Tranylcypromine Reduces Herpes Simplex Virus 1 Infection in Mice

Abstract

Herpes simplex virus 1 (HSV-1) infects the majority of the human population and establishes latency by maintaining viral genomes in neurons of sensory ganglia. Latent virus can undergo reactivation to cause recurrent infection. Both primary and recurrent infections can cause devastating diseases, including encephalitis and corneal blindness. Acyclovir is used to treat patients, but virus resistance to acyclovir is frequently reported. Recent in vitro findings reveal that pretreatment of cells with tranylcypromine (TCP), a drug widely used in the clinic to treat neurological disorders, restrains HSV-1 gene transcription by inhibiting the histone-modifying enzyme lysine-specific demethylase 1. The present study was designed to examine the anti-HSV-1 efficacy of TCP in vivo because of the paucity of reports on this issue. Using the murine model, we found that TCP decreased the severity of wild-type-virus-induced encephalitis and corneal blindness, infection with the acyclovir-resistant (thymidine kinase-negative) HSV-1 mutant, and tissue viral loads. Additionally, TCP blocked in vivo viral reactivation in trigeminal ganglia. These results support the therapeutic potential of TCP for controlling HSV-1 infection.

INTRODUCTION

Herpes simplex virus 1 (HSV-1) infects about 80 to 90% of the human population worldwide (1–5). After replication at the initial inoculation site (abraded mucosa membrane, eye, or skin), neurotropic HSV-1 can spread to peripheral sensory ganglia and the central nervous system. Infectious virus is no longer detected in tissues about 2 weeks after infection, but some viruses establish latency by maintaining their genomes in neurons of sensory ganglia. Latent virus can reactivate periodically to cause recurrent infection. There are few effective therapies available to block viral reactivation. Both primary and recurrent infections can induce devastating diseases, including encephalitis and stromal keratitis.

HSV-1 can infect the brain to cause encephalitis, which is the most serious consequence of all HSV infections and also the most common cause of sporadic, fatal encephalitis, with an incidence of 1 in 200,000 individuals per year (3). The mortality rate of untreated patients is over 70% (3). Antiherpetic drugs, acyclovir, and related nucleoside analogues are used in patient treatment (3, 6). However, even with treatment, 30% of patients succumb to death (6). Survivors are often left with severe and permanent neurological sequelae, and only 2.5% of all patients return to normal neurological function (3). Due to the severity of HSV-1-induced encephalitis, more antiherpetic therapies are needed.

HSV-1 can infect the cornea to induce stromal keratitis, which is the leading cause of infection-induced corneal blindness in the Western world (7, 8). In the United States alone, more than 400,000 persons are affected, with 20,000 new cases per year (9). During the progression of herpetic stromal keratitis, viral replication in the cornea initiates angiogenesis and inflammation (7, 10, 11). Currently, a combination of antiherpetic drugs (acyclovir) and anti-inflammatory agents is used to treat patients (12–16). Unfortunately, some patients fail to respond to this regimen or develop viruses resistant to acyclovir (17–19). The majority (>90%) of acyclovir-resistant, clinical isolates contain mutations in the viral thymidine kinase, which is required to activate the drug (20, 21).

Infection of cells with DNA viruses, like HSV-1, can lead to the deposition of nucleosomes on viral genomes with viral DNA wrapped around histone proteins (22, 23). Furthermore, in vitro studies have found that HSV-1 can recruit lysine-specific demethylase 1 (LSD1) to enhance viral gene transcription by modifying histone methylation in viral gene promoters (24–26). LSD1 inhibitors, such as tranylcypromine (TCP), have been used in the clinic to treat Parkinson's disease, migraines, and psychiatric illnesses, such as depression and anxiety (27–33). As few reports have investigated the anti-HSV-1 activity of LSD1 in vivo, the present study was designed to address this issue. Murine models have been used to test therapies for HSV-1 infection in vivo, with regard to aspects of acyclovir resistance, encephalitis, corneal blindness, and reactivation. Using murine infection models, we showed that TCP significantly reduced HSV-1 acute infection and reactivation in vivo.

