Varicella-Zoster Virus ORF63 Inhibits Apoptosis of
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病菌学杂志 2006年第2期
Centre for Virus Research, Westmead Millennium Institute and University of Sydney
Dept. Infectious Diseases and Immunology, University of Sydney, P.O. Box 412, Westmead, 2145 NSW, Australia
Department of Pediatrics, Stanford University School of Medicine, Stanford, California 94305
Ophthalmology and Visual Science Research Center, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
ABSTRACT
Virus-encoded modulation of apoptosis may serve as a mechanism to enhance cell survival and virus persistence. The impact of productive varicella-zoster virus (VZV) infection on apoptosis appears to be cell type specific, as infected human sensory neurons are resistant to apoptosis, yet human fibroblasts readily become apoptotic. We sought to identify the viral gene product(s) responsible for this antiapoptotic phenotype in primary human sensory neurons. Treatment with phosphonoacetic acid to inhibit viral DNA replication and late-phase gene expression did not alter the antiapoptotic phenotype, implicating immediate-early (IE) or early genes or a virion component. Compared to the parental VZV strain (rOKA), a recombinant virus unable to express one copy of the diploid IE gene ORF63 (rOkaORF63) demonstrated a significant induction of apoptosis in infected neurons, as determined by three methods: annexin V staining, deoxynucleotidyltransferase-mediated dUTP-biotin nick end label staining, and transmission electron microscopy. Furthermore, neurons transfected with a plasmid expressing ORF63 resisted apoptosis induced by nerve growth factor withdrawal. These results show that ORF63 can suppress apoptosis of neurons and provide the first identification of a VZV gene encoding an antiapoptotic function. As ORF63 is expressed in neurons during both productive and latent infection, it may play a significant role in viral pathogenesis by promoting neuron survival during primary and reactivated infections.
INTRODUCTION
Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes varicella (chicken pox) during primary infection and herpes zoster (shingles) following reactivation of latent virus from the dorsal root ganglia (DRG) (16, 22). A severe and debilitating complication of herpes zoster is postherpetic neuralgia, which involves chronic pain persisting for months or years following the initial herpes zoster attack (21, 14). It has been proposed that the pain associated with herpes zoster and postherpetic neuralgia is due to neural tissue destruction caused by VZV reactivation (35, 36). However, we recently reported that VZV-infected neurons, but not VZV-infected human fibroblasts (HF), were resistant to apoptosis (20), suggesting that neuronal destruction in vivo may be a corollary of the host cell immune response. This neuron-specific antiapoptotic function could increase the ability of VZV to efficiently establish latency and allow augmented virion production for axonal transport to the skin and reactivation as zoster lesions.
Other alphaherpesviruses, i.e., herpes simplex virus type 1 (HSV-1) and bovine herpesvirus type 1, express latency-associated transcripts and the latency-related gene, respectively, during latent infection of neurons (10, 11, 13, 32, 33, 34). Both of these have been shown to promote neuronal survival by an antiapoptotic mechanism in the event of induced apoptosis (1, 6, 16, 18, 27, 31), although VZV does not encode a known homolog of either gene. However, during productive infection HSV-1 expresses a number of other genes with antiapoptotic functions: US3, ICP4, ICP22, ICP27, gD, and gJ (2, 3, 23, 25, 26, 32, 39), and several of these do share sequence homology with VZV genes. HSV-1 US3, ICP27, ICP4, and ICP22 share homology with ORF66, ORF4, ORF62, and ORF63, respectively, although any antiapoptotic functions encoded by these VZV genes have not been previously examined. In this study, we sought to identify a VZV gene product(s) that encodes an antiapoptotic function in neurons. Using three methods of apoptosis detection (annexin V staining, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end label[TUNEL] staining, and transmission electron microscopy [TEM]), we applied a combination of drug block experiments, infections with viral gene deletion mutants, and transient transfection-based assays to primary sensory neurons. We show that VZV ORF63, a gene expressed prominently in neurons during both the productive and latent phases of infection (12, 28, 29), encodes an antiapoptotic function, as deletion of this gene from the virus significantly increased the percentage of apoptotic neurons following infection and introduction of ORF63 to neurons protected them from nerve growth factor (NGF)-induced apoptosis. This study provides the first evidence that ORF63 promotes neuronal cell survival after VZV infection by modulating apoptosis.
MATERIALS AND METHODS
Culturing of dissociated human sensory neurons. Human fetal spines (14 to 20 weeks of gestation) were obtained after informed consent and approval by the University of Sydney Human Ethics Committee. DRG were dissected from the spine, dissociated, and cultured as previously described (20). Dissociated neuronal cultures were seeded to give a range of 200 to 500 neurons per well. Resulting cultures contained 80% neurons as determined by cell morphology, and on day 3 postplating, extensive axonal networks between neurons were observed. Rat DRG were dissected and processed as previously described (30).
Viruses. The VZV strains used in these studies were Schenke (a low-passage clinical isolate), recombinant strain rOka (generated using cosmids derived fromthe varicella vaccine strain) (7), the rOkaORF63 mutant, and the rOkaORF70 mutant (38). Viruses were propagated in HF grown in tissue culture medium (Dulbecco modified Eagle medium [DMEM]) (GIBCO) supplemented with 10% heat-inactivated fetal calf serum (CSL). For VZV infections, the degree of inoculating fibroblast infection was scored using a scale from 0 to 4+, where 0 corresponds to no detectable infection and 4+ corresponds to 100% cytopathic effect. Neuronal cultures were inoculated using HF showing cytopathic effect in a range between 2 and 3+.
Infection and PAA treatment. Human neuronal cultures were inoculated with VZV by adding 1 x 104 VZV-infected HF or mock infected by adding 1 x 104 uninfected HF. In some experiments, directly following a 2-h treatment with 300 μg/ml phosphonoacetic acid (PAA; Sigma), mock- or VZV-infected cells were added to the cultures for 2 h at 37°C with 5% CO2. Following this incubation, supplemented DMEM was added to the neuronal cultures.
Immunofluorescence staining of VZV antigens. Cells were immunostained with rabbit polyclonal antibodies directed against VZV ORF62, ORF4, or ORF29 or with VZV-immune immunoglobulin G (IgG)-purified polyclonal human serum as previously described (20). The VZV-immune IgG-purified polyclonal human serum reacts with glycoprotein E as the predominant target on infected cells (A. M. Arvin, personal communication). Primary antibodies were detected with fluorescein isothiocyanate (FITC)-conjugated goat anti-human IgG and FITC-conjugated goat anti-rabbit IgG. In all immunostaining experiments, isotype control antibodies were used on mock- and VZV-infected cells to control for nonspecific antibody binding.
Annexin V staining. Human neuronal cultures were assessed for annexin V staining by incubation with annexin V-Alexa Fluor 594 conjugate as previously described (20). Cells were washed in annexin V-binding buffer and mounted with SlowFade antifade mounting fluid containing a DAPI (4',6'-diamidino-2-phenylindole) nuclear counterstain (Molecular Probes). Positive control cells were incubated at 37°C with 5% ethanol prior to incubation with annexin V-binding buffer. Slides were analyzed using a Leica laser scanning confocal microscope. For determination of the percentage of viral antigen- or annexin V-positive cells, a total of 500 neurons were counted by randomly selecting fields of 50 neurons.
