ABSTRACT
Mechanisms of neuronal infection by varicella-zoster virus (VZV) have been challenging to study due to the relatively strict human tropism of the virus and the paucity of tractable experimental models. Cellular mitogen-activated protein kinases (MAPKs) have been shown to play a role in VZV infection of nonneuronal cells, with distinct consequences for infectivity in different cell types. Here, we utilize several human neuronal culture systems to investigate the role of one such MAPK, the c-Jun N-terminal kinase (JNK), in VZV lytic infection and reactivation. We find that the JNK pathway is specifically activated following infection of human embryonic stem cell-derived neurons and that this activation of JNK is essential for efficient viral protein expression and replication. Inhibition of the JNK pathway blocked viral replication in a manner distinct from that of acyclovir, and an acyclovir-resistant VZV isolate was as sensitive to the effects of JNK inhibition as an acyclovir-sensitive VZV isolate in neurons. Moreover, in a microfluidic-based human neuronal model of viral latency and reactivation, we found that inhibition of the JNK pathway resulted in a marked reduction in reactivation of VZV. Finally, we utilized a novel technique to efficiently generate cells expressing markers of human sensory neurons from neural crest cells and established a critical role for the JNK pathway in infection of these cells. In summary, the JNK pathway plays an important role in lytic infection and reactivation of VZV in physiologically relevant cell types and may provide an alternative target for antiviral therapy.
IMPORTANCE Varicella-zoster virus (VZV) has infected over 90% of people worldwide. While primary infection leads to the typically self-limiting condition of chickenpox, the virus can remain dormant in the nervous system and may reactivate later in life, leading to shingles or inflammatory diseases of the nervous system and eye with potentially severe consequences. Here, we take advantage of newer stem cell-based technologies to study the mechanisms by which VZV infects human neurons. We find that the c-Jun N-terminal kinase (JNK) pathway is activated by VZV infection and that blockade of this pathway limits lytic replication (as occurs during primary infection). In addition, JNK inhibition limits viral reactivation, exhibiting parallels with herpes simplex virus reactivation. The identification of the role of the JNK pathway in VZV infection of neurons reveals potential avenues for the development of alternate antiviral drugs.
INTRODUCTION
Varicella-zoster virus (VZV) is a neurotropic human alphaherpesvirus that has infected over 90% of people worldwide. Although primary infection by VZV results in the typically self-limited disease varicella (chickenpox), lifelong latent infection in cranial nerve and dorsal root ganglia ensues with the potential for reactivation later in life. While a live attenuated VZV vaccine is highly effective in preventing chickenpox, it too establishes latency and can reactivate, albeit at an apparently much reduced level (1, 2). Viral reactivation in the setting of diminished immunity can have severe consequences, including herpes zoster (shingles), encephalitis, myelitis, and inflammatory disorders of the eye (3, 4), and has recently been associated with giant cell arteritis (5). In many countries, including the United States, an aging population and the increasing adoption of novel and potent immunosuppressive treatments for autoimmune syndromes place individuals at growing risk for VZV-associated disease (6), highlighting the need for a better understanding of VZV infection, latency, and reactivation in neurons in order to develop more effective vaccines and antiviral agents.
Viruses have coevolved with their human hosts, and replication of VZV is dependent upon cellular, in addition to viral, proteins. In particular, the mitogen-activated protein kinase (MAPK) family of proteins, which consist of c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK), serve as responders to many environmental cues and have been implicated in the pathogenesis of a number of viruses (7). MAPKs are typically activated through a phosphorylation cascade that involves sequential activation of kinases, with eventual translocation of the activated MAPK to the nucleus and consequent effects on cellular transcription (8). VZV has been shown to activate the JNK pathway in both melanoma cells and fibroblasts, though with markedly differing consequences for viral pathogenesis. In melanoma cells, inhibition of JNK leads to an increase in VZV replication (9), while in fibroblasts, inhibition decreases VZV replication (10), indicating that the role of JNK in VZV pathogenesis depends upon the cell type infected.
Little, however, is known regarding the role of host factors, such as the JNK signaling pathway, in neuronal infection by VZV. A mechanistic understanding of VZV-neuronal interactions has been hampered by the relatively strict human tropism of the virus and lack of tractable human systems for experimentation. Studies of adult and fetal human dorsal root ganglia have provided some insights, while engraftment of human tissues into the SCID-hu mouse model has been utilized to examine replication in different cell types (11–14). An exciting recent advance has been the development of neuronal models of VZV infection based on human neural stem cells (15) and human embryonic stem (ES) cells (16). Such systems have recently been utilized to characterize axonal transport of the virus, to describe large-scale transcriptional changes in VZV-infected neurons, and to establish models of viral latency and reactivation (16–19). Here, we utilize several human neuronal systems to test the hypothesis that the JNK pathway plays a role in VZV infection and reactivation in neurons, the cell type in which VZV establishes latency.
