Med Microbiol Immunol DOI 10.1007/s00430-014-0333-6
Original Investigation
Characterization of coxsackievirus B3 replication in human umbilical vein endothelial cells A. Kühnl · C. Rien · K. Spengler · N. Kryeziu · A. Sauerbrei · R. Heller · A. Henke
Received: 29 October 2013 / Accepted: 26 February 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract After successful invasion of susceptible hosts, systemic distribution of coxsackievirus B3 (CVB3) most likely requires interactions with the endothelial system. Thereby, infection of endothelial cells occurs directly or viruses and/or virus-infected leukocytes migrate through the endothelial barrier. Many of these processes have not been studied so far. In order to analyze viral replication in the endothelium, human umbilical vein endothelial cells (HUVEC) were isolated and infected with CVB3. Time-course experiments revealed maximal viral replication at 10–24 h and viral RNA persistence up to 120 h post-infection (p. i.) without the induction of obvious general cytopathic effects or the loss of cellular viability. However, the application of the EGFP-expressing recombinant virus variant CVB3/EGFP revealed shrinkage and death of individual cells. Using infectious center assays, a noticeable CVB3 replication occurred on an average of 20 % of HUVEC at 10 h p. i. This may be in part due to a higher coxsackievirus/adenovirus receptor expression in a small subgroup of HUVEC (5–7 %) as analyzed by flow cytometry. Interestingly, CVB3 replication escalated and cellular susceptibility increased significantly after reversal of cell cycle arrest caused by serum deprivation indicating A. Kühnl and C. Rien have contributed equally to this manuscript. A. Kühnl · C. Rien · A. Sauerbrei · A. Henke (*) Institute of Virology and Antiviral Therapy, Jena University Hospital, Friedrich Schiller University Jena, Hans‑Knöll‑Str. 2, 07745 Jena, Germany e-mail: [email protected]‑jena.de K. Spengler · N. Kryeziu · R. Heller Institute for Molecular Cell Biology, Center for Molecular Biomedicine, Jena University Hospital, Friedrich Schiller University Jena, Hans‑Knöll‑Str. 2, 07745 Jena, Germany
that reactivation of cellular metabolism may help to promote CVB3 replication. Finally, CVB3-infected HUVEC cultures revealed increased DNA fragmentation, and inhibition of caspase activity caused an accumulation of intracellular virus particles indicating that apoptotic processes are involved in virus release mechanisms. Based on these observations, it is assumed that CVB3 replicates efficiently in human endothelial cells. But how this specific infection of the endothelium may influence viral spread in the infected host needs to be investigated in the future. Keywords Coxsackievirus B3 · Human umbilical vein endothelial cells · Replication · Coxsackievirus/adenovirus receptor · Serum deprivation · Apoptosis
Introduction Infections with different pathogens can lead to cardiovascular diseases such as acute/chronic myocarditis and dilated cardiomyopathy. Among others, viral myocarditis has been recognized as a cause of heart failure and premature death, but since its diagnosis is still challenging [1, 2], profound epidemiological data to evaluate this disease remain to be established. One of the pathogens, which provoke myocarditis, is coxsackievirus B3 (CVB3). CVB3 and the other five serotypes of the group B coxsackieviruses are enteroviruses (EV-B) and belong to the family of the Picornaviridae. Acute coxsackievirus-induced myocarditis is characterized by cardiac arrhythmias and heart failure. Chronic processes cause dilated cardiomyopathy, which may require heart transplantation or even lead to death. In addition to myocarditis, coxsackieviruses are also linked to other human diseases including aseptic meningitis and pancreatitis, which can trigger the onset of type 1 diabetes
13
mellitus [3, 4]. Currently, advances in laboratory techniques facilitate the diagnosis of such viral infections but so far treatments are entirely supportive [5] and common vaccines are not available [6, 7]. During the course of CVB3 infection, primary replication occurs in the cells of the nasopharynx and the Peyer’s patches of the intestine. Then, viruses spread via the blood circulation and interact inevitably with the vascular endothelium to reach target organs like the myocardium or the pancreas. So far, only a few publications describe that CVB3 infects murine [8, 9] and human endothelial cells [10] via the coxsackievirus/adenovirus receptor (CAR) [11]. In the latter study, drug-induced downregulation of CAR expression in human umbilical vein endothelial cells (HUVEC) decreased viral replication [11]. CVB3 may cause a persistent infection in endothelial cells [12], which in turn induces an increased expression of cytokines and adhesion molecules [13]. In addition, in CVB3-infected HUVEC, a reorganization of actin filaments, alterations of tight junctions, and an enhancement of permeability—possibly via p38 MAP kinase-mediated mechanisms—have been observed [14]. Moreover, the immunoregulatory response of CVB3-infected HUVEC might be of importance, especially in view of the attraction of inflammatory cells, which may become infected as well. In this context, it has been shown that human leukocytes, like monocytes and B cells, appear to be susceptible for CVB3 replication [15, 16], but so far, the interaction of CVB3-infected immune cells with the endothelium has not been demonstrated under in vivo conditions. Despite the fact that endothelial cells are known to be targets of CVB3, several important features of the infection process have not been investigated yet. For example, since a general virus-caused cytopathic effect is absent, it is not clear how CVB3 replicates in endothelial cells and whether damaging alterations are involved during the course of virus progeny production. In order to understand the progression of CVB3 replication in HUVEC, time-course experiments, single-cell studies, receptor analyses as well as apoptosis tests were performed. The obtained data show that CVB3 replicates in endothelial cells, and that this process is dependent on metabolic activity and that cellular apoptosis contributes to virus release from endothelial cells.