TUNEL staining. Human neuronal cultures were processed and assessed for TUNEL staining as previously described (20). Positive control coverslips were treated with 3 U of DNase I (GIBCO) for 30 min at 37°C prior to the addition of the 3'-OH labeling mix (1x terminal deoxynucleotidyltransferase [TdT] reaction buffer, 50 μM biotin-14-dCTP [GIBCO], and 0.2 U of TdT [GIBCO]). The negative control coverslips were incubated with a labeling mix containing no TdT. Cells were washed and incubated with 5 μg/ml of streptavidin-FITC (GIBCO) in 5% skim milk for 30 min at 37°C. Coverslips were washed and mounted onto slides by use of SlowFade antifade mounting fluid with propidium iodide (1:500) (Sigma) to determine cell viability. Cells were analyzed using a Leica laser scanning confocal microscope, and the percentage of TUNEL-positive cells was calculated as described above.
Transmission electron microscopy (TEM). Human neurons were fixed in modified Karnovsky's fixative for 1 h and processed as previously described (20). Briefly, cells were washed and postfixed in 2% buffered osmium tetroxide for 3 h and subsequently in 2% aqueous uranyl acetate (Fluka) for 1 h, dehydrated with ethanol, and embedded in Spurr resin (TAAB Laboratories Ltd). Polymerization occurred at 70°C for 10 h. Sections were cut using a Reichert-Jung Ultracut E microtome and stained with 1% uranyl acetate in 50% ethanol and Reynolds’ lead citrate. Sections were examined with a Philips CM120 BioTWIN transmission electron microscope at 80 kV.
Transfection of primary rat sensory neurons and NGF withdrawal. Primary rat neurons were dissociated as previously described (30) and, prior to being plated on Matrigel-coated coverslips, were transfected with plasmid pG310 or pG310 containing the VZV ORF63 gene. Transfection of primary rat neurons was performed using a rat neuron Nucleofector kit for rat DRG neurons (Amaxa Biosystems). A positive control included transfection of the green fluorescent protein (GFP) plasmid, pmaxGFP (Amaxa Biosystems). Rat neurons were transfected using program G-13 in the Nucleofector apparatus (Amaxa Biosystems), and the neuronal cell suspension was seeded onto Matrigel-coated glass coverslips at 500 neurons per well. Supplemented DMEM was added, and cells were incubated at 37°C with 5% CO2 for 48 h. In cultures that underwent NGF withdrawal following this incubation, the supplemented DMEM was aspirated, and supplemented DMEM without NGF but with the addition of an anti-NGF-neutralizing antibody (1:4,000) (Sigma) was added to the cultures, which were incubated for a further 48 h.
RESULTS
Effects of late viral gene products on the antiapoptotic phenotype of VZV-infected human neurons. A well-characterized cell-associated VZV inoculation method was used to infect dissociated human neuronal cultures (20). To identify the phase of viral gene expression responsible for antiapoptotic functions in VZV-infected neurons, prior to VZV infection the neuronal cultures were treated with PAA to inhibit viral DNA replication and subsequent late gene expression (19). At day 1 (D1) and D2 postinfection (p.i.) with VZV strain Schenke, neuronal cultures were harvested and analyzed for viral antigen expression of each kinetic class by use of antibodies specific to the following VZV gene products: IE62 and ORF4 (immediate-early [IE] gene products), ORF29 (early [E] gene product), and glycoproteins (late [L] gene products). In parallel, neuronal cultures were assessed for apoptosis using annexin V and TUNEL staining. To determine the proportions of VZV antigen-positive and apoptotic cells, a total of 500 neurons per coverslip were counted based on VZV antigen immunostaining, annexin V staining, or TUNEL staining. In these dissociated ganglionic cell cultures, neurons comprised 80% of cells and were clearly distinguished from nonneuronal cells on the following bases: their spherical or tearlike shapes; their sizes, which ranged from 10 to 50 μm in diameter; and their extensive uni- and bipolar axonal growth (20).
In experiments performed using dissociated human neuronal cultures generated from three different donors, VZV antigens from IE and E gene classes were readily detected, and similar increases in the rates of viral antigen-positive neurons were observed in untreated and PAA-treated cultures at D1 and D2 p.i. (Fig. 1). In contrast, L gene expressions of glycoproteins were similar for untreated and PAA-treated VZV-infected neurons only at D1 p.i.; by D2 p.i., they had increased 12-fold to 24% ± 1.5% (mean ± standard error of the mean [SEM]) in the untreated VZV-infected neurons compared to 2.3% ± 0.8% in PAA-treated VZV-infected neurons (Fig. 1). No VZV antigen-specific staining was observed in mock-infected neuronal cultures incubated with VZV-specific antibodies or in VZV-infected PAA-treated or untreated neurons incubated with isotype control antibodies (data not shown). These data establish that the PAA treatment and inhibition of L gene expression in VZV-infected neuronal cultures was successful.
In parallel, to determine whether late genes convey protection from virally induced apoptosis, the same human neuronal cultures were analyzed for annexin V staining and TUNEL. Neurons in VZV-infected or mock-infected cultures (data not shown), whether PAA treated or untreated, displayed no specific annexin V staining (Table 1), although positive control (5% ethanol-treated) neurons showed abundant annexin V staining ranging from 35.5% to 47.3% over the time course (Table 1). Similar results were also observed for VZV-infected and mock-infected PAA-treated and untreated neuronal cultures analyzed by use of TUNEL; no fragmented DNA was observed in any of the cultures, although 92.6% to 96% of positive control (DNase-treated) neurons showed TUNEL staining over the time course (data not shown). Combined, these results show that late gene expression was not responsible for the antiapoptotic phenotype observed during VZV infection of neurons and that a virion component and/or IE and/or E gene products were responsible for the ability of infected neurons to resist apoptosis.
Analysis of viral antigen expression and virus production in rOkaORF63- and rOka-infected human sensory neurons. VZV encodes a homolog for the known antiapoptotic HSV-1 immediate-early protein ICP22, VZV ORF63 (17). We evaluated the role of ORF63 in the antiapoptotic phenotype observed in neurons by use of a recombinant VZV strain with ORF63 deleted. ORF63 is duplicated in the VZV genome asORF70. Cosmid-based deletion of the diploid VZV genes ORF63/ORF70 revealed that at least one copy was necessary for VZV replication in vitro (34), although more recently, viruses with deletions in both ORF63 and ORF70 have been shown to be viable, albeit with impaired growth capabilities in cell culture (8, 9). Thus, for the following studies of apoptosis in which we directly compared parental and mutant viruses, weused viruses with the single ORF63 gene deletion, rOkaORF63 and rOkaORF70, which have parental virus patterns of replication and plaque formation (34). We initially assessed VZV infection and viral antigen expression. On days 1, 2, and 4 p.i., mock-, rOka-, rOkaORF63-, and rOkaORF70-infected neuronal cultures were assessed for the viral antigens IE62 and for glycoprotein expression.
