Life Sciences 120 (2015) 31–38

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Fungal metabolite myriocin promotes human herpes simplex virus-2 infection☆ Jingjing Wang a, Xuancheng Guo a, Ziying Yang a,1, Ren-Xiang Tan a, Xiaoqing Chen b,⁎, Erguang Li a,c,⁎⁎ a b c

Medical School and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, China Sino-French Hoffmann Institute of Immunology, Guangzhou Medical University, China State Key Laboratory of Natural Medicines, China Pharmaceutical University, China

a r t i c l e

i n f o

Article history: Received 26 July 2014 Accepted 3 November 2014 Available online 14 November 2014 Keywords: Herpes simplex virus HSV-2 Myriocin Sphingolipid Histone modification

a b s t r a c t Aims: Myriocin is a fungal metabolite with antiviral activity, including influenza, hepatitis B, and hepatitis C viruses. We investigated whether myriocin has activity against human HSV-2, one of the most prevalent pathogens of sexually transmitted disease. Main methods: Cell culture systems were used to evaluate myriocin effect on HSV-2 infection. Plaque forming assay and immunoblotting studies were used to determine virus production and viral protein expression, respectively. Key findings: Myriocin showed no cytotoxic effect at up to 5 μM. Myriocin treatment did not inhibit HSV-2 infection. Instead, the treatment resulted in accelerated replication of HSV-2 and increased titers of infectious virion. The effect was detected at concentrations as low as 3 nM and plateaued at approximately 30 nM. Myriocin at 30 nM increased HSV-2 production by approximately 1.7 logs. Myriocin also promoted HSV-1 infection but required higher concentrations. A time course study revealed that myriocin promoted HSV-2 infection by acceleration of virus replication. Unlike trichostatin A that promotes HSV-2 infection and histone modifications, myriocin treatment did not alter histone modifications. Myriocin is a well characterized inhibitor of sphingolipid biosynthesis pathway. Structurally different inhibitors of the pathway showed no effect on HSV-2 infection. Exogenous sphingolipids did not reverse the effect of myriocin on HSV-2 infection either. Significance: We found that myriocin promotes HSV-2 replication at nanomolar concentrations with yet unknown mechanisms. Further studies may uncover novel mechanisms regulating HSV replication and targets of myriocin action. This may have potential application in enhancing efficacy of oncolytic HSV for cancer therapy and other diseases. © 2014 Elsevier Inc. All rights reserved.

Introduction Herpes simplex viruses are double stranded DNA viruses that belong to the Herpesviridae family. There are two serotypes of human herpes simplex viruses, namely HSV-1 and HSV-2. HSV-1 is mainly associated with facial infections with visible cold sores or fever blisters and HSV2 in general is associated with genital infection and causes genital herpes [21,40], but both strains have the ability to cause infection in either area [3]. HSV can have both lytic and latent infection cycles. In the lytic infection, HSV infects mucosa epithelial cells after direct contact with an infected individual or body fluid from those individuals. The virus replicates with vigorous multiplication of immediate early (IE) genes within ☆ A portion of the work described here has been submitted to Nanjing University for the application of a master degree of science by Z. Yang. ⁎ Corresponding author. ⁎⁎ Correspondence to: E. Li, Medical School and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, China. Tel.: +86 25 83593193. E-mail address: [email protected] (X. Chen), [email protected] (E. Li). 1 Current address: The First Affiliated Hospital of Suzhou University, Suzhou, Jiangsu.

http://dx.doi.org/10.1016/j.lfs.2014.11.004 0024-3205/© 2014 Elsevier Inc. All rights reserved.

the first few hours of infection to “prime” host cells for further expression of viral genes and mobilize cellular transcriptional machinery including the NF-κB pathway for viral genome replication and viral structural protein expression of the early and late phase genes [1,14, 27,28]. After initial infection, HSV-1 and HSV-2 are transported along sensory nerves to the sensory nerve cell bodies, where they establish life-long latency of the human host. HSV exits latency periodically and is transported to the body surface where recurrent infection occurs. Several viral genes have been identified as critical for HSV replication [5,35], while few small molecules except trichostatin A (TSA), a well documented inhibitor of histone deacetylases, are known to promote HSV infection [9,10,16,20,25]. Myriocin is a fungal metabolite originally isolated from Mycelia sterilia with antibacterial and antifungal activities [18,26]. The compound is an atypical alpha amino acid that also resembles that of sphingosine. Myriocin is a well-characterized natural product with potent activity against serine palmitoyltransferase (SPT), a critical enzyme in de novo sphingolipid biosynthesis [7,22,38]. Recent studies show myriocin with potent activity against viral infection of human diseases,

