key: cord-1047114-m1ogpm4w
authors: Ooi, Linda S. M.; Ho, Wing-Shan; Ngai, Karry L. K.; Tian, Li; Chan, Paul K. S.; Sun, Samuel S. M.; Ooi, Vincent E. C.
title: Narcissus tazetta lectin shows strong inhibitory effects against respiratory syncytial virus, influenza A (H1N1, H3N2, H5N1) and B viruses
date: 2010-02-26
journal: J Biosci
DOI: 10.1007/s12038-010-0012-8
sha: 9037c67c7b4276e96838ee2f41ce08bedc07ea4e
doc_id: 1047114
cord_uid: m1ogpm4w

A mannose-binding lectin (Narcissus tazetta lectin [NTL]) with potent antiviral activity was isolated and purified from the bulbs of the Chinese daffodil Narcissus tazetta var. chinensis, using ion exchange chromatography on diethylaminoethyl (DEAE)-cellulose, affinity chromatography on mannose-agarose and fast protein liquid chromatography (FPLC)-gel filtration on Superose 12. The purified lectin was shown to have an apparent molecular mass of 26 kDa by gel filtration and 13 kDa by SDS-PAGE, indicating that it is probably a dimer with two identical subunits. The cDNA-derived amino acid sequence of NTL as determined by molecular cloning also reveals that NTL protein contains a mature polypeptide consisting of 105 amino acids and a C-terminal peptide extension. Three-dimensional modelling study demonstrated that the NTL primary polypeptide contains three subdomains, each with a conserved mannose-binding site. It shows a high homology of about 60%–80% similarity with the existing monocot mannose-binding lectins. NTL could significantly inhibit plaque formation by the human respiratory syncytial virus (RSV) with an IC(50) of 2.30 µg/ml and exhibit strong antiviral properties against influenza A (H1N1, H3N2, H5N1) and influenza B viruses with IC(50) values ranging from 0.20 µg/ml to 1.33 µg/ml in a dose-dependent manner. It is worth noting that the modes of antiviral action of NTL against RSV and influenza A virus are significantly different. NTL is effective in the inhibition of RSV during the whole viral infection cycle, but the antiviral activity of NTL is mainly expressed at the early stage of the viral cycle of influenza A (H1N1) virus. NTL with a high selective index (SI=CC(50)/IC(50)≥141) resulting from its potent antiviral activity and low cytotoxicity demonstrates a potential for biotechnological development as an antiviral agent.

Many plant lectins are known to possess antiviral properties against human enveloped viruses (Balzarini 2007a) . They play important biological roles both within and outside the plants. A majority of plant lectins are involved in defence. In many plants, lectins are usually associated with storage tissues such as seeds, tubers, bulbs, corms, rhizomes, root stocks and bark (Van Damme et al. 1995) . The antiviral activities of various lectins have been demonstrated against the human immunodefi ciency virus (HIV) (Balzarini et al. 1992; Balzarini 2006) , human cytomegalovirus (HCMV) (Balzarini et al. 1991 (Balzarini et al. , 1992 , hepatitis C virus (HCV) (Bertaux et al. 2007 ), herpes simplex virus type 1 (HSV-I) (Ooi et al. 2004a ), herpes simplex virus type 2 (HSV-2) (Luo et al. 2007) , infl uenza A (H1N1) virus (Ooi et al. 2004b) , poxvirus (Kaur et al. 2007 ), respiratory syncytial virus (RSV) (Ooi et al. 2004b ) and coronavirus (SARS-CoV) (Keyaerts et al. 2007 ). There has been increasing interest in recent years in the special characteristics of lectins derived from monocot plants (Van Damme et al. 1995) . The monocot mannose-binding lectins belong to a superfamily of structurally and evolutionarily related proteins (Barre et al. 1996) . This group of lectins shares a number of biological properties, such as rabbit erythrocyte haemagglutination, antiviral activity (Balzarini et al. 2006 (Balzarini et al. , 2007b Keyaerts et al. 2007 ) and insecticidal effect (Gatehouse et al. 1995; Hogervorst et al. 2006; Ohizumi et al. 2009 ).

