Herpes simplex virus 1 regulates β-catenin expression in TG neurons during the latency-reactivation cycle

Kelly S Harrison; Liqian Zhu; Prasanth Thunuguntla; Clinton Jones

doi:10.1371/journal.pone.0230870

. 2020 Mar 30;15(3):e0230870. doi: 10.1371/journal.pone.0230870

Herpes simplex virus 1 regulates β-catenin expression in TG neurons during the latency-reactivation cycle

Kelly S Harrison ¹, Liqian Zhu ^1,^2,^¤, Prasanth Thunuguntla ¹, Clinton Jones ^1,^*

Editor: Neal A DeLuca³

PMCID: PMC7105109 PMID: 32226020

Abstract

When herpes simplex virus 1 (HSV-1) infection is initiated in the ocular, nasal, or oral cavity, sensory neurons within trigeminal ganglia (TG) become infected. Following a burst of viral transcription in TG neurons, lytic cycle viral genes are suppressed and latency is established. The latency-associated transcript (LAT) is the only viral gene abundantly expressed during latency, and LAT expression is important for the latency-reactivation cycle. Reactivation from latency is required for virus transmission and recurrent disease, including encephalitis. The Wnt/β-catenin signaling pathway is differentially expressed in TG during the bovine herpesvirus 1 latency-reactivation cycle. Hence, we hypothesized HSV-1 regulates the Wnt/β-catenin pathway and promotes maintenance of latency because this pathway enhances neuronal survival and axonal repair. New studies revealed β-catenin was expressed in significantly more TG neurons during latency compared to TG from uninfected mice or mice latently infected with a LAT^-/- mutant virus. When TG explants were incubated with media containing dexamethasone to stimulate reactivation, significantly fewer β-catenin+ TG neurons were detected. Conversely, TG explants from uninfected mice or mice latently infected with a LAT^-/- mutant increased the number of β-catenin+ TG neurons in the presence of DEX relative to samples not treated with DEX. Impairing Wnt signaling with small molecule antagonists reduced virus shedding during explant-induced reactivation. These studies suggested β-catenin was differentially expressed during the latency-reactivation cycle, in part due to LAT expression.

Introduction

More than 50% of adults in the United States are latently infected with herpes simplex virus 1 (HSV-1), reviewed in [1–3]. HSV-1 infections at the surface of the eye or oral cavity lead to life-long latent infections within sensory neurons of trigeminal ganglia (TG) as well as neurons within the central nervous system [4, 5]. Sporadic virus reactivations occur during the life of an infected individual resulting in virus shedding at the periphery, which can trigger recurrent disease.

In contrast to productive infection where more than 70 viral transcripts are readily detectable, including the latency-associated transcript (LAT), LAT is the only viral transcript abundantly expressed during latency [2, 3]. Deleting LAT coding sequences or LAT promoter sequences reduce the efficiency of reactivation in latently infected rabbits and mice [1–3]. This complex locus expresses multiple transcripts, six micro-RNAs, and two small non-coding RNAs [6–8]. LAT protects neurons from cell death, partly by inhibiting apoptosis and viral gene expression [9–13]. The anti-apoptosis functions of LAT are dependent on an active serine/threonine protein kinase (AKT) [14, 15]. LAT also interferes with granzyme B mediated apoptosis [16] and increases CD8+ T cell exhaustion during latency [17]. The anti-apoptosis functions of LAT are important in the context of the latency-reactivation cycle because inserting anti-apoptosis genes into the LAT locus of a LAT^-/- mutant virus restores reactivation to WT like levels [18–21]. As LAT is generally considered to express non-coding RNAs, LAT may also influence expression of cellular factors that promote the establishment and maintenance of latency.

Cellular genes associated with the Wnt/β-catenin signaling pathway are differentially expressed during the bovine herpesvirus 1 (BoHV-1) latency-reactivation cycle [22–24]. For example, during latency signficantly more TG neurons express β-catenin relative to TG from uninfected calves or during reactivation from latency. When BoHV-1 reactivation from latency is induced by the synthetic corticostroid dexamethasone, the Wnt/β-catenin signaling pathway is turned off, in part because expression of several soluble Wnt antagonists are stimulated [37, 38]. The Wnt/β-catenin signaling pathway is comprised of 19 related Wnt receptors, 7 Wnt co-receptors, many downstream effectors of this pathway, and two distinct families of Wnt antagonists [37, 38]. In the absence of the Wnt ligand or in the presence of a Wnt antagonist, a multi-protein β-catenin destruction complex, which includes Axin, APC (adenomatous polyposis gene), glycogen synthase kinase 3 beta (GSK3β), and casein kinase alpha (CKIα), hyper-phosphorylates β-catenin. This leads to poly-ubiquitination of β-catenin and degradation via the proteasome. Binding of Wnt to one of its receptor/co-receptor complexes disrupts the β-catenin destruction complex, the transcription factor β-catenin levels increase, β-catenin enters the nucleus, and interacts with TCF (T cell factor) family members bound to DNA. β-catenin binding to a TCF family member displaces transcriptional co-repressors and recruits co-activators to activate Wnt target genes. This signaling pathway promotes neuronal development and maturation, axonal growth and targeting, and neuronal survival [25–30], functions crucial for maintaining latency. Since BoHV-1 is a neurotropic α-herpesvirinae subfamily member, it is reasonable to suggest the canonical Wnt/β-catenin signaling pathway may also regulate certain aspects of the latency-reactivation in additional neurotropic herpesviruses.

In this study, we provide evidence that significantly more TG neurons expressed the β-catenin protein in mice latently infected with HSV-1 compared to TG from uninfected mice or mice latently infected with a LAT^-/- virus. Furthermore, β-catenin protein expression in TG neurons was reduced to levels similar to uninfected mice following TG explant for 4 or 8 hours if incubated with media containing the synthetic corticosteroid dexamethasone (DEX) to enhance reactivation. Virus shedding during explant-induced reactivation was significantly reduced relative to controls when TG from mice latently infected with WT HSV-1 were treated with a β-catenin specific inhibitor (iCRT14) or another Wnt antagonist (KYA1797K). Collectively, these studies suggest β-catenin expression in TG neurons may play a role during the latency-reactivation cycle.

Materials and methods

Virus and cell lines

HSV-1 strain McKrae dLAT2903R (WT; LAT^+/+) and dLAT2903 (LAT^-/-) were kindly gifted from Steven Wechsler and grown in Vero cell (ATCC CCL-81) monolayers in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), 2mM L-glutamine, 100 I.U./mL Penicillin, 100μg/mL Streptomycin at 37°C, 5% CO₂ until >80% CPE was observed. Viral aliquots were obtained through serial freeze-thawing of cells and subsequent centrifugation to remove cellular debris. Virus was titered on Vero cell monolayers to determine PFU/mL for each stock prior to animal infections.