There was a significant increase in the number of rOkaORF63- and rOkaORF70-infected neurons detected over the time points tested, and the rate of increase of viral antigen-positive neurons was comparable to that observed in rOka-infected neuronal cultures. In a total of three replicate experiments using different donors to generate neuronal cultures, IE62 gene expression at D4 p.i. was observed in 36.3% ± 2.7% rOka-infected neurons, 42.7% ± 4.4% rOkaORF63-infected neurons (Fig. 2), and 59.0% ± 1.2% rOkaORF70-infected neurons (data not shown). For rOka- and rOkaORF63-infected neurons, similar levels of glycoprotein expression at D4 p.i., 43.6% ± 2.3% and 44.3% ± 1.2%, respectively (Fig. 2), were also observed, and the level was 67.3% ± 2.0% for rOkaORF70-infected neurons (data not shown). The rate of viral antigen expression in neurons was similar to that in our previous experiments using neuronal cultures infected with VZV strains Schenke and rOka (20). No IE62 or glycoprotein expression was observed in mock-infected neuronal cultures (data not shown). The subcellular localization of these viral gene products in VZV-infected neurons was consistent with that found by our previous assessment of productive VZV infection of human neurons, which showed that ORF62 was localized to the nucleus and cytoplasm of neurons and that the late viral antigens (glycoproteins) were localized predominantly to the cell surface of neurons. Furthermore, the early gene products ORF4 and ORF29 were localized to the cytoplasm and subcompartmental cytoplasmic vacuoles, respectively (data not shown) (20). Due to the cell-associated VZV-infected HF inoculum, it was not possible to perform an infectious center assay to directly determine whether new infectious virions were generated in VZV-infected neurons. However, we utilized TEM to demonstrate that numerous unenveloped virions in the nucleus and enveloped virions on the surface of neuronal cell bodies were present in both rOka and rOkaORF63 VZV-inoculated neuronal cultures (see Fig. 5). These data demonstrate that rOkaORF63 could infect, synthesize viral proteins, and assemble virions in primary sensory neurons, and that the kinetics of productive infection of the parent virus, rOka, and of rOkaORF63 were comparable.
Assessment of phosphatidylserine translocation in rOkaORF63-infected neurons by annexin V staining. Human neuronal cultures were mock, rOka, rOkaORF63, or rOkaORF70 infected and on days 1, 2, and 4 p.i. were analyzed for apoptosis by use of annexin V binding to phosphatidylserine. Positive controls included mock-, rOka-, rOkaORF63-, and rOkaORF70-infected neurons incubated with 5% ethanol for 30 min at 37°C prior to staining.
In three replicate experiments, mock-infected and rOka-infected neuronal cultures showed <6.5% of neurons as annexin V positive at any time point tested (see Fig. 4A). In contrast, there was a significant increase in the number of annexin V-positive neurons in the rOkaORF63- and rOkaORF70-infected cultures at all time points tested. Specifically, at D4 p.i. there was a three- to fourfold increase, with 23.0% ± 2.1% rOkaORF63-infected and 27.3% ± 1.9% rOkaORF70-infected neurons being annexin V positive (see Fig. 4A). The positive control neuronal cultures showed 54.0%± 3.0% staining in mock-infected, 60.0% ± 2.0% staining in rOka-infected (Fig. 3A), 58.0% ± 2.0% staining in rOkaORF63-infected (Fig. 3C), and 44.3% ± 1.2% staining in rOkaORF70-infected neuronal cultures. Therefore, despite the detection of similar and significant numbers of VZV antigen-positive neurons at D4 p.i. with rOka (43.6% ± 2.3%), rOkaORF63 (44.3% ± 1.2%), and rOkaORF70 (67.3% ± 2.0%), significant annexin V staining was detected only in the rOkaORF63- (Fig. 3D) and rOkaORF70-infected neurons and was not detected in rOka-infected neurons (Fig. 3B). These results demonstrate that VZV infection of human neurons can induce characteristic apoptotic membrane changes in the single deletion viruses, rOkaORF63 and rOkaORF70, but cannot do so in the parental rOka virus.
Analysis of DNA fragmentation in rOkaORF63-infected neurons by TUNEL staining. We employed TUNEL staining, a late marker of apoptosis that detects cleavage of DNA into nucleosomal fragments. Mock-, rOka-, rOkaORF63-, and rOkaORF70-infected neuronal cultures harvested on days 1, 2, and 4 p.i. were processed for viral antigen expression and TUNEL staining. Negative and positive controls were mock-, rOka-, rOkaORF63-, and rOkaORF70-infected neuronal cultures incubated with 3'-OH DNA labeling mix containing no TdT or treated with DNase prior to TdT labeling, respectively.
In mock-infected and rOka-infected neuronal cultures, neurons showed no TUNEL staining at D2 or D4 p.i. (Fig. 4B). In stark contrast, there was a 13- to 18-fold increase in the number of TUNEL-positive neurons in rOkaORF63-infected (13% ± 2.5%) (Fig. 4B) and rOkaORF70-infected (18% ± 2.4%) cultures at D4 p.i. The positive control-treated neurons showed 95.0% ± 4.0%, 96.0% ± 1.0%, 97% ± 1.0%, and 97.6% ± 0.3% staining in mock-infected, rOka-infected (Fig. 3E), rOkaORF63-infected (Fig. 3F), and rOkaORF70-infected neuronal cultures, respectively, at D4 p.i. No staining of apoptotic nuclei was detected in negative control mock-infected, rOka-infected (Fig. 3I), rOkaORF63-infected (Fig. 3J), or rOkaORF70-infected neuronal cultures. Therefore, while VZV antigen expression at D4 p.i. was detected in 43.6%± 2.3% of rOka-, 44.3% ± 1.2% of rOkaORF63-, and 67.3%± 2.0% of rOkaORF70-infected neurons, significant TUNEL staining was observed only in rOkaORF63- (Fig. 3H) and rOkaORF70-infected neurons. These results were replicated in three independent experiments with human neuronal cultures generated from different donors. These experiments demonstrate that infection of neurons with the single deletion viruses, rOkaORF63 and rOkaORF70, but not with the parental rOka virus, induces detectable DNA fragmentation. Thus, combined with the annexin V results, these data suggest the VZV IE gene ORF63 is involved in protecting neurons against apoptosis during VZV infection.
Effects of rOkaORF63 infection on neuronal cellular morphology determined by TEM. On day 4 p.i., mock-, rOka-, and rOkaORF63-infected neurons were processed for TEM to assess specific ultrastructural changes in cellular morphology that are characteristic of apoptosis. rOka-infected neurons appeared normal; intact double nuclear membranes, normal chromatin distribution, and organelles, specifically mitochondria, endoplasmic reticulum, and Golgi apparatus (Fig. 5A), were present. There were numerous unenveloped virions in thenucleus (Fig. 5C) and enveloped virions budding from thesurface of the neuronal cell body. In contrast, the rOkaORF63-infected neurons displayed typical ultrastructural apoptotic features, including a complete loss of the double nuclear membrane, condensation of chromatin, and an increased number of lipid vesicles (Fig. 5B). The presence of a neuron axonal cross section adjacent to the cell confirmed its neural origin (Fig. 5D). Enveloped virions appeared in the cytoplasm of the infected neuronal cell (Fig. 5E). Investigation of numerous rOka- and rOkaORF63-infected neurons showedsimilar intracellular localizations and abundances of viral particles; however, ultrastructural changes indicative of apoptosis were observed only in rOkaORF63-infected neurons. Mock-infected neurons did not display any cellular ultrastructural changes associated with apoptosis (data not shown). These results confirm that apoptosis, as indicated by ultrastructural modifications, was observed in rOkaORF63-infected but not in rOka-infected neurons, despite comparable amounts of enveloped VZV virions in both cultures.
