Antiviral activity against enterovirus 71 of sulfated rhamnan isolated from the green alga Monostroma latissimum

Accepted Manuscript Title: Antiviral activity against enterovirus 71 of sulfated rhamnan isolated from the green alga Monostroma latissimum Authors: Shuyao Wang, Wei Wang, Cui Hao, YunjiaYu, Ling Qin, Meijia He, Wenjun Mao PII: DOI: Reference:

S0144-8617(18)30859-2 https://doi.org/10.1016/j.carbpol.2018.07.067 CARP 13867

To appear in: Received date: Revised date: Accepted date:

10-6-2018 20-7-2018 23-7-2018

Please cite this article as: Wang S, Wang W, Hao C, YunjiaYu, Qin L, He M, Mao W, Antiviral activity against enterovirus 71 of sulfated rhamnan isolated from the green alga Monostroma latissimum, Carbohydrate Polymers (2018), https://doi.org/10.1016/j.carbpol.2018.07.067 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.

Antiviral activity against enterovirus 71 of sulfated rhamnan isolated from the green alga Monostroma latissimum

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Shuyao Wanga, Wei Wanga,b*, Cui Haoc, YunjiaYua, Ling Qina, Meijia Hea, Wenjun Maoa,b*

Key Laboratory of Marine Drugs of Ministry of Education, Shandong Provincial Key Laboratory of

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Glycoscience and Glycotechnology, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China

Laboratory for Marine Drugs and Bioproducts of Qingdao National Laboratory for Marine Science

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Institute of Cerebrovascular Diseases, Affiliated Hospital of Qingdao University Medical College,

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and Technology, Qingdao 266237, China

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Qingdao, 266003, China.

Corresponding author. Tel.: +86 532 8203 1560; fax: +86 532 8203 3054. E-mail address:

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[email protected] (W.Wang), [email protected] (W.-J.Mao)

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Chemical compounds studied in this article

Guanidine hydrochloride (PubChem CID: 5742); Sulfo-NHS-SS-Biotin (PubChem CID: 71571496); Ribavirin (PubChem CID: 37542); Crystal violet (PubChem CID: 11057); Paraformaldehyde

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(PubChem CID: 712); DAPI dihydrochloride (PubChem CID:160166); Nitroblue tetrazolium chloride (PubChem CID: 9281); 5-Bromo-4-chloro-3-indoxyl phosphate (PubChem CID: 65409); Triton X-100 (PubChem CID: 5590); Tween-20 (PubChem CID: 443314)

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Highlights

► Sulfated polysaccharide PML was prepared from the green alga Monostroma latissimum.

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► PML effectively blocked EV71infection in vitro and showed excellent anti-EV71 activity

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in vivo.

►PML could strongly bind to VP1 protein of EV71 with evident dose dependence.

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►PML inhibit EV71 infection through targeting cellular EGFR/PI3K/Akt pathway.

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► PML could be a novel antiviral agent for therapy and prophylaxis of EV71 infection.

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Abstract

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Polysaccharide from Monostroma latissimum PML is a sulfated rhamnan, which consists of

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→3)-α-L-Rhap-(1→ and →2)-α-L-Rhap-(1→ residues with partial branches and sulfate groups at C-2 of →3)-α-L-Rhap-(1→ and/or C-3 of →2)-α-L-Rhap-(1→. The anti-enterovirus 71 (EV71) activity in vitro of PML was assessed by cytopathic effect inhibition and plaque reduction assays,

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and the results showed that PML was non-cytotoxic and significantly inhibited EV71 infection. The mechanism analysis of anti-EV71 activity demonstrated that PML largely inhibited viral replication before or during viral adsorption, mainly by targeting the capsid protein VP1. PML may also inhibit 2

some early steps of infection after viral adsorption by modulating signaling through the epidermal growth factor receptor (EGFR)/phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway. Moreover, PML markedly improved survival and decreased viral titers in EV71-infected mice. The investigation revealed that PML has potential as a novel anti-EV71 agent targeting the viral capsid

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protein as well as cellular EGFR/PI3K/Akt pathway.

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Keywords: Sulfated rhamnan; Enterovirus 71; Antiviral effect; VP1 protein; EGFR/PI3K/Akt pathway

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1. Introduction

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Enterovirus 71 (EV71) is a non-enveloped, positive-sense single-stranded RNA virus, which is one of the major causative agents of hand, foot and mouth disease in infants and young children. In

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some individuals, EV71 can infect the central nervous system and cause severe and life-threatening

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neurological disorders, including aseptic meningitis acute flaccid paralysis, myoclonus with autonomic dysfunction, encephalitis, and cardiac failure (Ooi, Wong, Lewthwaite, Cardosa, &

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Solomon, 2010; Tsai et al., 2012). According to the National Health and Family Planning Commission of the People’s Republic of China, EV71 caused at least 17,717,819 infections and

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3,509 deaths from 2009 to 2017 in China. Only a few vaccines and antiviral drugs have been approved for prophylaxis or treatment of EV71 infection (Pourianfar & Grollo, 2015). Hence, development of efficacious and non-toxic anti-EV71 drugs is urgently needed.

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A single enterovirus virion is typically composed of 60 copies of capsids, each of which is made up of four capsid proteins: VP1, VP2, VP3 and VP4 (Shia et al., 2002). It has been reported that VP1 might contain receptor-binding sites, and that it might determine cell tropism and regulate virus maturation. Thus, VP1 inhibitors have been proposed as candidate for antiviral against EV71.

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Moreover, the cellular PI3K/Akt signaling pathway plays important roles in cell survival, apoptosis, proliferation, migration and differentiation, as well as in metabolic regulation. This pathway has also

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been reported to be associated with virus uptake and virus induced autophagy (Diehl & Schaal, 2013; Lin et al., 2017). Thus, inhibitors of viral VP1 protein and cellular PI3K/Akt pathway may be used

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alone or in combination with other drugs to block both infection and replication of EV71.

