• Tiada Hasil Ditemukan

Enterovirus A71 uses cell surface heparan sulfate glycosaminoglycan as an attachment receptor

4.3 Enterovirus A71 uses cell surface heparan sulfate glycosaminoglycan as an

polysulfonate suramin (6.0 sulfate groups/molecule) were used. Previous studies have shown that suramin inhibits viruses such as DENV and HBV virus which bind to cell surface heparan sulfate (Chen et al., 1997; Schulze et al., 2007). Viral inhibition increased with concentration of the tested inhibitors, and at 1000 µg/ml the inhibition was 65.8 ± 6.7% for dextran sulfate and 63.4 ± 6.1% for suramin (Table 4.2). However, suramin was more effective as it was able to inhibit EV-A71 infection at a concentration as low as 20 µg/ml (Figure 4.16A). Inhibition of EV-A71 infection by the suramin analog NF449 was previously demonstrated by Arita et al. (2008b).

Pre-incubation of RD cells with heparin and dextran sulfate at concentrations from 100-1000 µg/ml before viral infection had no inhibitory effect, but enhanced the virus infectivity. The results demonstrated that the inhibitory effect was due to direct interaction of these compounds with the virus and not to the target cells (Figure 4.16A and 4.16B).

4.3.2 Inhibitory effects of anti-heparan sulfate peptide and poly-D-lysine peptide against enterovirus A71 infection

To assess the important role of negative charges carried on the cell surface in the binding of EV-A71, RD cells were pre-incubated with poly-D-lysine to neutralize cell surface negative charges before EV-A71 infection at a MOI of 0.1. As shown in Figure 4.16C, poly-D-lysine strongly decreased EV-A71 infection in a dose-dependent manner when applied from 1 to 5 µg/ml. At 5 µg/ml, the inhibition was significant, with viral RNA inhibition of 99.1 ± 0.2%. Concentrations higher than 5 mg/ml were found to be cytotoxic to RD cells. To ascertain whether the negative charge carried by heparan sulfate present on the cell surface plays a significant role in viral attachment, two anti-heparan sulfate peptides identified by Tiwari et al. (2011), G1 (LRSRTKIIRIRH) and G2 (MPRRRRIRRRQK) were evaluated for their inhibitory effects. The inhibitory

effect of G2 was significant with viral RNA inhibition of 76.5 ± 26.8% at 1000 µg/ml.

The G1 peptide did not inhibit EV-A71 infection (Figure 4.16C). These data confirmed that EV-A71 binds to cell surface heparan sulfate.

4.3.3 Inhibitory effect of heparin against enterovirus A71 clinical isolates

Heparan sulfate binding phenotype is often acquired through tissue culture adaptation as previously observed in Sindbis virus and FMDV (Jackson et al., 1996; Klimstra et al., 1998). To verify that the heparan sulfate binding phenotype of EV-A71 is not a result of tissue culture adaptation, the inhibitory effects of heparin against various laboratory EV-A71 strains (BrCr, 41, UH1/97 and SHA66/97) and low passage clinical EV-EV-A71 isolates from the Diagnostic Virology Laboratory, University Malaya Medical Center (EV-A71 strains 14716, 35017, 1657640 and 1687413) was evaluated. Heparin at 2500 µg/ml was observed to have an inhibitory effect on both laboratory strains and low passage EV-A71 isolates (Figure 4.17). The inhibitory effects varied between strains.

The inhibition of the laboratory strains ranged from 61.4-98.0%, while inhibition of the low passage isolates ranged from 30.4-78.4%. Interestingly, heparin failed to inhibit the PV vaccine strain even when tested at 2500 µg/ml, implying that PV does not use heparan sulfate as attachment receptor. The data suggested that the heparan sulfate-binding phenotypes were not likely acquired through tissue culture adaptation.

Figure 4.16: Inhibitory effects of GAGs and inhibitors. For the viral inactivation assay, various concentrations of (A) GAGs, polyanionic dextran sulfate and suramin were pre-incubated with EV-A71 for 1 hour at 37°C before infection of RD cells at room temperature. For the cell protection assay, various concentrations of (B) GAGs, (C) poly-D-lysine and anti-heparan sulfate peptides, designated as G1 and G2 peptides, were pre-incubated with RD cells for 1 hour at 37°C before EV-A71 infection. The viral RNA was extracted and quantified by TaqMan real-time PCR 24 hours post-infection. The data were obtained from at least two biological replicates and the error bars indicate the standard deviation of the mean.

