1 Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium
2 Laboratory of Immunology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium
Correspondence
Herman W. Favoreel
Herman.Favoreel{at}UGent.be
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ABSTRACT |
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INTRODUCTION |
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Viral DNA delivery to the nucleus of TG neurons may lead to either of two possible infection patterns. Either a full replication cycle is initiated, which occurs in a cascade-like manner: expression of immediate-early and early genes gives rise to viral DNA replication, followed by expression of late genes at the final stages of the replication-cascade. Alternatively, a latent infection can be established at the very early stages of the virus replication cycle, before the onset of DNA replication. After establishment of latency, stress-related stimuli may lead to virus reactivation and initiation of a full virus replication cycle. Both during primary infection and reactivation, full alphaherpesvirus replication is thought to result in rapid cell death (reviewed by Preston, 2000).
However, under physiological conditions, TG neurons should be able to postpone alphaherpesvirus-induced cell death following a full virus replication cycle long enough to allow newly produced virus to travel long distances towards axon termini. Furthermore, based on several indirect data, it has already been suggested that TG and other neurons may survive (limited) virus replication during primary infection or reactivation, and enter or resume latency afterwards (Aleman et al., 2001; Geiger et al., 1995
; Perng et al., 2000
; Simmons & Tscharke, 1992
). Taken together, this may suggest that TG neurons might have a higher resistance towards alphaherpesvirus-induced cell death compared with other cell types. However, the kinetic time-course of an alphaherpesvirus infection and the possible resulting cell death in TG neurons of its natural host has, to date, never been studied.
Therefore, the aims of this study were to examine the interaction between PRV, a swine alphaherpesvirus, and primary TG neurons of its natural host in vitro. More specifically we determined (i) whether a PRV infection in porcine TG neurons in vitro generally results in a typical full virus replication as assessed by evaluating the kinetics of expression and processing of viral glycoproteins and comparing these with similar kinetic studies in different other porcine cell types and, if so, (ii) whether TG neurons can resist cell death induced by PRV to some extent.
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METHODS |
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Cells
Swine kidney (SK) and swine testicle (ST) cells.
Cells were seeded on glass coverslips and grown to confluency in minimum essential medium (MEM) supplemented with 10 % fetal bovine serum (FBS), 0·3 mg L-glutamine ml1, 100 U penicillin ml1, 0·1 mg streptomycin ml1 and 0·1 mg kanamycin ml1 (basic culture medium).
Superior cervical ganglion (SCG) cells.
Primary neuronal cultures were prepared from SCG from porcine fetuses collected at the slaughterhouse (gestation stage between 6·5 and 10 weeks) as adapted from Wang et al. (1995).
TG cells.
Primary neuronal cultures were prepared from TG from 4 to 10-week-old piglets. TG were dissected and longitudinally cut into two pieces before starting enzymic digestion with 0·2 % collagenase-A (Roche). Every 30 min, dissociated cells were harvested by centrifugation at 200 g for 5 min and the collagenase solution was re-used for further digestion of TG. After full digestion of the ganglia, the collected cell suspension was centrifuged at 200 g for 10 min. The pellet was resuspended in culture medium (basic culture medium without glutamine and supplemented with 30 ng nerve growth factor ml1) and cells were plated on poly-D-L-ornithine- and laminin-coated glass coverslips. One day after seeding, cells were washed extensively to remove non-adherent cells and fresh culture medium was added. Culture medium was replaced every 34 days.
Epithelial kidney cells.
Primary porcine epithelial cells were isolated from kidneys from 4 to 10-week-old piglets. Briefly, after removal of the capsule and the upper layer of the cortex, tissue originating from the mid part of the cortex was collected and cut into small pieces. Repetitive incubation of the tissue pieces with 2·5 mg trypsin ml1 resulted in a single-cell suspension, which was centrifuged at 400 g for 10 min. Cells were resuspended and seeded in basic culture medium supplemented with 0·5 % lactalbumin hydrolysate. All experiments were performed on first passage cells, seeded on glass coverslips and grown to confluency. Purity of epithelial cells was analysed by fluorescent staining with the anti-cytokeratin 7 monoclonal antibody (Dako), a marker for simple ductal epithelia found in kidneys. Approximately 90 % of the cells expressed cytokeratin 7.
