHV-1. E and F. Cells infected with HHV- 2. Drp1, green fluorescence; mitochondria, red fluorescence; nuclei, blue fluorescence. Objective magnification, x63 orchestrating the apoptotic pathway, we measured the mito- with dysfunctional mitochondria; JC-1 was present in the chondrial potential using the JC-1 method. The principle of cytosol in its monomeric form, emitting green fluorescence. the test depends on the fact that JC-1, a mitochondrial-poten- During infection with both HHV-1 and HHV-2 (2 and 24 h tial-sensitive dye, accumulates in the matrix of mitochon- p.i.), we observed changes in mitochondrial potential. The dria by forming J-aggregates with red fluorescence when most significant decrease was observed in HaCaT cells at the mitochondrial potential is high and becomes a monomer 2 h p.i. However, at 24 h p.i., a decrease in the mitochon- with green fluorescence when it is low. HaCaT cells treated drial potential had also occurred, but it was not statistically with CCCP served as a positive control (Fig.  6A IV). In significant and was similar to the values obtained with the these cells, a low mitochondrial potential was observed. negative control (Fig. 6A, C). It was also found that HHV-1 Cells treated with CCCP exhibited the characteristics of cells and HHV-2 infection caused a reduction in the vitality of 1 3 Disruption of mitochondrial dynamics by human herpesviruses 2671 Fig. 6 A and C. Mitochondrial membrane potential (ΔΨ) of HaCaT HHV-1 at 24 h p.i.; V, HaCaT cells infected with HHV-2 at 2 h p.i.; cells. Uninfected control cells have a high mitochondrial potential VI, HaCaT cells infected with HHV-2 at 24 h p.i. B and D. Cell (I). CCCP-treated HaCaT cells (positive control) have a low mito- vitality assay. HaCaT cells were stained with VitaBright -48 ™ (VB- chondrial potential (IV). The level of green fluorescence in cells 48™), acridine orange (AO), and propidium iodide (PI). I, control is indicated as a percentage (**, P < 0.01; *, P < 0.05). II, HaCaT cells; II, HaCaT cells infected with HHV-1 at 24 h p.i. III, HaCaT cells infected with HHV-1 at 2 h p.i.; III, HaCaT cells infected with cells infected with HHV-2 at 24 h p.i. D. Level of dead and live cells HaCaT cells at 24 h p.i. (Fig. 6B and D). The decrease in confirmed by qPCR analysis, caused morphological changes vitality was more pronounced in the case of HHV-1 infection in infected cells (Fig. 1). These changes were manifested as than after infection with HHV-2. a loss of intercellular connections between HaCaT cells and the presence of syncytial cells at 48 h p.i., which was also observed in previous studies [12]. Overall, the cytopathic effect caused by HHV-1 and HHV-2 in the late stages of Discussion infection was very similar. In this study, we performed an analysis of mitochondrial The major target cells during primary and recurrent HHV-1/ dynamics during HHV-1 and HHV-2 infection in human HHV-2 infections are cells of epithelial and neuronal ori- keratinocytes. Changes in the mitochondrial network were gin. During the initial exposure, HHV-1 and HHV-2 use apparent in the early stage of infection with HHV-1 or HHV- mucosal epithelial cells, including epidermal keratinocytes, 2. We also observed the migration of mitochondria to the as the primary portal of entry, and infection then spreads perinuclear area, both in the early and late stages of infec- through the epithelium. Additionally, after episodic reactiva- tion. Similar results were obtained previously, and it was tion from latency established in neurons, newly replicating also shown that microtubules are responsible for the trans- viruses infect epithelial cells, often leading to recurrent her- port of mitochondria. Moreover, the migration of mitochon- petic lesions. The productive cycle of HHV-1 and HHV-2, dria to the perinuclear area during infection with HHV-1 was 1 3 2672 M. Chodkowski et al. inhibited after addition of the microtubule inhibitor nocoda- The assay provides a reliable and fast way to evaluate cell zole [10]. These results have provided new insights into the viability, as an appearance of free thiols is the early indica- various strategies of cytoskeleton utilization that are used tor of apoptosis [14]. In our study, we did not observe a during herpesviral infections. Apparently, the cytoskeleton is significant change in the level of free thiols after infection not only necessary for transport of the virions to the nucleus, with either HHV-1 or HHV-2. This may indicate that only a as has been described before, but also supports spatial rear- small percentage of infected HaCaT cells undergo apoptosis. rangement of cellular organelles in order to facilitate the In conclusion, our results suggest that productive HHV-1 replication cycle [5, 15]. and HHV-2 infections cause changes in the mitochondrial After HHV-1 or HHV-2 infection, we also observed network in comparison to uninfected control cells. These changes in mitochondrial morphology. In control cells, the changes