Effects of NGF withdrawal on ORF63-transfected primary sensory neurons. We transfected plasmids containing the ORF63gene, the parental plasmid (pG310), or the GFP plasmid into primary rat sensory neurons and induced apoptosis by NGF withdrawal. In three replicate experiments, the transfection efficiency in neurons, as detected by GFP positivity, was 30% ± 1.8%. Rat sensory neuronal cultures were analyzed for annexin V and TUNEL staining. Neurons transfected with pG310 showed 83% ± 4.7% annexin V positivity and 63% ± 10% TUNEL positivity. In contrast, ORF63-transfected neurons showed 63% ± 3.1% annexin V positivity and only 6% ± 1.3% TUNEL positivity. These results show that ORF63 can suppress apoptosis of human neurons and further support our findings that the gene can protect neurons from apoptosis during VZV infection.
DISCUSSION
In our previous work investigating the apoptotic effect of VZV in human sensory neurons and human fibroblasts, we found a cell-type-specific response whereby virus-induced apoptosis was present in HF but not in VZV-infected sensory neurons (20). This phenotype was observed despite the majority of sensory neurons being antigen positive following infection with VZV strains Schenke or rOka. In the present study, we identified a VZV gene encoding an antiapoptotic function in human sensory neurons during the productive phase of infection. Using a targeted approach, we found that rOkaORF63-infected neurons were more susceptible to apoptosis than parental rOka-infected neurons. This indicates that during active VZV infection, the IE gene ORF63 encodes a protective mechanism against apoptosis in neurons. This is the first demonstration of a VZV gene encoding an antiapoptotic function; the observation was made by use of three independent methods of apoptosis detection, namely, annexin V staining, TUNEL staining, and TEM. Furthermore, VZV ORF63-transfected rat primary sensory neurons showed low levels of apoptosis induced by NGF withdrawal compared to the control plasmid, further supporting our contention that VZV ORF63 encodes an antiapoptotic function in these cells. Colocalization studies of VZV ORF63 and apoptosis markers will determine whether the antiapoptotic effect of ORF63 applies only to infected neurons and not to bystander cells also, although it is expected that ORF63 would act directly only on infected neurons, as it remains localized in the cytoplasm of productively infected neurons and does not appear to be secreted.
Neurons play a pivotal role in the pathogenesis of VZV, serving as a site of latency and also of virus replication and spread during reactivation to cause herpes zoster. Given that cell death terminates viral replication, it would be advantageous for VZV to postpone the death of the cell in which it is replicating. Prolonging neuronal life during productive infection would enable more virions to be produced, an outcome unfavorable to the host but advantageous to the virus. Thus, ORF63 protein may exert an antiapoptotic effect that enhances replication in neurons and the subsequent characteristic dermatomal rash that occurs during zoster.
The success of lifelong latent infection in DRG neurons is dependent on neuronal viability. In addition to being expressed during productive infection, VZV ORF63 has also been shown to be expressed during latency (5, 12, 24, 28, 29). Indeed, it is the most abundantly expressed transcript and protein in latently infected neurons. Although our studies are restricted to productive infection, expression of ORF63 during latent infection raises the intriguing possibility that this gene product may exert an antiapoptotic function during neuronal latency or reactivation or, as it is expressed with immediate early kinetics, during initial infection and establishment of latency within these cells. Interestingly, in a model of VZV latent infection using cotton rats, ORF63 recently has been shown to play an important role in the efficient establishment of VZV latency in these animals, although the mechanism underlying this function remains to be defined (8, 9). Several novel systems for investigating the neurotropism of VZV in vivo using chimeric NOD-SCID mouse-human neural cells or intact DRG xenografts also recently have been described (4, 38). Studies using the chimeric NOD-SCID mouse-human neural cell model found that cells infected with the single-copy mutant of ORF63,rOkaORF70, showed the same cellular localization as pOka and vOka, which have two copies of ORF63 (4). This study also reported that a few rOkaORF70-infected human neural cells showed evidence of apoptosis detected by caspase-3. Ultimately, additional studies using models that mimic aspects of latency will determine whether ORF63 exerts any antiapoptotic function during this phase of infection.
The magnitude of the induction of apoptosis in neurons infected with either the ORF63 (rOkaORF63) or ORF70 (rOkaORF70) deletion viruses indicated that each of these genes was unlikely to be singularly responsible for the protection of neurons from apoptosis. Indeed, each of these viruses lacks only one copy of the duplicated gene (i.e., rOkaORF63 retains ORF70 and rOkaORF70 retains ORF63), and so deletion of either one may be partially compensated by the other. A determination of whether the antiapoptotic effect is dose dependent will require the purification of neurons from nonneuronal cells in dissociated ganglionic cell cultures and a quantitation of ORF63/ORF70 protein in parent and mutant virus-infected neurons to identify any correlation with the apoptotic response in these cells. In addition to ORF63/ORF70, other VZV genes, such as those with homology to known HSV antiapoptotic genes, may also play a protective role during neuronal infection. These include VZV IE62 and ORF4, which share homology with the HSV-1 antiapoptotic genes ICP4 and ICP27, respectively (15, 37). The assessment of these and other genes will help to determine the full repertoire of viral functions that modulate apoptosis.
The mechanism for apoptosis inhibition by VZV ORF63 remains to be determined. VZV does not encode homologs of the HSV-1 latency-associated transcript or the bovine herpesvirus type 1 latency-related gene, which have been shown to act on two major apoptotic pathways, the death receptor-mediated pathway and the mitochondrial pathway, respectively (1, 18). In addition, the antiapoptotic mechanism of action encoded by HSV-1 ICP22 (the homolog to VZV ORF63) is yet to be defined. Elucidation of the pathway by which VZV ORF63-mediated apoptotic inhibition transpires in human sensory neurons and of how this compares with the mechanism of action of HSV-1 antiapoptotic genes will be an important component of future studies.
Our in vitro results suggest that the marked neuropathology induced by VZV during herpes zoster may not be a result of VZV-induced apoptosis, but rather that it may be a consequence of the infiltrating immune cells, an area that merits further investigation. Preliminary data from our laboratory investigating VZV infection and immune infiltrates in postmortem human ganglia tissue from patients with herpes zoster support this hypothesis.
In summary, our results provide the first evidence that a VZV gene, ORF63, encodes an antiapoptotic function in productively infected human neurons. Definition of the mechanism of action of the antiapoptotic gene(s) encoded by VZV may help elucidate differences in the susceptibilities of neurons and other cells types to apoptosis and lead to a much better understanding of how VZV and other herpesviruses are able to persist successfully within the human host.
ACKNOWLEDGMENTS
This work was supported by NH&MRC Project Grants 211110 and 352341. C.H. was the holder of an Australian Postgraduate Award and Westmead Millennium Foundation Research Scholarship Stipend Enhancement Award. P.R.K. was supported by NIH grants EY09397 and EY8098 and by funds from the Eye and Ear Research Foundation and Research to Prevent Blindness Inc.