MATERIALS AND METHODS

Cells and viruses.

African green monkey kidney (Vero) cells, mouse neuronal (N18) cells, human lung epithelial (A549) cells, and human rhabdomyosarcoma (RD) cells were maintained and propagated according to the instructions of the American Type Culture Collection. The wild-type HSV-1 strains 294.1 (34–36), RE (37), McKrae, and KOS were used for experiments. In addition, tkLTRZ1 is a recombinant virus derived from KOS that lacks thymidine kinase activity due to an insertion within the gene (38). HSV-1 strains were propagated and titrated by plaque assay on Vero cell monolayers. Enterovirus 71 strain M2 was propagated and titrated on RD cell monolayers (39). The clinical isolate of type 3 adenovirus (kindly provided by Jen-Ren Wang in our college) was propagated and titrated on A549 cell monolayers.

In vitro antiviral assay.

N18 or A549 cell monolayers were infected with HSV-1 or enterovirus 71 at a multiplicity of infection (MOI) of 0.001 or adenovirus at one 50% tissue culture infectious dose for 1 h, treated with TCP (Sigma-Aldrich) or saline, and harvested 48 h postinfection (p.i.) unless otherwise indicated to determine viral titers by plaque assays.

Cell proliferation assay.

N18 and A549 cell monolayers were incubated with saline or TCP for 48 h. Cell viability was assessed using cell counting kit 8 (Dojindo Molecular Technologies) according to the manufacturer's instructions.

Infection and treatment of mice with TCP.

All mouse experimental protocols were approved by the Laboratory Animal Committee of National Cheng Kung University. Six-week-old mice were anesthetized and infected with HSV-1 or mock infected with lysates of uninfected Vero cells topically on the right eye following scarification of the cornea with a needle 20 times. Male ICR mice were infected with 1 × 10⁶ PFU/eye of tkLTRZ1 and treated with saline or TCP on the cornea (75 μg/eye) or both on the cornea and by intraperitoneal injection (10 mg/kg). Mice were given topical treatment 15 min before infection or 1 day p.i. to 3 days p.i. and systemic treatment 1 day before infection or 1 day p.i. to 3 days p.i. once daily. Male ICR mice were infected with 1 × 10⁵ PFU/eye of 294.1, treated with TCP on the cornea immediately before infection and by intraperitoneal injection 1 day before infection to day 10 p.i., and monitored for body weight and survival. Female C57BL/6N mice were infected with 5 × 10⁵ PFU/eye of RE, treated with TCP on the cornea immediately before infection and by intraperitoneal injection 1 day before infection to day 9 p.i., and monitored for corneal opacity and angiogenesis as described in our previous reports (37, 40). The corneal opacity was graded on a scale of 0 to 5 as follows: 0, normal cornea; 1, mild corneal haze; 2, moderate corneal opacity or scarring; 3, severe corneal opacity, iris visible; 4, opaque cornea, iris invisible; and 5, necrotizing cornea with vascularization. Corneal angiogenesis was scored by measuring the length of neovessels in corneas with grades between 0 (no neovessels) and 4 (neovessels in the corneal center) in increments of about 0.4 mm (the radius of the cornea is about 1.5 mm). The cornea was divided into 4 quadrants, and the angiogenesis score for each cornea (ranging from 0 to 16) was the sum of four quadrants. Female C57BL/6N mice latently infected with 6 × 10⁵ PFU/eye of McKrae were given saline or TCP by intraperitoneal injection at days 27 and 28 p.i. Ten hours later, mice were placed in a 43°C water bath for 10 min and given saline or TCP 6 h after hyperthermic stimulation. Mouse trigeminal ganglia were collected 19 h after hyperthermic stimulation, placed in tubes with 1 ml medium, and frozen. Tissues were thawed, homogenized, and frozen again. Tissue homogenates were thawed and sonicated. The homogenate of one trigeminal ganglion was combined with 2 ml medium before plating onto one well of confluent Vero cell monolayers. Cultures were incubated, and the culture medium was changed after 3 days. The Vero cell monolayers were inspected daily for 6 days to detect cytopathic effect. Cultures were then collected and frozen. Frozen cultures were thawed, sonicated, and plated onto fresh, confluent Vero cell monolayers. The next day, culture medium was removed, and Vero cell monolayers were overlaid with medium containing methylcellulose, cultured for 3 more days, and stained with crystal violet to detect plaques for confirmation.