Animals

The mouse studies performed in this study were reviewed by the Oklahoma State University Institutional Animal Care and Use Committee. The mouse studies were approved and the animal use protocol is VM15-26. Female, 6–8 week-old Swiss-Webster mice were purchased from Charles River Laboratories. Mice were acclimated to standard laboratory conditions (12 hrs. light/dark cycles, 5 animals/cage) for 1 week prior to ocular infections. For infections, mice were anesthetized via standard isoflurane/oxygen vaporization and infected with ~2x10⁵ PFU wt HSV-1 (n = 50) or the LAT^-/- mutant (dLAT2903, n = 50) in 2μL MEM per eye without scarification as described previously [31]. Ocular infections can result in herpes simplex encephalitis (HSE) of approximately 50% of animals during the first 15 days after infection. Consequently, mice were monitored twice daily for the duration of the experiments and, if any HSE-related symptoms such tremors, swollen forehead, or weight loss were observed, animals were promptly and humanely euthanized via isoflurane overdose and subsequent cervical dislocation. Infected mice were not allowed to reach HSE-induced death as an endpoint. A subset of surviving animals commonly displayed symptoms of acute ocular herpes infections such as redness, discharge, and fur-loss at the site of inoculation and were therefore treated twice daily with Neomycin/polymyxin B/Bacitracin zinc triple antibiotic ophthalmic ointment (Bausch + Lomb, Tampa, FL) to prevent secondary bacterial infection and minimize pain and distress. Mice infected for at least 30 days were operationally defined as being latently infected.

Immunohistochemistry

TG from uninfected (U, n = 5), LAT^-/- (n = 25 mice, 5 mice/group: Latency; 4h; 4h +Dex; 8h; 8h+DEX) or WT latently infected mice (n = 25, 5 mice/group: Latency; 4h; 4h +Dex; 8h; 8h+DEX) were harvested at least 30 days after infection via humane euthanasia by isoflurane overdose followed by decapitation at the atlanto-occipital joint. TG were dissected out and either directly placed in formalin or explanted in MEM containing 2% stripped serum with or without DEX addition for 4 or 8 hours and then formalin fixed. Slide preparation and IHC was performed as previously described [31]. Briefly, 4–5 μm sections of paraffin-embedded TG were cut and mounted onto glass slides. Slides were incubated at 65°C for ~20 mins before deparffinization in Xylene and serial rehydration using decreasing concentrations of ethanol. Endogenous peroxidases were blocked by incubating slides in 0.045% H₂O₂ for 20 minutes. Antigen retrieval was performed using Proteinase K (Agilent Dako, Santa Clara, CA) and slides were blocked using Animal Free Blocking Solution (AFBS; Cell Signaling Technologies, Danvers, MA) for 45 minutes at room temperature (RT). Avidin and Biotin were blocked using Vector Labs Avidin/Biotin blocking kit as per manufacturer’s instructions (Vector Labs, Burlingame, CA). Slides were incubated overnight at 4°C with the indicated antibody in AFBS. The following day, Vectastain ABC HRP Kit was used according to manufacturer’s instructions (Vector Labs). Biotinylated secondary antibody was prepared in AFBS and slides were incubated for 30 mins at RT. Slides were then developed with NovaRed substrate (Vector Labs) and lightly counterstained with Mayer’s hematoxylin (Sigma Aldrich, St. Louis, MO). Slides were imaged using an Olympus BX43 microscope. The % of TG neurons stained by the respective antibodies were counted in a blinded fashion. Animal experiments were performed in duplicate at separate times for reproducibility.

Examining the effects of Wnt/β-catenin antagonists on explant-induced reactivation

Mice infected for ≥ 30 days were operationally defined as being latently infected. TG from latently infected mice were minced into 3–4 pieces each and explanted into 6-well dishes in MEM that contained 10% FBS and incubated at 37°C, 5% CO₂. Certain TG explants were incubated with 10 μM water soluble DEX (Sigma, D2915). Where indicated, 25 μM iCRT14 (Santa Cruz Biotechnology, CAS: 677331-12-3) or 10μM KYA1797K (Selleckchem, CAS: S8327) reconstituted in DMSO was added to TG explants. Each day 100μL media was removed and used to perform plaque assays on Vero cell monolayers as previously described [31].

Statistical analysis

All graphs and comparisons were performed using GraphPad Prism software (v7.0d). P values less than 0.05 were considered significant for all calculations.

Results

More neurons express β-catenin in TG of mice latently infected with wt HSV-1

The Wnt signaling pathway enhances HSV-1 productive infection in cultured cells [32] and Wnt-associated genes are differentially expressed during the BoHV-1 latency-reactivation cycle [23]. Specifically, the transcription factor β-catenin is expressed in more TG neurons of latently infected calves when compared to TG neurons of uninfected calves or calves treated with DEX to induce viral reactivation [22–24]. In humans, the Wnt pathway has 19 genes encoding ligands and 15 receptor- or coreceptor-encoding genes [33]. It is not known which ligand activates each receptor; nor is it known which Wnt ligands or receptors are expressed in TG neurons. When a Wnt ligand engages its receptor, β-catenin protein levels generally increase, which was the rational for examining β-catenin as a marker for active canonical Wnt signaling. For these studies, we examined the effect of a LAT^-/- mutant (dLAT2903) relative to wild-type (wt) HSV-1. LAT is the only viral gene product abundantly expressed in latently infected neurons and thus was an important comparison to wt LAT^+/+ HSV-1 [34]. The dLAT2903 mutant virus used for this study contains a deletion from -161 to +1667 relative to the start of the primary 8.3-kb LAT: consequently, dLAT2903 does not express detectable levels of LAT, and reactivation from latency in rabbits and mice is significantly reduced [34–37]. The LAT^-/- mutant (dLAT2903) was marker repaired back to wt (dLAT2903R) and has identical properties as the parental wt McKrae strain of HSV-1 [34, 35]: therefore, dLAT2903R is referred to as wt HSV-1 throughout this study.

When compared to TG from uninfected mice (Fig 1; panel denoted as U), β-catenin+ TG neurons were readily detected in mice latently infected with wt HSV-1(WT panels; black arrows denote β-catenin+ TG neurons). Strikingly, β-catenin+ TG neurons were not readily detected in mice latently infected with dLAT2903 (panels denoted LAT^-/-). When IHC was performed with TG sections from mice latently infected with wt HSV-1, β-catenin+ TG neurons were not observed if the primary antibody was omitted (Fig 2A). Significantly more β-catenin+ TG neurons were detected in mice latently infected with wt HSV-1 when compared to TG sections prepared from mice latently infected with dLAT2903 and uninfected mice (Fig 2B). Approximately 60% of TG neurons were β-catenin+ compared to less than 20% in TG from mice latently infected with dLAT2903 and uninfected mice.

