Inhibition effects of novel polyketide compound PPQ-B against influenza A virus replication by interfering with the cellular EGFR pathway

Accepted Manuscript Inhibition effects of novel polyketide compound PPQ-B against influenza A virus replication by interfering with the cellular EGFR pathway Miaomiao Wang, Shuyao Wang, Wei Wang, Yi Wang, Hui Wang, Weiming Zhu PII:

S0166-3542(16)30723-9

DOI:

10.1016/j.antiviral.2017.04.007

Reference:

AVR 4056

To appear in:

Antiviral Research

Received Date: 24 November 2016 Accepted Date: 12 April 2017

Please cite this article as: Wang, M., Wang, S., Wang, W., Wang, Y., Wang, H., Zhu, W., Inhibition effects of novel polyketide compound PPQ-B against influenza A virus replication by interfering with the cellular EGFR pathway, Antiviral Research (2017), doi: 10.1016/j.antiviral.2017.04.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Inhibition Effects of Novel Polyketide Compound PPQ-B Against Influenza A Virus Replication by Interfering with the

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Cellular EGFR Pathway

Miaomiao Wang1,#, Shuyao Wang1,#, Wei Wang1,2,*, Yi Wang1, Hui Wang1,

Key Laboratory of Marine Drugs, Ministry of Education, Ocean University of China,

Qingdao 266003, PR China. 2

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Weiming Zhu1,3,*

Shandong Provincial Key Laboratory of Glycoscience and Glycotechnology, Ocean

University of China, Qingdao 266003, PR China.

Laboratory for Marine Drugs and Bioproducts of Qingdao National Laboratory for

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Marine Science and Technology, Qingdao, 266237, P. R. China.

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*Corresponding author:

Mailing Address: School of Medicine and Pharmacy, Ocean University of China,

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Qingdao 266003, PR China. Tel: +86 532 8203 1268; Fax: +86 532 8203 1268; E-mail: [email protected]; [email protected].

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These authors contributed equally.

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Abstract Development of anti-influenza A virus (IAV) drugs with novel targets and low toxicity is critical for preparedness against influenza outbreaks. In the current study, our results indicated

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that the novel polyketide compound purpurquinone B (PPQ-B) derived from acid-tolerant fungus Penicillium purpurogenum strain JS03-21 suppressed the replication of IAV in vitro with low toxicity, and may block some stages after virus adsorption. PPQ-B could inhibit H1N1 (A/Puerto PR8),

H1N1

(A/California/04/2009;

Cal09)

and

H3N2

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Rico/8/34;

(A/swine/Minnesota/02719/2009) virus replication in vitro, suggesting that PPQ-B possesses

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broad-spectrum anti-IAV activities. PPQ-B’s antiviral activity may be largely related to its inhibition of some steps that occur 0-4 h after adsorption. Oral administration of PPQ-B could decrease pulmonary viral titers and improve survival rate in IAV infected mice. PPQ-B also significantly decreased the production of inflammatory factors TNF-α, IL-6, RANTES and KC in IAV infected lungs and A549 cells, suggesting that PPQ-B may also attenuate the inflammatory

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responses caused by IAV infection. PPQ-B may down-regulate the NF-κB and MAPK pathways to inhibit both virus replication and inflammatory responses. In summary, PPQ-B has the

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potential to be developed into a novel anti-IAV drug targeting host EGFR pathway in the future.

Keywords: Polyketide; Influenza A virus; EGFR pathway; Virus replication; Inflammatory

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responses

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1. Introduction Influenza pandemic is often associated with significant morbidity and mortality and is a continuing worldwide public health problem (Palese, 2004). In late April 2009, a novel influenza

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A (H1N1) virus caused a pandemic within a short period of time, which attracted great attention all over the world (Neumann, Noda, and Kawaoka, 2009). However, there are currently only two classes of anti-influenza A virus (IAV) drugs approved for the treatment of influenza, which are

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directed against the viral M2 protein (amantadine and rimantadine) and neuraminidase (zanamivir, oseltamivir, and peramivir), respectively (Krug and Aramini, 2009; Moscona, 2005). Nonetheless,

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there remain concerns regarding drug efficacy, resistance, toxicity, and cost (Hayden, 2006; Lagoja and De Clercq, 2008; Moscona, 2009). Hence, the development of novel antiviral agents with new target and low toxicity is of high importance.

Receptor tyrosine kinases (RTKs) are a group of growth factor receptors that regulate a variety of cellular activities related to growth, metabolism, and differentiation (Lemmon and

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Schlessinger, 2010). Many recent studies have shown that RTK molecules such as epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor (FGFR), and its downstream signaling pathways such as Raf/MEK/ERK, NF-κB and p38MAPK pathways can promote the

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invasion and replication processes of IAV (Eierhoff et al., 2010; Karlas et al., 2010; Konig et al., 2010; Kumar et al., 2008; Lee et al., 2005; Mazur et al., 2007; Pleschka et al., 2001). Kumar and

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co-workers reported that the two kinds of RTK inhibitor AG879 and A9 will not only inhibit the nuclear export of the vRNP complex, but also block viral RNA synthesis and virus release (Kumar et al., 2011a, 2011b). Pinto and co-workers found that inhibition of IAV-induced NF-κB and Raf/MEK/ERK activation can reduce both virus titers and cytokine expression in vitro and in vivo (Pinto et al., 2011). Moreover, specific inhibitors that block the activation of RTK pathway can impair IAV propagation through interfering with the cellular functions instead of viral proteins, thus do not easily lead to emergence of resistant virus variants (Ludwig et al., 2004). 3

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Therefore, RTKs may be used as novel anti-IAV targets and inhibitors of RTKs may be developed as potential anti-IAV agents. Our previous studies showed that a new polyketide compound, purpurquinone B (PPQ-B),

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derived from acid-tolerant fungal strain JS03-21, exhibited significant antiviral activity against influenza A virus H1N1 in vitro (Wang et al., 2011a). In the current study, we have extended our previous findings by investigating the anti-IAV effects and mechanisms of this compound in vitro

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and in vivo. Our results indicated that PPQ-B may block some stages after virus adsorption. Oral administration of PPQ-B could decrease pulmonary viral titers and improve survival rate in IAV

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infected mice. PPQ-B may down-regulate NF-κB and MAPK signal pathways to inhibit virus replication and inflammatory responses caused by IAV infection.