RESULTS
VZV infection causes activation of JNK in neurons.To investigate the potential role of the JNK pathway in VZV infection of neurons, we utilized human embryonic stem cell (HES)-derived neurons. Since only a fraction of neurons are infected by VZV under the conditions that we employed, we performed immunocytochemistry to assess infection and JNK activation. Infection by cell-free pOka (parental Oka) VZV resulted in induction of the VZV immediate-early protein IE62 by 48 h postinfection (p.i.), with increasing amounts of IE62 at 72 h p.i. (Fig. 1A). Expression of the VZV structural protein glycoprotein E (gE) lagged behind expression of IE62 and was predominantly observed at 72 h p.i. To examine JNK activation, we assessed for phosphorylation of JNK, which was seen to be increased from baseline as early as 48 h p.i. and further increased at 72 h p.i. (Fig. 1A and B). We also examined phosphorylation of c-Jun, a protein that is phosphorylated by activated JNK. We found that phosphorylated c-Jun was increased at 48 and 72 h p.i. in the same cells that exhibited increased expression of phosphorylated JNK (Fig. 1A and C). Immunocytochemistry revealed no differences in expression of total JNK or total c-Jun following infection, nor was there evidence of activation of two other mitogen-activated protein kinase pathways, as neither levels of phosphorylated or total ERK or those of p38MAPK were affected by VZV infection (data not shown). Following infection with cell-free VZVORF66GFP (see Materials and Methods), 64.2% ± 5.4% (mean ± standard error of the mean [SEM]) of green fluorescent protein (GFP)-expressing neurons demonstrated increased expression of phosphorylated JNK (pJNK), and 71.2% ± 4.9% of GFP-expressing neurons demonstrated increased expression of phosphorylated c-Jun (pc-Jun), indicating that the JNK pathway was activated in infected cells (Fig. 1C). Taken together, these data provide evidence for specific induction of the neuronal JNK pathway following VZV infection.
Activation of the JNK pathway in neurons following VZV infection. (A) Human ES-derived neurons were infected with cell-free pOka VZV. (Top two rows) Immunostaining for IE62 and gE (red) revealing expression at 48 h p.i. and 72 h p.i., respectively (scale bar, 50 μm). (Bottom two rows) Immunostaining for pJNK (red) and pc-Jun (magenta) demonstrating upregulation beginning at 48 h p.i. Parallel images are depicted in each row (scale bar, 20 μm). The arrows indicate examples of individual cells or cell clusters with increased expression. Nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole) (blue). (B) Analysis of the percentages of pOka VZV-infected cells expressing 1.5-fold-higher intensity than mock-infected cells, revealing increased expression of pJNK and pc-Jun at 48 h p.i. and 72 h p.i. (*, P < 0.05; **, P < 0.01; ***, P < 0.001 compared to mock infection; analysis of variance [ANOVA] with Bonferroni test). Bars represent the mean ± SEM. (C) Immunostaining of neurons infected with VZVORF66GFP at 48 h p.i. demonstrating GFP expression in several cells expressing increased amounts of pJNK (red) and pc-Jun (purple). Scale bar, 10 μm. Neuron density, 2.5 × 104/cm2; VZV pOkaORF66GFP, multiplicity of infection (MOI) = 0.001.
Inhibition of JNK activation blocks VZV protein expression in a dose- and time-dependent manner.To assess the role of JNK activation in VZV infection of neurons, we infected neurons with VZV in the presence of SP600125, a competitive binding inhibitor of JNK. We observed a dose-dependent decrease in VZV gE expression in the presence of SP600125 (Fig. 2A). Next, neurons were infected with VZV in the absence or presence of SP600125 (18 μM; maximum dose in Fig. 2A), and expression of gE was assayed over time. We observed a marked difference in VZV gE expression by 72 h p.i., and the differences persisted up to the final time point analyzed at 96 h p.i. (Fig. 2B and C). Moreover, neurons infected with VZVORF66GFP in the presence of SP600125 exhibited much less expression of VZV gE and GFP (Fig. 2D). As expected, pJNK expression was diminished in the setting of SP600125 (Fig. 2E). We did not observe changes in phospho-ERK or phospho-p38MAPK levels by Western blotting or immunohistochemistry in infected neurons upon treatment with 18 μM SP600125 (data not shown). Taken together, these results demonstrate that JNK phosphorylation is induced by VZV infection in human neurons and that this activation of JNK is essential for efficient viral replication and protein expression.