manifest themselves mainly as defragmentation mitochondria were elongated and branched. Murata et al. of the mitochondrial network, an increase in the number also observed the changes in the mitochondrial area, but they of punctate mitochondria, and a decrease in the number did not characterize these changes [10]. According to ImageJ of tubular mitochondria. In addition, in the early stage of analysis, mitochondria were significantly affected by HHV-1 infection, mitochondrial membrane permeabilization occurs, or HHV-2 infection. Primarily, we observed defragmenta- which may be associated with rearrangement of the network. tion of the mitochondrial network and an increased number These results indicate that mitochondria are utilized in the of punctate mitochondria. We also detected colocalization early stages of HHV-1 and HHV-2 replication, as evidenced of punctate mitochondria with viral antigen. In the case of by the increased transport of punctate mitochondria to the HHV-1, in the late stage of infection, there was a decrease replication site and colocalization of mitochondria with viral in the number of mitochondrial networks, but we did not antigens in the perinuclear area. It is, however, worth stress- observe a significant change in the case of HHV-2 infection. ing that viral infection does not destroy the mitochondrial These changes were not observed in cells in which viral anti- network completely, which ensures survival of the cell and gen was not detected. Similar changes, including an increase enables viruses to complete their replication cycle. in the number of punctate mitochondria, were also observed Acknowledgements This work was funded by KNOW (Leading previously in the case of infection with Human herpesvirus National Research Centre) Scientific Consortium “Healthy Animal— type 5 (HHV-5) [8]. Our results suggest that HHV infection Safe Food”, decision of Ministry of Science and Higher Education causes increased fission of the mitochondrial network. More- No. KNOW2016/SGGW/PRO1/01/10, and by Internal Research Grant No. 505-10-023400-P00350-99 from WULS-SGGW in Warsaw. The over, we observed a translocation of Drp1 to the perinuclear authors would like to thank Prof. Zdzisław Gajewski, WULS-SGGW, area and binding of this protein to mitochondrial outer mem- Warsaw, Poland, for providing us access to the Leica TCS SP8-WWL brane. This may indicate that viral infection increases the confocal microscope. activity of this protein, stimulating the defragmentation of mitochondrial network. Compliance with ethical standards It is believed that one of the early indicators of apopto- sis is the permeabilization of the mitochondrial membrane. Conflict of interest The authors declare no conflict of interest. For that reason, we evaluated the mitochondrial potential Open Access This article is distributed under the terms of the Crea- using JC-1 dye. We believe that the internal mitochondrial tive Commons Attribution 4.0 International License (http://creat iveco potential decreased as a consequence of changes in the mmons.or g/licenses/b y/4.0/), which permits unrestricted use, distribu- mitochondrial network. It was the result of rearrangements tion, and reproduction in any medium, provided you give appropriate of the network as well as changes in the distribution of credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. mitochondria in the early stages of infection. Interestingly, the mitochondrial potential had been stabilized by 24 h p.i. At 2 h p.i., we observed a large loss in the mitochondrial potential after infection with both HHV-1 and HHV-2. References These results showed that the most significant changes 1. 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Namvar L, Olofsson S, Bergstrom T, Lindh M (2005) Detection 326. https ://doi.org/10.1016/j.acthi s.2017.03.001 and typing of herpes simplex virus (HSV) in mucocutaneous sam- ples by TaqMan PCR targeting a gB segment homologous for HSV types 1 and 2. J Clin Microbiol 43:2058–2064. https ://doi. org/10.1128/JCM.43.5.2058-2064.2005 1 3 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Archives of Virology Springer Journals

Human herpesvirus type 1 and type 2 disrupt mitochondrial dynamics in human keratinocytes

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Biomedicine; Virology; Medical Microbiology; Infectious Diseases
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Abstract