Figure 4.17: Inhibitory effect of heparin against EV-A71 isolates and the PV vaccine strain. EV-A71 and PV viral particles were pre-treated with heparin at a final concentration of 2500 µg/ml for 1 hour at 37°C before RD cell infections. The low passage EV-A71 isolates (14716, 35017, 1657640 and 1687413) and the PV vaccine strain were obtained from the Diagnostic Virology Laboratory, University Malaya Medical Center. The viral titers of EV-A71 and the PV vaccine strain were quantified 24 hours post-infection by TaqMan real-time PCR and plaque assays, respectively. The data were obtained from at least two biological replicates and the error bars indicate the standard deviation of the mean.

Table 4.2: Effect of GAGs, GAG variants and inhibitors tested on EV-A71 infection

Inhibitors a Inhibition (%)

Viral plaque reduction Viral RNA reduction

Heparin 95.5 ± 0.7 79.4 ± 2.9

Chondroitin sulfate 52.4 ± 16.4 38.4 ± 6.4

De-N-sulfated heparin 70.0 ± 1.1 39.7 ± 15.6

Dextran sulfate 95.2 ± 0.7 65.8 ± 6.7

Suramin 76.3 ± 2.4 63.4 ± 6.1

a The concentration of inhibitors used was 1000 µg/ml.

4.3.4 Characterization of the residues critical for the inhibitory properties

To establish if the degree of sulfation of heparin is critical for viral inhibition, different sulfated heparin variants were investigated. N-acetyl-de-O-sulfated heparin, which is completely de-sulfated (0 sulfate group/disaccharide), failed to abolish EV-A71 infection (Figure 4.18A). In contrast, de-N-sulfated heparin (0.8-1.2 sulfate group/disaccharide) showed moderate inhibition (39.7 ± 15.6%) and the wild-type heparin (2.4 sulfate group/disaccharide) exhibited the most significant inhibition (79.4 ± 2.9%) at 1000 µg/ml, indicating that the degree of sulfation within the GAG carbohydrate structure is functionally important. Inhibition of viral plaque formation by these GAGs and inhibitors are shown in Table 4.2.

To further confirm the role of sulfation of heparan sulfate in A71 attachment, EV-A71 infection of RD cells grown in medium containing 0-50 mM of sodium chlorate was carried out. Sodium chlorate inhibits cellular adenosine triphosphate sulfurylase, which reduces sulfation of heparan sulfate by up to 60% (Giroglou et al., 2001;

Guibinga et al., 2002). As shown in Figure 4.18B, RD cells treated overnight with sodium chlorate had significantly reduced EV-A71 infection in a dose-dependent manner in all the EV-A71 strains tested. Sodium chlorate inhibited EV-A71 strains 41, UH1, SHA66 and BrCr infections at 50 mM with viral RNA inhibition of 92.7 ± 2.7%, 79.0 ± 3.3%, 83.2 ± 4.2%, and 61.5 ± 5.5%, respectively. To rule out the possibility that the reduction of EV-A71 was due to the cytotoxicity of sodium chlorate, cytotoxicity of sodium chlorate was evaluated by the commercially available MTT assay. Sodium chlorate showed no toxicity at 10 mM and 30 mM, and minimal cytotoxicity at 50 mM (data not shown).

Figure 4.18: Identification of residues critical for the inhibitory effect. (A) EV-A71 was pre-incubated with various concentrations of heparin and heparin variants for 1 hour at 37 °C before infection of RD cells. N-acetyl-de-O-sulfated heparin is completely deN-acetyl-de-O-sulfated, and de-N-N-acetyl-de-O-sulfated heparin is partially desulfated compared to heparin. (B) Inhibitory effect of sodium chlorate on RD cells against different EV-A71 strains. RD cells were pre-treated with increasing concentrations (0 mM, 10 mM, 30 mM and 50 mM) of sodium chlorate for 24 hours before EV-A71 (strains BrCr, 41, UH1 and SHA66) infection at a MOI of 0.1. Data presented are obtained from at least two biological replicates. Error bars indicate standard deviation of the mean.