Mannose-binding lectins with antiviral activity have also been isolated from the leaves and bulbs of Narcissus tazetta var. chinensis, commonly known as the Chinese daffodil, which is a monocot perennial ornamental plant of the family Amaryllidaceae (Ooi et al. 1998 (Ooi et al. , 2000 . A potent antiviral lectin from N. tazetta (designated NTL) exists as a homodimer consisting of two identical subunits of 13 kDa (Ooi et al. 2000 (Ooi et al. , 2001 . In mouse, intraperitoneal injection of NTL modulates the gene expression of immunoregulatory cytokines such as interferon (IFN)-γ, transforming growth factor (TGF)-β and stem cell factor (SCF) in macrophages and/or spleen cells (Ooi et al. 2002) . Furthermore, NTL is shown to preserve haematopoietic stem and progenitor cells after 35 days of culture in a serum-and cytokinefree condition, and enhance ex vivo expansion of severe combined immunodefi ciency (SCID)-repopulating cells (Li et al. 2008) . The dual functions of NTL in preservation of early stem and progenitor cells, and expansion of lineagecommitted cells in prolonged culture could be developed for clinical expansion of CD34 + cells for transplantation and use of these cells for other cell therapy strategies. In this study, we investigated the inhibitory effect of a novel mannosebinding lectin isolated, purifi ed and cloned from the bulbs of the Chinese daffodil, Narcissus tazetta var. chinensis, against human RSV, and various strains of infl uenza A (H1N1, H3N2, H5N1) and infl uenza B viruses.

Lectins from the bulbs of the Chinese daffodil, Nar cissus tazetta var. chinensis, were isolated and purifi ed as described previously (Ooi et al. 1998 (Ooi et al. , 2000 , using ion exchange chromatography on diethylaminoethyl (DEAE)-cellulose, affinity column chromatography on mannose-agarose and fast protein liquid chromatography (FPLC)-gel filtration on a Superose 12 column. Haemagglutinating activity was determined using serially two-fold dilutions of the lectin solution in microtitre U-plates (50 μl) mixed with 50 μl of a 2% suspension of rabbit erythrocytes in pH 7.2 phosphatebuffered saline (PBS) at room temperature. Specifi c activity was regarded as the number of haemagglutination units per mg protein.

The crude proteins obtained by extraction with aqueous buffer, ammonium sulphate precipitation and DEAEcellulose column chromatography were dissolved in 20 mM MES buffer of pH 6.2 and chromatographed on a mannoseagarose (Sigma) column. Unadsorbed proteins (the M1 fraction) were eluted with MES buffer, and adsorbed proteins were desorbed with MES buffer containing 0.2 M mannose to give the M2 fraction. The M3 fraction was obtained after washing the column with MES buffer containing 0.5 M NaCl. The fractions were desalted and mannose was removed by dialysis and the proteins were lyophilized. Proteins with different molecular masses in the M2 and M3 fractions were further separated by gel fi ltration on a Superose 12 column using the FPLC system (Pharmacia), which gave rise to the relatively purifi ed fractions NTM2 and NTM3, respectively. The protein profi les of various fractions were studied and their molecular masses determined by sodium dodecyl sulphate (SDS)-PAGE. Briefl y, proteins were mixed with loading dye (1:1) with 2% SDS and 2% β-mercaptoethanol and denatured for 5 min at 95 o C. A low range SigmaMarker TM (Sigma) and the proteins were then resolved using 15% SDS-polyacrylamide gels. After electrophoresis, the gels were stained with Coomassie brilliant blue R-250 for 1 h and subsequently destained with 10% acetic acid and 40% methanol. Pure NTL (from the NTM2 fraction) was obtained after desalting the proteins with a PD-10 column (Bio-Rad, Hercules, CA, USA) and analysed by SDS-PAGE (fi gure 1). The N-terminal amino acid sequence was also determined using a Hewlett Packard (HP) G1000A Edman degradation unit and an HP 1000 HPLC system.

The cDNA-derived amino acid sequence of NTL was determined by molecular cloning as described previously (Ooi et al. 2001) . Its primary protein structure contains a mature polypeptide consisting of 105 amino acids and a C-terminal peptide extension beyond the C-terminal amino acids Thr-Gly (table 1). The homology model was built on the basis of amino acid sequence of the well-known lectin GNA (mannose-specifi c agglutinin from the bulbs of the snowdrop Galanthus nivalis) as the template (Gene Bank Database No. AAA33346.1). Its percentage of sequence identity with NTL is 67.619%. Three-dimensional (3D) structure predictions of the NTL polypeptide were conducted by using the SWISS-MODEL program (ExPASy proteomic tools, available from http://ca.expasy.org/tools/) and drawn by the RasMol software (fi gure 2).