Mitochondrial movement and distribution throughout the cytoplasm is crucial for maintaining cell homeostasis. Mitochon- dria are dynamic organelles but can be functionally disrupted during infection. Here, we show that the ubiquitous human pathogens HHV-1 and HHV-2 induce changes in the mitochondrial morphology and distribution in the early and late phases of productive infection in human keratinocytes (HaCaT cells). We observed a decrease in the mitochondrial potential at 2 h postinfection and a decrease in cell vitality at 24 h postinfection. Moreover, we found that mitochondria migrated to the perinuclear area, where HHV-1 and HHV-2 antigens were also observed, mainly in the early stages of infection. Positive results of real-time PCR showed a high level of HHV-1 and HHV-2 DNA in HaCaT cells and culture medium. Our data demonstrate that HHV-1 and HHV-2 cause mitochondrial dysfunction in human keratinocytes. Introduction simplex labialis, genital herpes, keratitis and encephalitis [3, 12]. Keratinocytes serve as the r fi st line of defense during Human herpesviruses types 1 and 2 (HHV-1 and HHV-2) skin infections. Their role includes recognition of infectious belong to the subfamily Alphaherpesvirinae of the family agents and initiation of innate immune response, which leads Herpesviridae. Infection with these viruses is widespread to production of cytokines and recruitment of neutrophils. in human populations all over the world. To enter its host, However, viruses have evolved multiple strategies that allow a virus must overcome a barrier of mucosal surfaces, skin them to escape from the immune response and complete or cornea. HHV-1 and HHV-2 target keratinocytes during their replication. One of these strategies includes utiliza- initial entry and establish a primary infection in the epithe- tion of host cell mitochondria. Mitochondria are organelles lium, which is followed by latent infection in neurons. Infec- that are involved in a variety of metabolic and cellular func- 2+ tions are usually mild but may spread to the central nervous tions, including Ca homeostasis, ATP production, and pro- system, causing serious neurological disorders. HHV-1 and grammed cell death. They also participate in the synthesis of HHV-2 have been identified as causative agents of various, key metabolites and are the primary source of endogenous mild and even life-threatening diseases, namely, herpes reactive oxygen species [6, 16]. Mitochondria form a network distributed throughout the cell [17]. They are dynamic organelles that constantly Handling Editor: Zhongjie Shi. change their shape, length and movement along cytoskel- * Joanna Cymerys etal tracks. There are two main processes responsible for jcymerys@op.pl mitochondrial homeostasis: fission and fusion. These are crucial for maintenance of a proper number of functional Division of Microbiology, Department of Preclinical mitochondria. Fusion allows the exchange of DNA, con- Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences, Ciszewskiego 8, tents and metabolites between neighboring mitochondria. 02-786 Warsaw, Poland Fission of the mitochondrial network enables the distribution Department of Physiological Sciences, Faculty of Veterinary and transport of mitochondria within and outside the cell Medicine, Warsaw University of Life Sciences, to a place where energy demand is high. Moreover, fission Nowoursynowska 159, 02-776 Warsaw, Poland adjusts apoptosis to eliminate damaged mitochondria [1, 13]. Military Institute of Hygiene and Epidemiology, Kozielska 4, The main proteins responsible for fusion are Mfn-1, Mfn-2 01-163 Warsaw, Poland Vol.:(0123456789) 1 3 2664 M. Chodkowski et al. and Opa-1. In mice, knockouts of the genes encoding these no. CRL1587). To produce the virus for the experiments, proteins results in lethality to embryos and mitochondrial the Vero cells were infected with HHV-1 or HHV-2 at 0.001 dysfunction. Cells with defective Mfn1 and Mfn2 have a plaque-forming units (PFU)/cell. One hour after infection disrupted mitochondrial network and many small, punctate at 37 °C, the inoculum was removed by aspiration, fresh mitochondria. The most important protein participating in culture medium was added, and the cells were cultured for the defragmentation of the mitochondrial network in the 3 days. Culture supernatants were harvested at 72 h after cell is dynamin-related protein 1 (Drp1), which has GTPase virus challenge, and after three cycles of freezing (-80 °C) activity. This protein migrates between the cytosol and the and thawing at RT, they were clarified by centrifugation at mitochondrial network and binds to the mitochondrial outer 800 g for 10 min and stored in small volumes at -80 °C. A membrane during fission. Dysregulation of mitochondrial virus stock suspension containing approximately 10 PFU/ motility has been observed in many human diseases, mainly ml was used in all the experiments. neurodegenerative disorders, cancer, diabetes, and arrhyth- HaCaT cells (10 cells per well) were infected with mias, and also during ageing [9]. HHV-1 or HHV-2 for 60 min at 37 °C. After adsorption, It is believed that mitochondria play a major role in viral the inoculum was removed by aspiration and fresh culture infections. Murata et al. [10] have shown that, in Vero cells, medium was added. The cells were then incubated for 2, 24 mitochondria are recruited to the site of viral replication and or 48 hours at 37 °C with 5% CO . morphogenesis. They have also shown that mitochondria migrate to the perinuclear area where HHV-1 tegument was