Quantitative real-time RT-PCR.

Mouse eyes were harvested, frozen, and homogenized in solution with guanidine thiocyanate (Sigma-Aldrich). One tenth of homogenate was used to extract DNA, and the rest of homogenate was used to extract RNA. In the samples, the amounts of viral (thymidine kinase gene) and cellular (adipsin gene) DNA were quantified using real-time PCR, and the amounts of ICP0, ICP27, Sp1, TBP, and β-actin RNA were quantified using real-time RT-PCR as previously described (24, 41). Viral DNA was normalized to adipsin gene DNA, and ICP0, ICP27, Sp1, and TBP transcripts were normalized to β-actin gene RNA. Additionally, normalized ICP0 and ICP27 RNA values were divided by the normalized viral genome value. The RNA values for saline-treated mice were set as 100%.

Histological and immunofluorescence staining.

Briefly, tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned. Sections (6 μm) were deparaffinized and stained with hematoxylin and eosin. In addition, deparaffinized sections were treated with 1% fetal bovine serum to block nonspecific binding before incubation with antibodies against HSV-1 (Dako) or NeuN (clone A60; Millipore) or with isotype-matched control antibodies overnight at 4°C. Subsequently, bound anti-HSV-1 antibody was detected with donkey anti-rabbit immunoglobulin G Alexa Fluor 488 (Invitrogen), and bound anti-NeuN antibody was detected with donkey anti-goat immunoglobulin G Alexa Fluor 594 (Invitrogen). Antibodies against HSV-1 antigens or NeuN yielded positive signals, whereas isotype-matched control antibodies failed to do so. Images were photographed using an Olympus DP12 digital microscope camera (Olympus).

Flow cytometry.

Corneas were cut into pieces, incubated with 82 units/cornea of type I collagenase (Sigma-Aldrich) at 37°C for 80 min, and passed through pipet tips (200 μl) to release cells. Cornea cells were stained with antibody against mouse CD31 (clone MEC13.3; BD Biosciences) or isotype-matched control antibody before incubation with the fluorescein isothiocyanate-conjugated secondary antibody. The stained cells were analyzed by a FACSCalibur (BD Biosciences) using WinMDI software.

Statistical analyses.

Data are expressed as means and standard errors (SE). For statistical comparison, viral titers produced by cell lines, optical densities, tissue viral loads, RNA values, and mouse body weights were analyzed by the Mann-Whitney U test. Kaplan-Meier survival curves were analyzed by the log-rank test. Corneal opacity scores and angiogenesis scores were analyzed by the Wilcoxon signed-rank test. CD31⁺ cell numbers were analyzed by the Student t test. HSV-1 reactivation frequencies were analyzed by Fisher's exact test. All P values are for two-tailed significance tests.

RESULTS

TCP treatment reduces the titers of wild-type and thymidine kinase-negative (TK⁻) HSV-1 strains in the human or mouse cell line.

Pretreatment of human cell lines at least 4 h before infection was used to evaluate the anti-HSV efficacy of TCP in previous reports (24–26). Here, we assessed TCP treatment after infection using a mouse neuronal cell line (N18) and a human epithelial cell line (A549). N18 cells were infected with wild-type HSV-1 strain 294.1 (MOI = 0.001) for 1 h, treated with saline or different concentrations of TCP, and harvested 48 h p.i. to determine viral titers. TCP at concentrations of 0.5 and 1 mM reduced HSV-1 titers in a dose-dependent manner (Fig. 1A), with a significant difference being found at the concentration of 1 mM compared with saline (P < 0.01, Mann-Whitney U test). In addition, TCP (1 mM) significantly decreased viral titers at all time points (24 to 72 h p.i.), which were examined (Fig. 1B) (P < 0.05, Mann-Whitney U test). We next evaluated the antiviral efficacy of TCP on the virus whose replication cycle is not dependent on DNA. N18 cells infected with a neurotropic RNA virus, enterovirus 71 (MOI = 0.001), for 1 h were treated with TCP. TCP at concentrations of 0.5 and 1 mM failed to decrease enterovirus 71 titers at 48 h p.i. (Fig. 1C).

