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. Author manuscript; available in PMC: 2025 Apr 15.
Published in final edited form as: J Neurovirol. 2024 Oct 4;30(5-6):513–523. doi: 10.1007/s13365-024-01229-4

Regulation of voltage-gated sodium channels by TNF-α during herpes simplex virus latency establishment

Qiaojuan Zhang 1, Shao-Chung Hsia 1, Miguel Martin-Caraballo 1
PMCID: PMC11998310  NIHMSID: NIHMS2036941  PMID: 39367281

Abstract

During lytic or latent infection of sensory neurons with herpes simplex virus type 1 (HSV-1) there are significant changes in the expression of voltage-gated Na+ channels, which may disrupt the transmission of pain information. HSV-1 infection can also evoke the secretion of various pro-inflammatory cytokines, including TNF-α and IL-6. In this work, we hypothesized that TNF-α regulates the expression of Na+ channels during HSV-1 latency establishment in ND7/23 sensory-like neurons. Latency establishment was mimicked by culturing HSV-1 infected ND7/23 cells in the presence of acyclovir (ACV) for 3 days. Changes in the functional expression of voltage-gated Na+ channels were assessed by whole-cell recordings. Our results demonstrate that infection of ND7/23 cells with the HSV-1 strain McKrae with GFP expression (M-GFP) causes a significant decrease in sodium currents during latency establishment. Exposure of ND7/23 cells to TNF-α during latency establishment reverses the effect of HSV-1, resulting in a significant increase in sodium current density. However, Na+ currents were not restored by 3 day-treatment with IL-6. There were no changes in the pharmacological and biophysical properties of sodium currents promoted by TNF-α, including sensitivity to tetrodotoxin and the current-voltage relationship. TNF-α stimulation of ND7/23 cells increases p38 signaling. Inhibition of p38 signaling with SB203580 or SB202190 eliminates the stimulatory effect of TNF-α on sodium currents. These results indicate that TNF-α signaling in sensory neurons during latency establishment upregulates the expression of voltage-gated Na+ channels in order to maintain the transmission of pain information.

Keywords: Sensory neuron, Sodium channel, Pain, Electrical excitability, Tumor necrosis factor-alpha

Introduction

Herpes simplex virus-type 1 (HSV-1) can trigger lytic infection of neurons and surrounding tissue, resulting in neuronal cell death and tissue damage. Alternatively, HSV-1 infection can result in latency establishment and undergo reactivation under certain conditions(Thellman and Triezenberg 2017). HSV-1 infection of sensory neurons also alters the neuronal excitability and the expression of voltage-gated ion channels (Oakes et al. 1981; Fukuda et al. 1983; Storey et al. 2002; Zhang et al. 2019, 2020a). During HSV-1 infection, downregulation of voltage-gated Na+ currents decreases neuronal excitability, resulting in increased viral replication (Storey et al. 2002; Zhang et al. 2005). Disruption in the functional expression of voltage-gated ion channel expression can interfere with the normal transmission of pain stimuli in HSV-1-infected neurons. We have previously demonstrated that HSV-1 infection of ND7/23 sensory-like neurons caused a significant reduction in the functional expression of voltage-gated Na+ and T-type Ca2+ channels during lytic infection (Zhang et al. 2017, 2019, 2020a). However, the mechanisms by which sensory neurons counteract this initial downregulation of ion channel expression to sustain sensory transmission during HSV-1 infection remain unexplored.

Transmission of pain signals relies on the flow of information between affected areas and the central nervous system. Changes in the functional expression of ion channels in the membrane can result in either diminished or enhanced pain signaling. HSV-1 can trigger a variety of pain sensations, including formication, paresthesia, or even numbness or tingling around the initial infection area, which has been reported to be associated with changes in neuronal excitability (Andoh et al. 1995). Voltage-gated Na+ channels are multimeric proteins that are involved in the generation of action potentials required for the transmission of pain signals. The main pore-forming α subunit of voltage-gated Na+ channels (Nav) is generated by at least 10 different genes, resulting in various functional channels (Goldin, 1999; de Lera Ruiz and Kraus 2015). Voltage-gated Na+ channels generated by Nav1.1 to Nav1.7 form TTX-sensitive channels, whereas channels formed by Nav1.8 or 1.9 subunits form TTX-insensitive channels (de Lera Ruiz and Kraus 2015). Neurons may express various Nav channel subunits (Yin et al. 2016; Lee et al. 2019). For example, Nav1.6 and Nav1.7 are the major subunits expressed in differentiated ND7/23 cells (Lee et al. 2019). Low-level expression of Nav1.1, Nav1.2, and Nav1.3 subunits is also detected but not Nav1.8 (Lee et al. 2019).