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including influenza virus, HBV, and HCV infection [2,33,34,36]. In this study, we investigated whether myriocin possessed antiviral effect against HSV-2. Unexpectedly, we found that myriocin treatment resulted in increased HSV infection at nanomolar concentrations. Histone deacetylase inhibitors are among the only group of small molecule compounds that have been observed with a similar effect on HSV infection. We found that the effect of myriocin treatment did not affect histone modifications. In addition, the effect seemed to be independent of sphingolipid pathway inhibition. The results are reported here. Materials and methods Cells and viruses African green monkey kidney epithelial Vero cells (ATCC CCL-81) and human neuroblastoma SK-N-SH cells (ATCC HTB-11) were purchased from the Cell Bank of the Chinese Academy of Science (Shanghai, China). The cells were cultured at 37 °C in DMEM (high glucose) supplemented with 10% heat-inactivated fetal bovine serum (Life Technologies, Carlsbad, CA), non-essential amino acids and sodium pyruvate in a humidified incubator with 5% CO2. The HSV-2 strain G was purchased from ATCC (Manassas, VA). HSV-1 strain 8F was kindly provided by Dr. Qihan Li, Institute of Medical Biology, Chinese Academy of Sciences (Kunming, China). The stock viruses were propagated in Vero cells and titrated by using plaque forming assay. Antibodies and reagents Myriocin (PubChem CID: 6438394), MTT (methylthiazolyl diphenyltetrazolium bromide), and D-sphingosine were purchased from SigmaAldrich (St. Louis, MO). All other chemicals were purchased from Enzo Life Sciences Inc. (Farmingdale, NY) or Cayman Chemicals (Ann Arbor, MI). Antibodies to viral gD and ICP0 of HSV-1 were purchased from Santa Cruz Biotechnologies (San Cruz, CA), to acetylated histone-3 at Lys9 and Lys14 (Ac-H3)from Millipore (Billerica, MA), to histone-3 (H3) and to Ser10 phosphorylated histone-3 (p-H3) from Beyotime Institute of Biotechnology (Haimen, China). Antibody to GAPDH was purchased from Bioworld Technology (Minneapolis, MN). An antiserum to ICP0 of HSV-2 was prepared using a commercial source (Abmart, Shanghai) by immunizing New Zealand rabbits with a synthetic peptide. IRDye 800 and IRDye 700DX-conjugated secondary antibodies were

obtained from Rockland Immunochemicals Inc. (Gilbertsville, PA). HRP-conjugated secondary antibodies were purchased from SigmaAldrich. Cytotoxic assay MTT assay was used to assay the cytotoxic effect of myriocin on host Vero cells as we previously used [8]. Briefly, cells in triplicates were treated with myriocin for 72 h. At the end of the treatment, MTT was added to each well to a final concentration of 0.5 mg/ml for the measurement of formazan formation, which can be extracted by DMSO and measured at 570 nm on a Versa Max microtiter plate reader (Molecular Devices, Sunnyvale, CA). Infection assay Monolayers of Vero cells or SK-N-SH cells were incubated with indicated concentrations of myriocin for 2 h and then uninfected or infected with HSV-2 or HSV-1 at an MOI of 0.3 or as indicated. For experiments that involved drug treatment, the compound was left in the medium once added. We used methods to determine the effect of myriocin on HSV-2 infection. We used MTT method to measure cell viability as a quick assessment of an infection since HSV-2 infection would lead to increased cell death due to cytopathic effect of an infection. We performed plaque forming assays to quantitatively determine infectious virion production by measuring plaque-forming units (PFUs) as we previously described [8]. Briefly, after an infection of an indicated hour, the cells and culture supernatants were collected and then freeze–thawed in liquid nitrogen to release infectious virus. After removal of cellular debris by centrifugation, the samples were series-diluted and used for titration by infecting nearly confluent Vero cells in 24-well plates, with each dilution in triplicate. After adsorption for 1 h, plates were washed and overlaid with DMEM containing 2% FBS and incubated for another 5 days. The plates were fixed with 3% paraformaldehyde and stained with 1% crystal violet for visualization of plaques. Virus titers are expressed as plaque forming units per ml (PFU/ml). In-cell western assay Monolayers of Vero cells in 96-well plate were treated as indicated. At 36 h PI, in-cell western assay was performed as described previously [29]. Briefly, cells were fixed with 3% paraformaldehyde for 30 min and permeabilization with 0.2% Triton X-100 for 5 min. After briefly washing