JNK blockade inhibits VZV gE protein expression and replication in neurons. (A) Western blotting demonstrating a dose-dependent decrease in VZV gE expression in neurons treated with increasing doses of the JNK inhibitor SP600125 (lane 1, mock infection; lanes 2 to 6, VZV infected; lanes 3 to 6, SP600125 at 1, 3, 9, and 18 μM, respectively). (B) Western blotting demonstrating that VZV gE expression increases to a far lesser extent over time in neurons treated with 18 μM SP600125. (C) Quantification of gE expression in panel B, relative to GAPDH expression. (D) Neurons infected with VZVORF66GFP in the presence or absence of SP600125 were subjected to immunostaining for gE (red) at the indicated time points postinfection. Scale bar, 10 μm. (E) Neurons were infected with VZVORF66GFP for 48 h and immunostained for pJNK (red). Scale bar, 20 μm. Neuron density, 2.5 × 104/cm2; VZVORF66GFP, MOI = 0.001.
JNK inhibition blocks the production of infectious virus in neurons.We next examined whether inhibition of JNK impacts the production of infectious virus in neurons. The 50% effective concentration (EC50) of the JNK inhibitor SP600125 in human fetal fibroblasts has been reported to be 8 μM (10), and the EC50 of acyclovir in MRC-5 cells was 40 μM (20). Neurons infected with a recombinant VZV, rVZVLUCBAC, for 72 h in the presence or absence of inhibitors were harvested, diluted, and used to infect MRC-5 cells to quantify virus production in human neurons. As expected, infection of neurons in the presence of acyclovir (60 μM), an agent known to inhibit VZV DNA replication, resulted in marked inhibition of infectious-virus production. Infection of neurons in the presence of 18 μM SP600125 also resulted in inhibition of infectious-virus production, though to a lesser extent than observed with acyclovir. Coincubation of neurons with SP600125 (18 μM) and acyclovir (60 μM) resulted in a modest further inhibition of viral production compared to either inhibitor alone (Fig. 3A).
JNK blockade inhibits the production of infectious virus and shows inhibitory effects additive to those of acyclovir on VZV infection in neurons. (A and B) Neurons (2 × 105 at a density of 5 × 104/cm2) were infected with rVZVLUCBAC at an MOI of 0.002 in the presence of inhibitors. Infected neurons at 72 h p.i. were harvested, diluted, and laid onto MRC-5 cells (1 × 105/cm2) at the dilution factors indicated on the left (1:1 corresponds to 1 × 105 infected neurons). The cells were fixed at 6 days p.i., and the plaques were visualized. (A) Vehicle (DMSO), JNK inhibitor SP600125 (SP) (18 μM), acyclovir (ACV) (60 μM), or a combination (SP + ACV) was used. (B) Vehicle (DMSO) and a combination of ACV (1 μM) and SP600125 (0, 4, and 8 μM) were used as inhibitors. Visualized plaques were counted. The experiment was performed twice, and each time triplicate wells were used. The error bars indicate SEM. *, P < 0.05; one-way ANOVA.
To test whether SP600125 has an inhibitory effect additive to that of acyclovir on VZV infection, the EC50s of acyclovir and SP600125 for human neurons were determined. The EC50 of SP600125 for human neurons was 4 μM, while the EC50 of acyclovir was 1 μM (data not shown). The concentration of acyclovir was fixed at 1 μM, and different concentrations of SP600125 were examined. The production of infectious virus from human neurons was effectively reduced by 1 μM acyclovir treatment, and 4 μM SP600125 showed additive inhibitory effects on VZV infection in human neurons (Fig. 3B). Thus, JNK inhibition blocks infectious-virus production in neurons at a lower dose than in fibroblasts and in a different manner from acyclovir.