Fig 2 — A) IHC on latently infected TG was performed with the primary antibody omitted as a control. B) β-catenin positive TG neurons were counted from approximately 500 total neurons from two separate animal experiments. Data are shown as mean ± SEM. Asterisks denote significant differences calculated by students t-test, ****p<0.0001.

While LAT expression appears to be critical for increased β-catenin staining in TG neurons during latency, reduced establishment of dLAT2903 versus wt McKrae may also influence the % of TG neurons that were stained by the β-catenin antibody. For example, ocular infection of C57Bl/6 mice (no corneal scarification) with LAT^+/+ McKrae establishes latency 3–4 times more efficiently relative to dLAT2903, as judged by viral DNA in TG [17]. Male Swiss Webster mice infected via ocular infection after corneal scarification with LAT^+/+ 17syn+ HSV-1 virus strain (17-AHR1) establishes latency approximately 4 times more efficiently compared to a LAT deletion mutant that lacked 1,964 bp encompassing the LAT promoter and first 828 bp of the 5’ end of LAT (17-AH) in [13]. Female Swiss Webster mice infected via the ocular cavity (no corneal scarification) with wt McKrae established latency approximately 2 times more efficiently than dLAT2903 (data not shown). In summary, the inability of dLAT2903 to establish latency as efficiently as wt McKrae (dLAT2903R) influenced the % of β-catenin+ TG neurons during latency: however, LAT expression clearly correlated with high levels of β-catenin expression in TG neurons during latency.

Examination of β-catenin expression in TG during explant-induced reactivation

To test whether β-catenin expression was altered during viral reactivation from latency, TG from mice latently infected with HSV-1 (wt and dLAT2903) were explanted. TG explants were incubated with MEM + 2% charcoal-stripped FBS in the presence or absence of DEX. FBS passed through a column containing “activated” charcoal removes hormones, lipid-based molecules, certain growth factors, and cytokines yielding stripped FBS. However, this process does not remove salts, glucose, and most amino acids. FBS contains bio-active corticosteroids because nearly all Neuro-2A cells incubated with MEM + 10% FBS [38] contain glucocorticoid receptor (GR) in the nucleus. Conversely, nearly all Neuro-2A cells contain GR in the cytoplasm when incubated with 2% stripped FBS [38]. Furthermore, corticosterone levels were significantly higher in FBS compared to stripped FBS [31]. Recent studies demonstrated explant-induced reactivation from latency is stimulated by the synthetic corticosteroid DEX and expression of viral regulatory proteins were expressed earlier when TG explants were incubated with DEX [31]. At 4 or 8 hours after TG explant without DEX, β-catenin+ neurons were readily detected in mice latently infected with wt HSV-1 (Fig 3; panels denoted WT 4 or WT 8). When TG explants were incubated with media containing DEX, β-catenin+ TG neurons were not frequently detected at 4 or 8 hours after explant (panels denoted WT 4+DEX or WT 8+DEX).

Fig 3 — TG from wt HSV-1 latently infected mice were explanted 4 or 8 hours with or without DEX, subsequently formalin fixed and paraffin embedded. TG thin sections were stained with a β-catenin antibody as described in Fig 1. Arrows denote β-catenin positive neurons and magnification of sections is 600x. Images are representative of two independent animal experiments.

TG from mice latently infected with dLAT2903 were explanted and examined for β-catenin protein expression by IHC. In contrast to the results with wt HSV-1, DEX treatment increased the number of β-catenin+ TG neurons at 4 hours after explant from mice latently infected with dLAT2903 (Fig 4; panels LAT^-/- 4 and LAT^-/- 4+DEX). At 8 hours after TG explant, weak staining of TG neurons was detected by the β-catenin antibody in the absence of DEX treatment whereas DEX treatment appeared to increase the number of β-catenin+ TG neurons (panels denoted LAT^-/- 8 and LAT^-/- 8+DEX). These studies also revealed β-catenin+ staining was dispersed outside of TG neurons at 8 hours after TG explant when DEX was added to MEM. Published studies concluded that the Wnt pathway is activated within the central nervous system when injured, including surrounding non-neuronal cell types such as oligodendrocytes and microglia [39, 40], which suggests similar events may occur following explant of mice latently infected with dLAT2903.

Fig 4 — TG from mice latently infected with a LAT-null HSV-1 (LAT^-/-) were either collected at ≥30 days after infection (latency) or explanted 4 or 8 hours in MEM containing 2% stripped FBS with or without DEX, subsequently formalin fixed and paraffin embedded. TG thin sections were stained with a β-catenin antibody as described in Fig 1. Arrows denote β-catenin positive neurons. Magnification of sections is 600x. Images are representative of two independent animal experiments.

As a control to TG from latently infected mice, β-catenin expression was performed in TG of uninfected mice. Following explant, β-catenin+ TG neurons were not readily detected at 4 hours after explant (panel U 4), but a few TG neurons were weakly stained 8 hours after explant (panel U 8; Fig 5). When DEX was added to cultures, more TG neurons from uninfected mice were weakly stained compared to TG explants incubated without DEX, or TG explants from mice latently infected with wt HSV-1 (Fig 2). Furthermore, the intensity of β-catenin staining in uninfected TG was not as strong relative to TG of mice latently infected with wt HSV-1 (Fig 3) suggesting more β-catenin was expressed in TG neurons of mice latency infected with wt HSV-1. Finally, we noted that following TG dissection, explant, and incubation in medium TG neurons in sections were not as pristine compared to when they were dissected and immediately fixed in formalin.

Fig 5 — TG from uninfected mice were explanted 4 or 8 hours with or without DEX, subsequently formalin fixed and paraffin embedded. TG thin sections were stained with a β-catenin antibody as described in Fig 1. Arrows denote β-catenin positive neurons and magnification of sections is 600x. Images are representative of two independent animal experiments.