HSV-1 infection of nerve and surrounding tissue often triggers the release of pro-inflammatory cytokines and chemokines (Wuest and Carr 2008). In human corneal epithelial cells, HSV-1 infection in vitro triggers the transcriptional upregulation and secretion of IL-6 and TNF-α within 8 h post-infection (Li et al. 2006). However, the precise role of pro-inflammatory cytokines and chemokines, released following HSV-1 infection, in exacerbating the impact of the virus on sensory neurons remains incompletely understood. Increased secretion of pro-inflammatory cytokines and chemokines may alter the function of voltage-gated Na+ channel conductances under normal and pathological conditions (Vezzani & Viviani, 2015). In nociceptive neurons, TNF-α increases the expression of voltage-gated Na+ channels, whereas it has the opposite effect on Ca2+ currents (Czeschik et al. 2008). In a motor nerve injury model, TNF-α enhances Na+ currents in dorsal root ganglion (DRG) neurons (Chen et al. 2011). We have previously demonstrated that IL-6 reverses the downregulation of T-type Ca2+ channels in differentiated ND7/23 cells caused by HSV-1 lytic infection (Zhang et al. 2019).

In this work, we tested the effect of TNF-α on the expression of voltage-gated Na+ channels in ND7/23 sensory-like neurons following latency establishment. Changes in voltage-gated Na+ channel expression may underlie specific sensory abnormalities in patients following HSV-1 infection. Those changes could be triggered not only by the direct effect of the virus on pain-transmitting neurons but also by the secretion of pro-inflammatory cytokines.

Methods

Cell culture, differentiation, and infection of ND7/23 cells

ND7/23 cells were obtained from Sigma-Aldrich (Cat.#92090903). As previously reported, the ND7/23 cells are derived by the fusion of mouse neuroblastoma and rat DRG cells, generating a more homogeneous cell population with sensory neuron-like properties (Wood et al. 1990). Differentiation of ND7/23 cells was performed as previously described (Wood et al. 1990; Zhang et al. 2017), using DMEM/F12 culture media (Millipore, Cat.#DF-041-B), supplemented with 0.5% fetal bovine serum (Invitrogen, Cat.#10437010), db-cAMP (1 mM, Sigma-Aldrich, Cat.#D0627), and NGF (50 ng/mL, Sigma-Aldrich, Ca.#N2513). Cells were grown either in poly-d-lysine-coated 6-well plates or on glass coverslips (for whole-cell recordings) on differentiation media for 4 days (d) before viral infection.

Viral infections were performed with the HSV-1 strain McKrae with GFP expression (M-GFP) under the control of a cytomegalovirus (CMV) promoter (David et al. 2012). Viral particles were propagated in African green monkey kidney (Vero) cells (ATCC, Cat.#CCL-81), cultured in MEM media (ThermoFisher, Cat.# 41090-036), supplemented with 10% fetal bovine serum. GFP expression was used to facilitate the identification of infected cells. To establish HSV-1 latency, differentiated ND7/23 cells were pretreated with 100 μM acycloguanosine (also known as acyclovir, ACV) overnight, followed by viral infection. Unbound viral particles were washed out after 1 h of viral exposure. The differentiation media, supplemented with 100 μM ACV (with or without TNF-α 40ng/mL) was applied for 3 d to allow for the establishment of latent HSV-1 infection.

Fluorescence microscopy

Images were obtained with a Nikon Eclipse Ti microscope with a 20× inverted objective and a Photometrics Cool-SNAP EZ cooled camera, using appropriate filters for phase contrast and GFP fluorescence. The intensity of the GFP fluorescence change was used to monitor the change in HSV-1 activity in infected cells.