Effects of JNK inhibition on acyclovir-resistant VZV.Since SP600125 has a different mechanism of action than acyclovir to inhibit VZV, we tested whether JNK inhibition would limit replication of a VZV isolate that is resistant to acyclovir (VZVr). Human neurons were pretreated with SP600125 (18 μM) and infected with either VZV pOka or VZVr. Acyclovir was far less effective in inhibiting virus formation in neurons infected with VZVr (Fig. 4A, first two columns) than in neurons infected with VZV-pOka (Fig. 4B). Inhibition of the JNK pathway, however, resulted in a marked decrease in plaques from VZVr-infected neurons (Fig. 4A, last two columns) to a degree similar to that with VZV-pOka (Fig. 4B). Thus, the acyclovir-resistant VZV isolate was as sensitive to the effects of JNK inhibition as acyclovir-sensitive VZV in neurons, and inhibition of the JNK pathway effectively limited infectious-virus production of either VZV isolate.
Acyclovir-resistant VZV is susceptible to inhibition of the JNK pathway. (A) Representative image of plaque assays from neurons (1 × 105) infected with two different input concentrations of VZVr at an MOI of 0.00073 (High) or 0.00036 (Low) and in the presence or absence of ACV or SP, as indicated. The dilution factors of infected neurons overlaid on MRC-5 cells are indicated on the left. (B) Quantification of plaque assays (*, P < 0.05; ***, P < 0.001; n.s., not significantly different; ANOVA with Bonferroni test) from neurons (1 × 105) infected with VZV-pOka (VZV) or VZVr at an MOI of 0.0073. ACV, acyclovir.
Role of JNK in infection of sensory neurons.Since sensory neurons represent a unique cellular niche for the establishment of VZV latency and reactivation in vivo, we next sought to determine whether the JNK signaling pathway is important for virus production in these cells. Neurons expressing markers of sensory neurons (Brn3a, peripherin, and MAP2) were derived from neural crest cells (Fig. 5A). Sensory neurons infected with VZVORF66GFP for 96 h expressed GFP (Fig. 5B) and VZV gE (not shown), demonstrating permissiveness to infection. Treatment of sensory neurons with SP600125 followed by infection with VZVORF66GFP resulted in substantially less GFP expression (Fig. 5B), indicating decreased viral gene expression. Moreover, plaque assays demonstrated a marked reduction in productive virus in sensory neurons treated with the JNK inhibitor (Fig. 5C).
JNK blockade inhibits VZV infection in sensory neurons. (A) Sensory neurons immunostained with antibodies to peripherin (red), which stains intermediate filaments, and Brn3a (green), which is a transcription factor in the nucleus of the cell. Scale bar, 50 μm. (B) Sensory neurons (2.5 × 104/cm2) were infected with VZVORF66GFP at an MOI of 0.001 for 4 days in the absence (top) or presence (bottom) of JNK inhibitor. Scale bar, 50 μm. (C) Quantification of plaque assays from 1 × 105 sensory neurons infected with VZVORF66GFP at an MOI of 0.001 in the absence (VZV) or presence (VZV+SP) of JNK inhibitor. **, P < 0.01; Student's t test. Plaque assays were performed as for Fig. 3 and 4. The error bars indicate SEM.
Role of JNK signaling in viral reactivation.As VZV can remain latent in neurons prior to reactivation, we next sought to determine whether JNK activation also plays a role in viral reactivation. We recently established an in vitro system that recapitulates elements of VZV latency and reactivation in vivo (19). In this system, neurons are cultured in microfluidic devices that allow axons to be fluidically isolated from their respective cell bodies. The axons are infected with cell-free VZV pOka, which results in a nonproductive infection with maintenance of the viral DNA genome in the neuronal soma in a circular configuration, consistent with virus latency (Fig. 6A, i and ii). Following treatment with antibodies to nerve growth factor (anti-NGF), productive virus infection was observed (Fig. 6A, iii and iv). We found that the JNK pathway is activated upon treatment of neurons with anti-NGF (Fig. 6B), raising the possibility that inhibition of the pathway may limit viral reactivation. In the absence of JNK inhibition, approximately 30% of the cultures demonstrated viral reactivation after addition of anti-NGF; however, in the presence of JNK inhibitor, only 8% of the cultures demonstrated reactivation (Fig. 6C). Thus, JNK is important for viral amplification in the setting of reactivation.