Dermal fibroblasts.
Primary cultures of dermal fibroblasts were established from skin of porcine fetuses collected at the slaughterhouse (gestation stage between 6·5 and 10 weeks). Briefly, dermal tissue was cut into small pieces and subjected to repetitive trypsinization (2·5 mg trypsin ml1). The resulting single-cell suspension was centrifuged at 400 g for 10 min and cells were resuspended and seeded in basic culture medium. All experiments were performed on first passage cells, seeded on glass coverslips and grown to confluency.
Arterial endothelial cells.
Primary porcine endothelial cells were isolated as described previously by Van de Walle et al. (2003).
Inoculation of cells.
Cells were infected with virus at an m.o.i. of 10 TCID50 in culture medium. After incubation for 1 h at 37 °C, the inoculum was aspirated and fresh culture medium was added.
Antibodies and reagents.
Porcine FITC-labelled polyclonal anti-PRV antibodies were used as described previously (Nauwynck & Pensaert, 1995). Porcine biotinylated polyclonal anti-PRV antibodies were prepared from anti-PRV hyperimmune (HI) serum according to the manufacturer's instructions (Amersham). The mouse IgG2a monoclonal antibody against PRV gB (1C11) was produced at the laboratory (Nauwynck & Pensaert, 1995
). Mouse IgG1 monoclonal antibodies, anti-neurofilament-68 and anti-Map2 (a+b), were purchased from Sigma and the rabbit polyclonal antibodies directed against active caspase-3 were obtained from R&D systems. The mouse IgG1 monoclonal antibody directed against giantin was kindly provided by Dr H.-P. Hauri (Biozentrum, University of Basel; Linstedt & Hauri, 1993
).
The following secondary antibodies were used. FITC-conjugated goat anti-mouse, FITC-conjugated goat anti-rabbit and Texas red-conjugated goat anti-mouse antibodies, Texas red-conjugated streptavidin, Alexa Fluor 350-conjugated streptavidin (Molecular Probes) and isotype-specific FITC-conjugated rat anti-mouse IgG1 and biotinylated rat anti-mouse IgG2a antibodies (Serotec).
DNA fragmentation was detected based on the TUNEL reaction by using the In Situ Cell Death Detection kit (fluorescein) obtained from Roche.
Immunofluorescent staining procedures.
After being washed in PBS, cells were fixed with either 4 % paraformaldehyde for 10 min at room temperature followed by permeabilization with 0·2 % Triton X-100 in RPMI 1640 (Gibco-BRL) for 3 min at room temperature or with methanol for 20 min at 20 °C for intracellular staining. For cell surface staining, cells were fixed with 4 % paraformaldehyde for 10 min at room temperature. Antibodies were always diluted in PBS: primary antibody dilutions ranging from 1 : 30 to 1 : 100, FITC-conjugated secondary antibody dilutions 1 : 100, Texas red-conjugated and Alexa Fluor-conjugated secondary antibody dilutions 1 : 50 and the isotype-specific biotinylated rat anti-mouse IgG2a antibody dilution 1 : 100. Cells were incubated with antibody for 1 h at 37 °C, washes were performed with PBS for 5 min at room temperature. The detection of fragmented DNA with the TUNEL reaction was performed according to the manufacturer's instruction (Roche). Non-fixed cells were assessed for viability using ethidium-monoazide-bromide (EMA) (Molecular Probes) as described previously (Riedy et al., 1991) and fixed afterwards as described above. Cells were mounted and analysed by fluorescence and confocal microscopy. In case of the viability assay, 150 or 200 cells were scored for each coverslip. Experiments were repeated three times and results are presented as mean percentages with corresponding standard deviations.