Fig. 1. Effects of myriocin on HSV-2 infection. A. Determination of maximal non-toxic concentration of myriocin in Vero cells. Vero cells in 96-well plates remained untreated or were incubated with myriocin at indicated concentrations for 72 h. DMSO at 0.2% was included as a solvent control since myriocin was dissolved in DMSO as a 1000× stock. Cell viability was determined by MTT assay. Data were presented as average optical densities at 570 nm (OD570 nm) ± standard deviation (SD) of triplicate samples. B. Pretreatment of Vero cells with myriocin causes more severe cytopathic effect in HSV-2-infected cells. Vero cells in 24-well plates were untreated or treated with myriocin (Myr) at 30 nM for 2 h prior to the infection. The cells were uninfected or infected with HSV-2 at 0.3 MOI for approximately 36 h. Represented fields of corresponding samples were photographed under an inverted microscope with an objective lens of 20× magnification. Con: untreated and uninfected Vero cells; HSV-2: untreated, but HSV-2 infected; Myr: uninfected but myriocin-treated; H+Myr: HSV-2 infected and myriocin-treated. The experiments were performed independently for at least 3 times. C. Myriocin treatment enhances infection-associated cell lysis. Vero cells in triplicate were mock-treated with DMSO or with myriocin (Myr) at indicated concentrations 2 h prior to the infection. The cells remained uninfected or were then infected with HSV-2 at 0.3 MOI for 36 h. Cell viability was then determined using a MTT assay. Open squares: myriocin-treated but uninfected controls (toxicity assay). Filled squares: HSV-2 infected that were treated with varying amounts of myriocin or untreated, which represents HSV-2 infected but untreated control (pointed with an arrowhead). Data are from an independent experiment of three separated studies. ** denotes a p ≤ 0.001, indicating a significant difference between myriocin-treated and HSV-2 infected samples to that of the infected but untreated controls (myriocin at zero concentration). D. Myriocin treatment on production of infectious HSV-2. Vero cells were mock-treated or treated with myriocin at indicated concentrations for 2 h, and then infected with HSV-2 at an MOI of 0.3. The cells and culture supernatants were collected at 36 h PI, and used for titration of infectious virion by a plaque forming assay. The data are presented as mean ± SD of triplicate samples. * and ** indicate statistically significant difference of infectious virion production in myriocin-treated samples compared to that of mock-treated controls (* and ** represent p ≤ 0.01 and p ≤ 0.001, respectively). The experiment was performed twice independently. E. Myriocin treatment on HSV-2 gene expression. Vero cells were treated with myriocin at varying concentrations 2 h prior to inoculation. The cells were infected with HSV-2 (MOI = 0.3) for 36 h and used for the detection of ICP0 and gD expression by immunoblotting assays. Numeric numbers under ICP0 and gD blots indicate relative intensities of protein expression in those samples compared to that of the infected but untreated controls. GAPDH was used as an internal control for the study. F. Myriocin treatment on late gene gD expression detected by in-cell western assay. Vero cells that were untreated or treated with 30 nM myriocin were infected with HSV-2 at MOIs as indicated. The cells were then fixed at 36 h PI with paraformaldehyde and stained with antibody against HSV-2 gD protein or GAPDH as a control, followed by fluorescence dye-labeled secondary antibodies. The experiment was performed twice independently. G. Myriocin treatment on HSV-2 gene expression determined by RT-PCR. Vero cells in 12-well plates were untreated or treated with 30 nM myriocin 2 h prior to inoculation. The cells were then infected with HSV-2 for indicated times. Total RNA was harvested and used for the detection of HSV-2 gene expression by RT-PCR. Myriocin treatment resulted in increased detection of HSV-2 TK and gD gene in corresponding samples. Numeric numbers under TK and gD gels indicate relative intensities of viral gene expression in the samples. The intensity of the untreated controls with a measureable band was arbitrarily assigned as 1 and used to calculate relative intensities of other samples. The boxed numbers highlight the difference between myriocin-treated and untreated samples. The experiments were performed twice independently.

