- freely available
Mar. Drugs 2013, 11(8), 3000-3014; doi:10.3390/md11083000
Published: 19 August 2013
Abstract: The antiviral activity of different structure fucoidans (α-l-fucans and galactofucans) was studied using two model viral systems based on a lentiviral vectors and a replication competent Moloney murine leukemia virus (Mo-MuLV). It was found that investigated fucoidans have no cytotoxic effects on Jurkat and SC-1cell at the concentration range of 0.001–100 µg/mL. Fucoidans with different efficiency suppressed transduction of Jurkat cell line by pseudo-HIV-1 particles carrying the envelope protein of HIV-1 and infection of SC-1 cells by Mo-MuLV. According to our data, all natural fucoidans can be considered as potential anti-HIV agents regardless of their carbohydrate backbone and degree of sulfating, since their activity is shown at low concentrations (0.001–0.05 µg/mL). High molecular weight fucoidans isolated from Saccharina cichorioides (1.3-α-l-fucan), and S. japonica (galactofucan) were the most effective inhibitors.
Virus entry into the cell is a key stage in the extension of viral infection. Most enveloped viruses enter the cell only after interaction of viral envelope proteins with the specific receptors on the cell surface. For many viruses it was found that the contact with the specific receptor is preceded by non-specific interaction with the primary receptor. It was shown that heparan sulfates located on the cell membrane are involved in primary cell binding of many viruses (herpes simplex virus, hepatitis C virus, human cytomegalovirus, human papilloma virus, HIV-1, murine leukemia virus) . The contact of retrovirus with the primary receptor is required for subsequent strong interactions with receptor/coreceptor specific for retroviruses of each group of interference [1,2].
Thus, the search for effective inhibitors that inhibit the interaction of viruses with cellular heparan sulfate is an important task, the solution of which can lead to the creation of a broad spectrum preparation effective for different groups of pathogenic retroviruses including HIV-1 .
Sulfated polysaccharides are known to demonstrate a high antiviral activity preventing penetration of DNA- and RNA-containing viruses to the cell . Their degree of activity directly depends on the following sulfated polysaccharide properties: molecular mass, structure of carbohydrate backbone, sulfation degree . For some classes of semisynthetic polysaccharide (e.g., dextran sulfate) it is known that their antiviral activity increases while the sulfation degree and/or molecular mass increases too [6,7,8].
Fucoidans—sulfated polysaccharides from brown algae—are heterogenic and represent the mixtures of structurally related polysaccharides with certain variations of carbohydrate units (l-fucopyranose and non-fucose ones) and non-carbohydrate substituents (mainly sulfate and acetyl groups) content. The detail structural analysis of fucoidans is difficult at present, so structural variety of fucoidans has not been investigated yet. The literature data analysis shows that most of known fucoidans fall into three structural types: the first type contains (1→3)-linked α-l-fucopyranose residues in the main chain; the second type alternating (1→3)- and (1→4)-linked residues of α-l-fucopyranose; and the third type is fucoidans containing galactose and fucose in comparable quantities (galactofucans). Besides fucose, many fucoidans contain some quantities of other monosaccharides [9,10]. Rarely found are fucoidans with the main chain represented by uronofucans [11,12].
In contrast to semisynthetic sulfated polysaccharides, natural algae polysaccharides do not show any discernible anticoagulant activity at therapeutically administrable doses . Advantages of the algae polysaccharides are their relatively low cost, wide spectrum of antiviral activity, absence of expressed cytotoxic effect, low drug resistance formation degree during long-term use, and good solubility .
At present, there are two main approaches to determine the efficiency of potential antiviral agents. The first is in vitro inhibitory analysis using purified viral enzymes . However, when working with purified enzymes, it is impossible to determine the ability of potential drugs to interact with the cell in vivo, in particular their interaction with cellular receptors, the ability to penetrate into the cell, stability, and the absence of cytotoxicity. The second approach involves working with infectious viruses, such as HIV. This approach can only be applied in a small number of laboratories that are specially equipped for work with human pathogens . Limitations in working with infectious viruses greatly complicate the search for and testing of antiviral drugs. Using the system designed at the Engelhardt Institute of Molecular Biology of the Russian Academy of Sciences, it is possible to investigate the activity of potential inhibitors that prevent the interaction of simple and complex retroviruses with the primary surface receptors of the cell thus preventing the infection of cells [15,16]. The aim of this work was to study the antiviral activity of a number of natural fucoidans and choose the most active fucoidans samples to be studied further as potential candidates for antiviral drug development.