Infectious centre assays.
Infectious centre assays were performed essentially as described previously (Van de Walle et al., 2003) using different sources of infectious virus.
Cell-free virus.
PRV 89V87 infectious virus particles (109 or 107) were neutralized using 20 % PRV-neutralizing porcine HI serum (serum neutralization titre 768) (Nauwynck & Pensaert, 1995) in MEM for 40 min on ice and subsequently 2x107 and 2x105 neutralized virus particles, respectively, were added to a monolayer of coverslip-grown ST cells.
Infected SK cells.
SK cells were inoculated with PRV 89V87 at an m.o.i. of 10 and cultivated in suspension as described previously (Favoreel et al., 2004). At 96 h post-inoculation (p.i.), cells were washed once and 106 SK cells were incubated in 20 % HI serum as described above. Next, 5x104 SK cells were added to a monolayer of coverslip-grown ST cells followed by gentle centrifugation for 2 min at 44 g.
Infected TG neurons and non-neuronal cells.
TG cultures were inoculated with PRV 89V87 at an m.o.i. of 10, and at 96 h p.i. detached using PBS without Ca2+ and Mg2+ containing 0·5 mM EDTA. Next, cells were washed once and 106 TG cells were incubated in 20 % HI serum as described above. Then, approximately 200 individual TG neurons or 3000 non-neuronal TG cells were manually picked, under phase-contrast microscopy, and transferred to a monolayer of coverslip-grown ST cells, followed by centrifugation for 2 min at 44 g.
At 20 h post-addition of cells or virus particles to the ST monolayer, cells were fixed with 4 % paraformaldehyde followed by methanol fixation as described above. Plaques were visualized using FITC-labelled anti-PRV antibodies, and when required a neuronal marker staining was performed as described above. All plaques on each coverslip were counted. Experiments were repeated three times and results are presented as mean values with corresponding standard deviations.
Confocal microscopy.
Confocal images were acquired using a Leica TCS SP2 confocal system (Leica Microsystems) or a Bio-Rad MRC-1024 confocal laser scanning microscope system (Bio-Rad) linked to a Nikon diaphot 300 inverted microscope (Nikon).
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RESULTS |
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Intracellular viral glycoprotein expression in PRV-infected TG neurons (Fig. 2a and b) was first observed at 6 h p.i. (in 22·4±1·9 % of TG neurons) and >90 % of TG neurons were positive at 12 h p.i., which was delayed by 24 h compared to the kinetic time-course of detection of intracellular viral glycoproteins in the other porcine cell types. The kinetic delay most likely reflects a delay at the very early stages of virus infection (e.g. virus entry, delivery of genome to the nucleus), since a similar delay was observed when analysing the expression of the immediate-early protein IE180 and the early protein US3 (data not shown).
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Cell survival following inoculation with PRV
The data presented in the former paragraph show that, at least in vitro, only marginal differences exist in the kinetics of PRV late protein expression and processing in porcine TG neurons versus other porcine cell types, which may lead to the speculation that only marginal differences are to be expected in the subsequent cell death. However, a significant number of data indirectly indicate that TG neurons, unlike other cell types, may possibly survive an ongoing alphaherpesvirus infection to some extent (Aleman et al., 2001; Geiger et al., 1995
; Hood et al., 2003
; Simmons & Tscharke, 1992
). To clarify this issue further, we examined whether, in our in vitro assay, TG neurons are more resistant to PRV-induced cell death compared to a broad range of other primary and immortalized porcine cell types. Therefore, primary porcine TG and SCG neurons, primary epithelial kidney cells, primary arterial endothelial cells, primary non-neuronal TG cells, primary dermal fibroblasts and immortalized SK cells were inoculated with PRV 89V87 and, at different times p.i., assessed for viability as described in Methods.