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Sulfated polysaccharides from green algae often possess potential antiviral activities (Ngo &

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Kim, 2013; Shi et al., 2017). An acidic polysaccharide purified from the green alga Coccomyxa

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gloeobotrydiformi was reported to exhibit the anti-influenza virus activity through preventing

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interactions between virus and host cells (Komatsu, Kido, Sugiyama, & Yokochi, 2013). A sulfated polysaccharide from Enteromorpha compressa showed significant anti-herpes simplex virus

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(HSV)-1 activity primarily by affecting viral replication (Lopes et al., 2017). Antiviral activities of

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sulfated polysaccharides from Monostroma sp. were also reported (Lee, Hayashi, Hayashi, Sankawa & Meada, 1999; Lee, Koizumi, Hayashi, & Hayashi, 2010). Rhamnan sulfate from Monostroma latissimum had potent antiviral activities against HSV-1 and human immunodeficiency virus type 1

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(HIV-1) (Lee, Hayashi, Hayashi, Sankawa &Meada, 1999). The sulfated polysaccharide from M. nitidum showed potent inhibition of HSV-2 adsorption and penetration (Lee, Koizumi, Hayashi, & Hayashi, 2010). The sulfated polysaccharides from green algae have potential as novel antiviral 4

agents. The green alga M. latissimum grows in upper part intertidal zone and widely cultivated as an edible alga (Lee, Yamagaki, Maeda, & Nakanishi, 1998; Li et al., 2011). Our previous study indicated that the polysaccharide, PML, isolated from M. latissimum was a novel sulfated rhamnan

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and possessed strong anticoagulant activity (Li et al., 2011). The purpose of this study was to investigate the inhibitory effects and mechanisms of PML against EV71. PML was found to be able

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to effectively inhibit EV71 activity in vitro with low cytotoxicity, markedly reduce viral loads and prevent death of EV71-infected mice. The anti-EV71 activity of PML might involve inhibition of

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EV71 invasion and replication by targeting the viral capsid protein and cellular EGFR/PI3K/Akt

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2. Materials and methods

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pathway. The results of our analyses are presented in this paper.

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2.1. Materials

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Sulfated polysaccharide PML was prepared from the green alga M. latissimum in our lab (Li et al., 2011). M. latissimum was provided by Yuhuan County, China. It was harvested in April 2005. The raw material was thoroughly washed with tap water, air dried and milled, and then kept in plastic

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bags at room temperature in a dry environment. Q Sepharose Fast Flow and Sephacryl S-400/HR were from GE Health care Life Sciences (Piscataway, NJ, USA). Dialysis membranes (flat width 44 mm, molecular weight cut off 3500) were from Lvniao (Yantai, China). Dulbecco's Modified Eagle's 5

medium (DMEM), Minimum Essential Medium (MEM), penicillin, and streptomycin were purchased from Gibco (Grand Island, NY, USA), fetal bovine serum (FBS) was obtained from Excell (Suzhou, China). EZ-Link™ sulfosuccinimidyl-2-[biotinamido]ethyl-1,3-dithiopropionate (Sulfo-NHS-SS)-Biotin was obtained from Thermo Scientific (Waltham, MA, USA). Guanidine was

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purchased from sigma (Shanghai, China). DyLight 649 conjugated secondary antibody was obtained from Abbkine (California, USA). BeaverBeads™ Streptavidin was purchased from Beaver

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Bioscience (Suzhou, China). Rabbit α-tubulin, glyceraldehyde 3-phosphate dehydrogenase

(GAPDH), p-PI3K, p-Akt, p- epidermal growth factor receptor (EGFR), p-nuclear factor-κB (NF-κB)

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monoclonal antibody was purchased from CellSignalling Technology (Boston, USA). The

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VP1-EV71 antibody was obtained from Immune Technology Corp. (Suzhou, China). 5× sample

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loading buffer, 4', 6-diamidino-2-phenylindole (DAPI) was purchased from Beyotime Biotechnology

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2.2. Cells culture and viruses

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(Nantong, China). All of the other reagents used were of analytical grade.

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African green monkey kidney (Vero) cells were cultured in MEM. Rhabdomyosarcoma (RD) cells were grown in DMEM. All media were supplemented with 10% (v/v) FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. EV71 strain BrCr-TR (GenBank accession number AB204852.1) was

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obtained from the Wuhan Institute of Virology, Chinese Academy of Sciences. Viruses were amplified in RD cells, and the virus titers determined by plaque assay and 50% tissue culture infectious dose (TCID50) assay in Vero cells (Smither et al., 2013). 6

2.3. Animals

The specific-pathogen-free ICR mice were purchased from Vital River Laboratory Animal

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Technology Co., Ltd. (Beijing, China), and maintained in the animal facility of the Ocean University of China. A breeding ICR mouse could give birth to 12–13 pups, which were kept in a single group.

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Newborn ICR mice were housed with their mothers in standard housing conditions with a 12-hour

light/dark cycle. Three-day-old ICR mice were the subjects of in vivo experiments. All animals had

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free access to water, except during test sessions. Animal experiments were approved by the

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institutional animal care and use committee of the Ocean University of China

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(OUC-YY-201801001).

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2.4. Preparation of the sulfated polysaccharide PML

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The extraction and purification of the sulfated polysaccharide PML from M. latissimum were

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carried out as described previously (Li et al., 2011). Briefly, the milled algae were dipped into 20 volumes of distilled water and kept at room temperature for 2 h, then filtrated. The residue was dipped into 20 volumes of distilled water, homogenized and refluxed at 100 °C for 2 h. After cooling

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to the room temperature, the supernatant was collected by centrifugation, concentrated, dialyzed in a cellulose membrane against distilled water at room temperature for three successive days. The retained fraction was recovered, concentrated under reduced pressure, precipitated by adding four 7

volumes of 95% ethanol (v/v) and dried. The protein in the fraction was removed by the method of Sevag. The crude polysaccharide was fractionated by anion-exchange chromatography on a Q Sepharose Fast Flow column and eluted with a step-wise gradient of 0, 0.5, 1.0, 2.0, 3.0 and 4.0 mol/L NaCl. The fractions eluted with 0.5 mol/L NaCl were pooled, dialyzed and further purified on

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a Sephacryl S-400/HR column eluted with 0.2 mol/L NH4HCO3. After these steps, a purified sulfated

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polysaccharide PML was obtained.

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2.5. Analytical methods for structural characteristics of polysaccharide

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Purity and molecular weight of polysaccharide were determined by high performance gel

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permeation chromatography (HPGPC) on a Shodex OHpak SB-804 HQ column, and the column

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calibration was performed with pullulan standards (Mw: 21.1, 47.1, 107, 200, 344, and 708 kDa,

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Showa Denko K.K., Japan) (Li et al., 2011). Total sugar content was determined by the phenol–sulfuric acid colorimetric method using rhamnose as the standard (Dubois et al., 1956).

