Department of Virology, School of Veterinary Science, University of Buenos Aires, Chorroarin 280, 1427 Buenos Aires, Argentina1
Author for correspondence: Gustavo Delhon. Fax +54 11 4524 8480. e-mail delhon{at}fvet.uba.ar
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Abstract |
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Introduction |
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Like herpes simplex virus 1 (HSV-1), the prototype member of the alpha subfamily of herpesviruses, BHV-1 establishes life-long, non-productive latent infections in ganglionic sensory neurons (Homan & Easterday, 1980 ; Rock et al., 1987
; for reviews see Rock, 1993
, 1994
; Jones, 1998
). During the first days following infection by BHV-1 or by HSV-1, some neurons are productively infected (acute phase). However, other neurons are not permissive to virus replication and become latently infected. After the acute phase subsides, these cells continue to harbour viral DNA without producing infectious virus (latent phase). During latency, gene expression is restricted to a small region of the genome known as the latency-related (LR) (BHV-1) or latency-associated (HSV-1) region (Rock et al., 1987
; Stevens et al., 1987
). Expression of LR sequences in cycling cells resulted in cell cycle arrest (Schang et al., 1996
) and in inhibition of apoptosis induced by sphingoid bases (Ciacci-Zanella et al., 1999
). A role for the latency-associated region in neuronal survival has been proposed recently. Infection of rabbits with HSV-1 mutants which do not express latency-associated transcripts (LATs) resulted in extensive neuronal apoptosis in the trigeminal ganglia (TG) (Perng et al., 2000). A significant loss of neurons was also observed after inoculation of mice with LAT null mutants. However, neuronal apoptosis was not detected in these animals (Thompson & Sawtell, 2001
). These results open the question as to whether apoptosis or another neuronal death mechanism is the target for LATs.
In this study, the susceptibility of ganglionic neurons to infection by BHV-1 was examined. Infection of cultured ganglionic neurons with BHV-1 resulted in features of apoptosis such as DNA fragmentation, chromatin condensation, membrane blebbing and cell shrinkage. We found that BHV-1 induces neuronal apoptosis in a viral DNA replication-dependent manner. Infection of rabbits with BHV-1 induced mononuclear cell infiltration and neuronophagia in the TG. Apoptosis in the infected TG was mainly confined to infiltrating mononuclear cells, but no morphological evidence of apoptosis was detected in neurons. Furthermore, DNA cleavage in neurons was very limited and sporadic.
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Methods |
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Nerve cell cultures.
Sensory neurons from newborn rabbits were prepared as described previously (Jones et al., 1990 ). Briefly, dorsal root ganglia (DRG) were collected, incubated with 0·25% collagenase (Sigma) in Eagles essential medium (MEM, Sigma) for 3 h at 37 °C, and mechanically dissociated. Cells were washed twice with PBS (pH 7·3), resuspended in growth medium, and plated onto collagen-coated coverslips or chamber slides at a density of 5x103 to 104 neurons per well. The growth medium consisted of MEM containing 25 µg/ml gentamicin, 1·4 mM glutamine, 12 mg/ml glucose, 50 ng/ml nerve growth factor (Collaborative Research, Bedford, Mass., USA), and 10% foetal bovine serum. To eliminate non-neural cells, cultures were maintained in growth medium supplemented with 10 µM fluorodeoxyuridine (Sigma) and 10 µM cytosine arabinoside (Sigma) for 10 days. This treatment consistently yields cultures with more than 95% of cells being neurons as judged by cell morphology. Neuronal cultures were maintained at 37 °C with 5% CO2.
Ganglionic non-neural cell (NNC) cultures.
DRG cell suspensions were obtained as described above and plated on plastic wells not covered with collagen. After 2 h incubation at 37 °C, 5% CO2, non- adherent cells were removed by gently pipetting the bottom of the well with fresh medium. Adherent cells were allowed to reach confluence, trypsinized, counted and split at a 1:3 ratio. This procedure eliminated virtually all neurons from the cultures.
Infection of cell cultures and virus titration.
After antimitotic treatment, nerve cell cultures were washed three times with PBS and maintained for 2 days in growth medium. Neuron (5000 cells per well) or NNC cultures (25000 cells per well) were infected with BHV-1 (m.o.i. 1·0) for 60 min at 37 °C. The cultures were rocked gently every 15 min to allow even spread of virus. After adsorption, the cultures were rinsed three times with PBS and once with growth medium, and refed with fresh growth medium. Virus was harvested at various days post-infection (p.i.) by three cycles of freezethawing the cells plus media. Viral titres were determined by standard plaque assays on MDBK cells. For each time-point, virus titres were the result of pooling three parallel cultures. For Fig. 1(a), the virus titres were normalized for the number of cells so that the given values are directly comparable.