4.3.5 Removal of cell surface heparan sulfate using enzymatic treatment

To identify the type of GAG which is responsible for EV-A71 binding to RD cells, the binding of EV-A71 to cells after enzymatic removal of cell surface GAGs with chondroitinase ABC and heparinase I/II/III digestion was examined. Heparinase I degrades heparin and highly sulfated domains in heparan sulfate (relative activity about 3:1) at the linkages between hexosamines and O-sulfated iduronic acids. Heparinase II cleaves heparan sulfate, and to a lesser extent heparin (relative activity about 2:1) at the 1-4 linkages between hexosamines and uronic acid residues. Heparinase III specifically degrades heparan sulfate (Ernst et al., 1995; Chen et al., 1997). Treatment of RD cells with each of the heparinases at 2.5 mIU/ml and 5.0 mIU/ml for 1 hour at 37˚C was found to significantly reduce the viral RNA (Figure 4.19A) 24 hours post-infection in a dose-dependent manner. Treatment of RD cells with heparinase I, II and III at 5.0 mIU/ml significantly inhibited EV-A71 viral RNA by 68.6 ± 6.2%, 91.7 ± 4.1% and 82.2 ± 11.7%, respectively (Figure 4.19A). Removal of cell surface chondroitin sulfate by chondroitinase ABC failed to inhibit EV-A71 infection (Figure 4.19A), even when tested at concentrations as high as 20 mIU/ml (data not shown). Removal of cell surface heparan sulfate significantly reduced the infectivity of the different EV-A71 strains tested, but not the PV vaccine strain (Figure 4.19B). Removal of surface heparan sulfate, but not chondroitin sulfate, significantly reduces EV-A71 attachment to the surface of RD cells (Figure 4.19C).

Figure 4.19, continued

Figure 4.19: Effect of heparinases and chondroitinase ABC treatment on EV-A71 infection. (A) Inhibitory effect of heparinase I, II, III and chondroitinase ABC on EV-A71 infection. RD cells were pre-treated with heparinases or chondroitinase ABC for 1 hour at 37˚C before EV-A71 infection at a MOI of 0.1. The viral RNA was extracted and evaluated by TaqMan real-time PCR. (B) Inhibitory effect of heparinase I against different EV-A71 strains and PV vaccine strain. PV vaccine strain was used as a control virus. (C) Confocal microscopy analysis (40X magnification) of EV-A71 binding assay after heparinase I and chondroitinase ABC treatments. EV-A71 viral particles were stained with monoclonal EV-A71 antibody and subsequently stained with Alexa Fluor 488 anti-mouse IgG. Nuclei were stained with DAPI. EV-A71 viral particles and nuclei are shown in green and blue, respectively.

4.3.6 Knockdown of heparan sulfate biosynthesis expression using small interference RNA

To further confirm that heparan sulfate plays an important role in EV-A71 infection, expression of the NDST-1 and EXT-1 genes were transiently knocked down using siRNA. NDST-1 is a heparan sulfate modification enzyme which removes N-acetyl groups from the selected N-acetylglucosamine (GlcNAc) residues and replaces them with sulfate groups (Presto et al., 2008). EXT-1 is a heparan sulfate polymerase that adds alternating units of glucuronic acid (GlcA) and GlcNAc to the non-reducing end of the chain. RD cells were transfected with various concentrations of NDST-1 and EXT-1 siRNAs for 24 hours using Lipofectamine 2000 reagent before infection with EV-A71 at a MOI of 0.1. As shown in Figure 4.20, transient knockdown of both NDST-1 and EXT-1 expression in RD cells significantly reduced EV-A71 infection with inhibition up to 80.1 ± 7.7% and 57.2 ± 19.1% at 20 nM, respectively. However, the scrambled negative control siRNA did not reduce EV-A71 infection.

4.3.7 Binding of enterovirus A71 to Chinese hamster ovary (CHO) cells that are variably deficient in glycosaminoglycan biosynthesis

CHO cells with defects in the biosynthesis of GAGs have been extensively used to demonstrate the involvement of heparan sulfate as the receptor for binding of various viruses (Summerford and Samulski, 1998; Goodfellow et al., 2001; Guibinga et al., 2002; Vlasak et al., 2005; Schulze et al., 2007). The mutant CHO-pgsD677 cells are deficient in N-acetylglucosaminyltransferase and glucuronosyltransferase activities required for heparan sulfate polymerization, and thus completely lack heparan sulfate.