FIG 1.

Effects of TCP treatment on the viral titers and proliferation of mouse and human cell lines. (A and C) Titers of HSV-1 294.1 (A) and enterovirus 71 (EV71) (C) in the mouse cell line N18 treated with saline or the indicated concentrations of TCP were determined at 48 h postinfection. (B) Titers of HSV-1 294.1 in N18 cells treated with saline or TCP (1 mM) were determined at the indicated times postinfection. (D) Relative titers of HSV-1 294.1 and a clinical isolate of type 3 adenovirus in the human cell line A549 treated with saline or the indicated concentrations of TCP were determined at 48 h postinfection. The titers of saline-treated samples were set at 100%. (E) Titers of the HSV-1 strains KOS, tkLTRZ1, and RE in A549 cells treated with saline or TCP (1 mM) were determined at 48 h postinfection. Uninfected N18 cells (F) and A549 cells (G) were cultured in medium containing the indicated concentrations of TCP for 48 h. Cell proliferation was determined by a WST-8 assay. Data are means and SE (error bars) for ≥5 samples per data point or group. *, P < 0.05, **, P < 0.01, and ***, P < 0.001 (Mann-Whitney U test), for comparisons with the saline-treated samples (A and D), with TCP-treated samples (B), or between indicated groups (D).

In A549 cells, TCP at concentrations of 0.5 and 1 mM significantly reduced the titers of both HSV-1 294.1 and a clinical isolate of type 3 adenovirus in a dose-dependent manner at 48 h p.i. compared with saline (Fig. 1D) (P < 0.01, Mann-Whitney U test). In addition, TCP (1 mM) significantly reduced the titers of two other wild-type HSV-1 strains (KOS and RE) and tkLTRZ1, which is the KOS-derived, TK⁻ mutant resistant to acyclovir (38), compared with saline at 48 h p.i. (Fig. 1E) (P < 0.01, Mann-Whitney U test). Collectively, the results for HSV-1, enterovirus 71, and adenovirus are consistent with the principle that TCP, which inhibits the histone-modifying enzyme LSD1, suppresses only the replication of DNA viruses. Our HSV-1 results show that TCP possesses antiviral activity against all the wild-type and TK⁻ HSV-1 strains in all of the cell lines that were tested.

We examined the effect of TCP on cell proliferation by incubating uninfected cells in medium containing TCP for 48 h. TCP at concentrations of 0.5 and 1 mM failed to significantly affect the growth of N18 and A549 cells compared with saline as determined by cell proliferation assay (Fig. 1F and G) (P > 0.05, Mann-Whitney U test). TCP treatment (1 mM) reduced the proliferation of A549 and N18 cells by 13% and 2%, respectively, suggesting that A549 cells may be more sensitive to TCP than N18 cells.

TCP treatment reduces the titers of TK⁻ HSV-1 mutant in mice.

We then determined the anti-HSV-1 efficacy of TCP in vivo. ICR mice were infected with tkLTRZ1 on scarified corneas, as inoculation of HSV-1 via the abraded eye mimics human infection in some individuals. Mice were given TCP once per day topically on the cornea (75 μg/eye) immediately before infection and systemically by intraperitoneal injection (10 mg/kg) 1 day before infection to day 3 p.i. Mice in the control group received saline. Like most authentic thymidine kinase mutants, tkLTRZ1 replication is restricted to the initial inoculation site, as the virus is incompetent in replication in neurons and in neuronal spread to other tissues (42). Mouse eyes were harvested to determine viral titers. TCP pretreatment reduced viral titers in mouse eyes by 0.7 and 1.5 log units at days 1 and 3 p.i., respectively, with a significant difference found at day 3 p.i. compared with saline treatment (Fig. 2A) (P < 0.001, Mann-Whitney U test). We also tested the effect of TCP pretreatment only on the cornea, which reduced the viral titer in mouse eyes by 0.9 log at day 3 p.i. (Fig. 2A).