Materials and methods Cells HUVEC were prepared from human umbilical cord veins by treatment with 0.05 % collagenase and cultured in M199 growth medium (Lonza, Switzerland) containing 15 % fetal calf serum (FCS) (Lonza, Switzerland), 5 % human serum (Lonza, Switzerland), and 7.5-μg/ml endothelial mitogen
13
Med Microbiol Immunol
(Sigma-Aldrich, USA) as described previously [17, 18]. The purity of cultures was >98 % as indicated by flow cytometric staining for platelet endothelial cell adhesion molecule-1 (PECAM-1). Experiments were performed only with cells of the first or second passage. Transfection and titration experiments as well as virus propagation and experiments to provide suitable controls for CVB3 replication were carried out with African green monkey kidney (GMK) cells in DMEM (Lonza, Switzerland) supplemented with 10 % FCS, 100 U/ml penicillin and 100 μg/ml streptomycin. Virus CVB3 and its recombinant EGFP-expressing variant CVB3/EGFP are cDNA-generated viruses obtained after transfection of GMK cells with plasmid DNA containing CVB3 [19] or CVB3/EGFP [20]. Viruses were propagated in GMK cells and quantified by standard TCID50 assays. CVB3/EGFP was constructed by cloning the EGFP sequence downstream of the CVB3 5′-NTR together with an artificial 3C protease cleavage site [20]. EGFP fluorescence is only detectable when viral translation occurs. Virus titration Confluent HUVEC cultures were infected with a multiplicity of infection (m.o.i.) of 10 for 1 h under serum-free conditions. As indicated individually, supernatants were obtained (extracellular virus) and cells (intracellular virus) were washed once with serum-free media before detachment. All samples were stored at −20 °C. After three freeze and thaw cycles of cells to release intracellular viruses, titers were determined by TCID50 titrations. Each sample was tested in triplicate. Infectious center assay (ICA) To determine the actual amount of productively infected HUVEC, cells were infected with CVB3 and incubated as indicated. Thereafter, cells were isolated, carefully separated, counted, and serial 10-fold dilutions were made. 100 μl of each dilution were added to GMK cell monolayers and cells were overlaid with 0.5 % agar-containing cell culture medium and 0.25 % FCS. After 72 h, cells were stained and plaques were counted. Each plaque indicates a single CVB3-positive HUVEC. The percentage of positive cells was calculated in comparison with the original cell number of each sample. WST‑1‑based viability test The standard WST-1 test was used following the manufacturer’s instructions (Roche Applied Science, Switzerland).
Med Microbiol Immunol
Briefly, 10 μl of the cell proliferation agent WST-1 was added to 100-μl medium of each cell culture (4 parallel samples) in a 96-well plate and incubated at 37 °C in a humidified atmosphere with 5 % CO2. Four hours later, the absorbance was measured at 450/650 nm and the percentage of viability was calculated in comparison with untreated controls (set as 100 %). In case of WST-1 measurement after release of serum deprivation, normal growth conditions were re-established and mitochondrial activity was measured at 1, 4, 10, and 24 h. Values were normalized to individual cell numbers and demonstrated as relative activity per 10,000 cells. RNA isolation Total RNA was isolated from cells using the RNeasy Mini Kit (Qiagen, Germany). After ethanol precipitation, RNA pellets were dissolved in diethyl pyrocarbonate-treated water and incubated with DNase I for 30 min at 37 °C to digest remaining DNA. DNase I was inactivated by heating at 65 °C for 15 min. Reverse transcription and PCR Equal RNA amounts of each sample were subjected to reverse transcription. After heating for 10 min at 70 °C, samples were placed on ice for 3 min. This was followed by incubation periods for 10 min at 25 °C and 50 min at 42 °C using 100 U Superscript II reverse transcriptase (Invitrogen, USA) and the appropriate buffer containing 10 mM DTT and 1 mM dNTP’s. The reaction was terminated by heating for 5 min at 90 °C. Thereafter, levels of relative gene expression of CVB3 in comparison with human β-actin as internal control were analyzed by common PCR and quantitative real-time SYBR Green-based (Qiagen, Germany) PCR (qRT-PCR) using the LightCycler 3 technology (Roche Applied Science, Switzerland). Primer combinations for common PCR were 5′-GGCC CAGTGGAAGACGCG-3′ (sense) and 5′-AAATGCGC CCGTATTTGTCATTG-3′ (anti-sense) for CVB3 and 5′-ATCTGGCACCACACCTTCTACAATGAGCTGCG-3′ (sense) and 5′-CGTCATACTCCTGCTTGCTGATCCA CATCTGC-3′ (anti-sense) for human β-actin. Primer combinations to generate specific amplicons for qRT-PCR were 5′-CCCTGAATGCGGCTAATCC-3′ (sense) and 5′-ATT GTCACCATAAGCAGCCA-3′ (anti-sense) for CVB3 [21] and 5′-TGGGCGACGAGGCCCAGAGCAAGAAG-3′ (sense) and 5′-GCTCATTGTAGAAGGTGTGGTGCCA G-3′ (anti-sense) for human β-actin. During initial experiments, amplification efficiency of the target genes (CVB3) and the reference gene (β-actin) was determined by analyzing dilution series of control cDNA according to the supplier’s instructions (Roche Applied Science, Switzerland).