These cells also produce three- to four-fold higher levels of chondroitin sulfate when compared to the wild-type K1 cells (Lidholt et al., 1992). The mutant CHO-pgsA745 cells are deficient in the enzyme

UDP-D-xylose:serine-1,3-D-xylosyltransferase, which catalyses the first sugar transfer reaction in GAG formation, and thus completely lack GAGs (Esko et al., 1985).

To investigate whether EV-A71 binds differently in these cell lines, cells were seeded in CellCarrier-96 and chamber slides, and infected with EV-A71 at an MOI of 100 for 1 hour at 4°C. As shown in Figure 4.21, significantly lower numbers of viral particles were attached to the CHO-pgsD677 and CHO-pgsA745 cells when compared to the wild-type CHO-K1 which expressed normal levels of heparan sulfate (P <0.001).

Results from Cellomics HCS VTI array scaning demonstrated that CHO-pgsD677 and CHO-pgsA745 cells showed reduced binding of 46.7% and 41.6%, respectively when compared to CHO-K1 cells. Interestingly, more viral particles were bound to RD when compared to CHO-K1 cells. This could result from the different levels of heparan sulfate expression or the sulfation phenotypes in RD and CHO-K1 cells.

4.3.8 Binding of enterovirus A71 to immobilized heparin sepharose beads

To characterize the interaction of EV-A71 particles to GAGs, virus-containing supernatant was applied to a heparin affinity chromatography column under normal physiological salt conditions (0.14 M NaCl) and eluted with 2M NaCl. The virus in each fraction collected was quantified by TaqMan real-time PCR and plaque assay for EV-A71 and PV, respectively. As depicted in Figure 4.22A, most of the EV-A71 viral particles were detected in all the eluates following application of 2M NaCl. The EV-A71 viral particles present in eluate 1 were concentrated by up to 4.1-fold. In a control experiment, a column packed with sepharose alone showed no binding of EV-A71 viral particles to the column (Figure 4.22B). The results confirm that the EV-A71 particles bind to heparin and were eluted by high salt concentrations. In contrast to EV-A71, the PV vaccine strain did not interact with heparin sepharose and most of the PV viral particles were detected in the flow-through fraction (Figure 4.22A). This result

confirmed that EV-A71 binds to cell surface heparan sulfate, and PV does not.

4.3.9 Enterovirus A71 three-dimensional structuring and prediction of heparan sulfate binding domains

To determine the possible heparan sulfate binding site(s) on the EV-A71 viral particles, the three-dimensional crystal structure of EV-A71 was built using DeepView Swiss PDB viewer (Guex and Peitsch, 1997). As shown in Figure 4.23A, amino acids Arg166, Lys242 and Lys244 are arranged symmetrically in the 5-fold axis of the EV-A71 pentamer structure. These amino acids are also located at positions which are highly exposed on the surface of the EV-A71 viral particle (Figure 4.23B). All these amino acids were highly conserved across all EV-A71 genotypes with the exception of Lys244, where lysine (K) was substituted with glutamic acid (E) in genotype A (Figure 4.23C).

These symmetrically arranged clusters of positively-charged amino acids could serve as the major binding site for heparan sulfate.

Figure 4.20: Effect of transient siRNA knockdown of NDST-1 and EXT-1 expression on EV-A71 infection. The NDST-1 and EXT-1 siRNA in lipofectamine 2000 reagent was transfected into RD cells for 24 hours before EV-A71 infection. The viral load was determined 24 hours post-infection by TaqMan real-time PCR. An siRNA with a nonsense sequence was used as a control. The data presented are means obtained from at least two biological replicates. Error bars indicate standard deviation of the mean.

Figure 4.21: Binding of EV-A71 to CHO-K1 and CHO mutant cells. CHO-K1 cells and CHO mutants defective in proteoglycan synthesis were assessed for their ability to bind to EV-A71. Cell line CHO-pgsA745 lacks heparan sulfate and chondroitin sulfate proteoglycans, while CHO-pgsD677 lacks heparan sulfate proteoglycan but produces 15% of normal proteoglycans.

Binding of EV-A71 to parental and mutant CHO cells was assayed using (A) confocal microscopic analysis (40X magnification) and (B) Cellomics HCS ArrayScan VTI SpotDetector BioApplication and verified by TaqMan real-time PCR. EV-A71 viral particles and nuclei are shown in green and blue, respectively. The data presented are means obtained from at least two biological replicates. Error bars indicate standard deviation of the mean.