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To determine whether APC affected cell numbers, neurons were grown in the presence or absence of 25 µM APC for 24 or 48 h and counted. To facilitate cell counting, a sterile needle was used to draw a grid on the surface of collagen-coated coverslips. Cell numbers per well were determined for four wells per condition and time-point in two parallel experiments. Individual values were analysed by one-way ANOVA.
In situ hybridization (ISH).
To estimate the number of neurons actively replicating viral DNA, neuronal cultures grown on coverslips were infected with 1·0 TCID50 per cell, incubated for 12, 24, 36 or 48 h p.i., and fixed with 95% ethanolglacial acetic acid (3:1) for 30 min at room temperature. ISH was performed essentially as described by Rock et al. (1992) . RNA was eliminated by incubation with 100 µg/ml DNase-free RNase (Promega). Cultures were hybridized with 2·5x105 c.p.m. of 3H-labelled BHV-1 DNA fragments for 72 h at 45 °C. For determination of the percentage of neurons actively replicating viral DNA, cell counts for two cultures per time-point were performed. A total of 500 cells per culture were counted by randomly selecting fields of about 15 to 40 neurons each (magnification x400). The results were expressed as the mean percentage of neurons positive for viral DNA replication from two cultures±standard error of the mean. Cells were considered positive for viral DNA replication when they exhibited grains too numerous to count.
Electron microscopy.
Neuronal cultures grown on chamber slides were infected with BHV-1 (m.o.i. 1·0) in the presence or absence of 25 µM aphidicolin. After 1, 24 or 48 h p.i., cells were fixed with 3% glutaraldehyde in 0·1 M sodium cacodylate pH 7·2, post-fixed in aqueous OsO4, dehydrated in a graded ethanol series and embedded in situ with Epon 12. Ultrathin sections were stained with uranyl acetate and lead citrate, and examined with a JEOL 100 apparatus.
TUNEL staining.
The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling (TUNEL) technique detects endonucleolytically cleaved DNA by addition of labelled dUTP to DNA ends in situ. Neuronal cultures were infected with infectious or heat inactivated BHV-1 as described above (m.o.i. 1·0). At various times p.i., cells were washed in PBS, fixed with 4% buffered paraformaldehyde (pH 7·3) for 20 min, and permeabilized by incubation with proteinase K (20 µg/ml in PBS) for 15 min at room temperature. Cells were stained with an in situ apoptosis detection kit (Apop Tag, Intergen Co., NY, USA) following the manufacturers instructions. The percentage of TUNEL-positive cells was calculated as described for ISH. TG sections were processed as described above except sections were previously deparaffinized and rehydrated.
DAPI staining.
For DNA staining, neuronal cultures were infected and fixed as described for the TUNEL technique. The cultures were incubated with 0·5% Triton X-100 for 10 min, washed with PBS (pH 7·3) and stained in a dark chamber with DAPI (2 µg/ml in absolute methanol; Sigma) for 2 min at room temperature. Coverslips were sequentially rinsed with absolute methanol and 70% ethanol, mounted with 20% glycerol in PBS and examined under UV light with a fluorescent microscope (Zeiss).
Animal inoculation.
Seven-month-old New Zealand White rabbits were tranquillized with acepromazine and inoculated in the right and left conjunctival sacs and nostrils with, in total, 106 TCID50 of BHV-1. The volume of the inoculum was 10 µl per site. Animals (two per time point) were sacrificed at 0, 1, 2, 3, 4, 5 or 6 days p.i. and the TG processed for standard histology or the TUNEL technique.
Histology.
The TG were fixed in 4% buffered paraformaldehyde (pH 7·4), dehydrated in a graded ethanol series, and embedded in paraffin. TG sections were deparaffinized, rehydrated, sectioned at 5 µm, and stained with haematoxylin & eosin (HE) or further processed for the TUNEL technique as described for neuronal cultures. Selected TG samples were embedded in Epon 12 resin, sectioned at 0·51 µm with an ultramicrotome, and stained with 1% Toluidine blue-Azur II.
RTPCR analysis.