We thank Ross Boadle for assistance with electron microscopy.
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Dept. Infectious Diseases and Immunology, University of Sydney, P.O. Box 412, Westmead, 2145 NSW, Australia
Department of Pediatrics, Stanford University School of Medicine, Stanford, California 94305
Ophthalmology and Visual Science Research Center, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
ABSTRACT
Virus-encoded modulation of apoptosis may serve as a mechanism to enhance cell survival and virus persistence. The impact of productive varicella-zoster virus (VZV) infection on apoptosis appears to be cell type specific, as infected human sensory neurons are resistant to apoptosis, yet human fibroblasts readily become apoptotic. We sought to identify the viral gene product(s) responsible for this antiapoptotic phenotype in primary human sensory neurons. Treatment with phosphonoacetic acid to inhibit viral DNA replication and late-phase gene expression did not alter the antiapoptotic phenotype, implicating immediate-early (IE) or early genes or a virion component. Compared to the parental VZV strain (rOKA), a recombinant virus unable to express one copy of the diploid IE gene ORF63 (rOkaORF63) demonstrated a significant induction of apoptosis in infected neurons, as determined by three methods: annexin V staining, deoxynucleotidyltransferase-mediated dUTP-biotin nick end label staining, and transmission electron microscopy. Furthermore, neurons transfected with a plasmid expressing ORF63 resisted apoptosis induced by nerve growth factor withdrawal. These results show that ORF63 can suppress apoptosis of neurons and provide the first identification of a VZV gene encoding an antiapoptotic function. As ORF63 is expressed in neurons during both productive and latent infection, it may play a significant role in viral pathogenesis by promoting neuron survival during primary and reactivated infections.
INTRODUCTION
Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes varicella (chicken pox) during primary infection and herpes zoster (shingles) following reactivation of latent virus from the dorsal root ganglia (DRG) (16, 22). A severe and debilitating complication of herpes zoster is postherpetic neuralgia, which involves chronic pain persisting for months or years following the initial herpes zoster attack (21, 14). It has been proposed that the pain associated with herpes zoster and postherpetic neuralgia is due to neural tissue destruction caused by VZV reactivation (35, 36). However, we recently reported that VZV-infected neurons, but not VZV-infected human fibroblasts (HF), were resistant to apoptosis (20), suggesting that neuronal destruction in vivo may be a corollary of the host cell immune response. This neuron-specific antiapoptotic function could increase the ability of VZV to efficiently establish latency and allow augmented virion production for axonal transport to the skin and reactivation as zoster lesions.
Other alphaherpesviruses, i.e., herpes simplex virus type 1 (HSV-1) and bovine herpesvirus type 1, express latency-associated transcripts and the latency-related gene, respectively, during latent infection of neurons (10, 11, 13, 32, 33, 34). Both of these have been shown to promote neuronal survival by an antiapoptotic mechanism in the event of induced apoptosis (1, 6, 16, 18, 27, 31), although VZV does not encode a known homolog of either gene. However, during productive infection HSV-1 expresses a number of other genes with antiapoptotic functions: US3, ICP4, ICP22, ICP27, gD, and gJ (2, 3, 23, 25, 26, 32, 39), and several of these do share sequence homology with VZV genes. HSV-1 US3, ICP27, ICP4, and ICP22 share homology with ORF66, ORF4, ORF62, and ORF63, respectively, although any antiapoptotic functions encoded by these VZV genes have not been previously examined. In this study, we sought to identify a VZV gene product(s) that encodes an antiapoptotic function in neurons. Using three methods of apoptosis detection (annexin V staining, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end label[TUNEL] staining, and transmission electron microscopy [TEM]), we applied a combination of drug block experiments, infections with viral gene deletion mutants, and transient transfection-based assays to primary sensory neurons. We show that VZV ORF63, a gene expressed prominently in neurons during both the productive and latent phases of infection (12, 28, 29), encodes an antiapoptotic function, as deletion of this gene from the virus significantly increased the percentage of apoptotic neurons following infection and introduction of ORF63 to neurons protected them from nerve growth factor (NGF)-induced apoptosis. This study provides the first evidence that ORF63 promotes neuronal cell survival after VZV infection by modulating apoptosis.
MATERIALS AND METHODS
Culturing of dissociated human sensory neurons. Human fetal spines (14 to 20 weeks of gestation) were obtained after informed consent and approval by the University of Sydney Human Ethics Committee. DRG were dissected from the spine, dissociated, and cultured as previously described (20). Dissociated neuronal cultures were seeded to give a range of 200 to 500 neurons per well. Resulting cultures contained 80% neurons as determined by cell morphology, and on day 3 postplating, extensive axonal networks between neurons were observed. Rat DRG were dissected and processed as previously described (30).
Viruses. The VZV strains used in these studies were Schenke (a low-passage clinical isolate), recombinant strain rOka (generated using cosmids derived fromthe varicella vaccine strain) (7), the rOkaORF63 mutant, and the rOkaORF70 mutant (38). Viruses were propagated in HF grown in tissue culture medium (Dulbecco modified Eagle medium [DMEM]) (GIBCO) supplemented with 10% heat-inactivated fetal calf serum (CSL). For VZV infections, the degree of inoculating fibroblast infection was scored using a scale from 0 to 4+, where 0 corresponds to no detectable infection and 4+ corresponds to 100% cytopathic effect. Neuronal cultures were inoculated using HF showing cytopathic effect in a range between 2 and 3+.
Infection and PAA treatment. Human neuronal cultures were inoculated with VZV by adding 1 x 104 VZV-infected HF or mock infected by adding 1 x 104 uninfected HF. In some experiments, directly following a 2-h treatment with 300 μg/ml phosphonoacetic acid (PAA; Sigma), mock- or VZV-infected cells were added to the cultures for 2 h at 37°C with 5% CO2. Following this incubation, supplemented DMEM was added to the neuronal cultures.
Immunofluorescence staining of VZV antigens. Cells were immunostained with rabbit polyclonal antibodies directed against VZV ORF62, ORF4, or ORF29 or with VZV-immune immunoglobulin G (IgG)-purified polyclonal human serum as previously described (20). The VZV-immune IgG-purified polyclonal human serum reacts with glycoprotein E as the predominant target on infected cells (A. M. Arvin, personal communication). Primary antibodies were detected with fluorescein isothiocyanate (FITC)-conjugated goat anti-human IgG and FITC-conjugated goat anti-rabbit IgG. In all immunostaining experiments, isotype control antibodies were used on mock- and VZV-infected cells to control for nonspecific antibody binding.
Annexin V staining. Human neuronal cultures were assessed for annexin V staining by incubation with annexin V-Alexa Fluor 594 conjugate as previously described (20). Cells were washed in annexin V-binding buffer and mounted with SlowFade antifade mounting fluid containing a DAPI (4',6'-diamidino-2-phenylindole) nuclear counterstain (Molecular Probes). Positive control cells were incubated at 37°C with 5% ethanol prior to incubation with annexin V-binding buffer. Slides were analyzed using a Leica laser scanning confocal microscope. For determination of the percentage of viral antigen- or annexin V-positive cells, a total of 500 neurons were counted by randomly selecting fields of 50 neurons.