2. Materials and methods 2.1. Compounds and reagents

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Purpurquinone B (PPQ-B) was isolated from the fermentation broth of the acid-tolerant fungus Penicillium purpurogenum strain JS03-21 (Wang et al., 2011a). The purity of PPQ-B was determined by HPLC (Agilent 1260 HPLC system, Agilent Technologies, CA, USA), and the data

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indicated that PPQ-B had purity more than 98%. PPQ-B was dissolved in DMSO to prepare a solution with the concentration of 10 mg/ml, and stored under 4 ºC avoiding strong light. Rabbit

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anti-phosphorylated NF-κB-p65, PKCα, ERK1/2, Akt, and p38MAPK polyclonal antibodies and anti-β-actin, α-tubulin and GAPDH monoclonal antibodies were purchased from Cell Signaling Technology (Danvers, USA). Mouse anti-IAV NP antibody and alkaline phosphatase (AP)-labeled secondary antibodies were obtained from Santa Cruz Biotechnology (USA). FITC labeled secondary antibodies were obtained from Boster (Wuhan, China). ELISA kits of mouse TNF-α, IL-6, KC, RANTES, and ELISA kits of human TNF-α and IL-6 were purchased from Dakewei (Bejing, China). Ribavirin injection (50 mg/mL) was purchased from LuKang Cisen (Jining, 4

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China). Oseltamivir carboxylate was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Oseltamivir phosphate was obtained from Roche (Shanghai, China).

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2.2. Cell culture and virus infection

Madin-Darby canine kidney (MDCK) cells were grown in RPM1640 medium supplemented with 10% FBS, 100 U/mL of penicillin and 100 µg/mL of streptomycin. Human lung epithelial

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cells (A549 cells) were cultivated in F12 medium containing 10% FBS and 2 mM L-glutamine. Influenza virus (A/Puerto Rico/8/34 [H1N1]; PR8) was propagated in 10-day-old embryonated

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eggs for three days at 36.5 ºC. Influenza H1N1 virus A/California/04/2009 (Cal09) and H3N2 strain A/swine/Minnesota/02719/2009 were propagated in MDCK cells for 3 days at 37 °C. For virus infection, virus propagation solution was diluted in PBS containing 0.2% bovine serum albumin and was added to cells at the indicated multiplicity of infection (MOI). Virus was allowed to adsorb 60 min at 37 ºC. After removing the virus inoculum, cells were maintained in

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infecting media (RPM1640, 4 µg/mL trypsin) at 37 ºC in 5% CO2 (Wang et al., 2011b).

2.3. Cytopathic effect (CPE) inhibition assay

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The antiviral activity was evaluated by the cytopathic effects (CPE) inhibition assay as described previously (Hung et al., 2009; Leibbrandt et al., 2010). In brief, MDCK cells in 96-well

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plates were firstly infected with IAV (MOI=0.1), and then treated with different compounds in triplicate after removal of the virus inoculum. After 48 h incubation, the cells were fixed with 4% formaldehyde for 20 min at room temperature (RT). After removal of the formaldehyde, the cells were stained with 0.1% crystal violet for 30 min. The plates were washed and dried, and the intensity of crystal violet staining for each well was measured at 570 nm. The virus inhibition (%) was calculated by the equation: Virus inhibition (%) = [(Asample 570 − Avirus 570) / (Amock 570 − Avirus 570)] × 100; Where, Amock 570 was the absorbance without virus infection, Asample 570 was 5

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absorbance with virus infection and drug treatment, Avirus 570 was absorbance with virus infection but without drugs.

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2.4. Plaque assay

Confluent cell monolayers in 6-well plates were incubated with 10-fold serial dilutions of IAV at 37 °C for 1 h. The inoculum was removed; cells were washed with PBS and overlaid with

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maintenance DMEM medium containing 1.5% agarose, 0.02% DEAE-dextran, 1 mM L-glutamine, 0.1 mM non-essential amino acids, 100 U/ml penicillin, 100 µg/ml streptomycin

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and 1 µg/ml TPCK-treated trypsin. After incubation for 3 days at 37 °C in a humidified atmosphere of 5% CO2, cells were fixed with 0.05% glutaraldehyde, followed by staining with 1% crystal violet in 20% ethanol for plaque counting.

2.5. Hemagglutination (HA) assay

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The hemagglutination (HA) assay was performed as previously reported (Sriwilaijaroen et al., 2015; Wang et al., 2012). Standardized chicken red blood cell (cRBC) solutions were prepared according to the WHO manual. Virus propagation solutions were serially diluted 2-fold in round

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bottomed 96-well plate and 1% cRBCs were then added at an equal volume. After 60 min incubation at 4 ºC, RBCs in negative wells sedimented and formed red buttons, whereas positive

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wells had an opaque appearance with no sedimentation. HA titers are given as hemagglutination units/mL (HAU/mL).

2.6. Indirect immunofluorescence assay After drug treatment, MDCK cells were fixed with 4% PFA for 15 min, and permeabilized with 0.5% (v/v) Triton X-100 for 5min before incubated with 2% BSA/PBS for 1 h at 37 ºC. After washing, cells were then incubated consecutively with anti-IAV NP antibody and 6

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FITC–conjugated secondary antibody. Finally, cells were washed and directly observed using an inverted fluorescence microscope (DMI6000B; Leica, Germany) equipped with a cooled CCD

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camera.

2.7. Western Blot assay

Confluent A549 cell monolayers were firstly infected with IAV (MOI=1.0), and then treated

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with or without indicated compounds after virus adsorption. After incubation for 16 h, cell lysate was separated by SDS-PAGE and transferred to nitrocellulose membrane. After being blocked in

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Tris-buffered saline containing 0.1% Tween 20 (v/v) and 5% bovine serum albumin (w/v) at RT for 2 h, the membranes were rinsed and incubated at 4 ºC overnight with anti-phosphorylated NF-κB, PKCα, ERK1/2, Akt, and p38MAPK antibody or anti-β-actin and α-tubulin antibody as control. The membranes were washed and incubated with AP-labeled secondary antibody (1:2000 dilutions) at RT for 2 h. The protein bands were then visualized by incubating with the

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developing solution [p-nitro blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate toluidine (BCIP)] at RT for 30 min. The relative densities of proteins were all

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determined by using ImageJ (NIH) v.1.33u (USA).

