Analysis of Compounds That Interfere with Herpes Simplex VirusâHost

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Analysis of compounds that interfere with herpes simplex virushost receptor interactions using surface plasmon resonance Subash C.B. Gopinath, Kyoko Hayashi, Jung-Bum Lee, Akiko Kamori, Cai-Xia Dong, Toshimitsu Hayashi, and Penmetcha K.R. Kumar Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac4025522 • Publication Date (Web): 30 Sep 2013 Downloaded from http://pubs.acs.org on October 10, 2013

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Analytical Chemistry

Analysis of compounds that interfere with herpes simplex virus-host receptor interactions using surface plasmon resonance Subash C.B. Gopinath1,2, Kyoko Hayashi3, Jung-Bum Lee3, Akiko Kamori3, Cai-Xia Dong3, Toshimitsu Hayashi3 and Penmetcha K.R. Kumar1*

1

Biomedical Research Institute, Central 6, 2Nanoelectronics Research Centre, Central 4,

National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba City 305-8566, Ibaraki, 3Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan

Running title: HSV-host receptor interfering compounds study by SPR

*

Correspondence should be addressed to:

Dr. Penmetcha K. R. Kumar Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1 Higashi, Tsukuba City 305-8566, Ibaraki, Japan. Tel./Fax: 81-298-61 6773 Email: [email protected]

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ABSTRACT: The entry of herpes simplex virus into host cells involves a complex series of events that require concerted inputs from multiple HSV glycoproteins. Among these glycoproteins, the gD protein of HSV-1 and HSV-2 plays an important role for host receptor binding and membrane fusion. In the present study, we evaluated the ability of different sulfated saccharides to interfere with gD-host receptor (HVEM) interactions using our recently reported molecular assay (Gopinath et al., 2012, J. Virol. 86, 67326744). Initially, we tested the ability of heparan sulfate to interfere with the HVEM-HSV1 gD interaction and found that heparan sulfate is able to interfere efficiently, with an apparent EC50 of 2.1 µM. In addition, we tested different synthetic sulfated polysaccharides and natural sulfated polysaccharides from an edible alga, Sargassum horneri, after fractionation into different sizes and sulfate and uronic acid contents. Six polysaccharides isolated from S. horneri were found to efficiently interfere with the HVEM-gD interaction. Three others caused moderate interference, and five caused weak interference. These results were confirmed with plaque assays, and good agreement was found with the results of the SPR assay for the identification of compounds that interfere with HVEM-HSV-1 gD binding. These studies suggest that our molecular assay based on surface plasmon resonance is not only useful for the analysis of viral-host protein interactions but is also applicable for the routine screening of compounds to identify those that interfere with the first step of viral entry, thus facilitating the rapid development of novel antiviral compounds that target HSV.


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Enveloped viruses express glycoproteins on their surfaces and use these proteins to recognize their cognate receptors on the surfaces of host cells. These glycoproteins also facilitate membrane fusion. Both of these events involve specific interactions between the viral envelope glycoproteins and the host cell receptors, and these events can be divided broadly into three steps: the interaction of viral glycoproteins with host cell receptors, the triggering of the fusion process and the completion of membrane fusion. All three steps are regulated by viral glycoproteins in concert with host receptors during their final journey into the host cells. A number of viruses utilize this approach, including herpes simplex virus (HSV), hepatitis C virus (HCV), and enterovirus 71 (EV71). HSV entry into target cells is a complex process involving different glycoproteins. The entry of HSV into the host cell begins with the binding of the viral proteins gC and gB to proteoglycans on the host cell surface. This binding interaction is then followed by the coordinated action of four essential glycoproteins: gD, gB, gH, and gl.1,2 gD initially binds to a specific cellular receptor and sends a signal to the downstream glycoproteins to initiate the membrane fusion process. gD is known to interact with a wide range of cellular receptors, including a member of the TNF receptor family (herpes virus entry mediator, HVEM),3 cell adhesion molecules of the immunoglobulin superfamily [nectin-14 and nectin-25] and 3-O-sulfated heparan sulfate.6 Because of the broad specificity of the gD of HSV, this virus can infect a wide range of cell types, such as epithelial cells, fibroblasts, and neurons. Depending on the cell type, HSV enters the host cell via the fusion of the viral envelope with either the plasma membrane or endocytic membranes.7 The structure of the gD protein of HSV-1 has been solved in the absence and presence of its cognate receptors HVEM and nectin-1.8-11 All these structural studies showed that gD contains a V-like Ig fold between residues 56 and 184. This structural feature is commonly found in cell surface adhesion molecules. N- and C-terminal extensions flank this fold and are unfolded and disordered in the absence of the gD receptor. The structure of the gD-HVEM complex and functional studies indicate that the N-terminal region of gD, including residues 7-15 and 24-32, interact with the HVEM receptor (PDB 1JMA).12,13 Similarly, the molecular interactions between gD and nectin-1 have been revealed by crystallographic studies.10,11 All these structure-based analyses 3 ACS Paragon Plus Environment