The number of β-catenin+ TG neurons in the respective samples were quantified and these results are shown in Fig 6. TG from mice latently infected with wt HSV-1 contained similar levels of β-catenin+ TG neurons at 4 and 8 hours after TG explant (approximately 60% were β-catenin+; Fig 6A). When DEX was added to TG explants from mice latently infected with wt HSV-1, significantly fewer TG neurons were β-catenin+. At 4 hours post-explant (no DEX treatment), TG from mice latently infected with dLAT2903 contained significantly more β-catenin+ TG neurons relative to latency (Fig 6B). Interestingly, mice latently infected with dLAT2903 had approximately 60% β-catenin+ TG neurons when explants were treated with DEX for 4 hours, which was similar to TG from mice latently infected with wt HSV-1. At 8 hours post-explant, β-catenin expression decreased to levels similar to mice latently infected with dLAT2903 (~10%, Fig 6B); however, DEX treatment increased β-catenin expression 3-fold. Overall, TG from uninfected mice was more similar to TG from mice latently infected with dLAT2903 than wt HSV-1 (Fig 6C). While the intensity of β-catenin expression in uninfected mice (Fig 5) was clearly less than latently infected TG (Figs 3 and 4), approximately 80% of TG neurons were β-catenin+ at 4 hours after explant when incubated in MEM containing DEX (Fig 6C; 4 + DEX). At 8 hours after explant in the presence of DEX, the number of β-catenin+ TG neurons was reduced to approximately 60%. In summary, these studies indicated explant-induced reactivation in the presence of DEX dramatically altered β-catenin expression in TG neurons. While these studies suggested LAT expression influenced β-catenin expression during explant-induced reactivation, published reports concluded RNA encoding ICP0, ICP4, and/or thymidine kinase can be detected in TG of mice latently infected with HSV-1 [41, 42]. If these lytic cycle proteins are expressed, they may have differential impacts on β-catenin expression, depending on whether LAT is expressed.

Antagonists of the Wnt-signaling pathway significantly reduced DEX-induced virus reactivation from latency in TG explants

The data above revealed β-catenin was differentially expressed during the latency-reactivation cycle. However, these studies did not address whether the Wnt/β-catenin signaling pathway influenced virus shedding during explant-induced reactivation. To examine the effect the Wnt/β-catenin signaling pathway had on explant-induced reactivation from latency, two different Wnt-pathway antagonists were used: iCRT14 and KYA1797K. iCRT14 is a thiazolidinedione inhibitor that impairs β-catenin binding with a T-cell factor (TCF) family member and subsequent TCF binding to DNA: consequently, β-catenin dependent transcription is not induced [43]. KYA1797K enhances formation of the β-catenin destruction complex through binding of Axin, which stimulates GSK3β activation [44]. For each antagonist, cytotoxicity studies using both Vero cells and a mouse neuroblastoma cell line (Neuro-2a, ATCC CCL131) were performed, showing no effect with concentrations up to 50μM [32] and data not shown.

TG from mice latently infected with wt HSV-1 were explanted in media containing 10% FBS and either iCRT14, KYA1797K or DMSO (Fig 7). Each day after TG explant, 100μL of the supernatant was removed and used to measure levels of reactivated virus. Relative to the vehicle control, a 2-3-log decrease was detected in cultures treated with either antagonist (Fig 7), and this trend continued through 9 days post-explant. At early timepoints, KYA1797K was not as effective compared to iCRT14 (approximately 10³ vs 10¹, respectively). However, both exhibited a plateau in virus shedding around day 7 at ~10³ PFU from TG explants. TG explants incubated with just FBS exhibited peaked levels of virus shedding at day 6 with 10⁶ PFU/mL and remained at this level until day 9, which was consistent with previous studies [31].

Given the most dramatic effect was observed using iCRT14 at 5 days post-explant, this antagonist and timepoint were used to analyze the effect on reactivation of dLAT2903 infected TG. Since dLAT2903 displays significantly reduced reactivation following explant, latently infected TG were explanted in both 10% FBS or 2% stripped serum + DEX to compare virus reactivation [31]. Regardless of the treatment, virus reactivation was significantly reduced when compared to WT, ranging between 10² to 10^3- PFU/mL (Fig 8). Upon addition of 25μM iCRT14, virus shedding was reduced approximately 2-log when TG were explanted in 10% FBS (<10²); but, was not detected when explanted in 2% stripped serum + DEX (Fig 8, N.D: none detected). Collectively, this data demonstrated that the Wnt/β-catenin signaling pathway enhanced explant-induced reactivation from latency in mice latently infected with wt HSV-1 and the LAT^-/- mutant.

Fig 8 — Peak inhibition of WT virus reactivation was observed using 25μM iCRT14, 5 days post-explant; therefore, this antagonist and time point were used to assess inhibition of LAT^-/- (dLAT2903) reactivation. LAT^-/- infected TG were harvested 30dpi (latency) in either 10% FBS or 2% stripped FBS with DEX and 25μM iCRT14. Untreated controls (either 10% FBS only or 2% stripped serum with DEX) were included as positive controls for reactivation. 5 days post-explant, 100μL supernatant was removed and used to measure viral shedding (PFU/mL). Data are shown as mean ± SD for duplicate wells. Asterisks denote significant difference calculated by students t-test, *p<0.05, **p<0.005; N.D. = None detected.

Discussion

These studies revealed that more β-catenin+ TG neurons and higher levels of β-catenin were detected in mice latently infected with wt HSV-1 relative to TG from uninfected mice or mice latently infected with a LAT^-/- mutant suggesting LAT, in part, plays a role in mediating β-catenin expression during latency. It is assumed LAT does not encode a functional protein that regulates the latency-reactivation cycle [3]. Hence, the dogmatic belief is small non-coding RNAs mediate key events during the latency-reactivation cycle, including micro-RNAs located within the LAT locus [6, 7]. Certain LAT encoded miRNAs interfere with expression of viral transcripts that encode key regulatory proteins. For example, miR-H3 [6] interferes with expression of the key viral transcriptional regulator ICP4. ICP34.5, an important lytic neurovirulence factor, is targeted by two viral miRNAs, miR-H3 and miR-H4 [6]. A mutant virus that does not express the LAT-encoded miR-H2 has increased neurovirulence and reactivation [45], in part because this miRNA interferes with ICP0 expression [6, 46]. Two small non-coding RNAs, which are not micro-RNAs, are expressed in TG of mice latently infected with wt HSV-1; interestingly, sequences encoding these small non-coding RNAs are deleted from dLAT2903 [8]. These LAT small non-coding RNA interfere with apoptosis and ICP4 expression [47] suggesting they also play a role in the latency-reactivation cycle. Analysis of the known miRNAs encoded by the human herpesviruses are reported to preferentially target the Wnt signaling pathway [48], which supports the premise that stabilizing β-catenin expression in TG neurons during latency is important for maintaining a life-long latent infection in sensory neurons.