2.8. In vivo experiments

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Four-week-old female Kunming mice (average weight, 16.0 ± 2.0 g) were housed and studied under protocols approved by the Animal Care and Use Committee of Ocean University of China (OUCYY-2016003). 10 mice per group were inoculated intranasally with PR8 (2LD50/mouse; 500 PFU/mouse) diluted in 40 µL of 1×PBS, and randomly divided into experimental groups. Two hours after inoculation, mice received oral therapy of either PPQ-B (5 or 10 mg/kg/day) or Oseltamivir phosphate (10 mg/kg/day), and the treatments were repeated once daily for the entire experiment. Mice were weighed and killed on day 4 after inoculation, and the lungs were then 7

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removed, weighed, and homogenized in 1×PBS for determination of viral titers by plaque assay. Histopathological analysis was performed using H&E and IHC staining (using an anti-NF-κB antibody as the primary antibody) on samples collected on 4 days post infection (dpi) as

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described previously (Fukushi et al., 2011). In the survival experiments, 10 mice per group were intranasally infected with PR/8 virus (4LD50/mouse; 1000 PFU/mouse) at Day 0. The drugs administration was repeated once daily

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during the experiment, and survival was assessed in all groups for 14 days after infection. A group of mice received an oral dose of Oseltamivir phosphate (10 mg/kg/day once daily for the

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entire experiment) was used as the positive control (Abed, Pizzorno, and Boivin, 2012; Smee et al., 2006). Mice were monitored daily for weight loss and clinical signs. If a mouse lost body weight over 25% of its pre-infection weight, it was defined as dead and humanely euthanized immediately; the rest of the mice were sacrificed at the end of experiment on 14 dpi.

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2.9. Statistics

All data are representative of at least three independent experiments. Data are presented as mean ± S.D. Statistical significance was calculated by SPSS 10.0 software using one-way

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ANOVA with Turkey's test or using nonparametric Wilcoxon rank-sum test, with P values < 0.05

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considered significant.

3. Results

3.1. Inhibition of influenza A virus multiplication in vitro by polyketide compound PPQ-B

Our previous studies showed that PPQ-B (Fig. 1) derived from acid-tolerant fungal strain JS03-21 exhibited significant antiviral activity against influenza A virus H1N1 in vitro (Wang et al., 2011a). Herein, we further explored the anti-IAV effects and mechanisms of this compound in 8

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vitro and in vivo. Firstly, the cytotoxicity of PPQ-B in MDCK cells was evaluated by MTT assay. The results showed that PPQ-B exhibited no significant cytotoxicity at the concentrations from 12.5 to 200 µg/ml (Fig. 2A). The CC50 (concentration required to reduce cell viability by 50%)

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value for PPQ-B was about 661.7 ± 12.2 µg/ml. The results were used to determine the dose range of PPQ-B for the subsequent experiments.

PPQ-B was then assayed for its ability to inhibit IAV multiplication in vitro using CPE

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inhibition assay and HA assay, as previously described (Leibbrandt et al., 2010; Wang et al., 2011b). Briefly, MDCK cells were firstly infected with PR8 virus (MOI=0.1), and then treated

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with compounds at the indicated concentrations after removal of the virus inoculum. After 48 h, virus titers of the culture media were determined by virus yield reduction assay, and the cell viability was measured by CPE inhibition assay. As shown in Fig. 2B and C, PPQ-B could significantly reduce the virus titer and promote cell viability when used at the concentration of more than 50 µg/ml (P < 0.05). The 50% inhibitory concentration (IC50 value) of PPQ-B was

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about 30.6 ± 2.8 µg/ml and the selectivity index (CC50/IC50) for PPQ-B was approximate 21.6, suggesting that PPQ-B possessed anti-IAV activities in vitro. To explore whether PPQ-B exerts broad antiviral spectrum, the plaque reduction assay was

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used to evaluate the inhibition of PPQ-B against two other clinical isolates of influenza viruses, H1N1 strain (A/California/04/2009) (Cal09) and H3N2 strain (A/swine/Minnesota/02719/2009).

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Briefly, approximately 50–100 PFU/well of Cal09 or H3N2 was pre-incubated with PPQ-B (0, 50, 100, 200 µg/ml) for 1 h at 37 °C before infection, respectively. Then the virus-PPQ-B mixture was transferred to MDCK cells, incubated at 4 °C for 1 h, and subjected to plaque reduction assay after removal of inoculum. As shown in Fig. 2D and E, PPQ-B could inhibit the plaque formation in Cal09 and H3N2-infected cells at the concentrations from 50 to 200 µg/ml, and could significantly reduce the plaque numbers when used at > 100 µg/ml (Fig. 2E), suggesting that PPQ-B possesses broad-spectrum anti-IAV activities.

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3.2. Influence of different treatment conditions of PPQ-B on IAV infection The time-of-addition assay was performed to determine the stage(s) at which PPQ-B exerted its inhibition actions in vitro. In brief, PPQ-B was added to MDCK cells at four distinct time points:

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pre-treatment of viruses, pre-treatment of cells, during virus adsorption, or after virus adsorption. Subsequently, the antiviral activity was determined by HA assay (Sriwilaijaroen et al., 2015; Wang et al., 2012). As shown in Fig. 3A, pretreatment of PR8 virus with 50 µg/ml PPQ-B for 1 h

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before infection significantly reduced the virus titer of IAV, compared to the non-drug treated virus control group (P<0.01), suggesting that PPQ-B may directly interact with IAV particles.