2. Results and Discussion
2.1. Characterization of the Fucoidans from Brown Algae
The antiviral activities of fucoidans from different species of brown algae isolated from the Far-East Russian coast were investigated. Fucoidans were represented with different structural types (Table 1). The polysaccharide chain of the α-l-fucan (ScF) isolated from Saccharina cichorioides was built up mainly of (1→3)-linked α-l-fucopyranose residues. Other α-l-fucan (FeF) isolated from Fucus evanescens contained the backbone with alternating (1→3) and (1→4) linkages. The galactofucans (GF) SgGF, AoGF, SjGF were obtained from brown algae Saccharina gurjanovae, Alaria ochotensis, Saccharina japonica, respectively.
|Table 1. Characteristics of polysaccharides fractions from brown algae.|
|Seaweed source||Fraction||Mw, |
|The linkage between Fuc residues||SO3Na− (%) *||Monosaccharide composition, mol%|
|F. evanescens||FeF **||620||α-1,3;1,4||23.2||77.9/9.5/4.2/0/8.4/0|
|S. gurjanovae||SgGF **||810||α-1,3||28.2||64.3/20.7/14.6/0.4/0/0|
|S. japonica||SjGF **||1800||α-1,3;1,4||23.3||49.9/44.1/5.3/0/1.1/0|
|C. costata||CcGF **||160||α-1,3||23.2||62.6/30.1/2.6/1.7/0/0|
* % of sample weight; ** acetated sample; *** Monosaccharide composition: Fuc = fucose, Gal = galactose, Man = mannose, Rha = ramnose, Xyl = xylose, Glc = glucose, SO3Na− = sulfate group.
All investigated α-l-fucans and galactofucans were highly sulfated. Gel-permeation chromatography investigation of fucoidan samples demonstrated their comparable elution profile and domination of the polysaccharide fractions with molecular weights (Mw) ranged from 300 to 800 kDa. The fucoidans with Mw higher than 1000 kDa were isolated from S. cichorioides, S. japonica. According to our recent studies, the α-l-fucan from F. evanescens and the galactofucans from L. gurjanovae, C. costata and S. japonica are not only sulfated but acetated also [17,18]. The presence of acetyl groups in fucoidans were detected by NMR spectroscopy. The removal of acetyl groups from FeF via alkaline treatment gives FeFDA with a low molecular weight of 20 kDa. The characteristics of fucoidans under study are presented in Table 1.
Previous studies showed that α-l-fucans from S. cichorioides and F. evanescens exhibited antiviral activity against Hantaan virus , galactofucan from S. japonica had antiviral activity against avian influenza A (H5N1) virus infection  and in case of experimental tick-borne encephalitis. However, the α-l-fucans from S. сichorioides and F. еvanescens and GF from C. costata showed moderate activity in the same experiment .
3. Experimental Section
3.1. Biological Material
Brown algae Saccharina cichorioides (two-year cycles of development) and Costaria costata were collected in July 2008 in the Troitsa Bay (Sea of Japan) at the Sea Experimental Station of Pacific Institute of Bioorganic Chemistry (PIBOC), Far-East Branch, Russian Academy of Sciences (Primorsky region of Russia). Brown algae Fucus evanescens and Saccharina gurjanovae were collected from the littoral at the west coast of the Iturup Island of the Sea of Okhotsk (Kuril Islands) in July 2011 and at the coast of the Island Big Shantar (Sea of Okhotsk) in July 2009, respectively. Brown algae Alaria ochotensis and Saccharina japonica were collected from natural habitats of Sakhalin Island (Okhotsk Sea) and Kunashir Island (Pacific Coast), respectively, in August 2008.
The algae species were identified by A. Skriptsova (A.V. Zhirmunsky Institute of Marine Biology FEB RAS, Russia).