JNK blockade inhibits viral reactivation. (A) Schematic of an in vitro model of viral latency and reactivation. Neurons (purple) were cultured in microfluidic devices whose microchannels (M) allow fluidic isolation of axons in the axonal compartment (A) from their neuronal cell bodies in the somal compartment (S). (i and ii) Selective addition of virus (red) to the axonal side did not lead to productive infection (i), though some neurons harbored viral DNA in a circular configuration compatible with latency (indicated by red soma) (ii). (iii and iv) Anti-NGF (blue) was added to cultures for 7 days (iii), resulting in viral reactivation from some latently infected neurons (3rd neuron from the top) (iv). (B) Immunostaining for pJNK revealed intense expression in the positive control (anisomycin at 25 μg/ml for 30 min, which is known to activate JNK) and in neurons treated with anti-NGF (50 μg/ml). Scale bar, 50 μm. (C) Addition of JNK inhibitor markedly decreased the observed reactivation following anti-NGF treatment. The red numbers represent the numbers of individual microfluidic cultures employed for each condition. Neuron density, 5 × 104/cm2; VZV pOka, 100 PFU/axonal compartment.
DISCUSSION
Cellular kinases may be coopted by viruses during infection. While previous reports have shown that VZV can activate the JNK pathway in nonneural cells (9, 10), it was not known whether this occurs in neurons and whether there are attendant consequences. Here, we have demonstrated the importance of the JNK pathway for VZV infection and reactivation in cells in which the virus establishes latency through the use of human embryonic stem cell-derived neurons. We found that VZV activates the JNK pathway in neurons, and we observed that pharmacologic blockade of the JNK pathway inhibits viral gene expression, lytic replication, and reactivation from latency. An acyclovir-resistant isolate of VZV was also inhibited by blockade of the JNK pathway. Furthermore, we found that the JNK pathway is critical for VZV infection of human sensory neurons.
Several viruses, including herpes simplex virus (HSV) and Epstein-Barr virus (EBV), activate the JNK pathway through a variety of mechanisms, with differing consequences for infected cells. For example, the ability of EBV to activate JNK via latent membrane protein 1 is essential for oncogenic activity of the virus (21, 22). Activation of the JNK pathway by HSV is mediated by HSV VP16, ICP0, and ICP27, which likely facilitates lytic infection (23–25). With respect to VZV, the consequences of JNK activation may be cell type dependent, since in melanoma cells, activation of JNK inhibits VZV replication, while in fibroblasts, where JNK associates with proteins in the virion tegument, JNK enhances virus replication (9, 10). Our results demonstrate that in neurons, sustained JNK activation occurs following infection, and this facilitates lytic infection.
The mechanism of herpesvirus reactivation has distinct differences from lytic infection. Unlike lytic infection, reactivation of HSV begins with a viral genome associated with a more compact chromatin structure, with heterochromatin associated with lytic genes and absence of tegument proteins, including VP16 (26, 27). Reactivation of HSV appears to follow a two-step response in which the first phase results in derepression of lytic genes with de novo synthesis of viral regulatory proteins, including tegument factors in the cytoplasm; in the second phase, these proteins translocate to the nucleus and activate widespread viral gene expression, genome amplification, and virus production (28, 29). Notably, activation of JNK signaling in the setting of cellular stress is a key feature of the first phase of HSV reactivation. JNK activation in neurons results in a histone methyl/phospho switch that activates HSV lytic promoters and allows transcription even in the presence of histone lysine modifications (29–31). While less is known about the mechanism of VZV reactivation, it has been proposed that a transcriptional deregulatory event (referred to as animation), similar to the first phase of HSV reactivation, may trigger generalized VZV transcription (32). Our findings that treatment of latently VZV-infected neurons with anti-NGF promotes JNK activation and viral reactivation while JNK inhibition blocks viral amplification in the setting of reactivation suggest the possibility of an role of the JNK pathway in VZV analogous to that in HSV in initiating virus reactivation from the latent state.
In order for latency to be established in vivo, sensory and autonomic neuronal ganglia, including dorsal root and trigeminal ganglia, must be infected. Little is known about mechanisms of VZV infection, latency, and reactivation in sensory neurons, in part because most human neuronal culture systems derived from HES or induced pluripotent stem cells (iPSCs) are typically comprised of few, if any, neurons expressing markers of sensory neurons (19). Recent experiments have shown that addition of small-molecule inhibitors of the SMAD signaling pathway to iPSC-derived neuronal cultures results in approximately 15% of the cells differentiating into Brn3a+ peripherin+ sensory neurons (33). These sensory neurons are capable of supporting VZV infection, as demonstrated by colocalization of viral proteins in cells that express Brn3a or peripherin (33). Here, we utilized a novel and highly efficient system to generate sensory neurons, resulting in approximately 95% of the cells coexpressing Brn3a and peripherin. This sensory neuronal differentiation protocol allows us to examine determinants of viral latency and reactivation in a physiologically relevant cell type.
