key: cord-0007115-tn9c8vld
authors: Harrison, Kelly S.; Zhu, Liqian; Thunuguntla, Prasanth; Jones, Clinton
title: Herpes simplex virus 1 regulates β-catenin expression in TG neurons during the latency-reactivation cycle
date: 2020-03-30
journal: PLoS One
DOI: 10.1371/journal.pone.0230870
sha: 9e2b96e706a68f22c702c70ea98713dafe617b5d
doc_id: 7115
cord_uid: tn9c8vld

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.

More than 50% of adults in the United States are latently infected with herpes simplex virus 1 (HSV-1), reviewed in [1] [2] [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 

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% CO 2 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.

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~2x10 5 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.

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. 

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

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] [23] [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] [35] [36] [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.

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.

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 explantinduced 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).

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.

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 βcatenin 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.

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-cate-nin+ 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.

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 10 3 vs 10 1 , respectively). However, both exhibited a plateau in virus shedding around day 7 at~10 3 PFU from TG explants. TG explants incubated with just FBS exhibited peaked levels of virus shedding at day 6 with 10 6 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 10 2 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 (<10 2 ); 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.

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-cate-nin+ 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 explantinduced 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 βcatenin 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 βcatenin 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. 

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