To examine bICP0 and gC transcription, neuronal cultures were infected with BHV-1 at 1·0 TCID50 per cell in the presence or absence of APC. Twelve or 48 h p.i. the cultures were washed twice with PBS and total RNA was extracted as described by Dyanov & Dzitoeva (1995) . RTPCR was performed by using a commercial kit (Access RTPCR, Promega) following the manufacturers instructions. Primers and PCR conditions for amplification of bICP0 and gC were described previously (Hossain et al., 1995
; Schang & Jones, 1997
). Two µg of total RNA was incubated with 1 U of RNase-free DNase I (Gibco BRL) in the presence of 7 U of RNasin (Promega) for 30 min at 20 °C. The reaction was terminated by addition of EDTA at a final concentration of 2 mM and incubation for 10 min at 62 °C. Twenty µl of DNase-treated total RNA was mixed with 30 µl of a solution containing 1x commercial reaction buffer, 200 µM dNTP mix, 1 µM of each primer, 1 mM MgSO4, 5 U of AMV reverse transcriptase (omitted in the RT control), 5 U of Tfl DNA polymerase, and 10% glycerol (omitted for gC). The reactions were covered with mineral oil and incubated 45 min at 48 °C (reverse transcription). The tubes were transferred to a thermocycler and hot started. Amplification was carried out for 40 cycles by denaturing 1 min at 95 °C, annealing 1 min at 65 °C and extending 2 min at 72 °C. For complete extension of the amplified products, a final incubation at 72 °C for 5 min was included. Amplified products were resolved by agarose gel electrophoresis. PCR primers for bICP0 were (sense) 5' AGT CGA GGC GCA CGC GGG 3' and (antisense) 5' TGG GGG GCG CGG AAA CTG 3'. Primers for gC were (sense) 5' GAG CAA AGC CCC GCC GAA GGA 3' and (antisense) 5' TAC GAA CAG CAG CAC GGG CGG 3'. The expected sizes of amplified products were 164 (bICP0) and 229 bp (gC).
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Results |
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Viral DNA replication in cultured neurons
To determine the number of neurons actively replicating viral DNA, neurons were grown on coverslips for 12 days and then infected with BHV-1 (m.o.i. 1·0). At various times p.i., neurons were processed for ISH and probed for viral DNA with 3H-labelled BHV-1 DNA fragments as described in Methods. Under these conditions, label was observed almost exclusively in the nuclei of infected neurons and ranged from sporadic grains compatible with background signal levels to dense accumulations of grains (Fig. 1b). Almost 50% of neurons were involved in active viral DNA replication by 36 hours p.i. (500 cells per time-point in two parallel experiments) (Fig. 1d
). Taken together these results are consistent with a delayed virus infectious cycle in neurons relative to NNC (Nichol et al., 1996
).
BHV-1 infection induces apoptosis in cultured neurons
Cleavage of DNA into nucleosomal fragments is characteristic of apoptosis. To determine whether BHV-1 infection is associated with DNA fragmentation in neurons, neuronal cultures were infected with BHV-1 (m.o.i. 1·0) and processed for TUNEL, a technique that detects free DNA ends in situ (Fig. 2). When mock-infected neuronal cultures were assayed with the TUNEL technique, less than 1% of the neurons were positive for TUNEL. Zero, 12 (±1), and 34 (±5)% of neurons were positive for TUNEL after 12, 24 and 48 h of infection with infectious BHV-1, respectively. The gross morphology of neurons remained unchanged up to 48 h p.i. (Fig. 2a
, c
). However, in the following days cell shrinkage, cytoplasmic blebbing and fragmentation of cells became evident (Fig. 2e
, f
). No TUNEL-positive neurons were detected at the same time-points when heat-inactivated BHV-1 was used as inoculum (not shown). Chromatin condensation, as judged by DAPI staining, was detected starting on day 3 p.i. in infected neurons (Fig. 3
). The results indicate that, under our experimental conditions, primary sensory neurons are killed by BHV-1 infection and that apoptotic cell death is involved in the process.