Figure 4.22: Binding of EV-A71 and PV to immobilized heparin-sepharose column.

(A) EV-A71 and PV supernatants were passed through a column of immobilized heparin-sepharose and eluted with 2M NaCl. The viral titers of EV-A71 and PV vaccine strain from each fraction were quantified by TaqMan real-time PCR and plaque assay, respectively. EV-A71 is presented as viral RNA/ml and the PV vaccine strain was presented as PFU/ml. (B) Binding of EV-A71 to the sepharose beads. Error bars represent means ± SD of each fraction.

Figure 4.23: Three-dimensional pentameric structure and sequence alignment of EV-A71. The structure of the EV-A71 pentamer was generated using DeepView Swiss PDB viewer. The molecular structure of EV-A71 VP1, VP2, VP3 and VP4 is represented by blue, green, red and purple, respectively. The amino acids Arg166, Lys242 and Lys244 are indicated in yellow, white and light blue, respectively. (A) Top view and (B) side view of the EV-A71 pentamer. (C) Histogram showing sequence consensus in the VP1 region of EV-A71. A total of 174 sequences were aligned and analyzed using ClustalW2. The arbitrary scale is depicted to the left of the histogram and 1.0 denotes perfect consensus at a given amino acid site across all entries. The alignment of Arg166, Lys242 and Lys244 of representative EV-A71 strains from genotypes A, B and C are shown above the histogram.

4.3.10 Enterovirus A71 receptors analysis

Multiple receptors have been discovered for EV-A71 infection, which include human SCARB2, PSGL-1, sialic acid, annexin II and vimentin (Nishimura et al., 2009;

Yamayoshi et al., 2009; Yang et al., 2009; Yang et al., 2011; Du et al., 2014). In this study, the sequential events of the SCARB2 and heparan sulfate usage were investigated.

RD cells in a chamber slide were incubated with EV-A71 viral particles at an MOI of 100 at 4°C for an hour. The cells were either immediately fixed or shifted to 37°C for 15 minutes before being fixed with 4% formaldehyde. The cells were then stained with anti-EV-A71 monoclonal antibodies and either anti-heparan sulfate monoclonal antibodies or anti-SCARB2 monoclonal antibodies. The nuclei were visualized by DAPI stain. At 4°C, most of the virus particles were co-localized with the heparan sulfate receptor (Figure 4.24A). However, when the temperature was shifted to 37°C for 15 minutes, most of the virus particles were now co-localized with SCARB2 receptor (Figure 4.24B). These results indicated that cell surface heparan sulfate serves as an attachment receptor and virus entry required further interactions with the SCARB2 entry receptor.

Cell surface sialic acid and heparan sulfate were removed using neuraminidase V and heparinase I/III blend, respectively, leading to the reduction of EV-A71 infection (Figure 4.25). Interestingly, the inhibition of EV-A71 infection was higher when cell surface heparan sulfate was removed, suggesting that heparan sulfate is more important compared to sialic acid. When both cell surface sialic acid and heparan sulfate were removed, a greater reduction of EV-A71 infection was observed. This data suggests that EV-A71 selectively binds to cell surface heparan sulfate and sialic acid, and heparan sulfate is functionally more important than sialic acid.

Figure 4.24: Colocalization analyses of EV-A71 receptor interactions. Confocal microscopy analysis (40X magnification) of EV-A71 binding to the RD cells was performed at the (A) attachment stage and (B) entry stage. EV-A71 was allowed to attach to the cell surface at 4°C for an hour, and viral entry was stimulated at 37°C. EV-A71 viral particles, heparan sulfate and SCARB2 were bound to their respective monoclonal antibodies and stained with the appropriate Alexa Fluor-conjugated IgG antibodies. Nuclei were stained with DAPI. EV-A71 viral particles and nuclei are stained green and blue, respectively. Both heparan sulfate and SCARB2 are stained red. Co-localization is indicated in yellow.

Figure 4.25: Effect of heparinase I/III and neuraminidase V treatment on EV-A71 infection. RD cells were pre-treated with heparinase I/III, neuraminidase V or both for 1 hour before infection of EV-A71 at a MOI of 0.1. The virus titers were determined 24 hours post-infection by plaque assay. The data presented are means of two biological replicates. Error bars indicate standard deviation of mean.

4.4 Inhibition of enterovirus A71 infections by octaguanidinium-conjugated