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Sulfate content was measured according to Therho and Hartiala (1971). Monosaccharide

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composition was measured by reversed–phase high performance liquid chromatography (HPLC) after pre-column derivatization and UV detection (Sun et al., 2009). Sugar identification was done by comparison with reference sugars. Desulfation of the sulfated polysaccharide was performed

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according to the method of Falshaw and Furneaux (1998). The effectiveness of desulfation procedure was confirmed by determination of residual sulfate in the polysaccharide. Methylation analysis was performed according to the method of Hakomori (1964). The partial 8

methylated alditol acetates were analyzed by gas chromatography-mass spectrometry (GC–MS) on a HP6890II/5973 instrument using a DB 225 fused silica capillary column (0.25 mm × 30 m). Identification of partially methylated alditol acetates was carried out on the basis of retention time and mass fragmentation patterns. The Fourier-transform infrared (FTIR) spectrum was recorded on a

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Nicolet Nexus 470 spectrometer with a KBr pellet. 1H and 13C nuclear magnetic resonance (NMR) spectroscopy were performed at 23 °C on a JEOL ECP 600 MHz spectrometer. Acetone was used as

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internal standard (2.225 ppm for 1H and 30.7 ppm for 13C).

Partial acid hydrolysis of PML was performed with 0.1 mol/L HCl at 60 °C for 8 h, and the

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supernatant and precipitate products were obtained by adding three-fold volume of 95% (v/v) ethanol

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and centrifugation (3600 × g, 10 min). The supernatant product was further purified on a Bio-Gel P-4

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column (1.6 cm × 100 cm) by elution with 0.2 mol/L NH4HCO3, and the oligosaccharide fractions

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were obtained. The sequence of the oligosaccharide was analyzed using negative-ion electrospray

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mass spectrometry (ESMS) on a Micromass Q-Tof Ultima instrument (Li et al., 2012).

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2.6. Cytopathic effect inhibition assay

The antiviral activity was evaluated by the cytopathic effect (CPE) inhibition assay (Hung et al., 2009). Briefly, Vero cells in 96-well plates were infected with EV71 at a multiplicity of infection

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(MOI) of 0.1, and then treated with different concentrations of PML (0.1, 1, 10, or 100 μg/mL) in triplicate after removal of the virus inoculum. After 16 h incubation, the cells were fixed with 4% formaldehyde for 15 min at room temperature. After removal of the formaldehyde, the cells were 9

stained with 0.1% (w/v) crystal violet for 30 min at room temperature. The plates were washed and dried, and the intensity of crystal violet staining for each well was measured at 570 nm.

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2.7. Plaque reduction assay

Inhibitory effect of virus infection was measured by the plaque reduction assay (Chiu, Chan,

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Tsai, Li, & Wu, 2012). Different concentrations (250, 125, 62.5, 31.25, or 15.625 μg/mL) of PML in 800 μL DMEM media were mixed with an equal volume of infectious EV71 (50–100 PFU/well) in

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DMEM, and incubated at 37 °C for 1 h. The virus-PML mixtures were transferred to confluent Vero

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cell monolayers in 6-well plates, and incubated at 37 °C for 1 h with gentle shaking every 15 min.

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After that, the inoculum was removed and each well was overlaid with 2 mL of agar overlay medium.

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After incubation for 3 days at 37 °C in a humidified atmosphere of 5% CO2, cells were fixed with 4%

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paraformaldehyde (PFA), followed by staining with 1% crystal violet for plaque counting.

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2.8. Indirect immunofluorescence assay

Antiviral activity in different drug-treatment conditions was evaluated by the indirect

immunofluorescence assay as described previously (Wang et al., 2017). Vero cells were infected

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with EV71 (MOI = 0.1) and treated with PML (10 μg/mL) under three different treatment conditions: (i) pre-treatment: EV71 was pre-treated with PML (10 µg/mL) at 37 °C for 1 h prior to infection. (ii) adsorption: Vero cells were infected in media containing 10 µg/mL of PML and, after 1 h at 37 °C, 10

were overlaid with PML-free media. (iii) post-adsorption: after removed the unabsorbed virus the infecting media containing 10 µg/mL of PML was added to cells. At 6 hours post infection (h p.i.), Vero cells were fixed with 4% PFA for 15 min, permeabilized with 0.25% (v/v) Triton X-100 for 8 min and blocked with 2% (w/v) bovine serum albumin (BSA) in PBS for 1 h at 37 °C. After washing,

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cells were incubated consecutively with anti-VP1 (EV71) monoclonal antibody and DyLight 649®-conjugated secondary antibody. The cell nuclei were stained with DAPI. Images were recorded

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using a Nikon confocal microscope, and analyzed by ImageJ (NIH) version 1.33u (USA).

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2.9. Pull-down and ELISA-binding assay

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Whether PML could directly bind to EV71 capsid proteins is further explored using a

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pull-down assay (Buck et al., 2006). Biotinylated PML (PML-biotin) was prepared by using

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EZ-Link™ Sulfo-NHS-SS-Biotin according to the manufacturer's instructions. Streptavidin-coupled magnetic beads were blocked with 0.5% BSA in PBS and complexed with biotinylated PML

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(PML-Biotin, 100 μg/mL). After washing three times with 0.2% BSA in PBS, the PML-beads were

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incubated with EV71 for 2 h, washed, and the bound proteins were eluted by incubation with 1× sample loading buffer at 100 °C for 10 min. Western blotting was used to detect VP1 protein in samples to indicate the presence of EV71.

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For ELISA based binding assay, the high-binding 96-well plates were firstly coated with

streptavidin (Kadam et al., 2017). After blocked with PBS containing 1% BSA and 0.1% Tween-20, the PML-Biotin (1, 10, or 100 μg/mL) was added and incubated at 37 °C for 1 h. After washing, 11

EV71 was added and incubated for another 2 h. Then, the anti-VP1 antibodies and HRP-labeled secondary antibodies were added sequentially and the TMB chromogenic Kit was used to determine the amounts of protein binding.