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However, addition of PPQ-B simultaneously with virus adsorption or pretreatment of cells did not significantly decrease the virus titers in vitro (Fig. 3A), suggesting that PPQ-B may not interact with MDCK cells directly. Surprisingly, addition of PPQ-B after adsorption also significantly reduced the virus titers (P<0.05) (Fig. 3A). Thus, PPQ-B might be able to inactivate virus particles directly or block some steps after adsorption.

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To further explore which viral stage after adsorption is inhibited by PPQ-B, another time course study was performed (Alam, 2006). Briefly, IAV (MOI = 1.0)-infected MDCK cells were treated with 50 µg/mL of PPQ-B for different time intervals, then the virus yields at 24 h p.i. were

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evaluated by HA assay (Sriwilaijaroen et al., 2015). The results showed that PPQ-B treatment for the first 2 h (0-2 h p.i.) or second 2 h (2-4 h p.i.) after adsorption significantly reduced the virus

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titers to about 13% and 25% of that in virus control group (P < 0.05), respectively (Fig. 3B), which suggested that PPQ-B may be able to inhibit some steps after virus adsorption. However, PPQ-B treatment at later time intervals (4-8 h p.i. or 8-24 h p.i.) could not significantly decrease the virus titers (P > 0.05) (Fig. 3B), suggesting that PPQ-B’s antiviral activity may be largely related to its inhibition of virus life-cycle steps that occur 0-4 h after adsorption. Moreover, we also explored whether PPQ-B could interact with virus surface HA protein by using the HA inhibition (HI) assay. The results showed that the anti-HA antibodies significantly inhibited the PR8 virus-induced aggregation of chicken erythrocytes at the concentrations of 10

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0.625-5 µg/ml (Fig. 3C). However, PPQ-B could not inhibit virus-induced aggregation of chicken erythrocytes even at a concentration of 50 µg/ml (Fig. 3C), suggesting that PPQ-B may have no direct interaction with virus HA protein. Then the interaction between PPQ-B and NA protein was

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evaluated by NA inhibition assay (Hashem et al., 2009). The results showed that PPQ-B could not significantly inhibit the activity of NA protein (Inhibition percentage < 30%) at the concentrations of 12.5-100 µg/ml, suggesting that virus NA protein may be not the target of PPQ-B (Fig. 3D).

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Furthermore, the effect of PPQ-B on IAV protein expression and localization after adsorption was evaluated by immunofluorescence assay. As shown in Fig. 3E-H, in IAV-infected cells

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without drug treatment, the fluorescence of viral NP proteins could be obviously found in cell nucleus and cytoplasm (Fig. 3F), while after treatment with PPQ-B for 2 h, only little fluorescence could be found in cell cytoplasm and nucleus (Fig. 3H), similar to that in Ribavirin (50 µg/ml) treated cells (Fig. 3G). In addition, PPQ-B treatment significantly inhibited the virus RNP release from cell nucleus at 4 h p.i. (Fig. 3K) as compared to that in virus control cells (Fig.

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3I). However, at 6 h p.i., the fluorescence of viral NP proteins could also be found in cell cytoplasm (Fig. 3L), suggesting that PPQ-B may be able to delay the export of viral RNP

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complex from nucleus.

3.3. The inhibition of PPQ-B on cellular EGFR pathways in host cells

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It was reported that some cellular signaling receptors such as epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor (FGFR) are indispensable for IAV entry process (Eierhoff et al., 2010; Karlas et al., 2010; Konig et al., 2010). Thus, the influence of PPQ-B on the activation of downstream signal molecules of EGFR pathway, such as NF-κB and PKCα, was explored by western blot. As shown in Fig. 4A and B, the phosphorylated NF-κB was significantly increased to 3.7-fold higher than normal control group after IAV infection for 16 h (P < 0.01). However, after treatment with PPQ-B (50, 25, 12.5 µg/ml) for 16 h, the level of

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phosphorylated NF-κB protein was significantly reduced from 3.7 to about 0.6, 1.4 and 2.9-fold of normal control group (P < 0.05), respectively (Fig. 4B). Moreover, treatment with PPQ-B (50, 25, 12.5 µg/ml) for 16 h also significantly inhibited the activation of PKCα from 1.4 to about 0.2,

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0.3 and 0.5-fold of normal control group, respectively (P < 0.01) (Fig. 4A and B). These data indicated a crucial role of NF-κB signaling in the anti-IAV mechanism of PPQ-B in vitro.

Moreover, the downstream MAPK and PI3K/Akt signaling of EGFR pathway was reported to

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be required for efficient vRNP export from nucleus and IAV endocytosis, and the inhibitors of MAPK and Akt could reduce IAV replication and inflammatory symptoms (Hirata et al., 2014;

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Lee et al., 2005; Ludwig et al., 2004; Pinto et al., 2011; Pleschka et al., 2001). In this study, ERK1/2 and p38MAPK was significantly activated in virus control group to about 1.5 and 8.5-fold higher than normal control group at 16 h p.i, respectively. (Fig. 4C and D). However, treatment with PPQ-B (50, 25, 12.5 µg/ml) for 16 h significantly decreased the expression level of phosphorylated ERK1/2 and p38MAPK proteins from over 1.5 and 8.5 to less than 1.0 and

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3.0-fold of normal control group (P < 0.01), respectively (Fig. 4C and D). In addition, treatment with PPQ-B (50, 25 µg/ml) after adsorption also significantly decreased the expression levels of p-Akt from about 12.0 to about 6.7 and 7.1-fold of the normal control group (P < 0.01),

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respectively (Fig. 4C and D). Thus, cellular MAPK and PI3K/Akt pathways may also be involved in the anti-IAV actions of PPQ-B in vitro.

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To further verify the inhibition of PPQ-B on IAV induced activation of EGFR pathway, the western blot assay of p-NF-κB in Oseltamivir (50 µg/ml) or PPQ-B (50, 25 µg/ml) treated cells at 4 h p.i. was performed. The results showed that PPQ-B treatment after adsorption could significantly inhibit the phosphorylation of NF-κB at 4 h p.i. (P < 0.01), while Oseltamivir did not significantly reduce the levels of p-NF-κB in IAV infected cells (Fig. 4E and F), suggesting that PPQ-B can truly inhibit the activation of NF-κB pathways in IAV infected cells. Moreover, treatment with PPQ-B (50, 25 µg/ml) for 6 h significantly decreased the expression of virus NP protein as compared to the virus control group (P < 0.01) (Fig. 4G and H). Ribavirin (50 µg/ml) 12

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also significantly reduced the expression of NP protein (P < 0.05) at 6 h p.i (Fig. 4H). Therefore, PPQ-B may be able to interfere with the activation of cellular EGFR pathways to inhibit IAV

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replication in vitro.