Fig. 5(a) demonstrates that the percentage of dead SCG neurons increased rapidly during infection resulting in 79·7±1·2 % dead PRV-infected SCG neurons at 16 h p.i., the final time point measured. No further time points could be included because of detachment of cells. Dermal fibroblasts, arterial endothelial cells and non-neuronal TG cells showed a comparable increase in dead cells at early stages of infection (73·4±1·7, 80·7±2·9 and 67·4±7·2 %, respectively, at 24 h p.i.) and at 48 h p.i., the vast majority of these cells had succumbed due to infection (94·8±0·2, 99·0±0·6 and 84·8±1·0 %). Immortalized and primary SK cells, however, were somewhat more resistant to PRV-induced cell death at early stages of infection (49·7±1·6 and 40·8±2·5 % dead cells, respectively, at 24 h p.i.). Nevertheless, 97·9±0·4 and 94·8±1·6 % of the PRV-infected immortalized and primary SK cells had died because of infection at 48 and 72 h p.i., respectively.
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To assess the sensitivity of our viability assay, as a positive control, PRV-infected TG neuronal cell cultures were incubated with genistein, which has been shown to be neurotoxic to primary rat cortical neurons in a dose-dependent manner (Linford et al., 2001). Addition of 200 genistein µM to TG cultures at 24 and 48 h p.i. increased the percentage of dead TG neurons significantly (Fig. 5a
). In conclusion, a significant fraction of porcine TG neurons exhibited unusually high resistance to PRV-induced cell death in comparison with a broad range of other porcine cell types including SCG neurons.
Surviving TG neurons are still able to transmit infectious virus
To determine whether the observed surviving TG neurons (96 h p.i.) are still able to transmit infectious virus, an infectious centre assay was performed (Van De Walle et al. 2003). Control SK cells and TG neuronal test cultures were infected with PRV for 96 h p.i., before cells were collected. SK or TG cells (106) were then incubated for 40 min with 20 % PRV-specific HI serum to neutralize extracellular virus. As a control, 109 or 107 PRV infectious virus particles were treated with 20 % PRV-specific HI serum for 40 min. Afterwards, cells or infectious virus particles were put on top of an ST monolayer, further incubated at 37 °C for 20 h and fixed and stained for viral antigens. The addition of up to 2x107 and 2x105 neutralized infectious virus particles, 50 000 SK cells (96 h p.i.) or 3000 non-neuronal TG cells (96 h p.i.), resulted in no or only very few plaques (Table 1
). In contrast, the addition of only ±200 TG neurons to ST cells produced 91·6±35·9 plaques. Fig. 6
shows that SK cells (96 h p.i.) are unable to induce plaque formation upon co-cultivation with ST cells, whereas TG neurons (96 h p.i.) can induce plaque formation.
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A PRV strain without the anti-apoptotic US3 protein does not induce substantial apoptosis in TG neurons
Over the last few years, it has been demonstrated that both HSV serotypes and bovine herpesvirus 1 can counteract apoptosis via viral-encoded anti-apoptotic genes in a cell type-specific way (Lovato et al., 2003 and references therein). Additionally, such anti-apoptotic activity of specific alphaherpesvirus genes has been suggested to promote the survival of infected TG neurons and has even been suggested to possibly allow re-establishment of latency (Lovato et al., 2003
; Perng et al., 2000
). The US3 protein kinase, which is conserved among all alphaherpesviruses, appears to be one of the most potent anti-apoptotic HSV-encoded proteins. Recently, we showed that the PRV US3 protein kinase is able to suppress virus-induced apoptosis in ST cells (Geenen et al., 2005
), and US3 is the only anti-apoptotic protein identified in PRV thus far. To assess whether PRV US3 has a role in the remarkable resistance of TG neurons towards PRV-induced cell death by protecting them from virus-induced apoptosis, TG neuronal cultures were inoculated with either wild-type (WT) or US3 null PRV at an m.o.i. of 10. At 24, 48 or 72 h p.i., cells were fixed and apoptotic cell death was determined and quantified using anti-active caspase-3 antibodies (Fig. 7a and b
) or the TUNEL reaction (Fig. 7c and d
).