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Ten-day-old neuronal cultures were treated with APC for 2 days before virus infection (m.o.i. 1·0) and maintained in the presence of the drug throughout the length of the experiment. Decreasing low levels of infectious virus could be recovered from culture supernatants for the first 2 days p.i., but not on day 3 or beyond, suggesting that detected virus represents inoculum virus and not progeny virus (not shown). At various times p.i. in the presence of APC, neuronal cultures were processed for the detection of viral DNA by ISH and scored as positive or negative for ongoing viral DNA replication as described above. No positive neurons were observed after 24, 36, 48 or 72 h p.i. (Fig. 1c). When parallel cultures were stained with the TUNEL technique, or DAPI, no TUNEL-positive cells or cells with condensed chromatin were detected (Fig. 2b
, d
; not shown). Transmission electron microscopy of APC-treated neurons infected with BHV-1 demonstrated that virus was able to attach to neurons in the presence of the inhibitor (Fig. 4D
). These results indicate that viral DNA replication plays a role in the induction of neuronal apoptosis by BHV-1 in vitro.
Transcription from the bICP0 gene is not prevented in APC-treated neurons
Using RTPCR, we observed that gC, a late virus gene, is never transcribed at detectable levels in APC-treated infected neurons (Fig. 5). However, it is theoretically possible that virus genes expressed before the initiation of viral DNA replication are transcribed in APC-treated neurons and, therefore, could participate in or be responsible for the induction of apoptosis. This is especially significant for genes like bICP0, the BHV-1 homologue of HSV-1 ICP0 (Wirth et al., 1989
, 1992
). The bICP0 gene is toxic when expressed in mammalian cells in the absence of other viral proteins (Ciacci-Zanella et al., 1999
; Inman et al., 2001
). Using RTPCR, we explored the possibility that the dual kinetics bICP0 gene is expressed under circumstances where viral DNA replication is pharmacologically prevented. Following infection with BHV-1 (m.o.i. 1·0), total RNA from APC-treated or untreated neurons was extracted, reverse transcribed and PCR amplified. No DNA amplification resulted from RNA extracted from uninfected cultures (Fig. 5
, lane 4) or from infected cultures when the RT step was omitted (Fig. 5
, lane 2). An amplified DNA fragment of the expected size was obtained from infected cultures at 12 h p.i. in the absence of APC (Fig. 5
, lane 3). Transcription from the bICP0 gene was consistently detected in APC-treated neuronal cultures after 12 but not 48 h p.i. (Fig. 5
, lanes 5 and 6), supporting the idea that virus is internalized by neurons in the presence of APC. These results indicate that transcription from the bICP0 gene is not prevented in APC-treated neurons.
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Discussion |
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BHV-1 causes apoptosis in a number of cell systems (Devireddy & Jones, 1999 ; Eskra & Splitter, 1997
; Hannon et al., 1996
, 1997
, 1998
; Winkler et al., 1999
). Requirements for induction seem to be cell-type specific. For example, both infectious and inactivated BHV-1 are able to induce apoptosis in mitogen-stimulated PMBCs (Hannon et al., 1996
) or activated CD4+ cells (Eskra & Splitter, 1997
). Attachment of virus particles is sufficient for induction in PMBCs (Hannon et al., 1998
). Whereas infectious BHV-1 induced apoptosis in epithelial cell lines, inactivated virus was unable to efficiently induce apoptosis (Devireddy & Jones, 1999
). The fact that virus attachment to the cell membrane per se is not followed by apoptosis in cultured neurons was indicated by ultrastructural examination of APC-treated cultures inoculated with BHV-1 (Fig. 4D
).
In cultured neurons infected with BHV-1, we found that 0, 12 and 34% of neurons exhibited a TUNEL-positive signal at 12, 24 and 48 h p.i., respectively. These values represent roughly half of the neurons supporting viral DNA replication at the corresponding time-points (Fig. 1d), suggesting that the apoptotic programme is triggered after viral DNA replication is initiated. The observation that not all neurons supporting viral DNA replication exhibit a TUNEL-positive signal could reflect the fact that a critical stage in viral DNA replication (one that is not discerned by ISH) should be achieved before apoptosis is triggered in neurons. Another possibility is that template amplification above a critical threshold is required for the induction of apoptosis.
It was previously shown that rabbit neuronal cultures can be maintained for several weeks in the presence of APC without any obvious secondary toxic effects (Delhon, 1990 ). APC inhibits eukaryotic DNA polymerases, including viral polymerases, thus blocking DNA replication at an early stage (Marheineke & Hyrien, 2001
). In this report, BHV-1-induced apoptosis was completely prevented when viral DNA replication was blocked with APC (Fig. 2b
, d
). Although at this time we cannot rule out the possibility that other factors associated with virus infection are involved in the induction of apoptosis, the fact that treatment with APC did not preclude virus attachment to the cell surface (Fig. 4D
), nor viral gene expression (Fig. 5
), supports the idea that viral DNA replication plays an important role in BHV-1-induced neuronal apoptosis in vitro.