Acyclovir is a nucleoside analog that is phosphorylated by the viral thymidine kinase (TK) and cellular kinases to form a triphosphate moiety that blocks viral DNA polymerase. While acyclovir resistance is uncommon in healthy individuals, in immunocompromised individuals with recurrent or chronic VZV infections, resistance can emerge and occur, predominantly due to mutations in the viral TK gene (34–36). Mutations may also occur in the viral DNA polymerase, which is the target of second-line drugs, such as foscarnet and cidofovir (37). In addition, resistance of HSV to drugs that target the viral helicase and primase has been reported (38, 39). In light of increasing resistance and the substantial toxicity of current second-line drugs, there is growing interest in developing newer inhibitors to treat herpesvirus infections. Recently, several lysosomotropic nonnucleoside agents have been identified that inhibit lytic infection by several herpesviruses, including HSV and VZV, presumably by inhibiting viral packaging and maturation (40). However, the effects of these drugs on infection of neurons by VZV were not reported. We found that inhibition of JNK activation resulted in a marked reduction in the production of acyclovir-resistant VZV in neurons. Thus, inhibition of the JNK signaling pathway to limit neuronal infection could potentially provide an alternative target for herpesvirus antiviral therapy. A potential barrier to the translation of JNK inhibitors to human therapy is the involvement of multiple JNK isoforms in a wide variety of cellular processes, including several directly relevant to viral infection, such as apoptosis and cytokine production. Notably, several recently developed compounds that target the JNK signaling pathway have exhibited high potency and good oral bioavailability and can penetrate the central nervous system (CNS), potentially making them attractive candidates for further evaluation as antivirals for VZV (41, 42).
MATERIALS AND METHODS
Cell culture.Tissue culture wells were treated with poly-d-lysine (PDL), followed by Matrigel or CellStart (1:100) (catalog no. A1014201; Life Technologies) prior to use. HES (H9)-derived neural stem cells (NSCs) were cultured in proliferating medium consisting of Knockout D-MEM/F-12 (catalog no. 12660-012; Gibco) base medium supplemented with 2 mM GlutaMax-I (catalog no. 35050-061; Life Technologies), 20 ng/ml basic fibroblast growth factor (bFGF) (catalog no. 13256-029; Invitrogen), 20 ng/ml of epidermal growth factor (EGF) (catalog no. PHG0311; Invitrogen), and 2% StemPro neural supplement (catalog no. A10508-01; Invitrogen). NSCs were differentiated into neurons by utilizing a neuronal differentiation medium prepared in neurobasal base medium (catalog no. A24775-01; Gibco) with 2% B-27 serum-free supplement (catalog no. 17504-044; Invitrogen) and 2 mM GlutaMax-I supplement (catalog no. 35050-061; Life Technologies). Human NSCs were seeded at a density of 5 × 104 to 10 × 104 cells/cm2. The cells were plated in proliferation medium for 2 days and were then differentiated for 11 days or more before experiments were performed. Under these conditions, the density of neurons in each well was approximately 2.5 × 104 to 5 × 104 cells/cm2. Human lung (MRC-5) fibroblasts were cultured in minimum essential medium (MEM) (Life Technologies) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (FBS) (Sigma–Aldrich) and GlutaMax-I (2 mM).