Human larynx epidermoid carcinoma cell line (HEp-2) cells, Madin-Darby canine kidney cell line (MDCK) cells and RSV (Long strain) were purchased from the American Type Culture Collection (ATCC). All infl uenza A (three strains, i.e. H1N1, H3N2 and H5N1) and infl uenza B viruses were clinical isolates obtained from the Department For RSV culture, 1.75×10 4 HEp-2 cells in 100 μl growth medium were seeded into each well of a 96-well tissue culture plate and incubated for 24 h. After removing the growth medium, the RSV stock was diluted using EMEM supplemented with 1% FBS (maintenance medium) and added to the subconfl uent HEp-2 cells (100 μl/well). The plate was observed daily under the microscope for any cytopathic effects (CPE) induced by the virus. When the maximal formation of syncytia was observed, the supernatant was collected and centrifuged at 4 o C, 2000xg for 10 min to remove cell debris. Aliquots of the supernatant were frozen at -80 o C until use.

The cytotoxicity of NTL on MDCK cells was measured quantitatively by the reduction of formazan dye using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay (Mosmann 1983) . MDCK cells were seeded at 80% confl uence in a 96-well microtitre plate (Nunc, Denmark) with 10-fold dilutions of NTL in culture medium. Morphological changes on the MDCK cells were observed microscopically after 24 h of incubation (fi gure 3).

For studying the cytotoxicity of NTL on HEp-2 cells using MTT assay, 2-fold serially diluted NTL samples were added to cells at 25% confl uence and incubated for 48 h. After removal of the medium, 50 μl of MTT (0.5 mg/ml in PBS) was added into each well and incubated for 3 h at 37°C. Dimethyl sulphoxide (DMSO) (100 μl/well) was then added to dissolve the water-soluble formazan. The optical density (OD) was quantifi ed by a micro-spectrophotometer at 540/ 630 nm. The reduction of formazan at each concentration was determined as the percentage of cell control. Cytotoxicity is expressed as 50% cytotoxic concentration (CC 50 ), which is the concentration of the substance that inhibits up to 50% of viable cells (table 2). The percentage of cytotoxicity was calculated using the following formula: Cytotoxicity (%) = (Exp. value -low control) / (High control -low control) ×100. The full-length amino acid sequence of NTL contains a mature polypeptide domain consisting of 105 amino acids and a C-terminal peptide extension beyond Thr-Gly (as indicated by an arrow).

3 × 10 5 cells/well were seeded in 12-well plates and incubated for 24 h. After removing the growth medium, 100 μl of RSV at about 80 PFU and 100 μl of NTL were added to each well with a fi nal concentration of 10 μg/ml. For virus control wells and the wells of condition 3, 200 μl RSV was added at a fi nal concentration of 80 PFU. After further incubation for 2 h, unbound RSV and samples were washed away by rinsing the cells twice with PBS. The cells were overlaid with 1% agarose in the maintenance medium (1 ml/well). After the agarose had solidifi ed, 1 ml of the sample at double concentration (20 μg/ ml) was added to each well of conditions 2 and 3. For each of the virus control wells and the wells of condition 1, 1 ml of maintenance medium was added. After plaque development, the number of plaques was counted and the percentage under different conditions as compared with the virus control was calculated. The results were analysed with one-way ANOVA followed by the Tukey multiple comparison test by the GraphPad Prism 4.0 software.