Western blot analysis

Immunoblot analysis was conducted as previously described by Zhang et al. (2017, 2020a), using specific antibodies (Table 1). Briefly, cell lysates were combined with Bolt LDS sample buffer (ThermoFisher, Cat.#B0007), supplemented with reducing agent (ThermoFisher, Cat.#B0004), and boiled for 10 min at 80 °C. Proteins were separated on pre-cast SDS-PAGE 8% gels (Bolt Bis-Tris Plus Gels, ThermoFisher, Cat.# NW00080BOX). Proteins were transferred to nitrocellulose membranes using the Invitrogen iBlot system, followed by incubation in SuperBlock blocking buffer for 45 min (ThermoFisher, Cat.#37535) before overnight incubation with the primary antibodies. BupH Tris-buffered saline (ThermoFisher, Cat.#28379) was used to wash the membranes following incubation with the antibodies. Blots were analyzed using a horseradish peroxidase-conjugated anti-rabbit secondary antibody (Jackson ImmunoResearch Lab, Cat.#211-035-109) and a chemiluminescent substrate (SuperSignal West Pico, ThermoFisher, Cat.#34580). To control for equal loading of protein in each sample, membranes were stripped using Restore Plus stripping buffer (ThermoFisher, Cat.#46430) for 30 min at room temperature and reprobed with a tubulin-specific antibody (1:2000 dilution) followed by incubation with a peroxidase-conjugated anti-mouse secondary antibody (Jackson ImmunoResearch Lab, Cat.# 115-035-146) and immunodetection. Signals were captured using a ChemiDoc™ XRS + documentation system (Bio-Rad). Changes in protein expression were determined by densitometry analysis using Image Lab software (Bio-Rad).

Table 1.

List of antibodies

Antibody Supplier
p-p38 Cell Signaling, Cat.#8690
p38 Cell Signaling, Cat.#AB5210
Anti-PAN Millipore, Cat.#AB5210
Tubulin Millipore, Cat.#05-829
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Quantitative RT-PCR

Quantification of thymidine kinase (TK) and Nav subunit expression was performed as previously described (Zhang et al. 2020a). Briefly, RNA was isolated using the iScript sample preparation reagent (BioRad, Cat.#170–8898). Synthesis of cDNA by RT reaction was performed with iScript RT Supermix (Bio-Rad, Cat.#17008841). The SsoAdvanced Universal SYBR green supermix (Bio-Rad, Cat.#1725271) was used to perform the qPCR reactions on triplicate samples using specific primers (Table 2). qRT-PCR reactions were carried out at 98 °C for 3 min, 98 °C for 10 s, followed by 58 °C for 30 s (39 cycles), 65 °C for 5 s. TK and Nav transcripts expression was normalized to that of peptidylpropyl isomerase A (PPIA, Table 2).

Table 2.

Quantitative RT-PCR primers

Target gene Sequence
TK 5’-ATG GCT TCG TAC CCC TGC CAT-3’
5’-GGT ATC GCG CGG CCG GGT A-3’
PPIA 5’-GGT GGC AAG TCC ATC TAC GG-3’
5’-CTT GCC ATC CAG CCA CTC A-3’
Nav1.1 5’- AGA GTG GAG AGA TGG ACG CT -3’
5’- CCC GCC CTT GAG TTT GTT CT -3’
Nav1.2 5’- GAG GAG GTG TCT GCT ATT GTC – 3’
5’- GTC TTC TTT GAT GGG CGT TCC – 3’
Nav1.3 5’- CTT CGG CTC GTT CTT CAC TCT – 3’
5’- TCT GGT CAT CCG TTT CCA CC -3’
Nav1.6 5’- GAG AGG CGT ATT GCC GAG AG -3’
5’- CAC TGT TTG GCT TGG GCT TG -3’
Nav1.7 5’- TGG CGA GGA AAA GGG TGA TG -3’
5’- TGC TGA GTG GTG ACT GGT TG -3’
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Electrophysiology

A Nikon Eclipse Ti inverted microscope equipped with Hoffman optics and epifluorescence filters was used to visualize differentiated ND7/23 cells expressing GFP during recordings. Infected cells were identified by the expression of GFP. Recordings were performed at room temperature using glass electrodes made from thin wall borosilicate glass (3–4 MΩ). The pipette solution consisted of (in mM) CsCl (120), MgCl2 (2), HEPES-KOH (10), EGTA (10), ATP (1), and GTP (0.1), pH 7.4 with CsOH. The composition of the normal external saline used for measurements of Na+ currents was (in mM) NaCl (145), KCl (5.3), CaCl2 (0.54), MgCl2 (5.7), HEPES (13), and glucose (5), pH 7.4 adjusted with KOH. Na+ currents were generated by applying a 50 ms-depolarizing step to various potentials from a holding potential of −100 mV. A MultiClamp 700 A amplifier and Pclamp software (Axon Instruments) were used to deliver voltage commands and to perform data acquisition and analysis. Pipette offset, whole cell capacitance, and series resistance were compensated automatically with the MultiClamp 700B Commander. Only cells with stable seals and series resistance (≤ 10 MΩ) were analyzed. Sampling rates were between 5 and 10 kHz. For quantitative analysis, cell size was normalized by dividing current amplitudes by cell capacitance, determined by integration of the transient current evoked by a 10-mV voltage step from a holding potential of −60 mV (Pachuau and Martin-Caraballo 2007; Zhang et al. 2020a).