Generation of human sensory neurons.Our strategy for derivation of neural crest stem cells was modified from a previously published protocol that we have experience with (43). However, we have employed a novel SOX10-based purification step, which provides a simpler and more reliable and precise method than surface marker staining-based purification or a transgenic Sox10::GFP bacterial artificial chromosome (BAC)-based line (43, 44). The HES (WA09/H9; WiCell)-based SOX10::GFP reporter line was cultured with mouse embryonic fibroblasts (ASF-113; Applied Stem Cell) in proliferation medium consisting of Dulbecco's modified Eagle's medium (DMEM)–F-12 (catalog no. 11330-057; Gibco) base medium supplemented with knockout serum (catalog no. 10828-028; Gibco), 2 mM l-glutamine (catalog no. 25030-081; Gibco), MEM nonessential amino acids (catalog no. 11140-050; Gibco), 5.5 mM 2-mercaptoethanol (catalog no. 21985-023; Gibco). To differentiate SOX10-expressing neural crest stem cells, 6 × 104 HES cells/cm2 on Geltrex (catalog no. A1413302; Gibco) were treated with dual SMAD inhibition and canonical WNT activation, as previously described (45). Briefly, 500 nM LDN193189 (Abcam) and 10 μM SB431542 (Cayman Chemical) were added for 3 days, followed by additional treatment with Chir 99021 (Tocris Bioscience) for 7 days, with gradual medium change from HES medium to neurobasal medium containing N2 (catalog no. 17502-048; Gibco), B27 (catalog no. 12587-010; Gibco), and 2 mM l-glutamine. Our previous studies have shown that this protocol results in differentiation into Brn3a+ Islet1+ peripherin+ sensory neurons with appropriate electrophysiological responses (46, 47). GFP-expressing cells could begin to be identified by day 7, and fluorescence-activated cell sorter (FACS) purification was performed at day 10. Sorted GFP+ SOX10-expressing neural crest-derived stem cells were replated and further differentiated into sensory neurons by incubating with neurobasal medium containing N2, B27, 2 mM l-glutamine, NGF, brain-derived neurotrophic factor (catalog no. 450-02; Peprotech), glial cell-derived neurotrophic factor (catalog no. 450-10; Peprotech), 100 μM sodium l-ascorbate (catalog no. A4034; Sigma), and 100 μM dibutyryl-cAMP (catalog no. D0627; Sigma).
Virus infection and plaque reduction assay.All the viruses used in this study are based on the pOka strain of VZV. A recombinant VZV, rVZVLUCBAC, was reconstituted in MRC-5 cells by transfection of VZVLUCBAC DNA (from Hua Zhu, New Jersey Medical School, Rutgers University, Newark, NJ) as described previously (48). Eleven days after differentiation, the neurons were infected with VZV pOka (from Michiaki Takahashi, Osaka University, Suita, Osaka, Japan), VZVORF66GFP (a recombinant VZV in which GFP is fused to VZV ORF66 derived from the pOka strain; from Paul R. Kinchington, University of Pittsburgh, Pittsburgh, PA) (49), acyclovir-resistant VZV (from Mark Prichard, University of Alabama, Birmingham, AL), or rVZVLUCBAC cell-free virus. Cell-free VZV was prepared as described previously (19). The cell-free VZV was added to neurons for 2 h, the inoculum was removed, and the cells were treated with sodium citrate buffer (40 mM sodium citrate, 10 mM potassium chloride, 135 mM sodium chloride [pH 3.2]) for 30 s and then washed with differentiation medium prior to adding fresh neuronal differentiation medium.
For the plaque reduction assay, infected neurons were plated onto MRC-5 cells to detect VZV cell-to-cell spread, because the virus does not form plaques on neurons. Neurons (2 × 105/well in a 12-well plate) at 11 or 12 days postdifferentiation were pretreated with various inhibitors at 37°C for 45 min and then infected with cell-free VZV as described above and cultured for 72 h in the presence of inhibitors. Infected neurons were harvested, diluted, plated onto MRC-5 cells (1 × 105/well in a 12-well plate 2 days prior to infection), and cultured for 6 days in the absence of any inhibitor. Plaques on MRC-5 cells were visualized as described previously (19) and counted. The competitive JNK inhibitor SP600125 (Sigma) was prepared as a 100 mM stock solution by dissolving it in dimethyl sulfoxide (DMSO) in sterile medium. Acyclovir was obtained from AAP Pharmaceuticals (Schaumberg, IL).
Immunocytochemistry.Neuronal cultures were washed with phosphate-buffered saline (PBS) and fixed for 20 min at room temperature with 4% paraformaldehyde (PFA). The cells were washed in PBS and incubated in blocking solution containing 0.25% Triton-X and 5% normal donkey serum for 1 h. Primary antibodies, which included anti-mouse pJNK (1:50; catalog no. SC6254; Santa Cruz), anti-rabbit pc-Jun (1:100; catalog no. 06-659; Millipore), anti-rabbit phospho-p38 MAPK (1:250; Cell Signaling), anti-mouse VZV gE (1:300; catalog no. MAB612; EMD Millipore), and anti-mouse VZV IE62 (1:100; catalog no. SC58211; Santa Cruz) were diluted in blocking solution and applied to the cells overnight at 4°C. The cells were washed three times in 1× PBS and incubated with appropriate secondary Alexa Fluor 488-conjugated donkey anti-rabbit or -mouse (1:250; Invitrogen) or Alexa Fluor 594-conjugated donkey anti-rat, -mouse, or -rabbit (1:250; Invitrogen) for 2 h at room temperature. Finally, the cells were incubated for 5 min with 1 μM DAPI (4′,6-diamidino-2′-phenylindoldihydrochloride) (Invitrogen) as a nuclear counterstain. A Zeiss live-cell inverted microscope (Axio Observer; Zeiss, Germany) was used for imaging.