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2.10. Western blotting

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Signaling pathway and the expression of viral VP1 protein were assessed by western blotting (Wang et al., 2017). Vero cells were infected with EV71 (MOI = 1.0) and treated with different

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concentrations of PML (12.5–50 μg/mL) after adsorption. Cell lysates were collected at 0.5 h

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p.i..Vero cell lysates were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis

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(SDS-PAGE) gels, and transferred onto nitrocellulose membranes. After blocking overnight at 4 °C

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in Tris-buffered saline containing 0.1% (v/v) Tween 20 and 5% BSA, the membranes were rinsed

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and incubated at 37 °C with antibodies against-phosphorylated PI3K, Akt, NF-κB, or EGFR. The membranes were washed and incubated with alkaline phosphatase(AP)-labeled secondary antibodies

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at room temperature for 1.5 h. Blots were developed with p-nitro blue tetrazolium chloride (NBT)

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and 5-bromo-4-chloro-3-indolyl phosphate toluidine (BCIP) solution at room temperature for 30 min. The relative densities of protein bands were determined by Image J (NIH) V.1.33u (USA).

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2.11. In vivo experiments

A breeding ICR mouse could give birth to 12–13 pups, which were kept in a single group. 12

Three-day-old neonatal ICR mice (n=10–12 per group) were intraperitoneally challenged with 105.5 TCID50 of EV71 (lethal dose) as reported (Zhang et al., 2017) followed by an intramuscular injection of guanidine (10 mg/kg), PML (5 or 10 mg/kg), or sterile phosphate-buffered saline (PBS) alone (infected control). Drugs were administered 12 h, 24 h and 48 h post-infection. The mice were

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monitored daily for 14 days to assess survival rate, clinical score and body weight. Body weight was determined and normalized according to the following equation: normalized body weight (%) =

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(Wn/W0) × 100, where Wn and W0 are the body weights of the newborn mice on day n and day 0,

respectively. Signs of disease were evaluated to obtain a graded clinical score (0, health; 1, lethargy;

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2, magersucht or hind limb weakness; 3, single limb paralysis; 4, double hind limb paralysis; 5,

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dying or death).

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The heart, brain, intestine and skeletal muscle of five groups of mice (mock infected,

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EV71-infected and treated with PBS, EV71-infected and treated with guanidine (10 mg/kg), or

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EV71-infected and treated with PML (5 or 10 mg/kg)) were obtained at five days post-challenge. These tissues were fixed by immersion in 4% (w/v) formaldehyde for 48 h at room temperature. The

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fixed tissues were embedded in paraffin and sectioned (4 mm), following by staining with

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hematoxylin and eosin (H & E).

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2.12. Statistical analysis

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All data are representative of at least three independent experiments. Data are presented as means ± standard deviations (SD). Statistical significance was in GraphPad Prism 7 software using one-way ANOVA with Turkey’s test. P values < 0.05 were considered significant.

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3. Results and discussion

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3.1. Characterization of the sulfated polysaccharide PML

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Sulfated polysaccharide PML was extracted from M. latissimum with hot water, and further

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purified by a combination of Q Sepharose Fast Flow column (Supplemental Fig. 1A) and Sephacryl

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S-400/HR column. PML appeared as a single and symmetrical peak in the HPGPC chromatogram,

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indicating its purity and corresponding to an average molecular weight of about 513 kDa

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(Supplemental Fig. 1B). Reversed-phase HPLC analysis demonstrated that PML mainly consisted of rhamnose (Supplemental Fig. 1C), and the content of the sulfate ester in PML was estimated to be

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26.1% (Li et al., 2011).

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The FTIR spectrum of PML showed the characteristic absorptions of sulfated polysaccharide, including two bands (851 and 1260 cm–1) corresponding to sulfate esters (Supplemental Fig. 2, Li et al., 2011). The signals at 3446–3445 cm–1 were assigned to the O–H stretching vibration. The signals

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at 2939–2938 cm–1 were assigned to the–CH stretching vibration. The signals at 1650–1649 cm–1 were due to the bending vibration of O–H, and the signals at 1059–1057 cm–1 were attributed to the stretch vibration of C–O and change angle vibration of O–H (Li et al., 2011). 14

Methylation analysis showed that PML mainly consisted of →3)-α-L-Rhap-(1→, →2)-α-L-Rhap-(1→ and →2,3)-α-L-Rhap-(1→ residues. Compared with the result of PML, increased amounts of →2)-α-L-Rhap-(1→ and →3)-α-L-Rhap-(1→, and reduced amount of →2,3)-α-L-Rhap-(1→ residues were detected in dsPML. Thus, the sulfate substitutions were

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deduced to be at the C-2 of →3)-α-L-Rhap-(1→ residues and/or the C-3 of →2)-α-L-Rhap-(1→ residues (Li et al. (2011). The identification and proportions of the methylated alditol acetates of

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PML and dsPML were listed in Supplemental Table 1.

In the 1H NMR spectrum of PML (Supplemental Fig. 3a,b, Li et al., 2011), five anomeric proton

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signals at 5.46 5.35, 5.25, 5.23, and 5.08 ppm classified to be α-L-rhamnopyranose were observed,

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and had relative integrals of 3:1:2:1:5. The signal at 1.35 ppm was attributed to the proton of CH3

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group of the rhamnose residues, and other signals were located at the region of 3.60–4.73 ppm (Li et

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al., 2011). In the anomeric region of 13C NMR spectrum (Supplemental Fig. 3c,d, Li et al., 2011), the

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main anomeric carbon signals occurred at 103.06, 101.48, 100.79 and 100.59 ppm, and confirmed that the rhamnopyranose units were α-anomeric configurations. The signal occurring at 17.99 ppm

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was assigned to the C-6 of the rhamnose residues, and other signals were located at the region of

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70.43–81.69 ppm (Li et al., 2011).

The 1H NMR spin systems of the polysaccharide were assigned by the 1H–1H COSY spectrum

(Supplemental Fig. 3e, Li et al., 2011). The direct C–H coupling was determined by the 1H–13C

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HMQC spectrum (Supplemental Fig. 3f, Li et al., 2011). Combined with the analysis of the 1H–13C HMQC spectrum and the comparison with the chemical shift data of similarly substituted sugar residues (Cassolato et al., 2008; Gargiulo et al., 2008), the assignment of the main signals of the five 15

sugar residues could be completed (Supplemental Table 2, Li et al., 2011). The anomeric proton signal at 5.46 ppm with its correlated anomeric carbon signal at 100.59 ppm was assigned to →3)-α-L-Rhap(2SO4)-(1→ residues. The anomeric proton signal at 5.35 ppm was related to the anomeric carbon signal at 100.79 ppm, and were ascribed to →2)-α-L-Rhap(3SO4)-(1→ residues.