Reagents

IL-6 (ThermoFisher, Cat.#PIRP8619), TNF-α (Cat.#210-TA), Tetrodotoxin (TXX, Sigma-Aldrich Cat.#554412), acyclovir (Sigma-Aldrich; Cat.#A4669), SB203580 (Cat.#13067) and SB202190 (Cat.#10010399) were purchased from Cayman Chemical.

Data Analysis

All electrophysiological data are presented as Mean ± SE. Statistical analyses consisted of Student’s unpaired t-test when single comparisons were made, and one-way ANOVA followed by post hoc analysis using Tukey’s honest significant difference test for unequal n for the more typical experimental designs that entailed comparisons between multiple groups (Statistica software, version 11). Throughout, p ≤ 0.05 was regarded as significant.

Institutional approval

All performed experiments using HSV-1 infection received institutional approval from the Institutional Biosafety Committee (#20110913-02).

Results

As previously reported, culture of ND7/23 cells in differentiation media for 4 d results in the formation of long neurite processes (Fg. 1 A). Treatment with ACV does not affect the overall neuronal morphology of differentiated ND7/23 cells (Fig. 1Ba). Lytic infection of differentiated ND7/23 cells causes significant cell death and loss of neuronal morphology. (Fig. 1Ca). However, culture of differentiated ND7/23 cells with ACV (with or without TNF-α) for 3 d following infection with M-GFP maintained cell morphology (Fig. 1Da–Ea). Fluorescent images of differentiated ND7/23 cells also reveal the presence of GFP fluorescent cells following viral infection with M-GFP even in the presence of ACV (Fig. 1Cb–Eb).

Fig. 1.

Fig. 1

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Morphology of differentiated ND7/23 cells following M-GFP infection. (A) Differentiated ND7/23 cells present long dendritic processes. (B, a-b) Culture of differentiated ND7/23 cells with acyclovir (ACV, 100 μM) for up to 4 d does not affect cell morphology or GFP expression. (C, a-b) Lytic infection of differentiated ND7/23 with M-GFP causes significant cell death and a reduction in dendritic outgrowth and GFP expression. (D-E, a-b) ACV-induced HSV-1 latency establishment in the presence or absence of TNF-α had minimal effect on dendritic outgrowth while showing GFP labeling of infected neurons. The arrows in Ca-b, Da-b, and Ea-b identify GFP-labeled cells