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with PBS, the cells were blocked for 2 h and incubated with primary antibodies against viral gD protein and GAPDH overnight at 4 °C. After washing with 0.1% Tween-20 in PBS, the cells were incubated with IRDye conjugated secondary antibodies for 2 h. The images were collected using an Odyssey Infrared Imager (LI-COR, Lincoln, NE). Immunoblotting analysis Total cell lysates were prepared by cell lysis with a buffer containing of 1% NP-40. Histones were isolated by acid-extraction as described [30].

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In brief, fractions containing nuclei were collected by centrifugation after being released by a hypotonic lysis buffer containing 10 mM Tris–HCl (pH 8.0), 1 mM KCl, 1.5 mM MgCl2, 1 mM DTT, 1 mM PMSF and a cocktail of protease inhibitors (Roche). Histones were then extracted using 0.2 M H2SO4 and acid-soluble proteins were separated by a 15% SDS-PAGE. The proteins were then transferred to PVDF membrane and proteins were identified by using specific antibodies and an ECL reagent kit (Pierce, Rockford, IL). The images were captured with the Alpha Innotech Flour Chem-FC2 imaging system (San Leandro, CA), and densities of corresponding bands were quantified using the pre-installed software.

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Reverse transcription polymerase chain reaction (RT-PCR) Total RNA from HSV-2 infected Vero cells was extracted using TRIzol reagent (Life Technologies). One microgram RNA was reverse transcribed into cDNA using AMV reverse transcriptase. Gene expression was determined by PCR assay using the following conditions: denaturing at 94 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 1 min. The RTPCR products were electrophoresed on 1.5% agarose gel and stained with ethidium bromide. The primers used for viral gene expression were: TK of HSV-2: 5′-GATAGGGTGCCGGTCGAAAA and 5′-GAGCCGATGA CTTACTGGCA; gD of HSV-2: 5′-GATGCACGCCCCCGCCTT and 5′-AGTTCGGGTTGCGT GGCGTT. GAPDH was used as a loading control: 5′-ATCTCCGCCCCTTCTGCCGA and 5′-CCACAGCCTTGGCAGCACCA. Statistical analysis Statistical comparison was performed using one-way ANOVA followed by a Dunnett's Multiple Comparison test. A p value ≤ 0.01 was considered significant. Results Myriocin promotes HSV infection

Fig. 2. Effect of myriocin on HSV-1 infection and on HSV infection in human SK-N-SH neuroblastoma cells. A. Effect of myriocin on HSV-1 infection in Vero cells. Vero cells were mock-treated or treated with myriocin at indicated concentrations. The cells were then infected with HSV-1 (MOI = 0.3). The samples were collected at 36 h PI and used for titration of infectious virus (upper panel) or used for detection of ICP0 expression by immunoblotting (lower panel). GAPDH was used as a loading control. ** indicates a significant difference (p ≤ 0.001) in virus production between myriocin-treated vs mock-treated samples. Numeric numbers under ICP0 blot indicate relative intensities of protein expression in treated samples compared to that of the infected but untreated controls. Both experiments were performed twice independently. B, C. Effect of myriocin on HSV-1 and HSV-2 infection in human SK-N-SH cells. SK-N-SH cells were pretreated with myriocin at indicated concentrations or mock-treated with DMSO prior to infection. The cells were infected with HSV-1 (B) or HSV-2 (C) at MOI = 0.3 for 36 h. Virus production in the samples was titrated by plaque forming assays. The data are presented as mean ± SD of triplicate samples. ** indicates significant difference (p ≤ 0.001) between treated and untreated samples. The experiments were performed twice independently.