We estimated that approximately 60% of TG neurons expressed β-catenin in mice latently infected with wt McKrae. A previous report concluded that approximately 30% of TG neurons in mice were latently infected with a virulent HSV-1 strain [49] suggesting a subset of β-catenin+ TG neurons were not infected. LAT miRNAs (miR-H3, miR-H5, and mIR-H6) are present in exosomes that are transported from productively infected cells to uninfected cells [50]. Hence, it is tempting to suggest exosomes containing LAT gene products are transported to surrounding uninfected TG neurons or non-neuronal support cells: consequently, β-catenin expression is activated in “bystander” cells, including neurons. However, it should be pointed out there is no current published study showing exosomes frequently transport LAT non-coding RNAs to uninfected TG neurons during latency. Extracellular factors regulate (positively as well as negatively) the Wnt/β-catenin signaling pathway [33, 51] suggesting mice latently infected with wt HSV-1 produce a soluble factor that stimulates β-catenin expression or increases its ½ life in bystander cells. Strikingly, Wnt16, a soluble Wnt agonist, is expressed at 54 times higher levels in TG of calves latently infected with BoHV-1 when compared to TG from calves treated with DEX for 30 minutes to initiate reactivation from latency [23]. Regardless of how the Wnt/β-catenin signaling pathway is activated in TG of mice latently infected with wt HSV-1, activation of this signaling pathway would promote neuronal survival and neurogenesis [26, 39, 52, 53], including TG neurons that do not contain viral genomes.

In general, increased corticosteroids, due to stress and depression, interfere with β-catenin in the nervous system [54]. Furthermore, stress induces expression of soluble Wnt antagonists that interfere with β-catenin expression, and induces neuronal death [51, 55]. While the findings with wt HSV-1 are in concordance with these findings, it was clear β-catenin+ TG neurons from uninfected mice or mice latently infected with dLAT2903 increased following explant. In particular, the number of β-catenin+ TG neurons in mice latently infected with dLAT2903 or uninfected mice increased when DEX was added. During explant-induced reactivation, the normal tropic support that TG receive in vivo is disrupted, which may impact how certain signaling pathways are regulated following explant. Additional evidence that explant-induced reactivation does not recapitulate all events during reactivation in vivo comes from a recent study that demonstrates apoptosis is induced in many TG neurons from latently infected mice within 4 hours after explant [31]. In contrast, TG neurons do not frequently undergo apoptosis during early stages of DEX induced reactivation in calves latently infected with BoHV-1 in vivo [56, 57]. It is also unlikely apoptosis occurs frequently in human TG neurons undergoing HSV-1 reactivation. While it seems clear that explant-induced reactivation in the mouse model has limitations, these studies indicated that LAT encoded products, directly or indirectly, influenced β-catenin expression in TG neurons during latency.

Since β-catenin was preferentially expressed during latency, it was surprising that inhibiting the Wnt/β-catenin signaling pathway consistently reduced virus shedding during explant-induced reactivation. However, iCRT14 reduced HSV-1 virus yields in productively infected non-neuronal cells (human fetal lung and Vero cells) and a late protein, VP16, enhanced β-catenin dependent transcription as well as stabilized β-catenin steady state protein levels [32]. An independent study revealed infected cells can be divided into transcriptionally unique subsets: furthermore, cells abortively infected were identified where antiviral signaling blunted productive infection [58]. In certain “highly infected cells” transcriptional reprogramming occurred, in part because β-catenin is recruited to the nucleus and viral replication compartments: hence, β-catenin promotes late viral gene expression and progeny production [58]. While the potential dual role for β-catenin during the latency-reactivation cycle may appear to be implausible, it is well established that the Wnt/β-catenin signaling pathway has different effects in a cell-type- and contextual-specific manner. For example, dysregulation of the Wnt/β-catenin pathway is crucial for development of certain types of cancer [59]: conversely, the same pathway mediates neurogenesis and cell survival in the nervous system [28, 29]. Based on these observations, we suggest that during establishment and maintenance of latency following infection with wt virus, β-catenin expression in TG neurons promotes neuronal survival [53] and neurogenesis [28, 29]. During early phases of reactivation in mice latently infected with wt virus, β-catenin may be recruited to replication complexes in certain latently infected TG neurons. This could be one of many transcriptional reprogramming events that culminates in stimulating lytic cycle viral gene expression and virus shedding, hallmarks of successful reactivation from latency.

While these studies suggested β-catenin plays a role during the HSV-1 latency-reactivation cycle, additional studies are necessary to unravel the mechanism by which 1) LAT regulates β-catenin expression during latency and 2) how β-catenin regulates the maintenance of latency but has a potential role during early stages of reactivation from latency.

Data Availability

All relevant data are within the paper.

Funding Statement

This research was supported by grants from the National Institute of Neurological Disorders and Stroke of the National Institutes of Health under Award Number R21NS102290 (CJ), USDA-NIFA Competitive Grants Program (16-09370 and 2018-06668) (CJ), the Sitlington Endowment (CJ), support from the Oklahoma Center for Respiratory and Infectious Diseases (National Institutes of Health Centers for Biomedical Research Excellence Grant # P20GM103648), and funds from the OSU CVM Research Advisory Council (KH). These studies were also partially supported by the Chinese National Science Foundation Grant (No. 31472172 and No. 31772743) (LZ) and National Key Research Program (No. 2016YFD0500704) (LZ).

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PLoS One. doi: 10.1371/journal.pone.0230870.r001

Decision Letter 0

Neal A DeLuca

14 Jan 2020

PONE-D-19-33585

Herpes simplex virus 1 regulates beta-catenin expression in TG neurons during the latency-reactivation cycle.

PLOS ONE

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Reviewer #1: Review of: Herpes simplex virus 1 regulates beta-catenin expression in TG neurons during the latency-reactivation cycle

By Harrison, Zhu, Thunuguntla and Jones,

In this manuscript, the authors show that β-catenin is upregulated in TG neurons latently infected with HSV-1 in a LAT dependent manner. The authors further establish that induction of reactivation by dexamethasone treatment results in dramatic downregulation/degradation of β-catenin. This decrease was not observed in TG infected with LAT null virus. Furthermore, expression of β-catenin is beneficial for virus because inhibition of this pathway during reactivation results in reduced viral shedding.

The findings reported here are very interesting and valuable for the advancement of the field, and only minor improvements are suggested as described below.

1. Line 68: How soon after reactivation does Wnt signaling decrease? In figure 6, no decrease in β-catenin expression is seen at 4hr, 8hr post ex-plantation. Why were these timepoints selected?

2. Figures 1 and 2: Was β-catenin detected exclusively in neurons? Did you observe any immune cells/supporting cells expressing β-catenin?

3. Line 200: Word “estimated” is a bit unclear- did you count number of β-catenin positive neurons over total number of neurons to determine percentage or “eyeball” percentage of β-catenin positive neurons?

4. Figure 4: LAT-/- 4 panel: cells surrounding β-catenin positive cells look different from other panels. Also, not necessary but if you can please replace it with a better image.