Whole-cell recordings of Na+ currents in differentiated ND7/23 cells demonstrate the presence of a transient current that undergoes fast inactivation (Fig. 2A). Those currents are greatly diminished following latency establishment (ACV + M-GFP) in differentiated ND7/23 cells; however, they can be observed following TNF-α treatment (Fig. 2A). To assess the effect of lytic infection and latency establishment on Na+ currents in differentiated ND7/23 cells we measured cell capacitances and changes in Na+ current densities (Fig. 2B–C). Lytic infection of differentiated ND7/23 cells caused a complete elimination of the Na+ currents compared with non-infected control cells without altering cell capacitance (Fig. 2B–C). Culture of differentiated ND7/23 with ACV did not affect the amplitude of Na+ current densities or cell capacitance compared with non-infected control cells (Fig. 2B–C). Infection of differentiated ND7/23 with M-GFP in the presence of ACV, representing latency establishment, caused a reduction in the Na+ current densities compared to ACV-treated cells without altering cell capacitance (Fig. 2B–C). The reduction in Na+ current densities during latency establishment (ACV + M-GFP treatment) was not significantly different from that occurring during lytic infection (Fig. 2C). However, infection of differentiated ND7/23 with M-GFP in the presence of ACV + TNF-α evoked a significant increase in Na+ current densities without changes in cell capacitance (Fig. 2B–C). The amplitude of the Na+ current densities measured in differentiated ND7/23 treated with ACV + M-GFP + TNF-α was not significantly different compared with the current densities in non-infected controls or ACV-treated cells, indicating that TNF-α maintained the functional expression of Na+ channels during latency establishment. We should point out that treatment of differentiated ND7/23 cells with TNF-α does not alter the functional expression of Na+ channels already present in the membrane since TNF-α treatment of differentiated ND7/23 cells did not affect the cell capacitance or Na+ current densities in control (non-infected) or TNF-α treated cells (cell capacitance control = 43.6 ± 3.2 pF (n = 13), TNF-α treated cells = 44.4 ± 3.6 pF (n = 15), p > 0.05; Na+ current densities control = 59.96 ± 9.5 pA/pF, TNF-α treated cells = 58.9 ± 6.8 pA/pF, p > 0.05). We also tested the potential effect of the cytokine IL-6 on Na+ currents in differentiated ND7/23 cells following M-GFP latency establishment. As previously reported, IL-6 treatment of differentiated ND7/23 cells prevents the loss of T-type Ca2+ channels following HSV-1 infection (Zhang el al. 2019). However, under our experimental conditions, Na+ current densities measured in differentiated ND7/23 cells treated with ACV + M-GFP + IL-6 were not significantly different compared with the current densities in ACV-M-GFP treated cells (Fig. 2C). IL-6 treatment of differentiated ND7/23 cells evoked a significant increase in cell capacitance compared to the capacitance in ACV- or ACV-M-GFP treated cells (Fig. 2B). To further assess the effect of TNF-α in maintaining Na+ currents during latency establishment, we compared the range of current densities recorded in ACV + M-GFP or ACV + M-GFP + TNF-α treated cells. As represented in Fig. 2D–E, more ACV + M-GFP + TNF-α treated cells expressed higher levels of current densities (in the range of 1–100 pA/pF and higher than 100 pA/pF) than cells treated with ACV + M-GFP. We should point out that the current-voltage relationship of the Na+ currents generated in differentiated ND7/23 cells treated with ACV alone or ACV + M-GFP + TNF-α shows a peak at −20 mV (Fig. 2F), suggesting no change in the biophysical properties of Na+ channels.

Fig. 2.

Fig. 2

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Effect of M-GFP infection on Na+ currents in differentiated ND7/23 cells. (A) Typical family of Na+ currents generated in a differentiated ND7/23 following a series of voltage steps from a holding potential of −100 mV. Recordings from control (non-treated) and treated cells were performed at the end of the 3 d period following latency establishment. In this and subsequent figures, the voltage step protocol is shown below the current trace. (B) Comparison of cell capacitance in differentiated ND7/23 cells following M-GFP infection and treatment with ACV, TNF-α, or IL-6. Note that infection of differentiated ND7/23 with M-GFP had no significant effect on cell capacitance even in the presence of ACV or TNF-α. However, following latency establishment and treatment with IL-6 there is a significant increase in cell capacitance (** denotes p ≤ 0.05 vs. ACV pre-treated cells). The number of cells recorded under each condition is presented in parenthesis from at least 3 different cell cultures. (C) Mean Na+ current densities generated in differentiated ND7/23 cells following M-GFP infection (with or without ACV) and treatment with TNF-α or IL-6. Na+ current densities were calculated from the peak current amplitude generated by depolarizing voltage steps from a holding potential of −100 mV (* denotes p ≤ 0.05 vs. control (non-treated) cells; ** denotes p ≤ 0.05 vs. ACV pre-treated cells; *** denotes p ≤ 0.05 vs. M-GFP infected cells pre-treated with ACV; ns denotes no significant difference). (D-E) Density plot of Na+ current densities generated in differentiated ND7/23 cells during latency establishment with or without TNF-α. Treatment of ND7/23 cells with TNF-α during latency establishment caused an increase in current densities in the range of 1 to < 100 or > 100 pA/pF. (F) Current-voltage relationship in ACV pre-treated cells and following latency establishment and treatment with TNF-α. There is no significant shift in the voltage dependence of Na+currents following treatment with TNF-α compared to ACV pre-treated cells

To assess whether TNF-α may alter the pharmacological properties of Na+ currents generated in differentiated ND7/23 cells with tested the effect of the specific Na+ channel blocker tetrodotoxin (TTX 250 nM, Fig. 3). Typical Na+ current traces generated with or without TTX in an ACV + M-GFP + TNF-α treated cell is represented in Fig. 3A. Both ACV or ACV + M-GFP + TNF-α treated cells express TTX-sensitive Na+ currents since application of TTX caused a near 100% inhibition of Na+ currents.

Fig. 3.