Quantification of pJNK and pc-Jun was performed by assessing intensity in each cell in 10 random fields per well using NIH Image J software (NIH, Bethesda, MD) to determine the integrated intensity (50). A threshold of 1.5-fold-higher expression than the average intensity of mock-infected cells was predefined as a positive cell. Experiments were performed in triplicate.
Protein isolation and Western blotting.Protein lysates from cells were prepared in radioimmunoprecipitation assay (RIPA) lysis and extraction buffer (catalog no. 89900; Thermo Scientific) with 1× protease inhibitor (catalog no. 78430; Thermo Scientific) and 1× phosphatase inhibitor (catalog no. 78420; Thermo Scientific) after removing the medium from the cells and washing with cold 1× PBS. Twenty micrograms of total protein was loaded in each lane, and proteins were separated by SDS-10% PAGE, followed by transfer onto a nitrocellulose membrane overnight. After blocking with 10% milk in PBS and Tween 20 for 30 min, the membranes were incubated for 3 h at room temperature with mouse antibody to VZV gE (1:3,000), with control anti-rabbit antibody to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (1:5,000; Cell Signaling; catalog no. 5174). After washing, horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit (1:5,000; GE Healthcare) secondary antibodies were applied for 45 min at room temperature. Antibody binding was detected using HRP chemiluminescence for 5 min and visualized using a Bio-Rad imager (Segrate, Milan, Italy). The immunoblotting intensity was quantified using NIH ImageJ software (NIH, Bethesda, MD).
Microfluidic system for viral latency and reactivation.Four-well polydimethylsiloxane (PDMS)-based devices were fabricated as previously described (19, 51). These devices allow selective axonal growth into one compartment, enable fluidic isolation of axons from their respective cell soma, and allow independent manipulation of axonal and somal compartments (51–53). The devices were plasma treated (Harrick Plasma, Ithaca, NY) to allow bonding to glass bottom dishes (WillCo, Denmark). Poly-d-lysine (200 μg/ml; catalog no. P6407; Sigma) was introduced into the devices through ports. The devices were then incubated overnight at 37°C in a humidified 5% CO2 incubator. The following day, the devices were washed twice with double-deionized water to remove unbound PDL. The devices were then filled with BD Matrigel matrix (catalog no. 354230; BD Biosciences) diluted 1:40 in plain neurobasal medium for >3 h prior to cell culture.
NSCs were seeded at a density of 1 × 104 to 2.5 × 104 cells/cm2 in proliferation medium for 2 days, followed by differentiation for at least 11 days, resulting in >98% neuron-specific beta-III tubulin-positive neurons (19). Axonal infection of these cells with VZV pOka resulted in a state closely resembling viral latency, with viral DNA present in the cells in a circular configuration with undetectable viral RNA (by reverse transcription-quantitative PCR) or infectious virus (19). Anti-NGF (50 μg/ml; AB1528SP; EMD Millipore) was subsequently added to promote reactivation. To assess for reactivation, neurons from individual devices were dissociated and added to MRC-5 cells, and plaques were detected as described above.
ACKNOWLEDGMENTS
We thank Paul Kinchington for providing VZVORF66GFP, Hua Zhu for providing VZVLUCBAC DNA, Michiaki Takahashi for providing VZV pOka, and Mark Prichard for providing acyclovir-resistant VZV.
T.S. was supported by the Japan Herpesvirus Infections Forum while at the National Institutes of Health and partially funded by the Takeda Science Foundation, Japan Society for the Promotion of Science (JSPS KAKENHI grant number JP17K008858), and the Ministry of Education, Culture, Sports, Science and Technology (MEXT KAKENHI grant number JP17H05816). T.S. and J.I.C. were supported by the intramural research program of the National Institute of Allergy and Infectious Diseases. Work in the G.L. laboratory was supported by grants from NIH (NINDS, R01NS093213, and NIAMS, R01AR070751, to G.L.), NSF (1547515 to G.L.), and Maryland Stem Cell Research Funding (MSCRF/TEDCO) (to G.L.).
FOOTNOTES
- Received 14 April 2017.
- Accepted 12 June 2017.
- Accepted manuscript posted online 21 June 2017.
- Copyright © 2017 American Society for Microbiology.