Real‑time cell growth analysis present. It is possible that mitochondria, as energy centers of the cell, provide the energy necessary for replication of the The growth kinetics, behaviour and morphology of HaCaT virus. It has also been shown that the mitochondrial potential cells infected with HHV-1 or HHV-2 were analysed using a is stable up to 6 hours postinfection (h p.i.) but decreases JuLI™ Br Live Cell Analyser system for a bright-field image during the late phase of infection [10]. In addition, many analysis (NanoEnTek, Korea) [4]. HaCaT cells (10 cells per types of viral proteins have been identified as responsible well) were seeded in a 6-well plate and infected with HHV-1 for the modulation of apoptosis. Alphaherpesviruses, like or HHV-2 as described above. Cell-growth images were cap- other large DNA viruses, encode proteins that interfere with tured for 48 h at 7-min intervals. Cell confluence analysis mitochondrial function and localization to block the apop- was done and a real-time cell growth curve was generated totic pathway. However, little is known about the role of using JuLI Br PC software. All images were captured at mitochondria in skin cells, which function as the primary objective magnification of ×4. barrier and, on the other hand, constitute a very important replication site. Real‑time PCR To the best of our knowledge, there are no data available concerning the effects of productive viral infection on mito- To determine the number of viral DNA copies per reac- chondria and the mitochondrial network in keratinocytes, tion, a standard curve was prepared as described previously which are crucial in the first steps of herpesviral infection. [7]. Briefly, fragments of glycoprotein B gene sequence of Therefore, in the present study we investigated the effect HHV-1 and HHV-2 were amplified using appropriate prim- of HHV-1 and HHV-2 infection on cell vitality, apoptosis, ers: HSV -1F ext (G T G A T G TT G AGG T CG A T G AAGG T) mitochondrial network rearrangement, and mitochondrial and HS V -1R e xt (A C A A CG CG A CGC A C A T C A A GG T) potential in human keratinocytes in vitro. a n d H SV - 2 F ex t ( C G T AC G A T G AG T T T G T G T T G G C G A ) and HSV -2R ext (T CA GCT GG T GAG AG T ACG CG T A). The products were cloned in pGEM-T Easy Vector. Serial Materials and methods dilutions of recombinant plasmids were prepared ranging from 10 to 10 copies per reaction. Real-time PCR was Cell culture and virus strains performed in 96-well plates using a 7500 Real Time PCR System thermocycler (Applied Biosystems) with TaqMan Human keratinocytes (HaCaT cells) were cultured as a mon- Universal Master Mix II (Applied Biosystems) and probes olayer using Dulbecco’s modified Eagle’s medium (DMEM; labelled with JOE as described previously [11]. Gibco) supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, 100 U of penicillin per ml and 100 mg of Immunofluorescent staining procedures streptomycin per ml, at 37 °C in a humidified atmosphere of 5% CO . Cells were grown to full confluence with a medium HaCaT cells seeded on glass coverslips in a 12-well plate change every 2 days. The McIntyre strain of HHV-1 and were infected with HHV-1 or HHV-2. At 2, 24 and 48 h p.i. HHV-2 strain 333 were grown in Vero cell cultures (ATCC 1 3 Disruption of mitochondrial dynamics by human herpesviruses 2665 (hour postinfection) coverslips were incubated with 100 nM images, obtained by confocal microscopy were used accord- MitoRed (Sigma-Aldrich) for 30 min at 37 °C, then washed ing to the protocol established by Valente et al. [18]. Each three times with culture medium and fixed in 3.7% para- analysis was performed on ten cells. formaldehyde/PBS (Sigma-Aldrich). The presence of viral antigens was detected by means of direct immunofluores- Image cytometry cence, using FITC-conjugated polyclonal rabbit anti-herpes simplex virus 1/2 serum (Dako, dilution 1:200). Cellular fluorescence was quantified using a NucleoCoun- Additionally, at 2 and 24 h p.i., HaCaT cells were stained for dynamin-related protein 1 (Drp1) detection. At the begin- ter NC-3000 image cytometer (ChemoMetec). The Nucle- oCounter system was used for evaluation of mitochondrial ning, cells were washed twice in PBS (Sigma-Aldrich) and fixed in 3.7% paraformaldehyde/PBS (Sigma-Aldrich) for transmembrane potential (Δψ) and cell vitality at 24 h p.i. In the mitochondrial potential assay, HaCaT cells were stained 30 min at room temperature (RT), then permeabilized with 0.5% Triton X-100 (Sigma-Aldrich) solution in PBS. Before with JC-1 (cationic dye 5,5,6,6-tetrachloro-1,1,3,3-tetraeth- ylbenzimidazol-carbocyanine iodide; ChemoMetec A/S). staining, fixed HaCaT cells on coverslips were blocked with PBS containing 1% bovine serum albumin (BSA) (Sigma First, the suspended HaCaT cells were diluted with PBS to a final concentration of 1.5 ×10 cells/mL. The samples Chemicals) for 30 min at room temperature. The presence of Drp1 was detected by using DNM1L polyclonal antibody were then