Myriocin (PubChem CID: 6438394) has been reported with antiviral activity against several viruses of human diseases [33,34,36]. In this study, we sought to investigate whether myriocin possessed anti-HSV-2 activity, one of the most prevalent pathogens of sexually transmitted disease (STD). To this end, we first determined the maximal nontoxic concentrations of myriocin to Vero cells and found that myriocin at up to 5 μM concentration was relatively nontoxic to those cells (Fig. 1A). The compound was therefore tested at varying concentrations for its effect on HSV-2 infection. Unlike compounds with antiviral activities, myriocin treatment did not block the cytopathic effect associated with HSV-2 infection that can be monitored under an inverted microscope. Instead, the treatment resulted in more profound morphological changes of Vero cells that resembled the effect of HSV infection at higher multiplicities of infection (MOIs) (Fig. 1B), suggesting that myriocin might have an effect in promoting HSV-2 infection. We therefore performed separate experiments to validate whether myriocin promoted HSV-2 infection. For a quick assessment of the effect of myriocin on HSV-2 infection, we measured cell viability since increased infection would lead to reduced cell viability that can be semiquantitatively determined using MTT (methylthiazolyl diphenyltetrazolium bromide) assay. As shown in Fig. 1C, treatment with myriocin at as little as 3 nM resulted in significant increase in cell lysis associated with HSV-2 infection. We next performed plaque-forming assay to determine whether the treatment resulted in increased production of infectious virus. Compared to that of the infected but untreated controls, HSV-2 titers in myriocin-treated samples were significantly increased (Fig. 1D). The increase was detected within a dose range of 3 to 100 nM, and maximized out at approximately 100 nM. Additionally, we detected for viral gene expression by measuring the immediate early gene ICP0 and late gene gD expression. As shown in Fig. 1E, myriocin treatment dose-dependently increased ICP0 and gD expression. The increase of gD expression was also demonstrated by in-cell immunoblotting study (Fig. 1F), as well as by RT-PCR experiments for gD gene expression (Fig. 1G). Those results together demonstrated that myriocin treatment promoted HSV-2 RNA synthesis, gene expression and infectious virus production. We found that the effect was not restricted to HSV-2 since myriocin treatment also increased ICP0 gene expression and infectious virus

J. Wang et al. / Life Sciences 120 (2015) 31–38

HSV-2 yield (log PFU/ml)

9

outcome of HSV-2 infection (Fig. 3, Tx-V), indicating that the compound did not target the inoculum directly for its effect on HSV infection.

HSV-2 inoculation

8 Myriocin treatment accelerates HSV-2 replication

7

6

5 HSV -2

0

1

3

6

12

24 Tx-V

Time of Myr addition (hr) Fig. 3. Time of drug-addition study of myriocin on HSV-2 infection. Vero cells in 24well plates were untreated or treated with 30 nM myriocin at times as indicated. The time of inoculation was counted as time 0, while −2 indicates that myriocin treatment started at 2 h prior to inoculation. In parallel experiments, HSV-2 in 30 μl culture medium was treated with 30 nM myriocin at RT for 60 min. The mixture, labeled as pretreatment of virus (Tx-V), was used to infect cells. The samples were collected at 36 h PI and used for titration of infectious HSV-2 production. The study was repeated two times and data are mean ± SD of triplicate samples from one independent experiment.

production of HSV-1 (Fig. 2A), though the effect was observed with higher concentrations of myriocin. In addition, myriocin treatment also promoted infectious HSV-1 and HSV-2 infection of SK-N-SH cells, a human nureoblastoma cell line (Fig. 2B, C).