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The anomeric proton signals at 5.25 and 5.23 ppm were correlated with the anomeric carbon signal at 101.48 ppm, and the signals were ascribed to (→2,3)-α-L-Rhap-(1→ and →2)-α-L-Rhap-(1→

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residues, respectively. The anomeric proton signal at 5.08 ppm was related to the anomeric carbon

signal at 103.06 ppm, and the signals were assigned to →3)-α-L-Rhap-(1→ residues (Li et al., 2011).

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From the 1H–13C HMQC spectrum, some of the correlations between carbon and proton signals

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within the sugar residues were also deduced. The major signals at 77.97 and 79.08 ppm were due to

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the C-2 and C-3 substituted rhamnose residues. The H-2 signal at 4.73 ppm and its correlated C-2

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signal at 77.97 ppm were attributed to →3)-α-L-Rhap(2SO4)-(1→ residues. The H-3 signal at 4.61 residues. The H-3

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ppm and C-3 signal at 79.08 ppm were assigned to →2)-α-L-Rhap(3SO4)-(1→

signal at 4.29 ppm was correlated with C-3 signal at 81.69 ppm, and were assigned to

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→3)-α-L-Rhap-(1→ and →2,3)-α-L-Rhap-(1→ residues. The proton signal at 4.37 ppm with

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corresponding carbon signal at 80.22 ppm were ascribed to H-2 and C-2 of →2)-α-L-Rhap-(1→ and →2,3)-α-L-Rhap-(1→ units, respectively (Li et al., 2011). These results demonstrated that PML consists of →3)-α-L-Rhap-(1→ and →2)-α-L-Rhap-(1→

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residues with partially branches, and the sulfate groups were at C-2 of →3)-α-L-Rhap-(1→ and/or C-3 of →2)-α-L-Rhap-(1→ residues (Li et al., 2011). To further determine the structural characteristics of PML, partial acid hydrolysis of PML was 16

performed, and the supernatant and precipitate products were obtained by ethanol precipitate and centrifugation. The monosaccharide composition of the precipitate product is similar to that of PML, whereas the supernatant product was composed of rhamnose. The supernatant product was further fractionated by gel filtration chromatography and the oligosaccharide fractions (F1, F2 and F3) were

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obtained. The molecular weights of the oligosaccharide fractions were determined by ES-MS. From the negative-ion ES-MS, F1 was a monosulfated rhamnose (Supplemental Fig. 4A). F2 was a

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monosulfated rhamno-disaccharide (Supplemental Fig. 4B). F3 was mixture of disaccharide,

trisaccharide and tetrasaccharide with or without sulfate ester (data not shown). R and S represented

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rhamnose and sulfate ester, respectively. Here, the detailed sequences of rhamno-oligosaccharide in

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the fractions F1 and F2 were further investigated by negative-ion electrospray tandem mass

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spectrometry with collision-induced dissociation (ES-CID-MS/MS).

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Structure of monosulfated rhamno-monosaccharide (RS) in the fraction F1 was deduced by the

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product-ion spectrum of m/z 243 in the negative-ion mode. In the ES-CID MS/MS spectrum (Supplemental Fig. 4C), the ion at m/z 139 which was originated from 0,2X indicated the 2-O-sulfated

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rhamnose (Tissot, Salpin, Martinez, Gaigeot, & Daniel, 2006). The ion at m/z 97 corresponded to the

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hydrogenosulfate anion HSO4-, and the ion at m/z 225 was produced by the dehydration. The 3-O-sulfated rhamnose might also be existent. The possible structures were shown in Supplemental Fig. 4C.

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Sequence of monosulfated rhamno-disaccharide (R2S) in the fraction F2 was analyzed. The ion

m/z 389 attributed to monosulfated rhamno-disaccharide was the major ion of the fraction F2. In the ES-CID MS/MS spectrum (Supplemental Fig. 4D), the ions m/z 225, 243, 285, 315 were assigned to 17

B1, C1 from glycosidic bond cleavages and 0,2X1/0,2X0, 0,3X1 from the cleavages in the saccharide ring, respectively. The sequence of R2S might be α-L-Rhap(2SO4)-(1→3)-α-L-Rhap and/or α-L-Rhap(3SO4)-(1→2)-α-L-Rhap. The possible structures were shown in Supplemental Fig. 4D. Above analyses revealed that the backbone of PML mainly consisted of 3-linked,

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2-linked-α-L-rhamnose residues with sulfate substitutions at C-2 of 3-linked-α-L-rhamnose and C-3 of 2-linked-α-L-rhamnose residues (Li et al., 2011). The branches were primarily composed of

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disaccharides in PML were listed in Fig. 1A (Li et al., 2011).

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unsulfated or monosulfated 3-linked, 2-linked-α-L-rhamnose. Structures of the main repeating

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3.2. Inhibition of EV71 infection in vitro by PML

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The cytotoxicity of PML in Vero, RD and HeLa cells was firstly evaluated by MTT assay

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(Senthilraja & Kathiresan, 2015). The results showed that PML had no significant cytotoxicity at concentrations from 0.1 to 5000 μg/mL (Fig. 1B). PML was then assayed for its ability to inhibit

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EV71 multiplication in vitro using CPE inhibition assay and plaque assay. Guanidine and ribavirin

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were used as the positive control drugs in this study. As shown in Fig. 1C and 1D, PML dose-dependently inhibited EV71-induced CPE and reduced virus yields at concentrations > 100 ng/mL (p < 0.01). The 50% inhibitory concentration (IC50) of PML for CPE was 461.0 ng/mL, and

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the selectivity index (SI; CC50/IC50) for PML was above 10000.0, which was superior to that of guanidine (SI = 33.9) and ribavirin (SI = 11.9) (Table 1). Considered that guanidine and ribavirin could significantly inhibit enterovirus RNA replication with IC50 values of 2.5 mmol/L (238.83 18

μg/mL) and 178.42 μg/mL, respectively (Yuan et al., 2018; Zhang et al., 2012), it was supposed that the EV71 strain BrCr-TR used in this study might be more sensitive to PML than the replication inhibitors guanidine and ribavirin. Moreover, CPE inhibition assays were also conducted using RD cells and HeLa cells to explore

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whether the inhibition of EV71 by PML was cell-type-specific. As shown in Fig. 1E, viral replication in RD and HeLa cells was also dose-dependently inhibited by PML, and PML significantly reduced

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EV71-induced CPE when used at the concentrations > 0.1 μg/mL (p < 0.05). To further explore whether PML had direct inhibitory effects on EV71 particles, the plaque reduction assay was

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performed. The results showed that pre-incubation of EV71 with PML at concentrations of

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15.625–250 μg/mL markedly reduced the number of EV71 plaques and protected Vero cells from

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infection (Fig. 1F), suggesting that PML may be able to inactivate EV71 particles directly.