3.4. Oral administration of PPQ-B significantly supports survival of mice infected with H1N1 virus.

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The anti-IAV effects of PPQ-B in vivo were also explored using mouse pneumonia model as described previously (Barnard, 2009). Oral administration of Oseltamivir (10 mg/kg/day) was

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used as the positive control as described previously (Abed, Pizzorno, and Boivin, 2012; Smee et al., 2006). Briefly, IAV-infected mice received oral administration of PPQ-B (5 and 10 mg/kg/day) or Oseltamivir (10 mg/kg/day) once daily for the entire experiment, and then sacrificed at 4 days p.i., respectively. Subsequently, the pulmonary viral titers were determined by plaque assay as described previously (Sriwilaijaroen et al., 2015). As shown in Fig. 5A and Table

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1, after treatment of PPQ-B (5 and 10 mg/kg/day) for 4 days, the pulmonary viral titers were significantly decreased compared to that in the virus control group (P < 0.05), suggesting that oral therapy with PPQ-B could effectively inhibit IAV multiplication in mice lungs. Oseltamivir (10

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mg/kg/day) treatment also showed significant reduction of virus titers in mice lungs (P < 0.05) (Fig. 5A and Table 1). Besides that, PPQ-B (5 or 10 mg/kg/day) also significantly prevented the

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weight loss of IAV infected mice compared to the virus control group (P < 0.05) (Table 1). Furthermore, the survival experiments were also performed to evaluate the effect of PPQ-B on the survival of IAV infected mice. As shown in Fig. 5B, oral administration with PPQ-B (10 mg/kg/day) could significantly increase survival rates as compared to the placebo-treated control group (P < 0.05). In the particular experiment shown in Fig. 5B, by day 14 p.i., only 30% of the individuals in the placebo group could survive whereas 80% of that in the PPQ-B (10 mg/kg/day) treated group survived, comparable to that in Oseltamivir treated group (80%). Oral 13

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administration with PPQ-B at the dose of 5 mg/kg/day also improved survival of IAV infected mice but without significance (Fig. 5B). In addition, the weights of the mice in virus control group (Placebo) began to decrease at 4 days p.i., losing up to about 23% of initial weight, before

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gradually recovering. In contrast, the PPQ-B (10 mg/kg/day) treated mice gradually increased their body weights without weight loss (Fig. 5C), comparable to the effect of Oseltamivir (10 mg/kg/day). In summary, these results suggested that PPQ-B also possessed good anti-IAV

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activities in vivo.

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3.5. PPQ-B significantly attenuated the inflammatory responses in vitro and in vivo. To further evaluate the effects of PPQ-B on viral pneumonia in mice, histopathology analysis was also performed. As shown in Fig. 5D-H, lung tissues in virus-control group showed marked infiltration of inflammatory cells in the alveolar walls and the presence of massive serocellular exudates in the lumen (Fig. 5E). However, mice treated with PPQ-B (5 or 10 mg/kg/day)

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following infection had intact columnar epithelium in the bronchiole even in the presence of some serocellular exudates in the lumen (Fig. 5G and H). Moreover, the lung tissues with Oseltamivir (10 mg/kg/day) treatment also showed intact columnar epithelium (Fig. 5F). PPQ-B also

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significantly reduced the lung index with an inhibition rate of 21.4% and 34.1% when used at a dose of 5 and 10 mg/kg/day (P < 0.05), respectively, comparable to that in Oseltamivir treated

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group (34.9%) (Table 1). Thus, PPQ-B may be able to attenuate pneumonia symptoms in IAV infected mice.

Since PPQ-B could inhibit MAPK and NF-κB signal pathways in A549 cells, we then investigated the inhibition effects of PPQ-B against virus induced cytokine secretion in mice lungs. As shown in Fig. 6A, the production of cytokine TNF-α and IL-6 significantly increased after IAV infection compared to that of the normal control group (P < 0.05). After oral treatment with PPQ-B (10 mg/kg/day) for 4 days, the production of TNF-α and IL-6 significantly decreased

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from over 550 pg/mL to about 200 and 300 pg/mL, respectively, as compared to that in the virus control group (P < 0.05) (Fig. 6A). In addition, after treatment with PPQ-B (10 mg/kg/day) for 4 days, the secretion of chemokine RANTES and KC also significantly decreased compared to that

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of the virus control group (P < 0.05) (Fig. 6B). The oral treatment of PPQ-B at 5 mg/kg/day could also significantly decrease the secretion of TNF-α and KC in IAV infected lungs (Fig. 6A and B). These results suggested that the anti-IAV effects of PPQ-B in vivo might also be related to its

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inhibition on inflammatory responses caused by IAV infection.

Moreover, the immunohistochemistry (IHC) assay was also performed to detect the production

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of NF-κB in lung tissues. Briefly, after treatment with PPQ-B (10 mg/kg/day) or placebo (PBS) for 4 days, the level of NF-κB in lung tissues was evaluated by IHC using anti-NF-κB antibody. As shown in Fig. 6D, the production of NF-κB significantly increased after IAV infection compared to that of the normal control group (Fig. 6C). After treatment with PPQ-B (10 mg/kg/day) for 4 days, the production of NF-κB markedly decreased compared to that of the virus

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control (PR8) group (Fig. 6F), similar to that in Oseltamivir (10 mg/kg/day) treated group (Fig. 6E). Thus, the inhibition of PPQ-B on virus induced inflammatory responses in vivo might be related to its inhibition of NF-κB signaling pathway.