TUNEL staining. Human neuronal cultures were processed and assessed for TUNEL staining as previously described (20). Positive control coverslips were treated with 3 U of DNase I (GIBCO) for 30 min at 37°C prior to the addition of the 3'-OH labeling mix (1x terminal deoxynucleotidyltransferase [TdT] reaction buffer, 50 μM biotin-14-dCTP [GIBCO], and 0.2 U of TdT [GIBCO]). The negative control coverslips were incubated with a labeling mix containing no TdT. Cells were washed and incubated with 5 μg/ml of streptavidin-FITC (GIBCO) in 5% skim milk for 30 min at 37°C. Coverslips were washed and mounted onto slides by use of SlowFade antifade mounting fluid with propidium iodide (1:500) (Sigma) to determine cell viability. Cells were analyzed using a Leica laser scanning confocal microscope, and the percentage of TUNEL-positive cells was calculated as described above.
Transmission electron microscopy (TEM). Human neurons were fixed in modified Karnovsky's fixative for 1 h and processed as previously described (20). Briefly, cells were washed and postfixed in 2% buffered osmium tetroxide for 3 h and subsequently in 2% aqueous uranyl acetate (Fluka) for 1 h, dehydrated with ethanol, and embedded in Spurr resin (TAAB Laboratories Ltd). Polymerization occurred at 70°C for 10 h. Sections were cut using a Reichert-Jung Ultracut E microtome and stained with 1% uranyl acetate in 50% ethanol and Reynolds’ lead citrate. Sections were examined with a Philips CM120 BioTWIN transmission electron microscope at 80 kV.
Transfection of primary rat sensory neurons and NGF withdrawal. Primary rat neurons were dissociated as previously described (30) and, prior to being plated on Matrigel-coated coverslips, were transfected with plasmid pG310 or pG310 containing the VZV ORF63 gene. Transfection of primary rat neurons was performed using a rat neuron Nucleofector kit for rat DRG neurons (Amaxa Biosystems). A positive control included transfection of the green fluorescent protein (GFP) plasmid, pmaxGFP (Amaxa Biosystems). Rat neurons were transfected using program G-13 in the Nucleofector apparatus (Amaxa Biosystems), and the neuronal cell suspension was seeded onto Matrigel-coated glass coverslips at 500 neurons per well. Supplemented DMEM was added, and cells were incubated at 37°C with 5% CO2 for 48 h. In cultures that underwent NGF withdrawal following this incubation, the supplemented DMEM was aspirated, and supplemented DMEM without NGF but with the addition of an anti-NGF-neutralizing antibody (1:4,000) (Sigma) was added to the cultures, which were incubated for a further 48 h.
RESULTS
Effects of late viral gene products on the antiapoptotic phenotype of VZV-infected human neurons. A well-characterized cell-associated VZV inoculation method was used to infect dissociated human neuronal cultures (20). To identify the phase of viral gene expression responsible for antiapoptotic functions in VZV-infected neurons, prior to VZV infection the neuronal cultures were treated with PAA to inhibit viral DNA replication and subsequent late gene expression (19). At day 1 (D1) and D2 postinfection (p.i.) with VZV strain Schenke, neuronal cultures were harvested and analyzed for viral antigen expression of each kinetic class by use of antibodies specific to the following VZV gene products: IE62 and ORF4 (immediate-early [IE] gene products), ORF29 (early [E] gene product), and glycoproteins (late [L] gene products). In parallel, neuronal cultures were assessed for apoptosis using annexin V and TUNEL staining. To determine the proportions of VZV antigen-positive and apoptotic cells, a total of 500 neurons per coverslip were counted based on VZV antigen immunostaining, annexin V staining, or TUNEL staining. In these dissociated ganglionic cell cultures, neurons comprised 80% of cells and were clearly distinguished from nonneuronal cells on the following bases: their spherical or tearlike shapes; their sizes, which ranged from 10 to 50 μm in diameter; and their extensive uni- and bipolar axonal growth (20).
In experiments performed using dissociated human neuronal cultures generated from three different donors, VZV antigens from IE and E gene classes were readily detected, and similar increases in the rates of viral antigen-positive neurons were observed in untreated and PAA-treated cultures at D1 and D2 p.i. (Fig. 1). In contrast, L gene expressions of glycoproteins were similar for untreated and PAA-treated VZV-infected neurons only at D1 p.i.; by D2 p.i., they had increased 12-fold to 24% ± 1.5% (mean ± standard error of the mean [SEM]) in the untreated VZV-infected neurons compared to 2.3% ± 0.8% in PAA-treated VZV-infected neurons (Fig. 1). No VZV antigen-specific staining was observed in mock-infected neuronal cultures incubated with VZV-specific antibodies or in VZV-infected PAA-treated or untreated neurons incubated with isotype control antibodies (data not shown). These data establish that the PAA treatment and inhibition of L gene expression in VZV-infected neuronal cultures was successful.
In parallel, to determine whether late genes convey protection from virally induced apoptosis, the same human neuronal cultures were analyzed for annexin V staining and TUNEL. Neurons in VZV-infected or mock-infected cultures (data not shown), whether PAA treated or untreated, displayed no specific annexin V staining (Table 1), although positive control (5% ethanol-treated) neurons showed abundant annexin V staining ranging from 35.5% to 47.3% over the time course (Table 1). Similar results were also observed for VZV-infected and mock-infected PAA-treated and untreated neuronal cultures analyzed by use of TUNEL; no fragmented DNA was observed in any of the cultures, although 92.6% to 96% of positive control (DNase-treated) neurons showed TUNEL staining over the time course (data not shown). Combined, these results show that late gene expression was not responsible for the antiapoptotic phenotype observed during VZV infection of neurons and that a virion component and/or IE and/or E gene products were responsible for the ability of infected neurons to resist apoptosis.
Analysis of viral antigen expression and virus production in rOkaORF63- and rOka-infected human sensory neurons. VZV encodes a homolog for the known antiapoptotic HSV-1 immediate-early protein ICP22, VZV ORF63 (17). We evaluated the role of ORF63 in the antiapoptotic phenotype observed in neurons by use of a recombinant VZV strain with ORF63 deleted. ORF63 is duplicated in the VZV genome asORF70. Cosmid-based deletion of the diploid VZV genes ORF63/ORF70 revealed that at least one copy was necessary for VZV replication in vitro (34), although more recently, viruses with deletions in both ORF63 and ORF70 have been shown to be viable, albeit with impaired growth capabilities in cell culture (8, 9). Thus, for the following studies of apoptosis in which we directly compared parental and mutant viruses, weused viruses with the single ORF63 gene deletion, rOkaORF63 and rOkaORF70, which have parental virus patterns of replication and plaque formation (34). We initially assessed VZV infection and viral antigen expression. On days 1, 2, and 4 p.i., mock-, rOka-, rOkaORF63-, and rOkaORF70-infected neuronal cultures were assessed for the viral antigens IE62 and for glycoprotein expression.