3.2. Isolation and Determination of the Fucoidan Structure
3.2.1. Extraction of Water-Soluble Polysaccharide from the Brown Algae
The fucoidans used in the present work are listed in Table 1. The procedure of the fucoidans isolation from S. cichorioides has been described earlier . The procedure includes the following stages: extraction of the dry defatted algal biomass with a dilute solution of HCl (pH 2.0–2.3), dialyzation and lyophilization or precipitation of polysaccharides with ethanol and an ion-exchange chromatography of the native polysaccharide performed to obtain the most sulfated fraction ScF. The same procedure was used to obtain fucoidans FeF from F. evanescens , SgGF from S. gurjanovae , AoGF from Al. ochotensis and SjGF from S. japonica  and CcGF from C. costata .
3.2.2. Analysis of Monosaccharide Composition
The monosaccharide compositions of fucoidans were determined as described previously . The monosaccharide composition of the polysaccharides after acidic hydrolysis was determined on a Biotronik IC-5000 carbohydrate analyzer (Germany) using a Shim-pack ISA-07/S2504 (0.4 × 25 cm) column. Elution was performed with a potassium borate buffer at an elution rate of 0.6 mL/min. Detection was carried out by the bicinchoninate method and integration on a Shimadzu C-R2 AX system. Monosaccharides (Rha, Man, Fuc, Gal, Xyl, and Glc) were used as standards.
3.2.3. Deacetylating of the Fucoidan
An alkaline treatment was used to remove acetyl groups from FeF giving FeFDA. Native polysaccharide FeF (500 mg) was dissolved in water (10 mL), than concentrated aqueous NH4OH (10 mL) was added to the solution. This mixture was kept at 60 °C for 4 h. A solution of polysaccharide was dialyzed by ultrafiltration (1 kDa cutoff) and lyophilized.
3.2.4. Sulfate Group Content
The sulfate group content was determined by the turbidimetric method after hydrolysis of the corresponding fractions with HCl (1 N) .
3.2.5. Anion-Exchange Chromatography
Anion-Exchange Chromatography: polysaccharide solutions made in 0.04 N HCl (1 g/20 mL) were applied onto a DEAE-cellulose column (Cl-form, 3 × 14 cm) equilibrated with 0.04 N HCl. Sulfated polysaccharides were successively eluted with liner gradient of 250 mL H2O/250 mL NaCl (2 M). The fractions were dialyzed and lyophilized to obtain the polysaccharide fractions.
3.2.6. Determination of Molecular Weight
The molecular weights (MW) of polysaccharides were determined by HPLC in a Shimadzu LC-20A instrument with a RID-10A refractometric detector. Polysaccharides were separated over successively connected columns of Shodex Asahipak GS-520 HQ and GS-620 HQ (7.5 mm × 300 mm) at 50 °C with elution by H2O (0.8 mL/min). Columns were calibrated using standard pullulans with MWs ranged from 180 to 667 kDa (Polymer Laboratories, Amherst, MA, USA) and blue dextran (Amersham, Uppsala, Sweden).
3.2.7. NMR Spectroscopy
NMR spectra for solutions of the fucoidan in D2O were obtained on a Bruker Avance DPX-500 NMR spectrometer with a working frequency of 75.5 MHz at 60 °C.
3.3. Biological Assays
3.3.1. Cell Culture
The following cell lines were used in this study:
HEK293 (human embryonic kidney cells), SC-1 (mouse embryonic fibroblasts) were cultivated in DMEM containing 10% fetal calf serum (FCS), 4 mM of l-glutamine, 100 U/mL of penicillin, and 100 μg/mL of streptomycin.
Jurkat (human T-lymphoblastic leukemia) were cultivated in RPMI-1640 containing 20% FCS, 4 mM of l-glutamine, 100 U/mL of penicillin, and 100 μg/mL of streptomycin.
The cells were grown at 37 °C in humid air containing 5% of CO2.
3.3.2. Generation of Jurkat Clone 4X4#3
Antibodies and FACS Analysis
Cells were stained with monoclonal antibodies against CD4 (#SK3), CD184 (#12G5) and CD195 (#2D7) or corresponding isotype controls (all BD, Heidelberg, Germany) according to the manufacturers’ instructions. FACS analysis was performed on a FACS CantoII (BD).