Time course of myriocin-addition on HSV infection We next performed a time course of drug-addition study to preliminarily determine whether myriocin targeted a particular event of HSV2 infection. Monolayers of Vero cells were treated with myriocin at 2 h prior to (−2 h), during (0 h), or within the first 24 h of inoculation (1, 3, 6, 12, and 24 h). Alternatively, an equal amount of HSV-2 virion was treated with 30 nM myriocin in 30 μl of DMEM at RT to determine if the compound targeted the virion directly. As shown in Fig. 3, addition of myriocin prior to HSV-2 infection, or within the first 6 h of inoculation strongly promoted HSV-2 infection. Co-incubation of HSV-2 with 30 nM myriocin prior to inoculation showed little effect in alternating the

HSV-2 PFU/ml (log10)

10 9

** 8 7 6

35

- Myr + Myr

**

5 4 24

36

48

60

72

(hr)

Fig. 4. Determination of time courses of HSV-2 production in the presence and absence of myriocin. Vero cells were mock-treated or treated with 30 nM myriocin 2 h prior to infection. The cells were infected with HSV-2 (MOI = 0.3). The cells and culture supernatants were collected at 24, 36, 48, 60, and 72 h PI. The viral titers were determined by PFU assay. Dotted line: without myriocin; solid line: myriocin-treated. The experiment was performed 3 times independently and data are presented as mean ± SD of triplicate samples from one experiment. ** indicates a significant difference (p ≤ 0.001) between myriocin-treated and mock-treated samples.

We next determined whether myriocin treatment simply accelerated HSV-2 replication or increased the overall yield of HSV-2 production. Vero cells were treated with 30 nM myriocin 2 h prior to HSV-2 infection. The samples were harvested at 24, 36, 48, 60, and 72 h PI and used for titration of virus production. We found that at 24 and 26 h PI, virus titers in myriocin-treated samples were significantly higher compared to those of untreated controls (Fig. 4). Although the highest virus titer in myriocin-treated samples was detected earlier than that of the untreated controls (at approximately 48 h PI vs 60 h PI for those samples, respectively), the difference among virus titers of those two groups became less significant, indicating that myriocin treatment might have simply improved HSV-2 infection efficiency.

Unlike trichostatin A that promotes HSV infection by increasing histone modifications, myriocin does not cause histone modifications Histone modification plays an important role in HSV gene transcription during productive infection [19]. Inhibitors of histone deacetylases (HDACi) like TSA are known to promote HSV infection by increasing histone modifications [4,10,16,24]. We therefore assessed whether myriocin promoted viral infection efficiency with a similar mechanism by immunoblotting for histone modifications. HSV-2 infection resulted in elevated acetylation and phosphorylation of histone (Fig. 5A). Unlike TSA that promoted both ICP0 expression and histone modifications, myriocin treatment only resulted in increased ICP0 expression, but not histone modifications (Fig. 5A). The conclusion on histone modification was substantiated with further experiments. As shown in Fig. 5B, addition of myriocin at varying times of HSV-2 infection did not significantly alter histone phosphorylation induced by HSV-2 infection. In addition, we found that myriocin treatment alone did not cause histone modifications (Fig. 5C). Those results suggested strongly that myriocin utilized a different mechanism from that of TSA in promoting HSV-2 infection. Myriocin promotes HSV infection independent of sphingolipid biosynthesis pathway Myriocin has been widely recognized as a specific inhibitor of de novo biosynthesis of sphingolipids since the compound is a selective inhibitor of SPT (Fig. 6A). Myriocin therefore has been used as a tool for depletion of sphingolipids in cell biology studies. To determine whether myriocin promoted HSV-2 infection through inhibition of sphingolipid biosynthesis, we first tested whether structurally different inhibitors of sphingolipid biosynthesis pathway, L-cycloserine and fumonisin B1, had a similar effect on HSV infection. Unlike myriocin, treatment with L-cycloserine or fumonisin B1 at concentrations known to suppress sphingolipid biosynthesis [13,39] showed no effect on HSV-2 infection (Fig. 6B). We then tested whether exogenous sphingolipids had the ability to reverse the effect of myriocin on HSV infection. To this end, Vero cells were pretreated with sphingosine, sphinganine, or C8ceramide, a cell permeable precursor of sphingosine derivative in the presence or absence of 30 nM myriocin. The cells were then infected with HSV-2. Treatment with exogenous sphingosine did not affect HSV-2 infection. Combined treatment with myriocin and a sphingolipid did not reverse the enhanced effect of HSV-2 infection by myriocin (Fig. 6C). The result therefore indicated that myriocin promoted HSV2 infection unlikely by targeting the sphingolipid biosynthesis pathway. In summary, we showed here that myriocin, a fungal metabolite, possessed selective activity in promoting HSV infection with yet undetermined mechanisms.