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3.3. Influence of PML treatment conditions on EV71 infection

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Time-of-addition assays were performed to determine the stage at which PML exerted its inhibitory actions in vitro. As shown in Fig. 2A, pre-treatment of EV71 with 100 ug/mL of PML for

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1 h prior to infection significantly reduced the virus titers compared to the virus control group (p < 0.01), suggesting that PML may have direct interaction with EV71 particles. Addition of PML during viral adsorption also significantly reduced the virus titers in Vero cells. Surprisingly, addition of PML

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after adsorption also significantly inhibited viral multiplication, suggesting that PML may also block some steps subsequent to adsorption. Moreover, the indirect immune fluorescence assay showed that the average fluorescence intensity of VP1 significantly decreased in PML (10 μg/mL)-treated

19

EV71-infected Vero cells under three treatment conditions (p < 0.01), as compared to that in the virus control cells at 6 h p.i. (Fig. 2B and C), suggesting that PML may inhibit some early stages of the EV71 life cycle after virus adsorption. To further explore which stage after adsorption is inhibited by PML, another time-course study

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was performed (Fig. 2D). In brief, EV71(MOI=0.1)-infected Vero cells were treated with 10 μg/mL of PML for different time intervals, then the virus yields at 16 h p.i. were evaluated by TCID50 assay.

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The results showed that PML treatment during the first 2 h (0–2 h p.i.), second 2 h (2–4 h p.i.), the

third 2 h (4–6 h p.i.) or the fourth 2 h (6–8 h p.i.) after adsorption all significantly reduced the virus

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titers (p <0.01), while PML treatment after 8 h (8–10 h p.i.) did not significantly inhibit EV71

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multiplication. Thus, PML may inactivate virions directly or block some early steps after viral

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adsorption.

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3.4. Effect of PML on adsorption EV71 particles

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Biotinylated PML (PML-biotin) was prepared and its inhibitory effect on virus infection was evaluated by CPE inhibition assay. PML possessed a little higher inhibition percentage than

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PML-Biotin at the concentrations of 0.1–100 μg/mL (Fig. 3A), indicating that biotinylation of PML slightly influenced its anti-EV71 activity. The streptavidin-coupled magnetic beads were complexed

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with PML-Biotin (1, 10, or 100 μg/mL). After washing with 0.2% BSA in PBS, the PML-beads were incubated with EV71 for 2 h, washed, and the bound proteins were detected by western blotting. As shown in Fig. 3B, PML-beads dose-dependently (1–100 μg/mL) bound to EV71 capsids while the

20

control beads without PML only unspecifically bound to the viral capsid. EV71 particles (104.5–106.5 PFU/mL) could also bind to PML-beads in a dose-dependent manner, suggesting that PML may be able to directly bind to EV71 capsids. Furthermore, the ELISA based binding assay showed that PML-Biotin (1–100 μg/mL) dose-dependently bound to EV71 particles (Fig. 3C), while the EV71

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particles (104.5–106.5 PFU/mL) also dose-dependently bound to PML-Biotin (Fig. 3D). Thus, PML is

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truly able to bind to the EV71 particles.

3.5. Involvement of EGFR/PI3K/Akt signaling pathways in the anti-EV71 actions of PML in Vero

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cells

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A large number of viruses rely on activation of the PI3K/Akt pathway for endocytosis and

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replication (Esfandiarei et al., 2004; Izmailyan et al., 2012; Soares et al., 2009; Zhang et al., 2003).

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The above results showed that PML may also inhibit some early stages of the EV71 life cycle after adsorption, thus the effects of PML on cellular PI3K/Akt pathway were evaluated by western

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blotting. As shown in Fig. 4A and 4B, PML could significantly reduce the levels of VP1 protein in a

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dose-dependent manner compared with the virus control group. In this study, it was found that phosphorylated Akt expression was significantly increased in EV71-infected cells (approximately 1.26-fold higher expression) compared with the uninfected cell control group at 0.5 h p.i. (Fig. 3C

A

and 3D). However, after treatment with PML (12.5–50 μg/mL) for 0.5 h, the expression of phosphorylated Akt significantly decreased from 1.26-fold to about 0.38-, 0.32- and 0.19 -fold of the uninfected cell control group, respectively (Fig. 4C and 4D). Moreover, treatment with PML 21

(12.5–50 μg/mL) for 0.5 h also significantly reduced the expression of phosphorylated PI3K from about 1.12-fold to about 0.78-, 0.71- and 0.62-fold of the uninfected cell control group, respectively (p < 0.01) (Fig. 4C and 4D). These data suggested that PML might inhibit the PI3K/Akt pathways to interfere with EV71 replication.

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The influence of PML on upstream signaling through EGFR and downstream activation of NF-κB, which are responsible for efficient virus endocytosis and replication, was also evaluated. As

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shown in Fig. 3E and 3F, expression of phosphorylated EGFR significantly increased (1.34-fold

higher than the control group) after EV71 infection for 0.5 h. Treatment with PML (12.5–50 μg/mL)

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significantly reduced the activation of EGFR from 1.34-fold to about 0.71-, 0.59-, and 0.56-fold of

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normal control group, respectively (p < 0.05) (Fig. 4F). However, treatment with PML (12.5–50

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μg/mL) for 0.5 h did not significantly decrease the expression level of phosphorylated NF-κB protein

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the anti-EV71 activity of PML in vitro.