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Furthermore, the inhibition of PPQ-B on IAV-induced production of cytokines TNF-α and IL-6 in A549 cells was also explored by ELISA. As shown in Fig. 6G, the production of TNF-α and

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IL-6 significantly increased after IAV infection as compared to that of the normal control group (P < 0.05). However, after treatment with PPQ-B (100, 50, 25 µg/ml) for 16 h, the production of TNF-α and IL-6 significantly decreased from about 76 and 21 pg/mL to less than 45 and 10 pg/mL, respectively, compared to that of the virus control group (P < 0.05) (Fig. 6G). Treatment of Oseltamivir (100 µg/ml) for 16 h could not significantly reduce the production of TNF-α and IL-6 in IAV infected cells (Fig. 6G). Moreover, PPQ-B (12.5, 25, 50, 100 µg/ml) could significantly decrease the nitrite production in LPS induced RAW264.7 cells (P < 0.05) (Fig. 6H), suggesting that PPQ-B may also inhibit the TLR4-dependent inflammatory responses. Therefore, 15

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PPQ-B may be able to attenuate the inflammatory responses in vitro and in vivo through interfering the activation of NF-κB and MAPK signaling pathways.

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4. Discussion

Recently, natural products (NPs) derived from fungi have been attracting increasing interest in developing potential anti-viral drugs (Peng et al., 2012; Wang et al., 2011a, 2011c). Our previous

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studies showed that a new NP derived from acid-tolerant fungal strain JS03-21 exhibited antiviral activity against IAV in vitro (Wang et al., 2011a). Herein, our results indicated that the novel

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polyketide compound purpurquinone B (PPQ-B) suppressed the replication of IAV in vitro with low toxicity (SI =21.6), and may block some stages after virus adsorption. PPQ-B could inhibit PR8 (H1N1), Minnesota (H3N2) and Cal09 (H1N1) virus replication in vitro, suggesting that PPQ-B possesses broad-spectrum anti-IAV activities. Oral administration of PPQ-B could decrease pulmonary viral titers and improve survival rate in IAV infected mice. Thus, PPQ-B has

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the potential to be developed into a novel anti-IAV drug in the future. The time-of-addition assay showed that pretreatment of PR8 virus with PPQ-B before infection markedly reduced the viral titers (Fig. 3), suggesting that PPQ-B may have direct inactivation

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effects on IAV particles. Interestingly, post-treatment of cells with PPQ-B after adsorption also significantly reduced the virus titers and viral protein expression, and PPQ-B could delay the

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export of viral RNP complex from nucleus, which suggested that PPQ-B may block some stages after virus adsorption (Fig. 3). Another time course study indicated that PPQ-B’s antiviral activity may be largely related to its inhibition of virus life-cycle steps that occur 0-4 h after adsorption (Fig. 3B). Moreover, PPQ-B did not directly bind HA protein to block virus-induced aggregation of chicken erythrocytes (Fig. 3C), and could not significantly inhibit the activity of NA protein (Fig. 3D), we supposed that PPQ-B may possibly interfere with the entry and replication of IAV through blocking the interaction of IAV particles with cellular membrane receptors or directly 16

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inhibiting the activation of intracellular signaling pathways. Receptor tyrosine kinases (RTKs) such as FGFR and EGFR and their downstream signaling pathways such as NF-κB and p38MAPK pathways are reported to be able to promote the invasion

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and replication processes of IAV (Eierhoff et al., 2010; Karlas et al., 2010; Konig et al., 2010; Kumar et al., 2008; Lee et al., 2005; Mazur et al., 2007; Pleschka et al., 2001). Pinto and co-workers found that inhibition of IAV-induced NF-κB and Raf/MEK/ERK activation could

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reduce both virus titers and cytokine expression in vitro and in vivo (Pinto et al., 2011). Herein, we found that PPQ-B significantly inhibited the activation of cellular PKCα, NF-κB, ERK1/2,

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Akt, and p38MAPK proteins in IAV infected cells (Fig. 4), which suggested PPQ-B may interfere with the activation of EGFR signaling pathway and its downstream NF-κB and MAPK pathway. Moreover, PPQ-B significantly decreased the production of cytokine TNF-α, IL-6 and chemokine RANTES and KC in IAV infected mice (Fig. 6), suggesting that PPQ-B may also attenuate the inflammatory responses caused by IAV infection. Moreover, PPQ-B rather than Oseltamivir

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could also significantly reduce the production of TNF-α and IL-6 in IAV infected A549 cells (Fig. 6G). Thus, PPQ-B may be able to attenuate the inflammatory responses in vitro and in vivo through interfering the activation of NF-κB and MAPK signaling pathways.

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PPQ-B’s in vitro antiviral effects were mirrored in a murine model of influenza. Oral treatment of PR8-infected mice with PPQ-B markedly improved their survival and decreased pulmonary

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virus titers, comparable to the effects of Oseltamivir (Fig. 5). PPQ-B treatment could also inhibit the infiltration of inflammatory cells and serocellular exudates in the lumen, and significantly decrease the secretion of inflammatory factors TNF-α, IL-6, RANTES and KC in IAV infected lungs (Fig. 6), suggesting that the anti-IAV effects of PPQ-B in vivo might also be related to its inhibition actions on inflammatory responses caused by IAV infection. In addition, PPQ-B (10 mg/kg/day) treatment also reduced the production of NF-κB in IAV infected lungs, which suggested that the inhibition of PPQ-B on inflammation in vivo might also be related to its downregulation of NF-κB pathway. Moreover, PPQ-B could also significantly decrease the nitrite 17

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production in LPS induced macrophages (Fig. 6H), suggesting that PPQ-B may also be able to interfere with the TLR4-dependent inflammatory responses in IAV infected mice. In conclusion, the compound PPQ-B derived from acid-tolerant fungal can significantly inhibit

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the replication of IAV in vitro and in vivo, and may block IAV infection through interfering the activation of cellular EGFR pathway such as NF-κB and MAPK pathways. Further studies of the antiviral effects of PPQ-B against highly pathogenic IAV strains (H5N1 or H7N9 strain) will be

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required to advance it for drug development. However, our studies indicated that the natural

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compound PPQ-B has the potential to be developed into a novel anti-IAV agent in the future.