To establish S2-compatible viral infection assays, various human T-cell lines were screened for expression of CD4 and HIV co-receptors CXCR4 and CCR5 by FACS analysis. Among three different Jurkat cell lines tested, one cell line (Jurkat JMP, kindly provided by Andreas Guse, University Medical Center Hamburg-Eppendorf, Hamburg, Germany) showed partial expression of CD4 in more than 50% of the cells in conjunction with CXCR4 (data not shown). To obtain cells with homogenous expression of both receptors, cells showing the highest expression of both CXCR4 and CD4 were isolated via fluorescence-activated cell sorting on a FACS ARIA (BD). Sorted cells were seeded as single cell cultures into 96 well plates, expanded for 4 weeks and tested for CD4 and CXCR4 expression. One clone (4X4#3), which was highly positive for CD4 and CXCR4 (>99.5%) and showed stable receptor expression for several months in culture, was selected as target for subsequent infections.
3.3.3. Obtaining of Pseudo-HIV-1 Particles
HEK293 cells seeded in Petri dishes with a diameter of 100 mm in the amount of 3.0–3.5 × 106 cells per dish 12–14 h prior to the transfection were used as packaging cells, in which the assembly of recombinant pseudo-HIV-1 particles occurs. DNA of the lentiviral vector containing the marker eGFP gene and the plasmids directing the synthesis of the proteins that are required for the formation of pseudo-HIV-1 particles were introduced into HEK293 cells via calcium phosphate transfection. The pseudo-HIV-1 particles were collected 24 h following transfection with a 12 h interval .
The pseudo-HIV-1 particles were titrated on Jurkat cells seeded to 24-well plates 24 h prior to transduction. The level of cell fluorescence was measured on Beckman Coulter Epics 4XL flow cytometer (USA) 48 h following the transduction.
The pseudo-HIV-1 particles titer was calculated using the formula T = NP/V, where N is the amount of seeded cells, P is the share of the transduced cells in the population, V is the amount of the added supernatant containing pseudo-HIV-1 particles (mL), and T is titer. The samples with titer of 5 × 105–5 × 106 were used in this study.
3.3.4. Obtaining Replication Competent Virus Particles
The assembly of replication competent retrovirus particles and their secretion into the culture medium was carried out in SC-1 cells, which were seeded on a Petri dish 100 mm in diameter with (3.0–3.5) × 106 cells per plate 12–14 h before transfection.
Retroviral vector containing the full length genome of Mo-MuLV, as well as the GFP marker gene, was introduced into SC-1 cells by Lipofectamine 2000 transfection. Replication competent retroviral particles were collected 48 h after transfection at an interval of 10–12 h. The virus was titrated on SC-1 cells seeded into 24 well plates (3 × 105 cells per well) 14–16 h before infection as described above for Jurkat cells.
3.3.5. Cytotoxicity Assay
Cytotoxicity of preparations for uninfected Jurkat and SC-1 cells was determined on changes in morphology and the number of viable cells using Trypan blue staining solution (Invitrogene Corp., Carlsbad, CA, USA). For this purpose, the preparation of polysaccharide up to concentration of 10 and 100 µg/mL was added in the medium. After 48 h, Jurkat cells were resuspended in the medium and stained with 0.4% solution of Trypan blue for 5 min. SC-1 cells were removed by trypsin, resuspended in the medium and stained with 0.4% Trypan blue solution for 5 min. Then, the number of viable (unstained) and nonviable (stained) cells were counted in a Neubauer chamber. The number of living cells in the population was estimated by the number of unstained cells (as a percentage of the total).
It can be concluded that antiviral activity of investigated fucoidans depends on the type of virus, more precise on the envelope protein species. The inhibitory activity of fucoidans is specific against viruses that use heparan sulfate as the primary cell receptor. The investigated fucoidans suppressed transduction of Jurkat cells by pseudo-HIV-1 particles that contain envelope protein HIV-1 regardless to structure of carbohydrate backbone. The data obtained allows us to consider all investigated natural high molecular weight fucoidans as potential anti-HIV agents, because their efficiency against lentiviral transduction was demonstrated at low concentrations (0.001–0.05 µg/mL). High molecular weight 1,3-α-l-fucan ScF and galactofucan SjGF had the best inhibiting effect. Investigation of these fucoidans’ antiviral activity will be continued.
The authors thank O.A. Stepanov for taking part in the range of experiments connected with Mo-MuLV. The authors are also grateful to A.V. Skriptsova for identification of algae species.
This work was supported by the RFBR (12-04-00669 and 11-04-01365) and the Program (Molecular and Cellular Biology) of the Presidium of Russian Academy of Sciences.
Conflicts of Interest
The authors declare no conflict of interest.
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