incubated with 12.5 mL of a 200 mg/mL solution of JC-1 for 10 min at 37 °C. After incubation, samples were (Invitrogen, dilution 1:500) and Alexa Fluor 488 goat anti- rabbit (Invitrogen; dilution 1:250). washed twice in PBS and resuspended in 250 mL of a 1 mg/ mL solution of 4′,6-diamidino-2-phenylindole in PBS. In Cell nuclei were stained with Bisbenzimidine/Hoechst 33258 according to manufacturer’s recommendations. the vitality assay (detection of changes in the intracellular level of thiols), HaCaT cells were stained with VitaBright-48 Afterwards, coverslips were mounted on microscope slides using anti-fade mounting medium (Sigma-Aldrich). Unin- (ChemoMetec A/S), acridine orange (ChemoMetec A/S), and propidium iodide (PI; ChemoMetec A/S). Suspended fected HaCaT cells served as a negative control. For Drp1 staining, HaCaT cells pre-incubated with Dynasore (GTPase HaCaT cells were diluted with PBS to a final concentra- tion of 2.0 × 10 cells/mL and were mixed with 5 mL of inhibitor; 80 µM/ml) for 60 min before infection served as a positive control. Results were evaluated using a confocal VitaBright-48·PI·acridine orange. Subsequently, the samples were examined using a Nucleo-Counter NC-3000 according microscope (Leica TCS SP8-WWL). to manufacturer’s instructions. The results were analyzed using the NucleoView NC-3000 software (details of the Confocal microscopy NucleoCounter NC-3000 design and capabilities are avail- able at www.chemo metec .com) [2]. Confocal images were acquired using a Leica white light A positive control for mitochondrial potential analysis laser scanning confocal microscope (Leica TCS SP8-WWL, was prepared by adding CCCP (carbonyl cyanide m-chlo- KAWA.SKA Sp. z o.o., Poland) with a 63x oil-immersion rophenyl hydrazone; 5 µl per ml of cell culture medium). lens, using excitation at 405 nm, 499 nm, 569 nm for Hoe- In parallel, uninfected HaCaT cells served as a negative chst, FITC and MitoRed, respectively. Images were captured control. and converted to 24-bit tiff files for visualization using the Leica Application Suite X (LAS X) software platform (Leica Microsystems). Statistical evaluation Analysis of mitochondrial morphology The results were statistically evaluated by one-way analysis of variance (ANOVA) using the Student–Newman–Keuls For mitochondrial morphology analysis, MiNa Single Image multiple comparisons test and the Tukey–Kramer multi- macro was used. This tool allows the number of individuals, ple comparisons test. This analysis was performed using number of networks, mean length of branches/rod, mean net- TM GraphPad Prism version 4.03 software (GraphPad Soft- work size, mean network size per branch, and mitochondrial ware Inc., San Diego, CA, USA). Statistical differences were footprint to be computed. In order to perform this analysis 1 3 2666 M. Chodkowski et al. Fig. 1 A and B. Morphological changes of HaCaT cells infected with Real-time PCR analysis of viral DNA copy number in HaCaT cells HHV-1. Cells were observed for 48 h using a JuLI™ Br Live Cell and cell medium during HHV-1 and HHV-2 infection. Statistical dif- Analyzer. CPE was manifested as cell destruction and fusion of cells, ferences were interpreted as significant at P < 0.05 (*) and P < 0.01 as confirmed by a growth curve. Objective magnification, x4. C. (**) interpreted as significant at P < 0.05 (*) and highly signifi- Fig. 2 The mitochondrial network in uninfected control HaCaT cells. ► A-F. Immunofluorescence staining of mitochondria (red fluorescence) cant at P < 0.01 (**). and the nucleus (blue fluorescence). A yellow arrow indicates tubular mitochondria. G. The original image was processed using “unsharp mask”, “CLAHE”, “median”, “binarize” and “skeletonize” for detec- Results tion different mitochondrial shapes and structures. H. Tubular mito- chondria. I. Punctate mitochondria. J. Branched mitochondrial net- work HHV‑1 and HHV‑2 replication in HaCaT cells During HHV-1 infection, a cytopathic effect (CPE) was medium at 24 h p.i. and 48 h p.i., which was most probably observed as morphology changes in HaCaT cells. After 24 the result of the release of progeny virions from the cell h p.i. and 48 h p.i., we observed a diffuse cytopathic effect, (Fig. 1C). manifesting as cell rounding, shrinking and lysis of indi- vidual cells. Interestingly, we did not observe large changes Changes in the mitochondrial network in the confluence of monolayer during the study. At 48 h p.i., during HHV‑1 and HHV‑2 infection the confluence has decreased to around 90% (Fig.  1A and B). Similar results were observed with cells infected with In uninfected HaCaT cells, the mitochondrial network was HHV-2 (data not shown). dense, branched and spread evenly throughout the cell The quantitative PCR analysis showed a statistically (Fig. 2). We observed many tubular, long and highly inter- significant increase in the DNA copy number of analyzed connected mitochondria localized in the subcellular region viruses in comparison to the uninfected control (Fig. 1C). and a small number of punctate mitochondria. Moreover, The highest, statistically significant, increase in the copy we observed fusion of mitochondria of dividing cells, number of viral DNA was observed at 48 h p.i. with HHV-1 which accumulated in close proximity to the chromosomes 7 7 and HHV-2 (2.8 ± 1.01 × 10 and 1.9 ± 1.31 × 10 cop- (Fig. 2A and D). In uninfected cells, we distinguished three ies/ml, respectively; P <0.01). We also found a signifi- types of mitochondrial shape: tubular (Fig. 2H), punctate cant increase in the viral DNA copy number in the culture (Fig. 2I) and branched mitochondrial network (Fig. 2 J). 