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(p > 0.05) (Fig. 4H). Therefore, the cellular EGFR/PI3K/Akt signaling pathway may be involved in

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3.6. The anti-EV71 activity in vivo of PML

Based on the strong anti-EV71 potency of PML in vitro, the protective efficacy of PML against

lethal EV71 infection in three-day-old ICR mice was further explored. Three-day-old ICR mice were

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intraperitoneally injected with EV71 BrCr-TR strain at a dose of 105.5 TCID50 followed by drug treatment. As shown in Fig. 5A, intramuscular administration of PML (5 or 10 mg/kg) significantly improved the survival rates of the infected mice as compared with the virus control group (0% 22

survival). There was no difference in survival between mice treated to 10 mg/kg PML or 10 mg/kg guanidine (100% survival). The body weights of infected ICR mice treated with PML (5 or 10 mg/kg) continued to increase until the end of the observation period, while those of animals in the virus alone control group did not increase after 6 days post-infection (Fig. 5B).

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Moreover, PML treatment (5 or 10 mg/kg) dramatically reduced the clinical scores of infected mice as compared to the virus control group, which was correlated with the effects of PML on

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survival rates (Fig. 5C). PML-treated (10 mg/kg) infected mice only showed some symptoms of

lethargy, did not show severe symptoms such as limb paralysis or death within 13 days. 5 mg/kg of

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PML or 10 mg/kg of guanidine treated infected mice showed limb weakness or slight limb paralysis

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newborn mice from lethal EV71 challenge.

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at 5 days post infection but survived far better than the virus control mice. Thus, PML could protect

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To further evaluate the inhibitory effects of PML in EV71-infected mice, the viral titers in the

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heart, brain, intestine, and muscle tissues of EV71-infected mice were determined by plaque assay 5 days post-challenge. In the challenged mice treated with PBS, a 1000-fold higher viral load was

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observed in the intestine compared with the brain (Fig. 5D), suggesting that EV71 is gut-tropic.

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Treatment of 10 mg/kg PML significantly reduced the viral load in the heart, brain, intestine, and muscle tissues (p < 0.05) compared with the control treatment (PBS); these reductions were greater in magnitude than those observed for mice treated with guanidine (10 mg/kg) (Fig. 5D). Treatment

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with a lower dose of PML (5 mg/kg) only significantly decreased viral titers in heart and intestine of EV71-infected mice (Fig. 5D). Thus, PML could effectively inhibit the propagation of EV71 in vivo.

23

The protective effects of PML against EV71-induced tissue damage were further investigated by histopathology analysis. As shown in Fig. 6A, F, K and P, no obvious abnormalities were observed in mock-treated ICR mice. By contrast, focal hemorrhages and submucosal inflammatory infiltration were observed in the hearts of PBS-treated virus control mice (Fig. 6B), while no obvious

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inflammatory symptoms occurred in the PML-treated mice (Fig. 6D and E). Moreover, focal and patchy hemorrhages were observed in the brains of the PBS-treated EV71-infected control mice,

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indicating damage caused by EV71, whereas no pathological changes occurred in the brains of

PML-treated mice (Fig. 6F-J). In the intestine, submucosal inflammatory infiltration was observed in

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the PBS-treated EV71-infected control mice as well as in guanidine-treated mice, but this improved

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after PML (10 mg/kg) treatment (Fig. 6K-O). Muscle tissues were widely damaged by EV71,

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showing visible degeneration, edema, hemorrhage, inflammatory infiltration, partial necrotic

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myositis and diffuse lesions (Fig. 6Q). After PML treatment (10 or 5 mg/kg), inflammatory

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infiltration and necrotic myositis were partially alleviated (Fig. 6S and T). Taken together, these results suggested that severe inflammatory infiltration and tissue damage might be the major causes

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survival rate.

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of death due to EV71 infection, and that PML can effectively alleviate tissue damage to improve

Above all results revealed that the sulfated rhamnan PML from M. latissimum was

non-cytotoxic and significantly inhibited EV71 infection in vitro and in vivo. PML possessed high

A

anti-EV71 activity in Vero cells, especially when virus was pre-treated with PML, suggesting that PML may directly interact with virus capsid proteins. It was also confirmed that PML could truly bind to EV71 particles directly. PML contains a high density of negative charge because of the 24

presence of sulfate ester residues, which may be able to interact with positively-charged domains of the viral capsid proteins involved in the attachment of the virion to the cell membrane (Callahan, Phelan, Mallinson, & Norcross, 1991). The cellular PI3K/Akt signaling pathway plays important roles in cell survival, apoptosis,

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proliferation, migration and differentiation, as well as in metabolic regulation. This pathway has also been reported to be associated with EV71 uptake and virus-induced autophagy (Diehl & Schaal,

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2013; Lin et al., 2017). PML significantly reduced the phosphorylation of PI3K and Akt, and

inhibited the activation of EGFR, which is responsible for efficient virus endocytosis and cytokine

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activation. However, PML did not significantly reduce the phosphorylation of downstream signal

N

NF-kB pathway. Thus, cellular EGFR/PI3K/Akt signaling pathway may be involved in the

A

anti-EV71 actions of PML in vitro.

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The in vitro anti-EV71 activity of PML was mirrored in EV71-infected newborn mice.

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Treatment of EV71-infected mice with PML markedly improved their survival and decreased the virus titers in different organs. PML treatment could significantly attenuate the severe symptoms

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such as limb paralysis and dying, and increase the body weights of EV71 infected mice, suggesting

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that PML may be able to protect newborn mice from lethal EV71 challenge. Moreover, the histopathological analysis indicated that PML treatment also significantly attenuated virus induced inflammatory infiltration and tissue damage in the newborn mice, superior to the effect of guanidine.

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Although like other high-molecular weight polysaccharides, PML may hardly cross the different barriers of the body by oral administration. Our studies showed that intramuscular therapy of PML could protect newborn mice from lethal EV71 challenge, superior to the effects to guanidine, 25

suggesting that PML may be used for prevention and treatment of EV71 infection by intramuscular administration.

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4. Conclusion

The sulfated rhamnan PML isolated from the green alga M. latissimum possessed strong

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anti-EV71 activities both in vitro and in vivo with low toxicity. PML may inhibit EV71 invasion and replication by targeting the viral capsid protein as well as the cellular PI3K/Akt pathway. Further

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studies of the anti-EV71 effects of PML against other EV71 strains (such as MS/7423/87, 18/Sin/97,

N

NCKU9822, SHZH98) in animal models and clinical research will be required to advance its

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prospects for drug development. Nevertheless, the sulfated rhamnan PML has the potential to be

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Acknowledgments

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developed into a novel antiviral agent for therapy and prophylaxis of EV71 infection.