Acknowledgments

This study was supported in part by National Natural Science Foundation of China (81302811, 81561148012, 81373298 and 31500646), NSFC-Shandong (U1406402) and -Guangdong (U1501221) Joint Funds, and the Promotive Research Fund for Excellent Young and

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Figure legends Figure 1. Schematic diagram of polyketide compound PPQ-B.

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Figure 2. PPQ-B effectively inhibited replication of IAV in vitro. (A) MDCK cells were exposed to different concentrations of PPQ-B in triplicate, and incubated at 37 ºC for 48 h. Then the cell viability was evaluated by MTT assay. The results were presented as a percentage of

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control group. Values are means ± S.D. (n=3). (B) IAV (A/Puerto Rico/8/34 [H1N1]; PR8) (MOI=0.1) infected MDCK cells were treated with PPQ-B at the indicated concentrations after

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adsorption. The antiviral activity was determined by the CPE inhibition assay at 48 h p.i. Results are expressed as percent of inhibition in drug-treated cultures compared with untreated. Values are means ± S.D. (n = 4). (C) IAV (MOI=0.1) infected MDCK cells were treated with PPQ-B at the indicated concentrations after removal of the virus inoculum. The antiviral activity was determined by plaque assay at 48 h p.i. Values are means ± S.D. (n = 4). Significance: *P < 0.05,

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**P < 0.01 vs. virus control group. (D) Approximately 50–100 PFU/well of H1N1 virus A/California/04/2009 (Cal09) or H3N2 strain A/swine/Minnesota/02719/2009 (H3N2) were pre-incubated with PPQ-B (0, 50, 100, 200 µg/ml) for 1 h at 37 °C before infection, respectively.

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Then the virus-PPQ-B mixture was transferred to MDCK cells, incubated at 4 °C for 1 h and subjected to plaque reduction assay after removal of inoculum. (E) Plaque number from plaque

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reduction assays (D) performed on MDCK cells infected with the two viruses and treated with the indicated concentrations of PPQ-B. Values are means ± S.D. (n = 3). *P < 0.05, **P < 0.01 vs. virus control group (PR8).

Figure 3. Influence of different treatment conditions of PPQ-B on IAV infection. (A) MDCK cells were infected with PR8 (MOI=0.1) by four different treatment conditions. i) Pretreatment of virus: IAV was pretreated with 50 µg/ml of PPQ-B at 37 °C for 1 h before infection. ii) 23

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Pretreatment of cells: MDCK cells were pretreated with 50 µg/ml of PPQ-B at 37 °C for 1 h before infection. iii) Adsorption: MDCK cells were infected in media containing 50 µg/ml of PPQ-B and, after 1 h adsorption at 4 °C, were overlaid with compound-free media. iv) After

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adsorption: after removal of unabsorbed virus the infecting media containing 50 µg/ml of PPQ-B were added to cells. At 48 h p.i., the virus yields were determined by plaque assay. Values are means ± S.D. (n = 3). *P < 0.05 vs. virus control group. (B) PR8 (MOI=1.0) infected MDCK cells

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were treated with 100 µg/mL of PPQ-B for the specified time period after adsorption, and then the media were removed and cells were overlaid with compound-free media. Then at 24 h p.i., the

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virus titers in the cell supernatants were determined by HA assay. Values are means ± S.D. (n = 3). *P < 0.05 vs. virus control group. (C) The inhibition effects of PPQ-B and anti-HA antibody on influenza virus PR8 induced aggregation of chicken erythrocytes were evaluated by hemagglutination inhibition (HI) assay. (D) Inactivated PR8 virus was incubated with indicated concentrations of PPQ-B or Zanamivir (10 µM), and the NA enzymatic activity was determined

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by a fluorescent assay. Values are means ± S.D. (n = 3). (E-H) IAV (MOI=1.0) infected MDCK cells were treated with or without 50 µg/ml of PPQ-B or Ribavirin after virus adsorption and then incubated at 37 ºC for 4 h. After that, NP protein expression was determined by

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immunofluorescence assay. Scale bar represents 25 µm. (I-L) IAV (MOI=1.0) infected MDCK cells were treated with or without PPQ-B (50 µg/ml) for 4 h or 6 h. After that, the localization of

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NP protein was detected by immunofluorescence assay. Scale bar represents 25 µm.

Figure 4. The influence of PPQ-B on cellular EGFR signaling pathways. (A) IAV (MOI = 1.0) infected A549 cells were treated with or without drugs at indicated concentrations after removal of virus inoculums. At 16 h p.i., the phosphorylation of NF-κB and PKCα proteins was determined by western blotting. Blots were also probed for β-actin protein as loading controls. (B) Quantification of immunoblot for the ratio of p-NF-κB or p-PKCα to β-actin. The ratio for normal control group was assigned values of 1.0 and the data presented as mean ± SD (n = 3). #P < 0.05, 24

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##P < 0.01 vs. normal control group; *P < 0.05, **P < 0.01 vs. virus control group. (C) IAV (MOI = 1.0) infected cells were treated with indicated compounds after removal of virus inoculums. At 16 h p.i., the phosphorylation of ERK1/2, p38 and Akt proteins was evaluated by western blot.

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Blots were also probed for α-tubulin or β-actin as loading controls. (D) Quantification of immunoblot for the ratio of p-ERK1/2, p-p38 to tubulin or p-Akt to actin. The ratio for normal control group was assigned values of 1.0 and the data presented as mean ± SD (n = 3). ##P < 0.01

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vs. normal control group; **P < 0.01 vs. virus control group. (E) IAV (MOI = 1.0) infected A549 cells were treated with PPQ-B (50, 25 µg/ml) or Oseltamivir (50 µg/ml) after removal of virus

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inoculums. At 4 h p.i., the phosphorylation of NF-κB was determined by western blot. (F) Quantification of immunoblot for the ratio of p-NF-κB to actin. The ratio for normal control group was assigned values of 1.0 and the data presented as mean ± SD (n = 3). ##P < 0.01 vs. normal control group; **P < 0.01 vs. virus control group. (G) IAV (MOI = 1.0) infected cells were treated with indicated compounds for 6 h, and then the expression of virus NP protein was

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evaluated by western blot. Blots were also probed for GAPDH as loading controls. (H) Quantification of immunoblot for the ratio of NP protein to GAPDH. The ratio for normal control group was assigned values of 1.0 and the data presented as mean ± SD (n = 3). ##P < 0.01 vs.