There was a significant increase in the number of rOkaORF63- and rOkaORF70-infected neurons detected over the time points tested, and the rate of increase of viral antigen-positive neurons was comparable to that observed in rOka-infected neuronal cultures. In a total of three replicate experiments using different donors to generate neuronal cultures, IE62 gene expression at D4 p.i. was observed in 36.3% ± 2.7% rOka-infected neurons, 42.7% ± 4.4% rOkaORF63-infected neurons (Fig. 2), and 59.0% ± 1.2% rOkaORF70-infected neurons (data not shown). For rOka- and rOkaORF63-infected neurons, similar levels of glycoprotein expression at D4 p.i., 43.6% ± 2.3% and 44.3% ± 1.2%, respectively (Fig. 2), were also observed, and the level was 67.3% ± 2.0% for rOkaORF70-infected neurons (data not shown). The rate of viral antigen expression in neurons was similar to that in our previous experiments using neuronal cultures infected with VZV strains Schenke and rOka (20). No IE62 or glycoprotein expression was observed in mock-infected neuronal cultures (data not shown). The subcellular localization of these viral gene products in VZV-infected neurons was consistent with that found by our previous assessment of productive VZV infection of human neurons, which showed that ORF62 was localized to the nucleus and cytoplasm of neurons and that the late viral antigens (glycoproteins) were localized predominantly to the cell surface of neurons. Furthermore, the early gene products ORF4 and ORF29 were localized to the cytoplasm and subcompartmental cytoplasmic vacuoles, respectively (data not shown) (20). Due to the cell-associated VZV-infected HF inoculum, it was not possible to perform an infectious center assay to directly determine whether new infectious virions were generated in VZV-infected neurons. However, we utilized TEM to demonstrate that numerous unenveloped virions in the nucleus and enveloped virions on the surface of neuronal cell bodies were present in both rOka and rOkaORF63 VZV-inoculated neuronal cultures (see Fig. 5). These data demonstrate that rOkaORF63 could infect, synthesize viral proteins, and assemble virions in primary sensory neurons, and that the kinetics of productive infection of the parent virus, rOka, and of rOkaORF63 were comparable.
Assessment of phosphatidylserine translocation in rOkaORF63-infected neurons by annexin V staining. Human neuronal cultures were mock, rOka, rOkaORF63, or rOkaORF70 infected and on days 1, 2, and 4 p.i. were analyzed for apoptosis by use of annexin V binding to phosphatidylserine. Positive controls included mock-, rOka-, rOkaORF63-, and rOkaORF70-infected neurons incubated with 5% ethanol for 30 min at 37°C prior to staining.
In three replicate experiments, mock-infected and rOka-infected neuronal cultures showed <6.5% of neurons as annexin V positive at any time point tested (see Fig. 4A). In contrast, there was a significant increase in the number of annexin V-positive neurons in the rOkaORF63- and rOkaORF70-infected cultures at all time points tested. Specifically, at D4 p.i. there was a three- to fourfold increase, with 23.0% ± 2.1% rOkaORF63-infected and 27.3% ± 1.9% rOkaORF70-infected neurons being annexin V positive (see Fig. 4A). The positive control neuronal cultures showed 54.0%± 3.0% staining in mock-infected, 60.0% ± 2.0% staining in rOka-infected (Fig. 3A), 58.0% ± 2.0% staining in rOkaORF63-infected (Fig. 3C), and 44.3% ± 1.2% staining in rOkaORF70-infected neuronal cultures. Therefore, despite the detection of similar and significant numbers of VZV antigen-positive neurons at D4 p.i. with rOka (43.6% ± 2.3%), rOkaORF63 (44.3% ± 1.2%), and rOkaORF70 (67.3% ± 2.0%), significant annexin V staining was detected only in the rOkaORF63- (Fig. 3D) and rOkaORF70-infected neurons and was not detected in rOka-infected neurons (Fig. 3B). These results demonstrate that VZV infection of human neurons can induce characteristic apoptotic membrane changes in the single deletion viruses, rOkaORF63 and rOkaORF70, but cannot do so in the parental rOka virus.
Analysis of DNA fragmentation in rOkaORF63-infected neurons by TUNEL staining. We employed TUNEL staining, a late marker of apoptosis that detects cleavage of DNA into nucleosomal fragments. Mock-, rOka-, rOkaORF63-, and rOkaORF70-infected neuronal cultures harvested on days 1, 2, and 4 p.i. were processed for viral antigen expression and TUNEL staining. Negative and positive controls were mock-, rOka-, rOkaORF63-, and rOkaORF70-infected neuronal cultures incubated with 3'-OH DNA labeling mix containing no TdT or treated with DNase prior to TdT labeling, respectively.
In mock-infected and rOka-infected neuronal cultures, neurons showed no TUNEL staining at D2 or D4 p.i. (Fig. 4B). In stark contrast, there was a 13- to 18-fold increase in the number of TUNEL-positive neurons in rOkaORF63-infected (13% ± 2.5%) (Fig. 4B) and rOkaORF70-infected (18% ± 2.4%) cultures at D4 p.i. The positive control-treated neurons showed 95.0% ± 4.0%, 96.0% ± 1.0%, 97% ± 1.0%, and 97.6% ± 0.3% staining in mock-infected, rOka-infected (Fig. 3E), rOkaORF63-infected (Fig. 3F), and rOkaORF70-infected neuronal cultures, respectively, at D4 p.i. No staining of apoptotic nuclei was detected in negative control mock-infected, rOka-infected (Fig. 3I), rOkaORF63-infected (Fig. 3J), or rOkaORF70-infected neuronal cultures. Therefore, while VZV antigen expression at D4 p.i. was detected in 43.6%± 2.3% of rOka-, 44.3% ± 1.2% of rOkaORF63-, and 67.3%± 2.0% of rOkaORF70-infected neurons, significant TUNEL staining was observed only in rOkaORF63- (Fig. 3H) and rOkaORF70-infected neurons. These results were replicated in three independent experiments with human neuronal cultures generated from different donors. These experiments demonstrate that infection of neurons with the single deletion viruses, rOkaORF63 and rOkaORF70, but not with the parental rOka virus, induces detectable DNA fragmentation. Thus, combined with the annexin V results, these data suggest the VZV IE gene ORF63 is involved in protecting neurons against apoptosis during VZV infection.
Effects of rOkaORF63 infection on neuronal cellular morphology determined by TEM. On day 4 p.i., mock-, rOka-, and rOkaORF63-infected neurons were processed for TEM to assess specific ultrastructural changes in cellular morphology that are characteristic of apoptosis. rOka-infected neurons appeared normal; intact double nuclear membranes, normal chromatin distribution, and organelles, specifically mitochondria, endoplasmic reticulum, and Golgi apparatus (Fig. 5A), were present. There were numerous unenveloped virions in thenucleus (Fig. 5C) and enveloped virions budding from thesurface of the neuronal cell body. In contrast, the rOkaORF63-infected neurons displayed typical ultrastructural apoptotic features, including a complete loss of the double nuclear membrane, condensation of chromatin, and an increased number of lipid vesicles (Fig. 5B). The presence of a neuron axonal cross section adjacent to the cell confirmed its neural origin (Fig. 5D). Enveloped virions appeared in the cytoplasm of the infected neuronal cell (Fig. 5E). Investigation of numerous rOka- and rOkaORF63-infected neurons showedsimilar intracellular localizations and abundances of viral particles; however, ultrastructural changes indicative of apoptosis were observed only in rOkaORF63-infected neurons. Mock-infected neurons did not display any cellular ultrastructural changes associated with apoptosis (data not shown). These results confirm that apoptosis, as indicated by ultrastructural modifications, was observed in rOkaORF63-infected but not in rOka-infected neurons, despite comparable amounts of enveloped VZV virions in both cultures.