Melting-curve analysis was performed to verify the accuracy of the amplicons. β-actin was quantified in parallel in every PCR. Relative gene expression levels of the target genes CVB3 in comparison with β-actin were calculated using Rest relative expression software [22]. Immunocytochemistry Non- or CVB3-infected HUVEC (m.o.i. of 10) were grown on sterile cover slips (Carl Roth GmbH, Germany) in 24-well cell culture plates. At different times p. i., cover slips were transferred into new wells, washed twice with pre-warmed PBS, and fixed with 4 % paraformaldehyde (Sigma-Aldrich, USA) for 20 min at 4 °C. Then, cells were washed twice with PBS and permeabilized with 0.1 % Triton-X100 (Sigma-Aldrich, USA) in PBS for 5 min at room temperature. Thereafter, samples were washed again and incubated with blocking solution containing 1 % BSA and 5 % goat or donkey serum (Santa Cruz Biotechnology, USA) in PBS for 30 min. This was followed by 1-h incubations with each primary monoclonal antibodies (CVB3: clone 31A2, 1:1,000, Mediagnost, Germany; CAR: clone H300, 1:500, Santa Cruz Biotechnology, USA) and secondary polyclonal antibody (CVB3: Cy3-conjugated goat anti-mouse IgG, 1:1,000, Jackson ImmunoResearch, USA; CAR: FITC-conjugated donkey anti-goat IgG, 1:1,000, Jackson ImmunoResearch, USA) diluted in blocking solution. Between all incubation procedures, cells were washed twice with PBS. In addition, cell nuclei were stained using 4 μM of fluorescence dye Hoechst-33342 (Sigma-Aldrich, USA) for 10 min at 37 °C. All cover slips were washed and embedded with mounting medium (Dako, Denmark), and positive cells were analyzed by fluorescence microscopy. Image analysis Virus-based production of EGFP by CVB3/EGFP was studied at the indicated time p. i. using a Carl Zeiss Axioskop 2 MOT microscope and a Carl Zeiss AxioCam color camera (Carl Zeiss Microscopy GmbH, Germany). In addition, cell nuclei were stained using the fluorescence dye Hoechst-33342 (Sigma-Aldrich, USA) at a concentration of 4 μM for 10 min at 37 °C. Cover slips were washed, embedded with mounting medium (Dako, Denmark), and positive cells were analyzed by fluorescence microscopy. Flow cytometric measurement of CAR HUVEC were washed with PBS, fixed with 0.5 % paraformaldehyde in PBS for 2.5 min, and dissociated by a combination of trypsinization (2 min) and scraping. The process was stopped by adding HEPES buffer containing
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10 % FCS. Cells were washed twice with PBS and finally resuspended in PBS containing 0.2 % BSA-c (Aurion, The Netherlands). Samples of 2 × 105 cells/50 μl were labeled with a rabbit polyclonal antibody to CAR (1:10) for 30 min (ABIN389372, antikoerper-online.de). Subsequently, cells were washed with PBS and incubated with Alexa Fluor 488-conjugated anti-rabbit secondary antibody (Life Technologies, 1:250, 30 min). After washing, cells were resuspended in 300 μl PBS. Cells without primary antibody labeling served as controls. The cell-associated fluorescence of 20,000 cells per sample was determined in a FACScanto flow cytometer (BD Biosciences, USA) using the FlowJo software and displayed in histograms. In addition, mean fluorescence intensities were calculated. Western blot analysis Samples were obtained using a buffer containing 50 mM Tris, 2 mM EDTA, 1 mM EGTA, 50 mM NaF, 1 mM DTT, 10 mM Na4P2O7, 1 mM Na3VO4, 1 % Triton-X100, 0.1 % SDS, 1 mM phenylmethylsulfonylfluoride, and 10 μl/ml protease inhibitor cocktail (Roche Applied Science, Switzerland). Protein concentrations were calculated by Bradford assays (Bio-Rad, USA). Protein samples (60–100 μg) were incubated for 5 min at 95 °C, separated by 10 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and blotted onto Protran® nitrocellulose membranes (Schleicher & Schuell, Germany). Nonspecific binding sites were blocked by incubation with skim milk solutions. Tris-buffered saline (150 mM NaCl, 10 mM Tris–HCl, pH 8.0, 0.05 % Tween 20) was used for all washing procedures. After separation proteins were transferred to a nitrocellulose membrane. Then, membranes were incubated with primary antibodies (CAR: clone H300, 1:200, Santa Cruz Biotechnology, USA; β-actin: clone 13E5, 1:1,000, Cell Signaling Technology, USA). Subsequently, AP-conjugated goat anti-rabbit IgG (1:1,000, Acris, Germany) secondary antibodies were applied. Proteins were visualized using the NBT/BCIP detection system (Roche Applied Science, Switzerland). Control experiments were performed without usage of primary antibodies.