5. Line 212- What does “GR” stand for?

6. Line 235 states that β-catenin staining was detected outside of TG neurons. Can you show this in Fig 4 and elaborate on the significance of this finding?

7. Line 251: Authors state that intensity of β-catenin was lower in uninfected TG than in infected TG. This suggests that while higher percentage of cells might stain positive for β-catenin, the overall expression levels may be lower. Could you elaborate on this?

8. Figure 6: Dex treatment in uninfected TG results in roughly two-fold higher β-catenin positive neurons when compared to LAT-/-, dex treated TG. These results suggest that viral factors other than LAT may contribute to β-catenin downregulation. Can you please comment on this possibility?

9. The finding that the percentage of cells expressing β-catenin is higher than the reported number of infected cells is intriguing, as it suggests that other, surrounding uninfected cells are also producing β-catenin. The authors propose this could be caused by miRNA/sncRNA encoded by LAT that are released in exosomes. Perhaps the authors could speculate the functional significance of causing β-catenin upregulation in surrounding cells (is it beneficial to the virus to have surrounding cells overexpress β-catenin?).

10. It seems the two sncRNAs may play a more important role in latency-reactivation than miRNA based on the authors work and Wechsler’s work. Perhaps the authors could elaborate on this?

11. Figure 7 suggests that inhibition of Wnt signaling results in decreased viral replication. This seems counterintuitive considering that Wnt pathway is turned off/ β-catenin expression is decreased during reactivation (as stated on lines 68)? Perhaps the authors could elaborate on this?

12. The authors are a bit cautious in their discussion, and could expand the discussion section a bit if they wish.

Reviewer #2: Harrison and colleagues have investigated the expression of beta-catenin in the context of herpes simplex virus 1 latency and reactivation using a mouse ocular infection model. The authors determined that beta-catenin expression was enhanced in trigeminal ganglia (TG) of mice latently infected with a wild type (WT) HSV-1 strain compared to uninfected animals, or animals latently infected with an HSV-1 LAT deletion mutant. Reactivation from latency was achieved by explanting latently infected TG in the presence of dexamethasone (DEX). Whereas beta-catenin expression was reduced in TG latently infected with WT virus upon explant in the presence of DEX, beta-catenin expression was significantly enhanced in explanted TG from both LAT infected and uninfected animals that had been treated with DEX. In a final set of experiments the authors show that compounds that inhibit the Wnt/beta-catenin signalling pathway are potent inhibitors of WT HSV-1 reactivation in explanted TG. The authors central conclusions are that the Wnt/beta-catenin signalling pathway is important for HSV-1 reactivation and that beta-catenin is differentially expressed during latency (high expression) and reactivation (low expression) and that LAT somehow regulates beta-catenin expression.

While the findings are potentially interesting, the study is incomplete, inadequately controlled, and the data are of variable quality. Thus, the findings are too preliminary to be particularly informative to researchers in this field.

Major issues:

1) Can the authors confirm that TG neurons are latently infected and quantify this? Are the TG neurons expressing beta-catenin the ones that are latently infected? What proportion of latently infected TG neurons express beta-catenin?

2) The uninfected controls in figure 5 do not look like the uninfected control sections in figure 1. Why? The uninfected TG explanted in DEX in figure 5 that are highlighted with arrowheads do not look to be beta-catenin positive. If the primary antibody was excluded in control experiments and the samples double-blinded could beta-catenin positive cells be identified with confidence?

3) Figure 6 and sentence on lines 347-349. Authors should include uninfected TG from mice that had not been explanted (i.e. fixed immediately after removal) to control for the effects of explant on beta-catenin expression.

4) Sentence on lines 279-280. What are the numbers of latently infected TG neurons in animals infected with WT virus compared to LAT mutant virus? Need to show this important control.

5) Figure 6. What is the degree of reactivation from TG explanted from WT and LAT mutant infected animals? Presumably, the medium associated with the explanted TG analyzed for beta-catenin expression shown in figure 6 was available for virus titration.

6) Figure 7. How do Wnt/beta-catenin inhibitors impact the reactivation of LAT mutants? How would DEX impact the action of the Wnt/beta-catenin inhibitors on virus reactivation?

7) Lines 341-343. This is an egregious over-interpretation of the data.

Minor issues:

1) It is curious that Wnt/beta-catenin agonists are so potent at inhibiting reactivation when the levels of beta-catenin are so low during the reactivation phase. Can the authors comment on this apparent inconsistency?

2) Line 27. Why highlight ocular disease here? Line 22 introduces ocular, nasal and oral infection. Consider "recurrent disease".

3) Line 35. "increased" compared to what?

4) Line 47. Odd phrase. Suggests oral disease upon reactivation after primary infection of the eye? This is confusing. Similar issues in abstract. Consider rephrasing.

5) Lines 49-50. This sentence implies LAT is not expressed during lytic infection. I don't think this is the point the authors are trying to make. Consider rephrasing.

6) Line 53. Why does the nature of LAT locus products "suggest" functions related to latency? Consider rephrasing.

7) Line 76. “Wnt binding” to what?

8) Line 170. “rational” should be “rationale”.

9) Line 177. “marker rescued” should read “repaired”.

10) Sentence on lines 308-309. Alternatively, the data may indicate that the inhibitors were toxic to the explants. Can the authors control for this? Is there a dose response to the inhibitors?

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2020 Mar 30;15(3):e0230870. doi: 10.1371/journal.pone.0230870.r002

Author response to Decision Letter 0

2 Mar 2020

With respect to the comments of Reviewer #1, the following changes in the revised manuscript were made.

Concern #1: Line 68: How soon after reactivation does Wnt signaling decrease? In figure 6, no decrease in β-catenin expression is seen at 4hr, 8hr post explantation. Why were these timepoints selected?

Response: �-catenin+ TG neurons were nearly undetectable after TG explants were incubated with MEM, 2% stripped FBS, and DEX. However, �-catenin+ TG neurons were readily detectable when there was no DEX added to TG explants. When TG explants are treated with DEX to stimulate reactivation from latency, there are significantly more caspase 3+ TG neurons at 4, 8 and 16 hours after TG explant relative to TG explants not treated with DEX, Refence #31. Since there was a clear-cut difference in �-catenin expression during early times after TG explant in the presence of DEX, we did not examine �-catenin expression at later times because of increased apoptosis.

Concern #2: Figures 1 and 2: Was β-catenin detected exclusively in neurons? Did you observe any immune cells/supporting cells expressing β-catenin?

Response: For mice latently infected with wt HSV-1, most β-catenin staining was detected in neurons. In TG explants from mice latently infected with dLAT2903, we saw staining observed in neurons and other cells, which is pointed out in lines 253-258 (Figure 4). Since we did not include any differential stain to establish what types of immune/supporting cells were expressing, it is not clear which cells types are expressing β-catenin.