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A

Con

H

H+M H+T

Ac-H3 rel. int. 1.0

3.5

3.5

9.2

3.0

2.9

11.2

rel. int. 1.0

4.1

3.9

p-H3 rel. int. 1.0 Total H3 ICP0

GAPDH

B

HSV-2 @ MOI = 0.3

Con p-H3

Total H3 Myr Tx (hr, PI): -

C

-2

0

1

3

6

12

24

Myr @ 30 nM (hr) Con

1

3

6

9 TSA

Ac-H3 p-H3

detected in both Vero cells and human neuroblastoma cells. The detection of increased ICP0 expression throughout the infection process in myriocin-treated samples is consistent with the reported role of ICP0 protein as an immediate-early gene driving HSV replication [5,6]. We have not been able to determine the mechanisms governing enhanced replication of HSV-1 and HSV-2 by myriocin. HDACi are among the only group of small molecules that are known to promote HSV-1 and HSV-2 infection [9,16,25]. Unlike TSA that promotes HSV-1 and HSV-2 replication by promoting histone modifications, myriocin enhanced HSV infection without interference with histone modifications. Myriocin is a fungal metabolite that has been demonstrated to bind directly to SPT and functions as a selective inhibitor of the sphingolipid biosynthesis pathway [7,12,22,32]. Recent studies uncovered new functions of the sphingolipid signaling and metabolism during pathogenic virus infections [33,37]. Myriocin regulates the sphingolipid biosynthesis. Whether this may lead to the creation of a cellular environment favorable for HSV replication is unknown since previous studies have implicated this pathway in suppression of infection by several viruses [33,34]. A time of drug-addition study showed that there was no significant difference on HSV-2 infection if myriocin was added within the first 6 h (Fig. 3). This suggested to us that myriocin likely targeted a post cell entry event since the compound did not target virion directly for its effect. Our data seemed to exclude the possibility that myriocin promoted HSV-2 infection by targeting SPT since exogenous sphingolipids did not reverse the effect of myriocin on HSV infection. In addition, treatment with inhibitors like L-cycloserine and fumonisin B1 of sphingolipid synthesis pathway did not affect HSV-2 infection. Although extensive progress has been made towards our understanding of the mechanisms regulating HSV infection, many aspects regarding HSV replication, viral latency, and reactivation remain to be investigated [27]. Our study only highlights the complicated nature of HSV replication as well as the unknown nature of natural products. Further studies are needed to uncover the mechanisms of myriocin on HSV-2 replication for the discovery of molecular targets regulating HSV replication and reaction.

Total H3 Conclusions Fig. 5. Effect of myriocin on HSV-2-induced histone modifications. A. Myriocin treatment does not affect histone modification patterns induced by HSV-2 infection. Vero cells in 6-well plates were uninfected or infected with HSV-2 in the presence or absence of 30 nM myriocin. Trichostatin A (T) at 150 nM was included as a positive control. Relatively high MOIs (10) were used for the detection of histone modifications during HSV-1 infection [23,25]. The samples were collected 6 h PI by acid extraction and histone modifications were detected by immunoblotting with antibodies recognizing acetylated histone-3 (Ac-H3) and phosphorylated histone-3 (p-H3), respectively. In parallel experiments, HSV-2 ICP0 expression was detected by immunoblotting analysis with specific antibodies. Numeric numbers under Ac-H3, p-H3 and ICP0 blots indicate relative intensities of protein expression in those samples. Intensity of relevant controls was assigned arbitrarily as 1. Histone-3 (H3) was used as a loading control for histone modifications, while GAPDH expression was used as a loading control for ICP0 expression. The experiments were performed independently 3 times. B. Time course of myriocin-addition on HSV-2 induced histone phosphorylation. Vero cells were uninfected or infected with HSV-2 at an MOI = 0.3. To test the effect of myriocin addition on HSV-2 induced histone phosphorylation, myriocin was added 2 h prior to (−2), during (0), or at varying times (1, 3, 6, 12, and 24) post inoculation. Histone modification was detected using an antibody against Ser10 phosphorylated histone-3 (p-H3). The experiment was performed twice independently. C. Myriocin treatment does not promote histone modifications. Vero cells were treated with myriocin at 30 nM for indicated times. Histone acetylation and phosphorylation were detected by immunoblotting assay. The trichostatin A (TSA, 150 nM)-treated sample was used as a positive control, and histone-3 (H3) expression was used as a loading control. The experiment was performed 3 times independently.