Med Microbiol Immunol
Detection of DNA fragmentation Cells were grown on sterile cover slips (Carl Roth GmbH, Germany) and were infected with CVB3 using a m.o.i. of 10. Control cells remained non-infected. At the indicated time p. i., samples were obtained and DNA fragmentation was analyzed using the DNA fragmentation imaging kit (Roche Applied Science, Switzerland) in accordance with the manufacturer’s instructions. Briefly, supernatants were removed and the remaining cells were fixed using a mixture of formaldehyde (4 %) and Triton-X (0.1 %) in purified water for 10 min at room temperature. Then, cells were washed once with PBS. Thereafter, DNA fragmentation was analyzed using the reaction solution containing both terminal deoxynucleotidyl transferase and fluorescent-dyelabeled nucleotide mixture. Samples were incubated for 1 h at 37 °C. Finally, nuclei were stained with Hoechst-33342 (Sigma-Aldrich, USA) for 5 min at room temperature. Prior to fluorescence analyses, cells were washed again with PBS and embedded in mounting medium (Dako, Denmark). Q‑VD‑OPH treatment HUVEC cultures were infected with CVB3 and simultaneously treated with the irreversible broad-spectrum caspase inhibitor Q-VD-OPH (Quinoline-Val-Asp-CH2-O-Ph; MP Biomedicals, Germany) [23, 24] at a final non-toxic concentration of 80 μM (DMSO as solvent control). While other pan-caspase inhibitors have an inhibitory effect on the enzymatic activity of the CVB3 proteases 2A and 3C, the non-methylated pan-caspase inhibitor Q-VD-OPH lacks this direct effect against viral proteases of CVB3 [25]. At the indicated times, samples were taken in triplicate and stored at −20 °C until further titration experiments. Statistical analysis The statistical comparisons were carried out using Mann– Whitney U-test. Each experiment was performed three to five times. The data shown represent the mean values of three to five different cell batches ± SD. A p value of ≤0.05 was considered as significant.
Induction of cellular quiescence by serum depletion
Results
HUVEC monolayers were washed twice with pre-warmed medium containing 0.25 % BSA. Then, growth medium without serum, with 0.5 % serum or with 20 % serum was added. After 24-h incubation and prior infection, the actual number of cells in each culture was determined. After infection, all cultures were cultivated in growth medium containing 20 % serum.
Infection of HUVEC with CVB3
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In order to establish conditions to investigate CVB3 replication in primary HUVEC cultures, cells were infected with CVB3 using a m.o.i. of 10. Then, samples were analyzed for the presence of infectious intra- as well as extracellular viruses by TCID50 titration assays as shown in Fig. 1a.
Med Microbiol Immunol
Virus concentration [TCID50/ml]
A
105
intracellular extracellular
104 103 102 101 0
0
10
20
30
40
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Time p. i. [h]
Ratio of CVB3-positive cells [%]
B 40
30
20
10
0
6
8
10
24
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Time p. i. [h]
Relative expression values
C
1.0
CVB3 β-actin
0.8
10
0.6
72 96 120 Time p. i. [h]
Co
0.4 0.2 0
Co
10
72
96
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Time p. i. [h]
Fig. 1 Infection of HUVEC with CVB3. a Kinetic of CVB3 replication in primary HUVEC cultures. Intra- and extracellular virus titers are demonstrated as mean ± SD generated from three different cell batches. b Single-cell analysis of CVB3 replication in HUVEC was performed to analyze the percentage of virus-positive cells up to 72 h p. i. Data are the mean ± SD of three different cell batches per time. c The presence of viral RNA was determined by quantitative and qualitative reverse transcription PCR up to 120 h p. i. Quantitative data are the mean ± SD of three different cell batches. Qualitative data represent results from one exemplary experiment. Analyses of β-actin expression was used as control
Clearly, a substantial virus replication occurs in HUVEC. After virus uptake, newly generated infectious viruses were detectable at 8 h p. i. and reached maximal intracellular concentrations at 10 h p. i. Extracellular viruses culminated
at 24 h p. i. In comparison with other susceptible cell lines (e.g., Raji, HeLa, or GMK) [26], virus progeny production was rather low in HUVEC (2–3 log levels less) indicating either minor replication in all cells or substantial replication in only a few cells. In order to analyze these possibilities, CVB3 infection of HUVEC was studied on the singlecell level using ICA. As shown in Fig. 1b maximal ratios of virus-positive HUVEC (18.2 ± 13.5 %) were observed at 10 h p. i. Thereafter, the percentage of virus-positive HUVEC declined till 72 h p. i. where
Original Investigation
Characterization of coxsackievirus B3 replication in human umbilical vein endothelial cells A. Kühnl · C. Rien · K. Spengler · N. Kryeziu · A. Sauerbrei · R. Heller · A. Henke