Concern #3: Agreed. Line 200: Word “estimated” is a bit unclear- did you count number of β-catenin positive neurons over total number of neurons to determine percentage or “eyeball” percentage of β-catenin positive neurons?

Response: We have replaced the word “estimated” with “counted”; total number of β-catenin positive neurons over total number of neurons to determine percentage because we did in fact count �-catenin+ TG neurons (lines 147-148 and 217-218).

Concern #4: Figure 4: LAT-/- 4 panel: cells surrounding β-catenin positive cells look different from other panels. Also, not necessary but if you can please replace it with a better image.

Response: A higher quality image has been used for LAT-/- panel in Figure 4.

Concern #5: Line 212- What does “GR” stand for?

Response: GR is glucocorticoid receptor, and this was defined in the revised manuscript (line 228-230). We have carefully examined the manuscript to ensure all acronyms have been defined.

Concern #6: Line 235 states that β-catenin staining was detected outside of TG neurons.

Response: Recent published studies demonstrated the Wnt pathway is activated within the CNS after injury, including surrounding non-neuronal cell types such as oligodendrocytes and microglia. Thus, the observation of β-catenin staining outside of neurons may be due to expression in supportive cells. This point is included in the revised manuscript (lines 255-258).

Concern #7: Line 251: Authors state that intensity of β-catenin was lower in uninfected TG than in infected TG. This suggests that while higher percentage of cells might stain positive for β-catenin, the overall expression levels may be lower. Could you elaborate on this?

Response: This statement was made because the intensity of staining did not appear to be as bright, which is difficult to quantify. However, it was clear staining was brighter, which suggested there is more β-catenin in these samples. These points were clarified in the revised manuscript (lines 273-276).

Concern #8: Figure 6: Dex treatment in uninfected TG results in roughly two-fold higher β-catenin positive neurons when compared to LAT-/-, dex treated TG. These results suggest that viral factors other than LAT may contribute to β-catenin downregulation. Can you please comment on this possibility?

Response: There have been reports that additional viral gene products can be detected in TG during latency; ICP0, ICP4, and thymidine kinase RNA for example. If these lytic cycle regulatory proteins are expressed (ICP0 and ICP4 in particular) they could influence cellular gene expression. This point is included in the revised manuscript (lines 303-309).

Concern #9: The finding that the percentage of cells expressing β-catenin is higher than the reported number of infected cells is intriguing, as it suggests that other, surrounding uninfected cells are also produce β-catenin. The authors propose this could be caused by miRNA/sncRNA encoded by LAT that are released in exosomes. Perhaps the authors could speculate the functional significance of causing β-catenin upregulation in surrounding cells (is it beneficial to the virus to have surrounding cells overexpress β-catenin?)

Response: We have expanded on the possibility of LAT-encoded non-coding RNAs being transported to surrounding TG neurons via exosomes and why this may impact surrounding neurons, and perhaps non-neuronal cells during latency (lines 393-410).

Concern #10: It seems the two sncRNAs may play a more important role in latency-reactivation than miRNA based on the authors work and Wechsler’s work. Perhaps the authors could elaborate on this?

Response: We have expanded this part of the discussion, as suggested (lines 372-391).

Concern #11: Figure 7 suggests that inhibition of Wnt signaling results in decreased viral replication. This seems counterintuitive considering that Wnt pathway is turned off/ β-catenin expression is decreased during reactivation (as stated on lines 68)? Perhaps the authors could elaborate on this?

Response: This section has been revised to explain how �-catenin may have dual roles during the latency-reactivation cycle. (lines 430-451).

Concern #12: The authors are a bit cautious in their discussion and could expand the discussion section a bit if they wish.

Response: After addressing Concerns 9-11, I think we have speculated as much as I feel comfortable until we pursue this project more in detail.

With respect to the comments of Reviewer #2, the following changes were made.

Concern #1: Can the authors confirm that TG neurons are latently infected and quantify this? Are the TG neurons expressing beta-catenin the ones that are latently infected? What proportion of latently infected TG neurons express beta-catenin?

Response: In the discussion, we proposed two mechanisms by which �-catenin may be expressed in latently infected TG neurons as well as a subset of TG neurons that are “bystanders” (lines 393-410). It is not trivial to accurately calculate the % of TG neurons that are latently infected and express ��catenin. To do this, one would have to cut consecutive sections and perform IHC on one section to identify �-catenin+ TG neurons and on the other section perform IHC to identify a viral protein during reactivation. We tried this approach and it was difficult to interpret because during explant-induced reactivation �-catenin+ TG neurons are nearly undetectable. It is possible to identify LAT+ TG neurons using in situ hybridization on one section; on the consecutive section perform IHC to visualize �-catenin+ TG neurons. There are a couple of issues with this approach, which would make it difficult to achieve a clear-cut and accurate result. First, it is well established there are HSV-1 genome+ TG neurons that do not express detectable levels of LAT during latency in mice: however, it is not clear what % of the total latently infected TG neurons do not express LAT. Secondly, TG sections from latently infected mice are very difficult to obtain superior consecutive sections. We routinely obtain excellent consecutive section from calves latently infected with bovine herpesvirus 1, but not mice latently infected with HSV-1. Finally, it is difficult to use two antibodies that are differentially stained on the same TG section, at least in our hands. We have done this with brain tissue, but when the same antibodies were used for TG sections the background was terrible. In summary, I do not believe these studies would yield convincing or accurate results. If necessary, these points can be added to the revised manuscript.

Concern #2: The uninfected controls in figure 5 do not look like the uninfected control sections in figure 1. Why?

Response: The uninfected sections presented in Figure 1 were dissected as intact TG and then directly placed into formalin post-euthanasia. Uninfected tissue in Figure 5 was minced, explanted in MEM containing 2% stripped FBS, and then incubated for 4 and 8 hours. These small TG pieces were then collected and placed in formalin and paraffin embedded. While the integrity of the tissue is not as good as intact TG immediately placed in formalin, they clearly show the general morphology of TG. This is stated in the revised manuscript (lines 276-278).

Concern #3: The uninfected TG explanted in DEX in figure 5 that are highlighted with arrowheads do not look to be beta-catenin positive. If the primary antibody was excluded in control experiments and the samples double-blinded could beta-catenin positive cells be identified with confidence?

Response: The TG neurons in Figure 5 are stained (albeit weakly) relative to the no primary antibody control. The no primary antibody control was performed using TG sections form mice latently infected with wt HSV-1: samples from explanted TG (no primary antibody control) also do not yield any staining: this is stated in the revised manuscript (lines 271-276). if necessary, we can add the no primary antibody TG sections for uninfected TG that was explanted to Figure 5. All TG sections in this study were analyzed in a blind fashion. This was stated in the materials and methods of the original manuscript (lines 147-148).