Fig. 3

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Effect of TTX on the Na+ currents generated in differentiated ND7/23 cells following latency establishment. (A) Examples of whole-cell Na+ currents generated in a differentiated ND7/23 cell following latency establishment and treatment with TNF-α in the presence of TTX. Note that the inward Na+ current generated in differentiated ND7/23 cells following latency establishment and treatment with TNF-α was eliminated by TTX (250 nM, arrow). (B) Comparison of cell capacitance in differentiated ND7/23 cells following latency establishment and treatment with TTX. (C) Overall effect of TTX on Na+ current densities in control (ACV-treated) cells and cells latently infected cells treated with TNF-α. TTX evokes a significant reduction in the current densities under all conditions tested. (* denotes p < 0.05 vs. ACV-treated cells; ** denotes p < 0.05 vs. ACV + M-GFP + TNF-α treated cells)

Several pore-forming subunits, including Nav1.1, Nav1.2, Nav1.3, Nav1.6, and Nav1.7 may form functional Na+ channels in differentiated ND7/23 cells (John et al., 2004; Yin et al. 2016; Lee et al. 2019). To assess changes in the mRNA expression of those subunits during latency establishment, we performed qRT-PCR analysis (Fig. 4). TNF-α alone appears to evoke a significant reduction in the expression of Nav1.3 and Nav1.7 transcripts (Fig. 4C, E). Following latency establishment, the Na+ channel transcripts for Nav1.7 underwent a significant reduction. Following latency establishment, TNF-α caused an increase in the expression of the Nav1.1, Nav1.2, and Nav1.6 Na+ channel subunits (Fig. 4A, B, D). These results suggest that TNF-α, latency establishment, or their combined actions can result in multiple changes in transcript expression for the various Na+ channel subunits.

Fig. 4.

Fig. 4

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Effect of latency establishment and TNF-α treatment on the expression of different sodium channel Nav transcripts as assessed by real-time PCR analysis. (A-F) Effect of ACV, ACV + TNF-α, latency establishment, and latency establishment with TNF-α treatment on the transcript expression of the Nav1.1 (A), Nav1.2 (B), Nav1.3 (C), Nav1.6 (D), and Nav1.7 (E) channel subunits. In ACV-treated cells, exposure of differentiated ND7/23 cells to TNF-α evoked a significant reduction in the expression of Nav1.3 and Nav1.7 channel transcripts (* denotes p < 0.05 vs. ACV-treated cells). Latency establishment (following ACV pre-treatment and M-GFP infection) resulted in a reduction in Nav1.7 transcripts compared to ACV-treated cells. Latency establishment followed by TNF-α treatment evokes a significant increase in the expression of the Nav1.1, Nav1.2, and Nav1.6 mRNA compared with latently infected cells. (* denotes p < 0.05 vs. M-GFP + ACV treated cells)

To assess changes in viral replication following latency establishment and treatment with TNF-α, we performed qRT-PCR analysis to measure the transcriptional expression of the thymidine kinase (TK) viral gene using specific primers (Table 2). As represented in Fig. 5A, infection of differentiated ND7/23 cells with M-GFP evokes a significant increase in TK expression. However, TK expression following M-GFP infection was significantly lower in ACV-pretreated ND7/23 cells compared with non-treated cells. TNF-α treatment during latency establishment does not alter the expression of TK compared with ACV + M-GFP treated cells (Fig. 5A).

Fig. 5.

Fig. 5

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Effect of latency establishment and TNF-α treatment on viral replication and p38 signaling. (A) M-GFP infection of differentiated ND7/23 cells generates an increase in the expression of TK viral transcripts, which is reversed by ACV treatment. TNF-α does not have any effect on TK expression following latency establishment (* denotes p ≤ 0.05 vs. M-GFP-infected cells; n = 4). (B) TNF-α treatment of differentiated ND7/23 cells evokes a significant increase in p38 signaling, without altering the level of sodium channel expression as detected with a pan-Na channel antibody. (C) Quantification of changes in p38 activation following latency establishment and TNF-α treatment (* denotes p ≤ 0.05 vs. ACV-treated cells; n = 4)

To assess the role of p38 signaling in promoting the functional expression of voltage-gated Na+ channels during latency establishment, we first performed western blot analysis to assess changes in p38 activity. As represented in Fig. 5B, ACV or ACV + TNF-α treatment of differentiated ND7/23 cells did not affect the levels of phosphorylated p38 (p-p38). However, latency establishment (ACV + M-GFP) caused a small increase in p-p38 expression, which was further enhanced by treatment with TNF-α. Those treatments had no significant effect on the total p38 expression (p38) or the levels of Na+ channel protein expression, detected with an anti-pan antibody (Fig. 5B).