In summary, we demonstrated that myriocin, a fungal metabolite, promotes human herpes simplex virus replication with a yet unidentified mechanism(s). Myriocin significantly accelerated HSV-2 replication/infection as well as viral gene expression at concentrations as low as 3 nM. The compound also promoted HSV-1 infection as well as HSV infection in human cells. Unlike other compounds known to promote HSV replication, myriocin treatment did not affect histone modifications. In addition, myriocin promotes HSV infection independent of sphingolipid biosynthesis. HSV-based vectors are currently tested for treatment of various diseases [11,15,17,41]. The discovery that myriocin has the ability to enhance HSV replication may lead to potential application of myriocin in tumor therapy using oncolytic HSV since chemicals with similar effects have the ability to enhance oncolytic effect of HSV-based vectors [16,24].

Conflict of interest statement None.

Acknowledgement Discussions We provide evidence from the following aspects demonstrating that myriocin promotes HSV-2 infection efficiency: 1) titration of increased production of infectious virion; 2) increased expression of viral proteins of both immediate-early (ICP0) and late (gD) stages. The effect was

The work was supported by grants from NSFC (81121062, 81371772, and 21132004). JJW and XCG were recipients of the Jiangsu Innovation Program for Predoctoral Students. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

J. Wang et al. / Life Sciences 120 (2015) 31–38

A

37

Serine + Palmitoyl-CoA Myr L-CS

Serine palmitoyltransferase (SPT)

Myriocin (Myr)

3-ketosphinganine 3-ketosphinganine reductase L-cycloserine (L-CS)

Sphinganine FB

Ceramide synthase

Dihydroceramide Fumonisin B1 (FB)

Dihydroceramide desaturase

Ceramide

C

9 8

**

PFU/ml (log10)

PFU/ml (log10)

B

7 6 5 HSV-2 Myr

L-CS

FB

9 8 7 6

5 Lipid: Myr:

- - +

Sa Sa Ce Ce So So - + - + - +

Fig. 6. Myriocin promotes HSV infection independent of sphingolipid biosynthesis. A. Diagram of de novo sphingolipid biosynthesis pathway. Myriocin (Myr) and L-cycloserine (LCS) are inhibitors of SPT, fumonisin B1 (FB) is an inhibitor of ceramide synthase [22,31,39]. B. Myriocin treatment, but not L-cycloserine or fumonisin B1, promotes HSV-2 infection. Vero cells were untreated or treated with myriocin (Myr, 30 nM), L-cycloserine (L-CS, 1 mM), or fumonisin B1 (FB, 10 μM) for 2 h before being infected with HSV-2 (MOI = 0.3). The samples were collected at 36 h PI and used for viral titer determination. The data are presented as vertical scatter plot of triplicate samples with mean values indicated by horizontal bars. No difference between L-CS and FB treated samples compared to that of untreated controls. ** denotes p ≤ 0.001. C. Exogenous sphingolipids do not reverse enhanced HSV-2 replication by myriocin treatment. Vero cells were untreated or treated with sphinganine (Sa, 2 μM), C8 ceramide (Ce, 2 μM), or sphingosine (So, 2 μM) in the presence or absence of 30 nM myriocin. The cells were then infected with HSV-2 at an MOI of 0.3 at 2 h post treatment. The samples were collected at 36 h PI and viral titers in those samples were determined by PFU assay. The data are presented as mean ± SD of triplicate samples. Solid bar: no myriocin; hatched bar: with 30 nM myriocin.

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