FIG 2.

Effects of TCP treatment on the viral load and expression of viral and cellular genes in the mouse eye. ICR mice corneally infected with HSV-1 tkLTRZ1 were treated with saline or TCP on the cornea before infection or both on the cornea before infection and by intraperitoneal (i.p.) injection 1 day before infection to day 3 postinfection. (A) Viral titers in the eye at the indicated days postinfection. Levels of viral (ICP0 and ICP27) RNA (B) and cellular (Sp1 and TBP) RNA (C) in the eyes of mice treated with saline or TCP on the cornea plus by intraperitoneal injection were determined at day 3 postinfection. The RNA levels of saline-treated mice were set at 100%. Data are means and SE (error bars) for ≥4 samples per data point or group. *, P < 0.05; ***, P < 0.001 (Mann-Whitney U test).

Previous in vitro studies showed that TCP pretreatment reduced the expression of HSV-1 immediate early genes, such as ICP0 and ICP27, but failed to affect the expression of cellular genes, such as Sp1 and TBP (24, 26). We examined the influence of TCP pretreatment on the expression of viral and cellular genes in vivo. The eyes of infected mice treated with saline or TCP on the cornea and by intraperitoneal injection were harvested at day 3 p.i., and the levels of ICP0, ICP27, Sp1, and TBP transcripts and viral genomes were determined. By comparison with saline treatment, TCP treatment reduced the levels of ICP0 and ICP27 RNA per viral genome by 95% and 71%, respectively, with a significant difference found in the ICP0 level (Fig. 2B) (P < 0.05, Mann-Whitney U test). Additionally, TCP slightly decreased the expression of Sp1 and TBP by 23% and 14% (Fig. 2C) (P > 0.05).

Lastly, we tested mice treated with TCP after infection. Mice infected with tkLTRZ1 were given TCP on the cornea and by intraperitoneal injection from days 1 to 3 p.i. TCP significantly reduced viral titers in mouse eyes by 0.8 and 1 log at days 2 and 3 p.i., respectively (Fig. 3) (P < 0.05, Mann-Whitney U test).

FIG 3.

TCP given after HSV-1 infection reduces eye viral loads. ICR mice corneally infected with HSV-1 tkLTRZ1 were treated with saline or TCP on the cornea and by intraperitoneal injection from days 1 to 3 postinfection. Viral titers in the eye at the indicated days postinfection are shown. Data are means ± SE (error bars) for ≥4 samples per data point. *, P < 0.05, and ***, P < 0.001 (Mann-Whitney U test), compared with the TCP-treated group.

TCP treatment reduces HSV-1-induced lethality and tissue viral loads in mice.

We tested the effect of TCP treatment in protecting mice from HSV-1-induced death. ICR mice were corneally infected with wild-type HSV-1 strain 294.1, which can induce encephalitis in mice (36). Infected mice were treated with saline on the cornea immediately before infection and by intraperitoneal injection from 1 day before infection to day 10 p.i. About 80% of saline-treated mice and 40% of TCP-treated mice showed severe signs of encephalitis, including hunched posture, lethargy, and ataxia. Compared with saline treatment, TCP treatment did not significantly affect the body weight of infected mice in the first 8 days but increased the body weight of infected mice 9 to 13 days p.i., when saline-treated mice succumbed to death (Fig. 4A). Of note, the final survival rate of TCP-treated mice (63%) was significantly higher than that of saline-treated mice (18%) by day 30 p.i. (Fig. 4B) (P < 0.05, log-rank test). Mouse eyes, trigeminal ganglia, and brains were harvested 1 to 9 days p.i. to measure viral titers, as HSV-1 replication was detected in these three tissues after corneal inoculation. The mean viral titers in these three tissues from TCP-treated mice were lower than those from saline-treated mice almost at all time points examined, with significant differences found at 1 or 2 time points in each tissue (Fig. 4C to E) (P < 0.05, Mann-Whitney U test). We examined the pathological changes in mouse brains harvested at day 8 p.i. Hematoxylin-and-eosin staining of the brain stem showed less inflammatory infiltrate and damage in infected mice treated with TCP than in infected mice treated with saline (Fig. 4F). Immunofluorescence staining of the brain stem showed less HSV-1 antigens and more neurons, as demonstrated by cells expressing NeuN (a neuron-specific marker), in infected mice treated with TCP than in infected mice treated with saline (Fig. 4G).