Received: 29 October 2013 / Accepted: 26 February 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract After successful invasion of susceptible hosts, systemic distribution of coxsackievirus B3 (CVB3) most likely requires interactions with the endothelial system. Thereby, infection of endothelial cells occurs directly or viruses and/or virus-infected leukocytes migrate through the endothelial barrier. Many of these processes have not been studied so far. In order to analyze viral replication in the endothelium, human umbilical vein endothelial cells (HUVEC) were isolated and infected with CVB3. Time-course experiments revealed maximal viral replication at 10–24 h and viral RNA persistence up to 120 h post-infection (p. i.) without the induction of obvious general cytopathic effects or the loss of cellular viability. However, the application of the EGFP-expressing recombinant virus variant CVB3/EGFP revealed shrinkage and death of individual cells. Using infectious center assays, a noticeable CVB3 replication occurred on an average of 20 % of HUVEC at 10 h p. i. This may be in part due to a higher coxsackievirus/adenovirus receptor expression in a small subgroup of HUVEC (5–7 %) as analyzed by flow cytometry. Interestingly, CVB3 replication escalated and cellular susceptibility increased significantly after reversal of cell cycle arrest caused by serum deprivation indicating A. Kühnl and C. Rien have contributed equally to this manuscript. A. Kühnl · C. Rien · A. Sauerbrei · A. Henke (*) Institute of Virology and Antiviral Therapy, Jena University Hospital, Friedrich Schiller University Jena, Hans‑Knöll‑Str. 2, 07745 Jena, Germany e-mail: [email protected]‑jena.de K. Spengler · N. Kryeziu · R. Heller Institute for Molecular Cell Biology, Center for Molecular Biomedicine, Jena University Hospital, Friedrich Schiller University Jena, Hans‑Knöll‑Str. 2, 07745 Jena, Germany
that reactivation of cellular metabolism may help to promote CVB3 replication. Finally, CVB3-infected HUVEC cultures revealed increased DNA fragmentation, and inhibition of caspase activity caused an accumulation of intracellular virus particles indicating that apoptotic processes are involved in virus release mechanisms. Based on these observations, it is assumed that CVB3 replicates efficiently in human endothelial cells. But how this specific infection of the endothelium may influence viral spread in the infected host needs to be investigated in the future. Keywords Coxsackievirus B3 · Human umbilical vein endothelial cells · Replication · Coxsackievirus/adenovirus receptor · Serum deprivation · Apoptosis
Introduction Infections with different pathogens can lead to cardiovascular diseases such as acute/chronic myocarditis and dilated cardiomyopathy. Among others, viral myocarditis has been recognized as a cause of heart failure and premature death, but since its diagnosis is still challenging [1, 2], profound epidemiological data to evaluate this disease remain to be established. One of the pathogens, which provoke myocarditis, is coxsackievirus B3 (CVB3). CVB3 and the other five serotypes of the group B coxsackieviruses are enteroviruses (EV-B) and belong to the family of the Picornaviridae. Acute coxsackievirus-induced myocarditis is characterized by cardiac arrhythmias and heart failure. Chronic processes cause dilated cardiomyopathy, which may require heart transplantation or even lead to death. In addition to myocarditis, coxsackieviruses are also linked to other human diseases including aseptic meningitis and pancreatitis, which can trigger the onset of type 1 diabetes
13
mellitus [3, 4]. Currently, advances in laboratory techniques facilitate the diagnosis of such viral infections but so far treatments are entirely supportive [5] and common vaccines are not available [6, 7]. During the course of CVB3 infection, primary replication occurs in the cells of the nasopharynx and the Peyer’s patches of the intestine. Then, viruses spread via the blood circulation and interact inevitably with the vascular endothelium to reach target organs like the myocardium or the pancreas. So far, only a few publications describe that CVB3 infects murine [8, 9] and human endothelial cells [10] via the coxsackievirus/adenovirus receptor (CAR) [11]. In the latter study, drug-induced downregulation of CAR expression in human umbilical vein endothelial cells (HUVEC) decreased viral replication [11]. CVB3 may cause a persistent infection in endothelial cells [12], which in turn induces an increased expression of cytokines and adhesion molecules [13]. In addition, in CVB3-infected HUVEC, a reorganization of actin filaments, alterations of tight junctions, and an enhancement of permeability—possibly via p38 MAP kinase-mediated mechanisms—have been observed [14]. Moreover, the immunoregulatory response of CVB3-infected HUVEC might be of importance, especially in view of the attraction of inflammatory cells, which may become infected as well. In this context, it has been shown that human leukocytes, like monocytes and B cells, appear to be susceptible for CVB3 replication [15, 16], but so far, the interaction of CVB3-infected immune cells with the endothelium has not been demonstrated under in vivo conditions. Despite the fact that endothelial cells are known to be targets of CVB3, several important features of the infection process have not been investigated yet. For example, since a general virus-caused cytopathic effect is absent, it is not clear how CVB3 replicates in endothelial cells and whether damaging alterations are involved during the course of virus progeny production. In order to understand the progression of CVB3 replication in HUVEC, time-course experiments, single-cell studies, receptor analyses as well as apoptosis tests were performed. The obtained data show that CVB3 replicates in endothelial cells, and that this process is dependent on metabolic activity and that cellular apoptosis contributes to virus release from endothelial cells.