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This work was supported by National Natural Science Foundation of China (41476108, 81741146, 31500646), Technological Innovation Project of Qingdao National Laboratory for Marine Science and Technology (2016ASKJ08, 2015ASKJ02), NSFC-Shandong Joint Fund (U1606403,

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U1706210), the Scientific and, and the Shandong Provincial Natural Science Foundation (ZR2017MH013).

26

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Figure Captions

Fig. 1. Structures of the main repeating disaccharides in PML and inhibition of EV71 infection in vitro by PML. (A) Structures of the main repeating disaccharides in PML (Li et al., 2011). a:

→3)-α-L-Rhap-(1→2)-α-L-Rhap(3SO4)-(1→;

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→3)-α-L-Rhap(2SO4)-(1→3)-α-L-Rhap-(1→; b: →3)-α-L-Rhap(2SO4)-(1→2)-α-L-Rhap-(1→; c:

d: →3)-α-L-Rhap(2SO4)-(1→2,3)-α-L-Rhap-(1→.

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(B) The cytotoxicity of PML in Vero, RD, HeLa cells was determined by MTT assay after 24 h incubation. (C) EV71 (MOI=0.1) infected Vero cells were treated with PML at the indicated

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concentrations for 16 h, then the antiviral activity was determined by CPE inhibition assay. (D) The

N

infectious virus titers in cell supernatants were evaluated by plaque assay after treatment of different

A

concentrations of PML for 16 h. (E) Anti-EV71 activities of PML in different cells were determined

M

by CPE inhibition assay. (F) Plaque reduction assay of PML pretreated EV71 in Vero cells. Values

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are mean ± SD (n=3). *P < 0.05, **P < 0.01 vs virus control group.

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Fig. 2. Influence of different treatment conditions of PML on EV71 infection. (A) Vero cells were

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infected with EV71 under three treatment conditions of PML, and the virus yields were evaluated by plaque assay. (B) Immunofluorescence assay of virus VP1 protein in EV71 infected Vero cells. Scale

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bar represents 10 μm. (C) The average fluorescence intensity of VP1 proteins in (B). (D) Time course study of PML with 2 h intervals determined by TCID50 assay. Values are mean ± SD (n=3). **P < 0.01 vs virus control group.

32

Fig. 3. Binding effect of PML to EV71. (A) The comparison of the anti-EV71 activities between PML and PML-Biotin by the CPE inhibition assay at 16 h p.i. Values are mean ± SD (n=3). #P < 0.05, ##P < 0.01 vs virus control group. (B) Pull-down assay of PML-Biotin coupled beads with EV71 particles (lgPFU/mL=4.5, 5.5, 6.5). (C) ELISA based binding assay of PML-Biotin (1, 10, 100

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μg/mL) with EV71 detected by using anti-VP1 antibody and HRP-labeled secondary antibodies. (D) EV71 with different dilution (lgPFU/mL=4.5, 5.5, 6.5) were incubated with 100 μg/mL of

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PML-Biotin, then the bound virus particles on the plates were detected by ELISA based binding

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assay. Values are mean ± SD (n=3). **P < 0.01 vs mock control without PML.

N

Fig. 4. Effects of PML on EGFR/PI3K/Akt signaling pathway in Vero cells. (A) PML inhibited the

A

expression of EV71 VP1. The relative VP1 protein level was shown in (B). α-tubulin was detected as

M

control. (C) The phosphorylation of Akt and PI3K was detected by Western blot. α-tubulin and

ED

GAPDH were detected as controls, respectively. The relative protein levels were shown as (D). (E) The phosphorylation of EGFR was detected by Western blotting. The relative protein level was

PT

shown as (F). (G) The expression of p-NF-κB was analyzed in whole-cell lysates by Western blot.

CC E

GAPDH was detected as control. The relative protein level was shown as (H). All the data presented as mean ± SD (n=3). ##P < 0.01 vs normal control group (Mock); *P < 0.05, **P < 0.01, ***P <

A

0.001 vs virus control group (EV71).

Fig. 5. Antiviral efficacy of PML against EV71 in newborn mice. Survival rates (A), body weights (B), clinical scores (C), and viral titers in heart, brain, intestine and muscle tissues (D) determined in 33

EV71 infected newborn mice. Values are mean ± SD (n > 10). *P < 0.05, **P < 0.01 vs virus control group (EV71).

Fig. 6. Histological analysis of different tissues in EV71-infected mice. Mice were sacrificed at 5

IP T

days post-challenge. Heart (A-E), Brain (F-J), intestine (K-O) and muscle (P-T) samples were then collected and stained with H & E. Representative images from each group are shown (n=5). Tissue

SC R

damage was identified and indicated by red arrows (magnification, 20×). Mock: non-infected tissues; EV71: EV71 infected tissues without drugs; Guanidine-10: EV71 infected tissues with Guanidine

U

(10 mg/kg) treatment; PML-10: EV71 infected tissues with PML (10 mg/kg) treatment; PML-5:

A

CC E

PT

ED

M

A

N

EV71 infected tissues with PML (5 mg/kg) treatment.

34

A ED

PT

CC E

Figure 1

35

IP T

SC R

U

N

A

M

A ED

PT

CC E

IP T

SC R

U

N

A

M

Figure 2

36

A

CC E

PT

ED

M

A

N

U

SC R

IP T

Figure 3

Figure 4

37

A ED

PT

CC E

IP T

SC R

U

N

A

M

Figure 5

38

A ED

PT

CC E

IP T

SC R

U

N

A

M

Figure 6

39

Tables

Table 1. Inhibition effects of different compounds on EV71 multiplication in vitro. Table 1 Sulfate content

IC50

CC50

(Da)

(%)

(μg/mL)a

(μg/mL)b

PML

513000

26.1

0.5 ± 0.3

> 5000

Ribavirin

244

—

172.9± 3.2

Guanidine

95.53

—

47.4± 9.6

> 10000.0

2064.7 ± 6.8

11.9

1605.1± 5.6

33.9

U

a Inhibition

(CC50/IC50)c

SC R

Compound

SI

IP T

Molecular weight

concentration 50% (IC50): concentration required to reduce the CPE of the virus by 50% at 16 h p.i.. concentration 50% (CC50): concentration required to reduce cell viability by 50%. c SI: Selectivity index is defined as the ratio of CC to IC (SI = CC /IC ). 50 50 50 50

A

CC E

PT

ED

M

A

N

b Cytotoxic

40