1 3 Disruption of mitochondrial dynamics by human herpesviruses 2667 1 3 2668 M. Chodkowski et al. HHV-1 and HHV-2 infection caused changes in the mor- plaques and multinucleated cells was also observed (Fig. 3B phology of the mitochondrial network. At 2 h p.i., with I and II). both HHV-1 and HHV-2, we observed the interaction of Examples of cells selected for mitochondrial morphology viral particles with the mitochondrial network. The viral analysis are shown in Fig. 4A-G’. At 2 h p.i., with HHV-1, antigens were located near the cell nucleus (Fig. 3A), and we observed a decrease in the number of mitochondrial net- interestingly, they partially colocalized with mitochondria works, together with an increase in the number of individual (Fig. 3A XIII and XIV). At 24 h p.i. with HHV-1 (Fig. 3B), mitochondrial objects. In addition, both the percentage of we observed changes in the shape of the mitochondrial net- cross-linked mitochondria and the length of the network work and its distribution within the cell in comparison to branches decreased. Moreover, the total area of mitochon- uninfected HaCaT cells. The mitochondrial network was dria decreased. At 24 h p.i., the number of mitochondrial organized near the nucleus. At the same time, we observed objects increased, and we observed a decrease in the number colocalization of some viral particles with mitochondria. of mitochondrial networks. We also observed a reduction in Similar observations were made in the case of infection with the total mitochondrial area. At 48 h p.i., we observed a sig- HHV-2. After 48 h p.i. we observed an increase in viral rep- nificant increase in the number of mitochondrial objects. The lication. During HHV-1 infection, the mitochondrial network mean number of branches per network was reduced at 2, 24, was completely fragmented. In the case of HHV-2 infec- and 48 h p.i., but the results were not statistically significant. tion, after 48 h we observed accumulation of viral antigens HHV-1 infection caused various changes in the morphology within the cells. Moreover, a cytopathic effect in the form of of the mitochondrial network in HaCaT cells. This analysis Fig. 3 Mitochondrial network organization in HaCaT cells infected effect in HaCaT cells during infection with HHV-2 at 48 h p.i. is vis- with HHV-1 or HHV-2 at 2 (A), 24 and 48 h p.i. (B). A yellow arrow ible. The red arrow in panel B (II) indicates the distribution of viral indicates colocalization of mitochondria with viral antigen (A, VIII). antigen along the edges of the cell, and the white arrow indicates A profile plot of fluorescence signal intensities along the yellow line interruption of the mitochondrial network between infected cells (B, visible in panel A XIV indicates the colocalization of mitochondria II) and viral antigen (A, XIII). In panel B (I), the syncytial cytopathic 1 3 Disruption of mitochondrial dynamics by human herpesviruses 2669 Fig. 4 A-G. Mitochondrial network analysis performed on 10 HaCaT at 24 h p.i; G’, a single cell infected with HHV-1 at 48 h p.i. H and I. cells. A’, a single uninfected control cell; B’, a single cell infected Summary statistics for all infected and control HaCaT cells. The box with HHV-1 at 2 h p.i; C’, a single cell infected with HHV-1 at 24 h plot shows median (horizontal lines), first-to-third quartile (box), and p.i; D’, a single cell infected with HHV-1 at 48 h p.i; E’, a single cell extreme values (%) (**, P < 0.01; *, P < 0.05). Each analysis was infected with HHV-1 at 2 h p.i.; F’, a single cell infected with HHV-1 performed on 10 cells shows that the mitochondrial network underwent s fi sion and mitochondria (Fig. 5B). During infection, we observed that was fragmented after infection (Fig 4H). Drp1 was partially translocated from the cytoplasm to the HHV-2 infection caused changes similar to those caused outer membrane of the mitochondria. After 2 h p.i., with by HHV-1. We observed a gradual increase in the number of both HHV-1 and HHV-2, we observed a colocalization of mitochondria, which reached a maximum at 48 h p.i. We also Drp1 with mitochondria localized in perinuclear area. After observed changes in mitochondrial cross-linking, as well as 24 h p.i. we observed a decrease in Drp1 protein expression a decrease in the length of the mitochondrial branches, but in infected cells together with progressive disintegration of these differences were statistically insignificant. At 24 and the mitochondrial network (Fig. 5C-F). 