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These results indicate that neither WT nor the strongly pro-apoptotic US3 null PRV is able to induce substantial apoptotic cell death in TG neurons.
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DISCUSSION |
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Here we show in vitro (i) that primary porcine TG neurons display a similar, albeit slightly delayed, pattern of viral late gene product expression upon infection with PRV, but (ii) that these cells are much more resistant towards PRV-induced cell death in comparison with a broad range of other porcine cell types (immortalized and primary epithelial kidney cells, primary arterial endothelial cells, primary non-neuronal TG cells, primary dermal fibroblasts, monocytes and primary SCG neurons).
We found that at 12 h p.i. at an m.o.i. of 10 over 90 % of the PRV-infected TG neurons displayed microscopically detectable levels of intracellular expression of viral glycoproteins, indicating that all TG neurons in our assay were permissive to PRV infection. Further, we showed that viral glycoproteins were processed through the Golgi and reached the cell surface, a typical pattern of late stages of infection, although with a slight, but consistent delay (±26 h) compared with other porcine cell types. Since neurons have limited concentrations of cellular transcription factors (Rajcani & Durmanova, 2000), it is likely that an extended time is needed for PRV-infected neurons to initiate viral transcription at early stages of infection, which could account for differences in expression kinetics between non-neuronal and neuronal cells. A possible explanation for the slower kinetics in TG versus SCG neurons may perhaps be the specific complexity of TG neurons, exemplified by the large size of the cell body (±65 µm for TG neurons versus ±15 µm for SCG neurons) and very complex structure of the Golgi-apparatus we have observed (Fig. 3
), which possibly may delay different aspects of the virus life cycle, including genome delivery at the nucleus and processing of newly formed viral glycoproteins.
It has been generally accepted that alphaherpesvirus replication ultimately results in cell death. However, indirect indications exist that alphaherpesvirus-infected neurons may survive replication to some extent, which could provide the time for virus spread to axon termini and perhaps even allow (re-)establishment of latency after virus replication has initiated (Aleman et al., 2001; Geiger et al., 1995
; Sawtell, 1997
; Simmons & Tscharke, 1992
). To clarify this issue further, we examined the kinetic time-course of cell death upon infection of different porcine cells with PRV. We showed that 48·9 % of PRV-infected TG neurons survived up to 4 days after infection (end of experiment), whereas in all other porcine cell types tested the vast majority of cells succumbed to PRV infection within 2 days. Moreover, we demonstrated that these surviving TG neurons (96 h p.i.) were still able to transmit infectious virus to other cells. This may suggests that the unusual resistance of TG neurons towards PRV-induced cell death may result in extended time of virus transmission in vivo, thereby promoting virus spread. Clearly, more research is required to test this hypothesis.