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normal control group; *P < 0.05, **P < 0.01 vs. virus control group.

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Figure 5. The anti-IAV effects of PPQ-B in vivo. (A) Viral titers in lungs. After oral treatment with PPQ-B (5 or 10 mg/kg/day) or Oseltamivir (10 mg/kg/day) for 4 days, the pulmonary viral titers were evaluated by plaque assay. The result shown is a representative of three separate experiments with similar results. Values are means ± S.D. (n = 5 mice/group). Significance: *P < 0.05 vs. virus control group. (B) Survival rate. IAV infected mice were received oral therapy with PPQ-B (5 or 10 mg/kg/day) or Oseltamivir (10 mg/kg/day) for the entire experiment. Results are expressed as percentage of survival, evaluated daily for 14 days. Significance: *P < 0.05 vs. control group (placebo). (C) IAV infected mice were received oral therapy with PPQ-B (5 or 10 25

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mg/kg/day) or placebo for the entire experiment. The body weights of ten mice in each group were monitored daily for 14 days and are expressed as a percentage of the initial value. The data represents the mean of ten mice in each group. (D-H) Histopathologic analyses of lung tissues on

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Day 4 p.i. by HE staining (×10). The representative micrographs from each group were shown (n = 5 mice/group). Mock: non-infected lungs; PR8: IAV infected lungs without drugs; PR8+Oseltamivir-10: IAV infected lungs with Oseltamivir (10 mg/kg/day) treatment;

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infected lungs with PPQ-B (10 mg/kg/day) treatment.

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PR8+PPQ-B-5: IAV infected lungs with PPQ-B (5 mg/kg/day) treatment; PR8+PPQ-B-10: IAV

Figure 6. PPQ-B decreased the secretion of cytokines and chemokines in vitro and in vivo. (A) After treatment with PPQ-B (5or 10 mg/kg/day) or placebo (PBS) for 4 days, the production of cytokines TNF-α and IL-6 in lung tissues was determined by ELISA assay. The result shown is a representative of four separate experiments with similar results. Values are means ± S.D. (n = 10

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mice/group). Significance: #P < 0.05 vs. normal control group; *P < 0.05, **P < 0.01 vs. virus control group. (B) After oral therapy with PPQ-B (5or 10 mg/kg/day) or placebo (PBS) for 4 days, the production of chemokines RANTES and KC in lung tissues was determined by ELISA. The

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result shown is a representative of three separate experiments with similar results. Values are means ± S.D. (n = 10 mice/group). Significance: #P < 0.05 vs. normal control group; *P < 0.05 vs.

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virus control group. (C-F) Immunohistochemistry analysis of lung tissues on Day 4 p.i. (×10) using anti-NF-κB monoclonal antibody as primary antibody. The representative micrographs from each group were shown (n = 5 mice/group). Mock: non-infected lungs; PR8: IAV infected lungs without drugs; PR8+Oseltamivir-10: IAV infected lungs with Oseltamivir (10 mg/kg/day) treatment; PR8+PPQ-B-10: IAV infected lungs with PPQ-B (10 mg/kg/day) treatment. (G) After treatment with PPQ-B (100, 50, 25 µg/ml) or Oseltamivir (100 µg/ml) for 16 h, the production of TNF-α and IL-6 in IAV infected A549 cells was determined by ELISA. Values are means ± S.D. (n = 3). #P < 0.05, ##P < 0.01 vs. normal control group; *P < 0.05, **P < 0.01 vs. virus control 26

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group. (H) After pre-treatment of RAW264.7 cells with different concentrations of PPQ-B (12.5, 25, 50, 100 µg/ml) for 2 h, the LPS (100 ng) was added to cells and incubated for 16 h. Then the content of nitrite in cell supernatant was determined by ELISA. Values are means ± SD (n = 5).

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##P < 0.01 vs. mock control; *P < 0.05, **P < 0.01 vs. LPS treated control.

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Tables

Table 1. The therapeutic effects of compounds on IAV infected mice. Dose Groups

Lung indexa

Inhibition

Pulmonary viral

(X±SD)

rateb (%)

titersc (PFU/mL)

—

—

0

(1.6 ± 0.05) x 104

Body weight (g) (mg/kg/day) —

22.11 ± 0.98

0.64 ± 0.05

Virus Control

—

16.99 ± 2.66##

1.26 ± 0.21##

Oseltamivir

10

20.20 ± 1.93*

0.82 ± 0.09*

34.9

(3.5 ± 0.07) x 102*

PPQ-B

5

19.48 ± 2.85*

0.99 ± 0.05*

21.4

(2.1 ± 0.07) x 103*

PPQ-B

10

21.53 ± 2.95**

0.83 ± 0.04*

34.1

(1.8 ± 0.17) x 102*

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Normal Control

Values are means ± S.D. (n = 10 mice/group). ##p < 0.01 vs. normal control group; *p < 0.05, **p < 0.01 vs. virus control

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group (nonparametric Wilcoxon test). Lung index = (lung weight (g) / mice weight (g)) × 100. The lung index was determined on Day 4 p.i.

b

Inhibition rate (%) = [(Lung index virus control − Lung index sample group) / Lung index virus control] × 100.

c

Pulmonary viral titers (PFU/mL): The pulmonary viral titers on Day 4 p.i. were evaluated by plaque assay.

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Figure 2

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Research highlights 1.

PPQ-B suppressed IAV replication in vitro with low toxicity, and possessed broad-spectrum anti-IAV activities. PPQ-B’s antiviral activity may be largely related to its inhibition of some steps that occur

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2.

0-4 h after adsorption.

Oral administration of PPQ-B decreased pulmonary viral titers and improved survival

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3.

rate in IAV infected mice.

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PPQ-B may down-regulate NF-κB and MAPK signaling pathways to inhibit virus

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replication.

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4.