To determine whether p38-evoked signaling by TNF-α promotes the functional expression of voltage-gated Na+ channels during latency establishment, we tested the effect of selective inhibitors of p38 MAP kinases, including SB203580 and SB202190 (10 μM) on whole-cell sodium currents. As represented in Fig. 6, treatment of differentiated ND7/23 cells with TNF-α during latency establishment caused a significant increase in Na+ current densities compared with ACV + M-GFP treated cells. However, pre-treatment with 10 μM SB203580 or SB202190 reversed the stimulatory effect of TNF-α on current densities during latency establishment (ACV + M-GFP + TNF-α compared with ACV + M-GFP + TNF-α + SB203580 (or SB202190) treated cells). None of these drugs had any effect on cell capacitance (Fig. 6A). Interestingly, SB202190, but not SB203580, appears to downregulate the expression of functional channels already present in the membrane of differentiated ND7/23 cells treated with ACV + TNF-α (Fig. 6B).

Fig. 6.

Fig. 6

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Effect of TNF-α signaling on the functional expression of Na+ channels in differentiated ND7/23 cells following latency establishment. (A) TNF-α stimulation of differentiated ND7/23 cells following ACV treatment or latency establishment does not affect cell capacitance. The p38 inhibitors SB203580 (10 μM) and SB202190 (10 μM) evoked no significant changes in cell capacitance under any treatment conditions. (B) Latency establishment (ACV + M-GFP) causes a significant reduction in the Na+ current densities compared to ACV-treated cells (* denotes p < 0.05 vs. ACV-treated cells). TNF-α stimulation of differentiated ND7/23 cells following latency establishment (ACV + M-GFP) evoked a significant increase in Na+ current densities compared to ACV + M-GFP-treated cells (** denotes p < 0.05 vs. ACV + M-GFP-treated cells). However, the stimulatory effect of TNF-α on Na+ channels during latency establishment was eliminated following treatment with SB203580 or SB202190 (*** denotes p < 0.05 vs. ACV + M-GFP + TNF-α treated cells)

Discussion

In this study, we examined the effect of TNF-α on voltage-gated Na+ channels during HSV-1 latency establishment in sensory-like neurons. Our results demonstrate that: first, HSV-1 latency establishment causes a significant reduction in the functional expression of voltage-gated Na+ channels in differentiated ND7/23 cells; second, TNF-α treatment of differentiated ND7/23 cells during latency establishment reverses the inhibitory effect of viral infection on the functional expression of voltage-gated Na+ channels, which can potentially regulate the electrical excitability of pain-transmitting neurons; and third, the stimulatory effect of TNF-α on increasing the functional expression of voltage-gated Na+ channels during latency establishment is regulated by p38 signaling.

Previous findings indicate that HSV-1 viral infection or reactivation triggers the release of cytokines, such as TNF-α and IL-6 from sensory neurons and surrounding cells (Shimeld et al. 1999; Li et al., 2005; Azher et al. 2017). Infection of trigeminal ganglion neurons with HSV-1 causes a persistent increase in the transcript expression of TNF-α and various cytokines (Halford et al. 1996); this effect can be prevented by pre-treatment with acyclovir (Halford et al. 1997). Radiation-evoked reactivation of latently infected trigeminal neurons also evokes a significant increase in the production and release of TNF-α and IL-6 from satellite cells (Shimeld et al. 1999). Increased production of TNF-α and other cytokines has also been demonstrated in corneal epithelial cells and macrophages following HSV-1 infection (Paludan and Mogensen 2001; Li et al. 2006). Thus, HSV-1 infection of sensory neurons and surrounding cells can evoke a significant increase in TNF-α production and release, which can further act in a paracrine and/or autocrine manner on sensory neurons.