Because quantification of transcription was not attempted here, differences in the level of bICP0 transcription between APC-treated and untreated neurons remain unknown. Previous work with HSV-1-infected primary neurons has shown that inhibition of viral DNA replication results in a dramatic decrease in the expression of all classes of viral genes, including the ICP0 gene (Nichol et al., 1996 ). Such dependence of viral gene expression on viral DNA replication has also been observed during infection of the mouse TG with thymidine kinase-negative HSV-1 mutants (Kosz-Vnenchak et al., 1990
, 1993
). bICP0 is toxic for cycling cells when expressed in the absence of any other viral gene (Ciacci-Zanella et al., 1999
), but does not directly induce apoptosis (Inman et al., 2001
). The observation that neither DNA cleavage nor chromatin condensation was detected in APC-treated neurons over a 3 day period (Fig. 2
; not shown), and that cell numbers were not decreased by APC, suggest that the level of bICP0 expression is not apoptotic or toxic in neurons under the effect of APC. It remains to be determined whether expression of bICP0 is apoptotic or toxic in transiently transfected neurons.
It is tempting to speculate about the role of viral DNA replication in the induction of apoptosis in neurons. Viral DNA replication could induce apoptosis through at least two mechanisms which are not mutually exclusive. One mechanism is that viral DNA replication per se triggers the apoptotic programme. The other mechanism is that viral DNA replication is required for maximal expression of viral genes, which in turn triggers the apoptotic programme. This could partially explain why we observed less TUNEL-positive neurons than neurons actively replicating viral DNA at the corresponding time-points. The idea of viral DNA replication as an apoptosis trigger in neurons is consistent with previous works showing that unscheduled DNA replication or activation of pro-proliferation genes in post-mitotic neurons leads to apoptosis instead of cell division (Freeman et al., 1994 ; Lee et al., 1994
; reviewed by Ross, 1996
). In this context, it is interesting that expression of cell cycle progression markers such as cyclins D1, E and A, is induced in TG neurons following acute infection by BHV-1 (Schang et al., 1996
; Winkler et al., 2000
).
TG from acutely infected rabbits but not from mock-infected animals showed pathological changes which increased in magnitude as a function of the time p.i. These changes included mononuclear cell infiltration, apoptosis and neuronophagia. Features of apoptosis such as chromatin condensation and DNA fragmentation were observed in mononuclear cells surrounding neurons (Fig. 6). In contrast, morphological hallmarks of apoptosis were absent in TG neurons. Furthermore, whereas neurons exhibiting pathological changes were observed in all the TG between 3 and 6 days p.i., TUNEL-positive neurons were exceedingly rare and sporadic, indicating that neuronal apoptosis is not a common event in BHV-1-infected TG. This low level of in vivo neuronal apoptosis in the TG is coincident with those observed in rabbit or mouse infected with wild-type HSV-1 (Perng et al., 2000
; Thompson & Sawtell, 2001
; Ahmed et al., 2002
), or in swine infected with pseudorabies virus (PRV) (Aleman et al., 2001
). As in PRV-infected TG, TUNEL-positive neurons in BHV-1-infected TG exhibited a well preserved morphology and were associated with moderate inflammatory reactions. Because virus detection was not attempted in this work, we do not know whether TUNEL-positive TG neurons (and mononuclear infiltrating cells) are virus-infected cells. These cells could represent virus-infected cells, cells under the effect of cytokines released in the ganglion during virus infection, or both (Allsopp & Fazakerley, 2000
; Chao et al., 1995
; Sei et al., 1995
; Shimeld et al., 1999
). In swine infected with PRV, the sporadic TUNEL-positive TG neurons were also positive for PRV antigens, suggesting that virus infection induced the apoptotic programme (Aleman et al., 2001
).
The results presented here indicate that ganglionic neurons are not intrinsically resistant to virus-induced apoptosis. However, neuronal apoptosis was very rare in the infected TG as reported for other two alphaherpesviruses. Therefore, mechanisms in the TG should exist to prevent neuronal apoptosis upon virus infection. If one function of the LR gene is to ensure neuronal survival and given that viral DNA replication is a major inducer of apoptosis, it will be of great interest to elucidate how and when the LR gene exerts its protective role.
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Acknowledgments |
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References |
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Received 20 December 2001;
accepted 16 April 2002.