Materials and methods Cells HUVEC were prepared from human umbilical cord veins by treatment with 0.05 % collagenase and cultured in M199 growth medium (Lonza, Switzerland) containing 15 % fetal calf serum (FCS) (Lonza, Switzerland), 5 % human serum (Lonza, Switzerland), and 7.5-μg/ml endothelial mitogen
13
Med Microbiol Immunol
(Sigma-Aldrich, USA) as described previously [17, 18]. The purity of cultures was >98 % as indicated by flow cytometric staining for platelet endothelial cell adhesion molecule-1 (PECAM-1). Experiments were performed only with cells of the first or second passage. Transfection and titration experiments as well as virus propagation and experiments to provide suitable controls for CVB3 replication were carried out with African green monkey kidney (GMK) cells in DMEM (Lonza, Switzerland) supplemented with 10 % FCS, 100 U/ml penicillin and 100 μg/ml streptomycin. Virus CVB3 and its recombinant EGFP-expressing variant CVB3/EGFP are cDNA-generated viruses obtained after transfection of GMK cells with plasmid DNA containing CVB3 [19] or CVB3/EGFP [20]. Viruses were propagated in GMK cells and quantified by standard TCID50 assays. CVB3/EGFP was constructed by cloning the EGFP sequence downstream of the CVB3 5′-NTR together with an artificial 3C protease cleavage site [20]. EGFP fluorescence is only detectable when viral translation occurs. Virus titration Confluent HUVEC cultures were infected with a multiplicity of infection (m.o.i.) of 10 for 1 h under serum-free conditions. As indicated individually, supernatants were obtained (extracellular virus) and cells (intracellular virus) were washed once with serum-free media before detachment. All samples were stored at −20 °C. After three freeze and thaw cycles of cells to release intracellular viruses, titers were determined by TCID50 titrations. Each sample was tested in triplicate. Infectious center assay (ICA) To determine the actual amount of productively infected HUVEC, cells were infected with CVB3 and incubated as indicated. Thereafter, cells were isolated, carefully separated, counted, and serial 10-fold dilutions were made. 100 μl of each dilution were added to GMK cell monolayers and cells were overlaid with 0.5 % agar-containing cell culture medium and 0.25 % FCS. After 72 h, cells were stained and plaques were counted. Each plaque indicates a single CVB3-positive HUVEC. The percentage of positive cells was calculated in comparison with the original cell number of each sample. WST‑1‑based viability test The standard WST-1 test was used following the manufacturer’s instructions (Roche Applied Science, Switzerland).
Med Microbiol Immunol
Briefly, 10 μl of the cell proliferation agent WST-1 was added to 100-μl medium of each cell culture (4 parallel samples) in a 96-well plate and incubated at 37 °C in a humidified atmosphere with 5 % CO2. Four hours later, the absorbance was measured at 450/650 nm and the percentage of viability was calculated in comparison with untreated controls (set as 100 %). In case of WST-1 measurement after release of serum deprivation, normal growth conditions were re-established and mitochondrial activity was measured at 1, 4, 10, and 24 h. Values were normalized to individual cell numbers and demonstrated as relative activity per 10,000 cells. RNA isolation Total RNA was isolated from cells using the RNeasy Mini Kit (Qiagen, Germany). After ethanol precipitation, RNA pellets were dissolved in diethyl pyrocarbonate-treated water and incubated with DNase I for 30 min at 37 °C to digest remaining DNA. DNase I was inactivated by heating at 65 °C for 15 min. Reverse transcription and PCR Equal RNA amounts of each sample were subjected to reverse transcription. After heating for 10 min at 70 °C, samples were placed on ice for 3 min. This was followed by incubation periods for 10 min at 25 °C and 50 min at 42 °C using 100 U Superscript II reverse transcriptase (Invitrogen, USA) and the appropriate buffer containing 10 mM DTT and 1 mM dNTP’s. The reaction was terminated by heating for 5 min at 90 °C. Thereafter, levels of relative gene expression of CVB3 in comparison with human β-actin as internal control were analyzed by common PCR and quantitative real-time SYBR Green-based (Qiagen, Germany) PCR (qRT-PCR) using the LightCycler 3 technology (Roche Applied Science, Switzerland). Primer combinations for common PCR were 5′-GGCC CAGTGGAAGACGCG-3′ (sense) and 5′-AAATGCGC CCGTATTTGTCATTG-3′ (anti-sense) for CVB3 and 5′-ATCTGGCACCACACCTTCTACAATGAGCTGCG-3′ (sense) and 5′-CGTCATACTCCTGCTTGCTGATCCA CATCTGC-3′ (anti-sense) for human β-actin. Primer combinations to generate specific amplicons for qRT-PCR were 5′-CCCTGAATGCGGCTAATCC-3′ (sense) and 5′-ATT GTCACCATAAGCAGCCA-3′ (anti-sense) for CVB3 [21] and 5′-TGGGCGACGAGGCCCAGAGCAAGAAG-3′ (sense) and 5′-GCTCATTGTAGAAGGTGTGGTGCCA G-3′ (anti-sense) for human β-actin. During initial experiments, amplification efficiency of the target genes (CVB3) and the reference gene (β-actin) was determined by analyzing dilution series of control cDNA according to the supplier’s instructions (Roche Applied Science, Switzerland).