Concern #4: Figure 6 and sentence on lines 347-349. Authors should include uninfected TG from mice that had not been explanted (i.e. fixed immediately after removal) to control for the effects of explant on beta-catenin expression.

Response: These images were included as the uninfected panels shown in Figure 1 of the original manuscript.

Concern #5: Sentence on lines 279-280. What are the numbers of latently infected TG neurons in animals infected with WT virus compared to LAT mutant virus? Need to show this important control.

Response: This topic was discussed in the revised manuscript (lines 193-206).

Concern #6: Figure 6. What is the degree of reactivation from TG explanted from WT and LAT mutant infected animals? Presumably, the medium associated with the explanted TG analyzed for beta-catenin expression shown in figure 6 was available for virus titration.

Response: Studies in Figure 7 and 8 show a comparison between efficiency of wt HSV-1 versus the LAT-/- mutant. The results demonstrated at least 2 logs reduction in the LAT-/- mutant relative to wt. These results are consistent with our recent studies, reference #31 in the revised manuscript.

Concern #7: Figure 7. How do Wnt/beta-catenin inhibitors impact the reactivation of LAT mutants?

Response: dLAT2903 explant-induced reactivation is significantly reduced compared to WT (reference 31 and Figures 7 and 8). Hence iCRT14 was assessed for LAT mutant reactivation at 5 days after explant (the peak effect of iCRT14 observed in WT HSV1 reactivation). dLAT2903 reactivation was decreased in the presence of iCRT14: Figures 7 and 8 (lines 349-369).

Concern #8: How would DEX impact the action of the Wnt/beta-catenin inhibitors on virus reactivation?

Response: New studies showing this data are presented in Figure 8.

Concern #9: Lines 341-343. This is an egregious over-interpretation of the data.

Response: Agreed. Our thought on this topic have been revised and are less speculative (lines 393-410).

Minor issues:

Concern #9: It is curious that Wnt/beta-catenin antagonists are so potent at inhibiting reactivation when the levels of beta-catenin are so low during the reactivation phase. Can the authors comment on this apparent inconsistency?

Response: The antagonists are measuring viral shedding in the supernatant 4-9 days post-explant. Under the conditions of our TG explant studies, this is when virus shedding consistently occurred (reference 31). Conversely, IHC analyzing beta-catenin expression was performed 4 and 8 hours post-explant. The integrity of the tissue after 16hrs post-explant is compromised and IHC results would not be a clear-cut. Two previous studies demonstrated �-catenin promotes HSV1 productive infection in cell culture (references 32 and 58). This was discussed in the revised manuscript (lines 430-451).

Concern #10: Line 27. Why highlight ocular disease here? Line 22 introduces ocular, nasal and oral infection. Consider "recurrent disease".

Response: This was modified (lines 29-30).

Concern #11: Line 35. "increased" compared to what?

Response: This error was corrected in the manuscript (lines 34-36).

Concern #12: Line 47. Odd phrase. Suggests oral disease upon reactivation after primary infection of the eye? This is confusing. Similar issues in abstract. Consider rephrasing.

Response: This was modified in the manuscript (lines 48-50).

Concern #13: Lines 49-50. This sentence implies LAT is not expressed during lytic infection. I don't think this is the point the authors are trying to make. Consider rephrasing.

Response: This has been corrected in the manuscript (lines 52-54).

Concern #14: Line 53. Why does the nature of LAT locus products "suggest" functions related to latency? Consider rephrasing.

Response: This sentence was modified (lines 55-57).

Concern #15: Line 76. “Wnt binding” to what?

Response: This has been corrected in the manuscript (lines 79-83).

Concern #16: Line 170. “rational” should be “rationale”.

Response: This has been corrected in the manuscript (lines 172-174).

Concern #17: Line 177. “marker rescued” should read “repaired”.

Response: This has been corrected in the manuscript (lines 180-182).

Concern #18: Sentence on lines 308-309. Alternatively, the data may indicate that the inhibitors were toxic to the explants. Can the authors control for this? Is there a dose response to the inhibitors?

Response: Previous data from our lab has shown iCRT14 does not have any cytotoxicity and no significant dose response in cell culture (31). We repeated this study and performed toxicity studies using KYA1797K in cell culture and found this does not have toxic effects in cell culture up to 50µM. This has been updated in the manuscript (lines 328-330).

PLoS One. doi: 10.1371/journal.pone.0230870.r003

Decision Letter 1

Neal A DeLuca

11 Mar 2020

Herpes simplex virus 1 regulates beta-catenin expression in TG neurons during the latency-reactivation cycle.

PONE-D-19-33585R1

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PLoS One. doi: 10.1371/journal.pone.0230870.r004

Acceptance letter

Neal A DeLuca

16 Mar 2020

PONE-D-19-33585R1

Herpes simplex virus 1 regulates beta-catenin expression in TG neurons during the latency-reactivation cycle.

Dear Dr. Jones:

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PERMALINK

Herpes simplex virus 1 regulates β-catenin expression in TG neurons during the latency-reactivation cycle

Kelly S Harrison

Liqian Zhu

Prasanth Thunuguntla

Clinton Jones

Roles

Abstract

Introduction

Materials and methods

Virus and cell lines

Animals

Immunohistochemistry

Examining the effects of Wnt/β-catenin antagonists on explant-induced reactivation

Statistical analysis

Results

More neurons express β-catenin in TG of mice latently infected with wt HSV-1

Fig 1. β-catenin expression is increased in latently infected TG.

Fig 2. Quantification of β-catenin expression.

Examination of β-catenin expression in TG during explant-induced reactivation

Fig 3. β-catenin protein expression in TG explants of WT latently infected mice.

Fig 4. β-catenin protein expression in TG explants of dLAT2903 latently infected mice.

Fig 5. β-catenin protein expression in TG explants of uninfected mice.

Fig 6. Quantification of β-catenin expression in explanted TG.

Antagonists of the Wnt-signaling pathway significantly reduced DEX-induced virus reactivation from latency in TG explants

Fig 7. Explant-induced reactivation from latency in the presence of Wnt antagonists.

Fig 8. TAiCRT14 inhibits explant-induced reactivation from latency in dLAT2903 infected TG.

Discussion

Data Availability

Funding Statement

References

Decision Letter 0

Neal A DeLuca

Roles

Author response to Decision Letter 0

Decision Letter 1

Neal A DeLuca

Roles

Acceptance letter

Neal A DeLuca

Roles

Associated Data

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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