HSV-1 infection of sensory neurons evokes significant changes in electrical excitability and the expression of voltage-gated ion channels (Oakes et al. 1981; Storey et al. 2002; Zhang et al. 2017). For example, HSV-1 infection of DRG or differentiated ND7/23 cells eliminates the functional expression of both voltage-gated Na+ and Ca2+ channels (Storey et al. 2002; Zhang et al. 2017). Reduced functional expression of voltage-gated Na+ channels can diminish the transmission of pain information through sensory neurons resulting in low pain signaling (Andoh et al. 1995; reviewed by Zhang et al. 2020b). Electrical excitability is an important regulator of viral replication, since inhibition of electrical activity increases viral replication (Zhang et al. 2005). Thus, it appears that HSV-1 infection promotes silencing of neuronal excitability in order to promote viral replication. On the other hand, immune responses to HSV-1 infection can trigger the release of inflammatory cytokines, which have the opposite effect on ion channel expression (present results, Zhang et al. 2019). These opposing factors likely lead to dysregulation in the functional expression of voltage-gated ion channels in infected neurons, resulting in significant changes in electrical excitability and changes in sensory perception. Indeed, it has been reported that HSV-1 infection can evoke a variety of pain sensations, including formication, paresthesia, or even numbness or tingling around the initial infection area, which can be associated with decreased or enhanced neuronal excitability (Andoh et al. 1995).

Our present results demonstrate that TNF-α treatment promotes the functional expression of voltage-gated Na+ channels during HSV-1 latency establishment in sensory-like neurons. Lytic infection of both DRG and differentiated ND7/23 cells evokes a significant reduction in the functional expression of voltage-gated Na+ channels (Storey et al. 2002; Zhang et al. 2017). Our present results also demonstrate that during latency establishment evoked by viral infection in the presence of ACV, the functional expression of voltage-gated Na+ channels is also reduced. We should mention that TNF-α treatment does not have any effect on voltage-gated Na+ channels already present in the membrane, since TNF-α treatment of non-infected neurons has no effect on Na+ current densities.

The stimulatory effect of TNF-α in promoting the functional expression of voltage-gated Na+ channels in differentiated ND7/23 cells during latency establishment requires p38 signaling. Consistent with this observation we demonstrated that inhibition of p38 signaling with SB203580 or SB202190 abrogates the stimulatory effect of TNF-α in promoting the functional expression of voltage-gated Na+ channels in differentiated ND7/23 cells during latency establishment. Previous findings indicate that TNF-α can alter the functional expression of voltage-gated Na+ channels in sensory neurons. For example, TNF-α upregulates the functional expression of Na+ channels generated by the Nav1.7 and Nav1.8 channel subunits in DRG neurons (Hudmon et al. 2008; Tyagi et al. 2024). Furthermore, TNF-α enhances the functional expression of voltage-gated Na+ channels following nerve injury or diabetic neuropathy (Chen et al. 2011; Huang et al. 2014). The stimulatory effect of TNF-α on voltage-gated Na+ channels following nerve injury involves increased expression of Nav1.3 and Nav1.8 sodium channel subunits in DRG neurons (He et al. 2010). Signaling via the p38 pathway is responsible for the cellular effect of TNF-α on sensory neurons (Chen et al., 2015; Tyagi et al. 2024). Thus, inhibition of p38 signaling prevents the stimulatory effect of TNF-α on voltage-gated Na+ channels. However, some Nav channel subunits may be differently regulated by p38 activation since increased p38 signaling downregulates Nav1.6 expressing Na+ channels (Wittmack et al. 2005). As previously reported, ND7/23 cells express various Nav sodium channel subunits (Yin et al. 2016; Rogers et al. 2016; Lee et al. 2019), with Nav1.6 and Nav1.7 as major contributors to functional Na+ channels. Our present results indicate that TNF-α stimulation of differentiated ND7/23 cells promotes the transcriptional expression of Nav1.1, Nav1.2, and Nav1.6 Na+ channel subunits following latency establishment, which is consistent with the generation of TTX-sensitive voltage-gated Na+ channels in ND7/23 cells in the presence of TNF-α.

Acknowledgements

This work was supported by funds provided by the UMES School of Pharmacy and grant R16AI182341 to SCH from NIAID/NIH. The content of this work is solely the responsibility of the authors and does not necessarily represent the official views of the NIAID/NIH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Abbreviations

d

Days

DRG

Dorsal root ganglion

GFP

Green fluorescence protein

h

Hour

HSV-1

Herpes simplex virus type 1

M-GFP

HSV-1 strain McKrae with GFP expression

IL-6

Interleukin-6

TNF-α

Tumor necrosis factor-α

Footnotes

Conflicts of interest The authors declare no potential conflicts of interest regarding the research, authorship, and/or publication of this article.

Data availability

No datasets were generated or analysed during the current study.

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Data Availability Statement

No datasets were generated or analysed during the current study.

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