Melting-curve analysis was performed to verify the accuracy of the amplicons. β-actin was quantified in parallel in every PCR. Relative gene expression levels of the target genes CVB3 in comparison with β-actin were calculated using Rest relative expression software [22]. Immunocytochemistry Non- or CVB3-infected HUVEC (m.o.i. of 10) were grown on sterile cover slips (Carl Roth GmbH, Germany) in 24-well cell culture plates. At different times p. i., cover slips were transferred into new wells, washed twice with pre-warmed PBS, and fixed with 4 % paraformaldehyde (Sigma-Aldrich, USA) for 20 min at 4 °C. Then, cells were washed twice with PBS and permeabilized with 0.1 % Triton-X100 (Sigma-Aldrich, USA) in PBS for 5 min at room temperature. Thereafter, samples were washed again and incubated with blocking solution containing 1 % BSA and 5 % goat or donkey serum (Santa Cruz Biotechnology, USA) in PBS for 30 min. This was followed by 1-h incubations with each primary monoclonal antibodies (CVB3: clone 31A2, 1:1,000, Mediagnost, Germany; CAR: clone H300, 1:500, Santa Cruz Biotechnology, USA) and secondary polyclonal antibody (CVB3: Cy3-conjugated goat anti-mouse IgG, 1:1,000, Jackson ImmunoResearch, USA; CAR: FITC-conjugated donkey anti-goat IgG, 1:1,000, Jackson ImmunoResearch, USA) diluted in blocking solution. Between all incubation procedures, cells were washed twice with PBS. In addition, cell nuclei were stained using 4 μM of fluorescence dye Hoechst-33342 (Sigma-Aldrich, USA) for 10 min at 37 °C. All cover slips were washed and embedded with mounting medium (Dako, Denmark), and positive cells were analyzed by fluorescence microscopy. Image analysis Virus-based production of EGFP by CVB3/EGFP was studied at the indicated time p. i. using a Carl Zeiss Axioskop 2 MOT microscope and a Carl Zeiss AxioCam color camera (Carl Zeiss Microscopy GmbH, Germany). In addition, cell nuclei were stained using the fluorescence dye Hoechst-33342 (Sigma-Aldrich, USA) at a concentration of 4 μM for 10 min at 37 °C. Cover slips were washed, embedded with mounting medium (Dako, Denmark), and positive cells were analyzed by fluorescence microscopy. Flow cytometric measurement of CAR HUVEC were washed with PBS, fixed with 0.5 % paraformaldehyde in PBS for 2.5 min, and dissociated by a combination of trypsinization (2 min) and scraping. The process was stopped by adding HEPES buffer containing
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10 % FCS. Cells were washed twice with PBS and finally resuspended in PBS containing 0.2 % BSA-c (Aurion, The Netherlands). Samples of 2 × 105 cells/50 μl were labeled with a rabbit polyclonal antibody to CAR (1:10) for 30 min (ABIN389372, antikoerper-online.de). Subsequently, cells were washed with PBS and incubated with Alexa Fluor 488-conjugated anti-rabbit secondary antibody (Life Technologies, 1:250, 30 min). After washing, cells were resuspended in 300 μl PBS. Cells without primary antibody labeling served as controls. The cell-associated fluorescence of 20,000 cells per sample was determined in a FACScanto flow cytometer (BD Biosciences, USA) using the FlowJo software and displayed in histograms. In addition, mean fluorescence intensities were calculated. Western blot analysis Samples were obtained using a buffer containing 50 mM Tris, 2 mM EDTA, 1 mM EGTA, 50 mM NaF, 1 mM DTT, 10 mM Na4P2O7, 1 mM Na3VO4, 1 % Triton-X100, 0.1 % SDS, 1 mM phenylmethylsulfonylfluoride, and 10 μl/ml protease inhibitor cocktail (Roche Applied Science, Switzerland). Protein concentrations were calculated by Bradford assays (Bio-Rad, USA). Protein samples (60–100 μg) were incubated for 5 min at 95 °C, separated by 10 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and blotted onto Protran® nitrocellulose membranes (Schleicher & Schuell, Germany). Nonspecific binding sites were blocked by incubation with skim milk solutions. Tris-buffered saline (150 mM NaCl, 10 mM Tris–HCl, pH 8.0, 0.05 % Tween 20) was used for all washing procedures. After separation proteins were transferred to a nitrocellulose membrane. Then, membranes were incubated with primary antibodies (CAR: clone H300, 1:200, Santa Cruz Biotechnology, USA; β-actin: clone 13E5, 1:1,000, Cell Signaling Technology, USA). Subsequently, AP-conjugated goat anti-rabbit IgG (1:1,000, Acris, Germany) secondary antibodies were applied. Proteins were visualized using the NBT/BCIP detection system (Roche Applied Science, Switzerland). Control experiments were performed without usage of primary antibodies.