The mechanism(s) of resistance of a significant part of TG neurons towards PRV-induced cell death in vitro is still speculative, but may perhaps depend on specific viral or cellular anti-apoptotic proteins. To date, only one viral protein of PRV has been demonstrated to display anti-apoptotic activity: the US3 protein kinase (Geenen et al., 2005). To evaluate the possible importance of PRV US3 in the resistance of TG neurons towards PRV-induced cell death, in vitro cultures of TG cells (consisting of neuronal and non-neuronal cells) were infected with WT or US3 null PRV. We found that WT PRV induced apoptotic cell death in the majority of non-neuronal TG cells at late stages of infection (
48 h p.i.), whereas infection with a US3 null virus resulted in much more frequent and earlier apoptosis in these cells (24 h p.i.). However, neither WT nor US3 null PRV induced substantial apoptosis in neuronal TG cells at any of the time points tested (24, 48 and 72 h p.i.). Thus, TG neurons show a marked resistance towards PRV-induced apoptosis, and do not depend on the viral anti-apoptotic US3 protein for this resistance. It will be interesting to explore whether the PRV orthologues of the other anti-apoptotic proteins described in HSV [e.g. ICP4, ICP27, ICP22, US5, US6 and especially latency-associated transcripts (LATs), reviewed by Aubert & Blaho, 2001
] display anti-apoptotic activity, and, if so, whether they are involved in the resistance of TG neurons towards PRV-induced cell death. On the other hand, TG neuron-specific cellular anti-apoptotic proteins may account for the unusual resistance of these cells to PRV-induced cell death. Interestingly, Brn-3a, a member of the IV-POU family of transcription factors, is highly expressed in sensory neurons. Brn-3a has been shown to protect sensory neurons (TG neurons and dorsal root ganglion neurons), but not sympathetic neurons (SCG) from apoptotic cell death by activating the expression of two anti-apoptotic members of the Bcl-2 family, Bcl-2 and Bcl-xL (Ensor et al., 2001
; Smith et al., 1998a
, b
, 2001
). This is in agreement with our findings that TG neurons, but not SCG neurons, show resistance to PRV-induced cell death. Future research will show whether this higher expression of Brn-3a in sensory versus sympathetic neurons may explain the resistance of TG neurons towards PRV-induced cell death. Although speculative, such possible involvement of Brn-3a in protecting TG neurons from PRV-induced cell death may perhaps also aid to explain why only about half of the TG neurons in our in vitro study survive PRV infection. In this context, it is interesting that Yang et al. (2000)
showed that all neuronal populations within the TG appear to be capable of supporting productive infection with HSV-1 as assayed by immunofluorescent staining using polyclonal antisera to HSV, but that some neuronal phenotypes are more permissive for establishment of latent infection with LAT expression than others. They found that a large percentage of latently infected sensory neurons expressed TrkA, the high affinity nerve growth factor-receptor. Since it has been demonstrated that the expression of TrkA is regulated by Brn-3a (Ma et al., 2003
), it will be interesting to explore whether the subpopulation of surviving TG neurons express high levels of TrkA.
Although speculative, the prolonged survival of PRV-infected TG neurons combined with the somewhat delayed kinetics of viral late protein expression and processing we observe in TG neurons may perhaps provide an idea on the possibility of latency (re-)establishment after limited virus replication in vivo. The slower viral protein expression kinetics and extended survival perhaps supply the time credit needed for innate and/or adaptive immune responses to develop and suppress virus replication thereby paving the way for the establishment of latency. Indeed, it has been shown that elements of the innate and adaptive immune response e.g. interferons (IFN), CD8+ T cells, tumour necrosis factor-, and antibodies are capable of suppressing alphaherpesvirus replication in neurons and that some of these components, like IFN-
, are directly linked to inhibition of virus-induced neuronal cell death and induction of a quiescent/latent state of infection (Cantin et al., 1995
; Geiger et al., 1995
, 1997
; Khanna et al., 2003
; Liu et al., 2001
; Oakes & Lausch, 1984
; Sainz & Halford, 2002
; Schijns et al., 1991
; Shimeld et al., 1997
; Simmons & Tscharke, 1992
). Using our homologous in vitro system of swine alphaherpesvirus infection in swine TG cultures that we describe here, we are planning experiments to clarify these issues further.
In conclusion, this study shows that using a unique homologous in vitro model based on the swine PRV and a primary neuronal culture derived from the porcine TG, viral glycoprotein expression, processing and cell surface transport could be observed in all TG neurons upon infection by PRV and that these TG neurons were remarkably more resistant to PRV-induced cell death compared with other porcine cell types.
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ACKNOWLEDGEMENTS |
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Received 12 November 2004;
accepted 4 February 2005.