FIG 4.

TCP treatment reduces HSV-1-induced lethality and tissue viral loads of mice. The relative body weights (A) and survival rates (B) of ICR mice infected with HSV-1 294.1 and treated with saline (n = 11) or TCP (n = 11) were monitored at the indicated days postinfection. Viral titers in the eyes (C), trigeminal ganglia (D), and brains (E) of mice treated with saline or TCP were determined at the indicated days postinfection. Data are means ± SE (error bars) for ≥3 samples per data point. *, P < 0.05, and **, P < 0.01 (Mann-Whitney U test), compared with the saline-treated group (A) or with TCP-treated groups (C to E). *, P < 0.05 (log-rank test) (B). Brain stems of mock-infected or infected mice treated with saline or TCP were harvested 8 days postinfection and stained with hematoxylin and eosin (F) or antibodies against HSV-1 or NeuN (G). Original magnification, ×200 (G). Data are representative of at least 6 samples per group from two independent experiments.

TCP treatment decreases the severity of HSV-1-induced corneal disease, eye viral load, and corneal angiogenesis and inflammatory infiltrate in mice.

We tested the effect of TCP treatment on reducing the severity of HSV-1-induced stromal keratitis. C57BL/6N mice were corneally infected with wild-type HSV-1 strain RE, which can induce stromal keratitis to result in corneal blindness in mice (37, 40). Infected mice were treated with saline or TCP on the cornea immediately before infection and by intraperitoneal injection from 1 day before infection to day 9 p.i. All mice survived after infection. Corneas of infected mice were monitored for opacity and angiogenesis, two important features of herpetic stromal keratitis. Infected corneas of saline-treated mice developed symptoms progressively, with moderate opacity and visible irises (an opacity score of 2) by day 7 p.i., and became opaque and necrotizing with invisible irises (opacity scores of 4 to 5) from days 14 to 28 p.i. (Fig. 5A). TCP treatment significantly reduced the corneal opacity of infected mice, with mean scores of 2 from days 14 to 28 p.i. (P < 0.01, Wilcoxon signed-rank test). Abundant and extended neovessels were detected in the infected corneas of saline-treated mice, with a mean angiogenesis score significantly higher than that in the infected corneas of TCP-treated mice by day 21 p.i. (Fig. 5B) (P < 0.05, Wilcoxon signed-rank test).

FIG 5.

TCP treatment decreases the severity of HSV-1-induced corneal disease, eye viral load, and corneal angiogenesis and inflammatory infiltrate in mice. C57BL/6N mice infected with HSV-1 RE were treated with saline (n = 19) or TCP (n = 17). (A and B) The corneal opacity scores of infected mice at the indicated days postinfection (A) and the corneal angiogenesis scores of infected mice at 21 days postinfection (B) are shown. *, P < 0.05, and **, P < 0.01 (Wilcoxon signed-rank test), compared with the TCP-treated group (A) or between the indicated groups (B). (C) Eyes of mock-infected and infected mice treated with saline or TCP were harvested 28 days postinfection and stained with hematoxylin and eosin. The corneal portion of representative samples is shown. (D) The corneas of infected mice treated with saline or TCP were harvested 28 days postinfection to quantify cells expressing CD31 by flow cytometry. Data are representative plots (left) and means plus SE (error bars) for ≥3 samples per group with ≥2 corneas per sample (right). (Left) The bottom plots are gated on the R1 regions in the top plots. The x axes of the top plots are linear, while those of the bottom plots are on a log scale. Cells in R2 regions are considered CD31⁺. *, P < 0.05 (Student t test). (E) Viral titers in the eyes of mice treated with saline or TCP were determined at the indicated days postinfection. Data are means ± SE (error bars) for ≥7 samples per data point. ***, P < 0.001 (Mann-Whitney U test), compared with the TCP-treated group.