Med Microbiol Immunol
Detection of DNA fragmentation Cells were grown on sterile cover slips (Carl Roth GmbH, Germany) and were infected with CVB3 using a m.o.i. of 10. Control cells remained non-infected. At the indicated time p. i., samples were obtained and DNA fragmentation was analyzed using the DNA fragmentation imaging kit (Roche Applied Science, Switzerland) in accordance with the manufacturer’s instructions. Briefly, supernatants were removed and the remaining cells were fixed using a mixture of formaldehyde (4 %) and Triton-X (0.1 %) in purified water for 10 min at room temperature. Then, cells were washed once with PBS. Thereafter, DNA fragmentation was analyzed using the reaction solution containing both terminal deoxynucleotidyl transferase and fluorescent-dyelabeled nucleotide mixture. Samples were incubated for 1 h at 37 °C. Finally, nuclei were stained with Hoechst-33342 (Sigma-Aldrich, USA) for 5 min at room temperature. Prior to fluorescence analyses, cells were washed again with PBS and embedded in mounting medium (Dako, Denmark). Q‑VD‑OPH treatment HUVEC cultures were infected with CVB3 and simultaneously treated with the irreversible broad-spectrum caspase inhibitor Q-VD-OPH (Quinoline-Val-Asp-CH2-O-Ph; MP Biomedicals, Germany) [23, 24] at a final non-toxic concentration of 80 μM (DMSO as solvent control). While other pan-caspase inhibitors have an inhibitory effect on the enzymatic activity of the CVB3 proteases 2A and 3C, the non-methylated pan-caspase inhibitor Q-VD-OPH lacks this direct effect against viral proteases of CVB3 [25]. At the indicated times, samples were taken in triplicate and stored at −20 °C until further titration experiments. Statistical analysis The statistical comparisons were carried out using Mann– Whitney U-test. Each experiment was performed three to five times. The data shown represent the mean values of three to five different cell batches ± SD. A p value of ≤0.05 was considered as significant.
Induction of cellular quiescence by serum depletion
Results
HUVEC monolayers were washed twice with pre-warmed medium containing 0.25 % BSA. Then, growth medium without serum, with 0.5 % serum or with 20 % serum was added. After 24-h incubation and prior infection, the actual number of cells in each culture was determined. After infection, all cultures were cultivated in growth medium containing 20 % serum.
Infection of HUVEC with CVB3
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In order to establish conditions to investigate CVB3 replication in primary HUVEC cultures, cells were infected with CVB3 using a m.o.i. of 10. Then, samples were analyzed for the presence of infectious intra- as well as extracellular viruses by TCID50 titration assays as shown in Fig. 1a.
Med Microbiol Immunol
Virus concentration [TCID50/ml]
A
105
intracellular extracellular
104 103 102 101 0
0
10
20
30
40
50
Time p. i. [h]
Ratio of CVB3-positive cells [%]
B 40
30
20
10
0
6
8
10
24
72
Time p. i. [h]
Relative expression values
C
1.0
CVB3 β-actin
0.8
10
0.6
72 96 120 Time p. i. [h]
Co
0.4 0.2 0
Co
10
72
96
120
Time p. i. [h]
Fig. 1 Infection of HUVEC with CVB3. a Kinetic of CVB3 replication in primary HUVEC cultures. Intra- and extracellular virus titers are demonstrated as mean ± SD generated from three different cell batches. b Single-cell analysis of CVB3 replication in HUVEC was performed to analyze the percentage of virus-positive cells up to 72 h p. i. Data are the mean ± SD of three different cell batches per time. c The presence of viral RNA was determined by quantitative and qualitative reverse transcription PCR up to 120 h p. i. Quantitative data are the mean ± SD of three different cell batches. Qualitative data represent results from one exemplary experiment. Analyses of β-actin expression was used as control
Clearly, a substantial virus replication occurs in HUVEC. After virus uptake, newly generated infectious viruses were detectable at 8 h p. i. and reached maximal intracellular concentrations at 10 h p. i. Extracellular viruses culminated
at 24 h p. i. In comparison with other susceptible cell lines (e.g., Raji, HeLa, or GMK) [26], virus progeny production was rather low in HUVEC (2–3 log levels less) indicating either minor replication in all cells or substantial replication in only a few cells. In order to analyze these possibilities, CVB3 infection of HUVEC was studied on the singlecell level using ICA. As shown in Fig. 1b maximal ratios of virus-positive HUVEC (18.2 ± 13.5 %) were observed at 10 h p. i. Thereafter, the percentage of virus-positive HUVEC declined till 72 h p. i. where