Effects of NGF withdrawal on ORF63-transfected primary sensory neurons. We transfected plasmids containing the ORF63gene, the parental plasmid (pG310), or the GFP plasmid into primary rat sensory neurons and induced apoptosis by NGF withdrawal. In three replicate experiments, the transfection efficiency in neurons, as detected by GFP positivity, was 30% ± 1.8%. Rat sensory neuronal cultures were analyzed for annexin V and TUNEL staining. Neurons transfected with pG310 showed 83% ± 4.7% annexin V positivity and 63% ± 10% TUNEL positivity. In contrast, ORF63-transfected neurons showed 63% ± 3.1% annexin V positivity and only 6% ± 1.3% TUNEL positivity. These results show that ORF63 can suppress apoptosis of human neurons and further support our findings that the gene can protect neurons from apoptosis during VZV infection.
DISCUSSION
In our previous work investigating the apoptotic effect of VZV in human sensory neurons and human fibroblasts, we found a cell-type-specific response whereby virus-induced apoptosis was present in HF but not in VZV-infected sensory neurons (20). This phenotype was observed despite the majority of sensory neurons being antigen positive following infection with VZV strains Schenke or rOka. In the present study, we identified a VZV gene encoding an antiapoptotic function in human sensory neurons during the productive phase of infection. Using a targeted approach, we found that rOkaORF63-infected neurons were more susceptible to apoptosis than parental rOka-infected neurons. This indicates that during active VZV infection, the IE gene ORF63 encodes a protective mechanism against apoptosis in neurons. This is the first demonstration of a VZV gene encoding an antiapoptotic function; the observation was made by use of three independent methods of apoptosis detection, namely, annexin V staining, TUNEL staining, and TEM. Furthermore, VZV ORF63-transfected rat primary sensory neurons showed low levels of apoptosis induced by NGF withdrawal compared to the control plasmid, further supporting our contention that VZV ORF63 encodes an antiapoptotic function in these cells. Colocalization studies of VZV ORF63 and apoptosis markers will determine whether the antiapoptotic effect of ORF63 applies only to infected neurons and not to bystander cells also, although it is expected that ORF63 would act directly only on infected neurons, as it remains localized in the cytoplasm of productively infected neurons and does not appear to be secreted.
Neurons play a pivotal role in the pathogenesis of VZV, serving as a site of latency and also of virus replication and spread during reactivation to cause herpes zoster. Given that cell death terminates viral replication, it would be advantageous for VZV to postpone the death of the cell in which it is replicating. Prolonging neuronal life during productive infection would enable more virions to be produced, an outcome unfavorable to the host but advantageous to the virus. Thus, ORF63 protein may exert an antiapoptotic effect that enhances replication in neurons and the subsequent characteristic dermatomal rash that occurs during zoster.
The success of lifelong latent infection in DRG neurons is dependent on neuronal viability. In addition to being expressed during productive infection, VZV ORF63 has also been shown to be expressed during latency (5, 12, 24, 28, 29). Indeed, it is the most abundantly expressed transcript and protein in latently infected neurons. Although our studies are restricted to productive infection, expression of ORF63 during latent infection raises the intriguing possibility that this gene product may exert an antiapoptotic function during neuronal latency or reactivation or, as it is expressed with immediate early kinetics, during initial infection and establishment of latency within these cells. Interestingly, in a model of VZV latent infection using cotton rats, ORF63 recently has been shown to play an important role in the efficient establishment of VZV latency in these animals, although the mechanism underlying this function remains to be defined (8, 9). Several novel systems for investigating the neurotropism of VZV in vivo using chimeric NOD-SCID mouse-human neural cells or intact DRG xenografts also recently have been described (4, 38). Studies using the chimeric NOD-SCID mouse-human neural cell model found that cells infected with the single-copy mutant of ORF63,rOkaORF70, showed the same cellular localization as pOka and vOka, which have two copies of ORF63 (4). This study also reported that a few rOkaORF70-infected human neural cells showed evidence of apoptosis detected by caspase-3. Ultimately, additional studies using models that mimic aspects of latency will determine whether ORF63 exerts any antiapoptotic function during this phase of infection.
The magnitude of the induction of apoptosis in neurons infected with either the ORF63 (rOkaORF63) or ORF70 (rOkaORF70) deletion viruses indicated that each of these genes was unlikely to be singularly responsible for the protection of neurons from apoptosis. Indeed, each of these viruses lacks only one copy of the duplicated gene (i.e., rOkaORF63 retains ORF70 and rOkaORF70 retains ORF63), and so deletion of either one may be partially compensated by the other. A determination of whether the antiapoptotic effect is dose dependent will require the purification of neurons from nonneuronal cells in dissociated ganglionic cell cultures and a quantitation of ORF63/ORF70 protein in parent and mutant virus-infected neurons to identify any correlation with the apoptotic response in these cells. In addition to ORF63/ORF70, other VZV genes, such as those with homology to known HSV antiapoptotic genes, may also play a protective role during neuronal infection. These include VZV IE62 and ORF4, which share homology with the HSV-1 antiapoptotic genes ICP4 and ICP27, respectively (15, 37). The assessment of these and other genes will help to determine the full repertoire of viral functions that modulate apoptosis.
The mechanism for apoptosis inhibition by VZV ORF63 remains to be determined. VZV does not encode homologs of the HSV-1 latency-associated transcript or the bovine herpesvirus type 1 latency-related gene, which have been shown to act on two major apoptotic pathways, the death receptor-mediated pathway and the mitochondrial pathway, respectively (1, 18). In addition, the antiapoptotic mechanism of action encoded by HSV-1 ICP22 (the homolog to VZV ORF63) is yet to be defined. Elucidation of the pathway by which VZV ORF63-mediated apoptotic inhibition transpires in human sensory neurons and of how this compares with the mechanism of action of HSV-1 antiapoptotic genes will be an important component of future studies.
Our in vitro results suggest that the marked neuropathology induced by VZV during herpes zoster may not be a result of VZV-induced apoptosis, but rather that it may be a consequence of the infiltrating immune cells, an area that merits further investigation. Preliminary data from our laboratory investigating VZV infection and immune infiltrates in postmortem human ganglia tissue from patients with herpes zoster support this hypothesis.
In summary, our results provide the first evidence that a VZV gene, ORF63, encodes an antiapoptotic function in productively infected human neurons. Definition of the mechanism of action of the antiapoptotic gene(s) encoded by VZV may help elucidate differences in the susceptibilities of neurons and other cells types to apoptosis and lead to a much better understanding of how VZV and other herpesviruses are able to persist successfully within the human host.
ACKNOWLEDGMENTS
This work was supported by NH&MRC Project Grants 211110 and 352341. C.H. was the holder of an Australian Postgraduate Award and Westmead Millennium Foundation Research Scholarship Stipend Enhancement Award. P.R.K. was supported by NIH grants EY09397 and EY8098 and by funds from the Eye and Ear Research Foundation and Research to Prevent Blindness Inc.
We thank Ross Boadle for assistance with electron microscopy.
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