NTL was isolated and purifi ed from the bulbs of N. tazetta var. chinensis as described previously (Ooi et al. 1998 (Ooi et al. , 2000 . Purifi ed NTL (derived from the NTM2 fraction) was obtained after FPLC-gel fi ltration followed by desalting with a PD-10 column, and its purity was analysed by SDS-PAGE (fi gure 1). It was determined to have a molecu lar mass of about 26 kDa by gel filtration and 13 kDa by SDS-PAGE. NTL is suggested to be a mannose-binding homodimer with two identical subunits of about 13 kDa. Molecular cloning revealed that the deduced amino acid sequence of the fulllength cDNA encoding NTL contained a mature polypeptide consisting of 105 amino acids and a C-terminal peptide extension beyond the C-terminal amino acids Thr-Gly (table 1) . There are two fixed-position cysteines within the protein domain (amino acids 29 and 52), which are probably involved in the disulphide-bond linkage within the molecules to confer the secondary structure of the mature lectin. One third of the deduced amino acid composition consisted of glycine, leucine and asparagines (table 1) . According to an NCBI conserved domain search, NTL protein possesses a β-lectin conserved domain, and 3 typical mannose-binding sites (Q_D_N_V_Y, from Q26 to Y34, Q57 to Y65 and Q89 to Y97, respectively) resembling many other monocot mannose-binding lectins, such as Galanthus nivalis (Van Damme et al. 1991; Hester et al. 1995; Barre et al. 2001) . Four conserved amino acid residues (Gln, Asp, Asn and Tyr) in the mannose-binding sites were predicted to form hydrogen bonds with O2, O3 and O4 of mannose, and hydrophobic residue Val to interact with C3 and C4 of mannose through hydrophobic interactions. The 3D structure prediction of the NTL protein using SWISS-MODEL indicates that NTL is composed of 28 alpha helixes, 52 extended strands and 83 random coils, and that its monomer contains three subdomains, each with a conserved mannose-binding site (fi gure 2). Two subdomains were orthogonal with four-stranded beta-sheets and the other was orthogonal with three-stranded beta-sheets. The three subdomain bundles were asymmetrically arranged as a β-prism, and extended strands in each subdomain were perpendicular to the axis of the prism.

The results of the MTT cytotoxicity assay showed no signifi cant reduction in viability of the MDCK cells (for culturing infl uenza) after 24 h incubation with NTL (fi gure 3), and no microscopic changes in cell morphology were observed. NTL also displayed low cytotoxicity (CC 50 =325.4 μg/ml) as determined by MTT assay towards HEp-2 cells (for culturing RSV) (table 2).

The inhibitory effect of NTL on the replication of various strains of infl uenza viruses was evaluated by the extracellular virus yield reduction assay. NTL could potently inhibit the replication of various infl uenza subtypes in a dose-dependent manner (fi gure 4), and the anti-infl uenza activities were prominent against the H1N1, H3N2 and infl uenza B viruses with IC 50 values of 1.33 μg/ml, 0.40 μg/ml, and 0.20 μg/ml, respectively. However, NTL exhibited only a moderate antiviral effect on the avian infl uenza A (H5N1) virus, with an IC 50 higher than 20 μg/ml.

The time of addition study for infl uenza A (H1N1) virus using HA assay showed that there was a reduction of 5 log 2 HA titre as compared with the controls when NTL was present throughout the entire virus adsorption and incubation period; whereas the reduction was 2 log 2 HA titre when NTL was added only at 1 h following virus adsorption. In an assessment for its anti-RSV activity using the plaque assay, NTL effectively inhibited plaque formation by RSV (IC 50 =2.30 μg/ml) in a dose-dependent manner (fi gure 5), and had a selective index value (SI=CC 50 /IC 50 ) of 141.36 (table 2). The result of a mechanistic study on anti-RSV action showed a signifi cant reduction in plaque formation by RSV. The inhibition rate was 76.4% when NTL was present during the whole infection cycle of the virus, but was 36.6% when NTL was present only during the fi rst 2 h of infection phase, and 45.7% if it was added only 2 h postinfection (fi gure 6).

Mannose-binding lectins with antiviral activity have been isolated from the leaves and bulbs of some monocot plants (Balzarini 2007a, b; Balzarini et al. 1991 Balzarini et al. , 1992 Ooi et al. 1998 Ooi et al. , 2004a Keyaerts et al. 2007 ). Among these, a novel mannose-binding lectin isolated from Narcissus tazetta var. chinensis, known as NTL, is a homodimer with two subunits of about 13 kDa (Ooi et al. 2000 (Ooi et al. , 2001 . Molecular modelling displays that the three-dimensional structure of NTL polypeptide contains three subdomains, each with a conserved mannose-binding site. Protein-protein BLAST (UCBI) of the deduced amino acid sequence of NTL shows high similarities with existing monocot mannosebinding lectins, for example, Allium ursinum (Smeets et al. 1997) , Amaryllis vittata (Wu et al. 2004) , Dioscorea batatas (Ohizumi et al. 2009 ), Zephyranthes candida (Wu et al. 2006) and Galanthus nivalis (Van Damme et al. 1991; Hester et al. 1995) .