We further assessed the opacity and neovascularization in corneas harvested at day 28 p.i. Results of hematoxylin and eosin staining showed that the infected corneas of saline-treated mice were thickened, with profound edema, inflammatory infiltrate, and vascularization with erythrocyte-filled vessels, especially in the stroma, compared with the infected corneas of TCP-treated mice (Fig. 5C). To further compare corneal angiogenesis, we quantified the number of cells positive for CD31, a marker specifically expressed on endothelial cells constituting the newly formed blood vessels (43). Flow-cytometric analysis showed that TCP treatment significantly reduced the mean number of CD31⁺ cells in infected corneas 6-fold at day 28 p.i. (Fig. 5D) (P < 0.05, Student's t test). The results of opacity scores, angiogenesis scores, hematoxylin-and-eosin staining, and flow-cytometric analysis were consistent and collectively showed that TCP treatment reduced HSV-1-induced corneal disease and corneal angiogenesis and inflammatory infiltrate. Next, we determined the effect of TCP on viral replication in the eye and found that TCP treatment significantly reduced the eye viral titer by 1.4 log units at day 1 p.i. (Fig. 5E) (P < 0.001, Mann-Whitney U test). Previous reports showed that the level of acute HSV-1 replication positively affects the severity of stromal keratitis by initiating leukocyte influx and neovascularization in the mouse eye (37, 44).

TCP treatment reduces the in vivo reactivation of HSV-1 in mice.

Previous studies used the ex vivo (explant) approach to test TCP treatment on HSV-1 reactivation (24–26). In the present study, we examined the effect of TCP treatment on blocking HSV-1 reactivation in vivo. Female C57BL/6N mice were inoculated with wild-type HSV-1 strain McKrae on the cornea. The virus established latency in mice at day 27 p.i., as demonstrated by the failure to detect infectious virus in any of 6 trigeminal ganglia harvested from infected mice by plaque assay. Latently infected mice were given saline or TCP at days 27 and 28 p.i. by intraperitoneal injection. Ten hours after the last saline or TCP treatment, mice were placed in a 43°C water bath for 10 min, because transient hyperthermia has been used to induce HSV reactivation in mouse trigeminal ganglia in vivo (45, 46). Mice were given saline or TCP 6 h after hyperthermic stimulation, and mouse trigeminal ganglia were harvested 19 h after hyperthermic stimulation to detect infectious virus. Figure 6 shows that trigeminal ganglia harvested from saline-treated mice yielded reactivated (infectious) virus with a frequency of 53% (10/19), and trigeminal ganglia harvested from TCP-treated mice yielded infectious virus with a frequency of 9% (1/11). TCP treatment significantly reduced HSV-1 reactivation in mouse trigeminal ganglia (P < 0.05, Fisher's exact test). We also tested treatment of latently infected mice with TCP before or after hyperthermic stimulation. Trigeminal ganglia harvested from mice treated with TCP only before or only after hyperthermic stimulation yielded infectious virus with frequencies of 33% (2/6) and 57% (4/7), respectively.

FIG 6.

TCP reduces HSV-1 reactivation in mice. C57BL/6N mice were infected with HSV-1 McKrae for 27 days and treated with saline (−) or TCP (+) before (pretreatment), after (posttreatment), or both before and after hyperthermic stimulation. Mouse trigeminal ganglia were harvested to detect infectious virus 19 h after hyperthermic stimulation. The numbers above the bars are the numbers of ganglia that were positive for reactivated virus per the number of ganglia tested. *, P < 0.05 (Fisher's exact test).