48 h p.i., a statistically significant decrease in the overall mitochondrial surface area was observed (Fig. 4I). The mitochondrial potential and vitality of HaCaT We then investigated the distribution of Drp1 in HaCaT cells during HHV‑1 and HHV‑2 infection cells. In uninfected control cells, Drp1, as well as the mito- chondrial network, was distributed evenly in the cytoplasm In this assay, changes in the mitochondrial potential that (Fig. 5A). After 24 h of treatment with Dynasore, which occurred during HHV-1 or HHV-2 infection were evaluated is an inhibitor of GTPases, we observed a decrease in the (Fig. 6A, C). Considering the pivotal role of mitochondria in Drp1 level in treated cells and the presence of tubular 1 3 2670 M. Chodkowski et al. Fig. 5 Localization of Drp1 in HaCaT cells. Immunofluo- rescence staining was used to examine mitochondrial translo- cation of Drp1 fission protein (white arrows indicate localiza- tion of Drp1). A. Uninfected HaCaT cells. B. Cells treated with Dynasore. C and D. Cells infected with HHV-1. E and F. Cells infected with HHV- 2. Drp1, green fluorescence; mitochondria, red fluorescence; nuclei, blue fluorescence. Objective magnification, x63 orchestrating the apoptotic pathway, we measured the mito- with dysfunctional mitochondria; JC-1 was present in the chondrial potential using the JC-1 method. The principle of cytosol in its monomeric form, emitting green fluorescence. the test depends on the fact that JC-1, a mitochondrial-poten- During infection with both HHV-1 and HHV-2 (2 and 24 h tial-sensitive dye, accumulates in the matrix of mitochon- p.i.), we observed changes in mitochondrial potential. The dria by forming J-aggregates with red fluorescence when most significant decrease was observed in HaCaT cells at the mitochondrial potential is high and becomes a monomer 2 h p.i. However, at 24 h p.i., a decrease in the mitochon- with green fluorescence when it is low. HaCaT cells treated drial potential had also occurred, but it was not statistically with CCCP served as a positive control (Fig.  6A IV). In significant and was similar to the values obtained with the these cells, a low mitochondrial potential was observed. negative control (Fig. 6A, C). It was also found that HHV-1 Cells treated with CCCP exhibited the characteristics of cells and HHV-2 infection caused a reduction in the vitality of 1 3 Disruption of mitochondrial dynamics by human herpesviruses 2671 Fig. 6 A and C. Mitochondrial membrane potential (ΔΨ) of HaCaT HHV-1 at 24 h p.i.; V, HaCaT cells infected with HHV-2 at 2 h p.i.; cells. Uninfected control cells have a high mitochondrial potential VI, HaCaT cells infected with HHV-2 at 24 h p.i. B and D. Cell (I). CCCP-treated HaCaT cells (positive control) have a low mito- vitality assay. HaCaT cells were stained with VitaBright -48 ™ (VB- chondrial potential (IV). The level of green fluorescence in cells 48™), acridine orange (AO), and propidium iodide (PI). I, control is indicated as a percentage (**, P < 0.01; *, P < 0.05). II, HaCaT cells; II, HaCaT cells infected with HHV-1 at 24 h p.i. III, HaCaT cells infected with HHV-1 at 2 h p.i.; III, HaCaT cells infected with cells infected with HHV-2 at 24 h p.i. D. Level of dead and live cells HaCaT cells at 24 h p.i. (Fig. 6B and D). The decrease in confirmed by qPCR analysis, caused morphological changes vitality was more pronounced in the case of HHV-1 infection in infected cells (Fig. 1). These changes were manifested as than after infection with HHV-2. a loss of intercellular connections between HaCaT cells and the presence of syncytial cells at 48 h p.i., which was also observed in previous studies [12]. Overall, the cytopathic effect caused by HHV-1 and HHV-2 in the late stages of Discussion infection was very similar. In this study, we performed an analysis of mitochondrial The major target cells during primary and recurrent HHV-1/ dynamics during HHV-1 and HHV-2 infection in human HHV-2 infections are cells of epithelial and neuronal ori- keratinocytes. Changes in the mitochondrial network were gin. During the initial exposure, HHV-1 and HHV-2 use apparent in the early stage of infection with HHV-1 or HHV- mucosal epithelial cells, including epidermal keratinocytes, 2. We also observed the migration of mitochondria to the as the primary portal of entry, and infection then spreads perinuclear area, both in the early and late stages of infec- through the epithelium. Additionally, after episodic reactiva- tion. Similar results were obtained previously, and it was tion from latency established in neurons, newly replicating also shown that microtubules are responsible for the trans- viruses infect epithelial cells, often leading to recurrent her- port of mitochondria. Moreover, the migration of mitochon- petic lesions. The productive cycle of HHV-1 and HHV-2, dria to the perinuclear area during infection