NTL possessed potent antiviral activities against RSV and various strains of infl uenza viruses. NTL could effectively inhibit RSV-induced plaque formation (IC 50 = 2.30 μg/ml). Its cytotoxicity against HEp-2 cells was low (CC 50 = 325.4 μg/ml) and thus it had a high SI value of 141.36. Similarly, the inhibition of replication of various infl uenza subtypes was clearly dose dependent. Viral infectivity with human infl uenza A (H1N1, H3N2) and infl uenza B viruses was strongly inhibited by NTL with EC 50 values ranging from 0.02 μg/ml to 1.33 μg/ml. However, NTL had weaker antiviral activity against the avian infl uenza (H5N1) virus than human infl uenza viruses, as no IC 50 for H5N1 virus can be determined only up to a concentration of 20 μg/ml. In parallel with antiviral activity, the cytotoxicity of NTL on MDCK cells was monitored by a colorimetric MTT assay. In concentrations of up to 500 μg/ml, no cell toxicity was observed by determining the effect on cell viability in MTT assay and cell morphology. The low cytotoxicity together with the high SI indicate that NTL possesses potential for biotechnological development as an antiviral agent against RSV and human infl uenza viruses, although potential application against avian infl uenza viruses remains to be determined.

Further investigation on its mode of action against RSV revealed that signifi cant reduction in the number of plaques was observed when NTL was present during the whole viral infection cycle (inhibition by 76.43%). The inhibition rate was 36.57% if NTL was present only during the early infection phase (i.e. the fi rst 2 h post-infection) and 45.73% if it was added in the later infection phase (i.e. sample only present 2 h post-infection). However, a similar investigation on the antiviral activity of NTL against the H1N1 virus (A/HongKong/CUHK-13003/2002) indicated that the target of NTL was mainly in the early phase (i.e. virus adsorption phase). As the early infection events of infl uenza viruses were inhibited, NTL might be virucidal or, like some other antiviral lectins, it might interact with the glycans on glycoproteins present on the envelope of the virus, which are crucial for attachment or fusion, and prevent them from functioning normally in the replication cycle. The results of mechanistic studies on the antiviral action of NTL against the two different viruses (RSV and Flu A) probably suggest that during different phases of the viral infection cycle, the addition of NTL at the time of adsorption and/or penetration is very important. It is also worth noting that the modes of antiviral action of NTL against RSV and infl uenza A (H1N1) virus are signifi cantly different. The inhibitory effect was mainly expressed in the early stage of the viral cycle in the H1N1 virus. Nevertheless, NTL actively inhibited RSV during the whole infection cycle of the virus.

It has been showed that Urtica dioica agglutinin (UDA) from U. dioica strongly binds to the glycans on the HIV-1 attachment protein gp120, and prevents HIV entry and transmission when co-cultivated with persistently HIVinfected and -uninfected cells in the lower μg/ml range (Balzarini 2006) . Some other lectins are also able to inhibit viral entry. Galanthus nivalis agglutinin (GNA), Hippeastrum hybrid agglutinin (HHA) and UDA have been found to be able to inhibit the entry of HCV and HIV-1 into their target cells (Bertaux et al. 2007) . Similarly, HHA inhibits SARS-CoV replication when added at the moment of infection (Keyaerts et al. 2007) . This is in line with the mode of action of the infl uenza A virus in the present study. Nonetheless, HHA is also able to inhibit the extracellular viral load when added up to 5 h post-infection (a time near the end of the virus replication cycle). Thus, HHA interacts both at virus entry and at virus release (Keyaerts et al. 2007 ). The result of our time of addition experiment of NTL against RSV is in agreement with that of the interaction of HHA with SARS-CoV. NTL is active in inhibiting RSV during the entire infection cycle. To inhibit RSV infection, the possible targets that NTL can bind to are the glycoproteins present on the RSV envelope. Three envelope glycoproteins are present on RSV, including the G protein, F protein and SH protein (Feldman et al. 2001) . Both the G and F proteins may be involved in the initial attachment of RSV to the host cell, and the F protein is largely responsible for the subsequent fusion process, though the highest level of fusion was observed when all three glycoproteins were present (Feldman et al. 2001) . This interaction may be blocked if the RSV glycoproteins are bound by NTL, and thus the spread of RSV would be affected. In the present study, NTL was also found to be able to reduce plaque formation when added during the later infection phase. Whether NTL interferes with RSV spread by binding to viral glycoproteins or inhibits other events during the process of viral replication or assembly remains to be elucidated. Ooi 

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