DISCUSSION

In this study, we provide evidence that TCP treatment of mice significantly reduces the severity of encephalitis and corneal blindness induced by wild-type viruses, viral reactivation from trigeminal ganglia, and infection by the TK⁻ HSV-1 mutant. Furthermore, our in vitro results show that TCP treatment given after infection significantly decreases the titers of TK⁻ mutant and several wild-type virus strains in the human cell line. Our findings support the potential of TCP to control HSV-1 infection in patients.

Previous in vitro reports demonstrated that TCP pretreatment restrained the transcription of herpesviruses, varicella zoster virus, and human cytomegalovirus by reducing the activity of the histone-modifying enzyme LSD1 (24–26). Here we demonstrate that TCP treatment given after infection inhibits HSV-1 and adenovirus, but not an RNA virus (enterovirus 71), in the mouse or human cell lines. These studies demonstrate the potential of TCP against DNA viruses. For HSV-1 (strain 294.1), our results show that TCP has comparable 50% effective concentrations (EC₅₀s) in the mouse (N18) and human (A549) cell lines (0.57 and 0.61 mM, respectively), suggesting that TCP inhibits both mouse and human enzymes. For adenovirus, our results show that TCP has an EC₅₀ of 0.49 mM in A549 cells. We are not aware of any previous study testing TCP on adenovirus.

In the clinic, 30 to 60 mg/person/day of TCP is prescribed for patients with neurological disorders (31, 32). We used TCP at a dose of about 10 mg/kg/day to treat HSV-1-infected mice, because previous neuroscience reports found promising in vivo results in mice with 10 to 6 mg/kg/day of TCP (47–50). The TCP dose used for our mouse study is higher than those used for humans but is less than the acyclovir dose (10 to 20 mg/kg every 8 h) used to treat HSV-1-infected patients by intravenous injection (51). LSD1 has been hypothesized and demonstrated to be critical for the initial stage of HSV infection by reducing repressive chromatin to enhance transcription in promoters of viral immediate early genes (24, 26). Therefore, pretreatment with TCP, an LSD1 inhibitor, has been shown to suppress HSV infection in previous in vitro and ex vivo studies (24–26). Our results revealed that TCP treatment given before and after infection or hyperthermic stimulation significantly reduced the tissue viral loads, lethality, corneal blindness, and reactivation in HSV-1-infected mice. Moreover, we found that TCP given after infection also significantly decreased the titers of TK⁻ HSV-1 in mice. These results imply that TCP has both preventative and therapeutic potential.

All antiherpetic drugs currently available for patients are directed against viral proteins to prevent viral replication (3). However, their extensive use has led to the emergence of resistant mutant viruses. For acyclovir, the most effective anti-HSV-1 therapy, drug resistance is estimated to occur in ∼6% of treated patients (20, 52). There is a clear need to develop novel antivirals. Agents targeting cellular factors to reduce viral infection may be another option to prevent drug resistance. In the future, studies selecting HSV-1 mutants resistant to TCP are needed to address whether virus can mutate to circumvent cellular inhibition and whether TCP would unlikely select for resistance mutations in host cell LSD1. TCP with the capacity to reduce LSD1 activity was originally designed to inhibit monoamine oxidases A and B for the treatment of severe psychiatric disorders, such as depression and anxiety (27–30, 33). The EC₅₀ of TCP obtained in the present study (0.5 to 0.6 mM) is comparable to that obtained in a previous study (26) but is higher than that of acyclovir (3 to 9 μM) (53). This has promoted a recent study to design chemicals with a high specificity for LSD1 (26), and one compound, with promising anti-HSV-1 activity and low EC₅₀s (3 to 10 μM), OG-L002, was found. However, until drugs such as OG-L002 are approved for human use, clinicians may consider the use of TCP or combinations thereof for treatment of patients with drug-resistant HSV-1.