with HHV-1 was 1 3 2672 M. Chodkowski et al. inhibited after addition of the microtubule inhibitor nocoda- The assay provides a reliable and fast way to evaluate cell zole [10]. These results have provided new insights into the viability, as an appearance of free thiols is the early indica- various strategies of cytoskeleton utilization that are used tor of apoptosis [14]. In our study, we did not observe a during herpesviral infections. Apparently, the cytoskeleton is significant change in the level of free thiols after infection not only necessary for transport of the virions to the nucleus, with either HHV-1 or HHV-2. This may indicate that only a as has been described before, but also supports spatial rear- small percentage of infected HaCaT cells undergo apoptosis. rangement of cellular organelles in order to facilitate the In conclusion, our results suggest that productive HHV-1 replication cycle [5, 15]. and HHV-2 infections cause changes in the mitochondrial After HHV-1 or HHV-2 infection, we also observed network in comparison to uninfected control cells. These changes in mitochondrial morphology. In control cells, the changes manifest themselves mainly as defragmentation mitochondria were elongated and branched. Murata et al. of the mitochondrial network, an increase in the number also observed the changes in the mitochondrial area, but they of punctate mitochondria, and a decrease in the number did not characterize these changes [10]. According to ImageJ of tubular mitochondria. In addition, in the early stage of analysis, mitochondria were significantly affected by HHV-1 infection, mitochondrial membrane permeabilization occurs, or HHV-2 infection. Primarily, we observed defragmenta- which may be associated with rearrangement of the network. tion of the mitochondrial network and an increased number These results indicate that mitochondria are utilized in the of punctate mitochondria. We also detected colocalization early stages of HHV-1 and HHV-2 replication, as evidenced of punctate mitochondria with viral antigen. In the case of by the increased transport of punctate mitochondria to the HHV-1, in the late stage of infection, there was a decrease replication site and colocalization of mitochondria with viral in the number of mitochondrial networks, but we did not antigens in the perinuclear area. It is, however, worth stress- observe a significant change in the case of HHV-2 infection. ing that viral infection does not destroy the mitochondrial These changes were not observed in cells in which viral anti- network completely, which ensures survival of the cell and gen was not detected. Similar changes, including an increase enables viruses to complete their replication cycle. in the number of punctate mitochondria, were also observed Acknowledgements This work was funded by KNOW (Leading previously in the case of infection with Human herpesvirus National Research Centre) Scientific Consortium “Healthy Animal— type 5 (HHV-5) [8]. Our results suggest that HHV infection Safe Food”, decision of Ministry of Science and Higher Education causes increased fission of the mitochondrial network. More- No. KNOW2016/SGGW/PRO1/01/10, and by Internal Research Grant No. 505-10-023400-P00350-99 from WULS-SGGW in Warsaw. The over, we observed a translocation of Drp1 to the perinuclear authors would like to thank Prof. Zdzisław Gajewski, WULS-SGGW, area and binding of this protein to mitochondrial outer mem- Warsaw, Poland, for providing us access to the Leica TCS SP8-WWL brane. This may indicate that viral infection increases the confocal microscope. activity of this protein, stimulating the defragmentation of mitochondrial network. Compliance with ethical standards It is believed that one of the early indicators of apopto- sis is the permeabilization of the mitochondrial membrane. Conflict of interest The authors declare no conflict of interest. For that reason, we evaluated the mitochondrial potential Open Access This article is distributed under the terms of the Crea- using JC-1 dye. We believe that the internal mitochondrial tive Commons Attribution 4.0 International License (http://creat iveco potential decreased as a consequence of changes in the mmons.or g/licenses/b y/4.0/), which permits unrestricted use, distribu- mitochondrial network. It was the result of rearrangements tion, and reproduction in any medium, provided you give appropriate of the network as well as changes in the distribution of credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. mitochondria in the early stages of infection. Interestingly, the mitochondrial potential had been stabilized by 24 h p.i. At 2 h p.i., we observed a large loss in the mitochondrial potential after infection with both HHV-1 and HHV-2. References These results showed that the most significant changes 1. 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Archives of VirologySpringer Journals

Published: Jun 5, 2018

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