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demonstrated that both the N- and C-terminal regions of gD are essential not only for the recognition of its receptors on the target cells but also for mediating the membrane fusion process. Because the gD protein is essential for HSV entry into host cells and because it is an easily accessible target for biological interventions, as it is extracellular, gD is potentially an attractive target in the development of therapeutics that interfere with the interactions between gD and host receptors such as HVEM and nectin-1. A few biological reporter assays have been developed as an alternative to plaque assays to facilitate the efficient screening of inhibitors of HSV infection.14-18 Some of these reporter assays can be readily adapted for automated high-throughput screening.14,19 However, all of these assays employ either the native or recombinant whole herpes simplex virus and thus cannot be used in laboratories that do not have a cell culture facility. To overcome these limitations, we developed a molecular assay that directly monitors the interaction between the host receptor and the gD protein using surface plasmon resonance (SPR) (Fig. 1).20 In this assay, the host receptor (for example, HVEM) is immobilized on the sensor chip, and gD is allowed to pass over the HVEM surface as an analyte. Upon the binding of gD to the immobilized HVEM, a response is generated that can be used to analyze the binding kinetics. This binding assay can be modified into a competitive assay in which increasing concentrations of gD-specific binders (inhibitors) progressively decrease the binding of gD to its cognate receptor. We used this approach to evaluate our selected aptamer that binds efficiently to the gD of HSV-1, and we found that that the aptamer specifically interferes with the gD-HVEM and gD-nectin-1 interactions.20 Nearly five decades ago, heparan sulfate was reported to inhibit HSV by effectively blocking the initial phase of the infection—viral adsorption on the host cells.21,22,23 Further investigations revealed that 3-O-sulfated heparan sulfate is expressed on the host cells and serves as an entry receptor for HSV-1 and HSV-2 by interacting directly with the glycoproteins of these viruses.6,24-26 In addition, 3-O-Sheparan sulfate was shown to bind to the gD protein.27 All of these studies suggest that the binding of heparan sulfate to gD prevents its interactions with host cell receptors. 4 ACS Paragon Plus Environment

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However, due to the absence of a structure for the gD-heparan sulfate complex, the anti-HSV-1 and anti-HSV-2 activities of heparan sulfate have remained elusive. Previously, one of us showed that a sulfated polysaccharide isolated from the sporophyll of Undaria pinnatifida (Mekuba) was able to efficiently inhibit the infection of host cells by HSV-1 and HSV-2.28,29 Similarly, polysaccharides isolated from Sargassum horneri were also found to exhibit anti-HSV-1 activity.30 All of the above studies suggest that sulfated polysaccharides have the potential to bind to the gD proteins of HSV-1 and HSV-2 and block their interactions with host cell receptors such as HVEM. In the present study, we used our recently reported molecular assay20 to determine whether sulfated polysaccharides have the ability to interfere with the gD-HVEM interaction. Our studies showed that heparan sulfate binds to gD and efficiently interferes with the HVEM-gD interaction; however, other shorter synthetic sulfated saccharides failed to interfere with this interaction. Next, we analyzed fractionated sulfated polysaccharides isolated from S. horneri to determine their ability to interfere with the HVEM-gD interaction. A total of 14 natural polysaccharides were isolated and tested. Among these polysaccharides, 6 were found to interfere efficiently, 3 moderately and 5 weakly. These analyses showed that sulfated polysaccharides isolated from an edible alga, S. horneri, efficiently inhibited the HSV-1 gD-HVEM interaction, most likely by binding to the gD of HSV-1. To assess the suitability of the above molecular assay for wider use in the screening of compounds for inhibitors that block

the

gD-HVEM

interaction,

we

tested

these

isolated

and

fractionated

polysaccharides for anti-HSV-1 activity using plaque assays. Importantly, the polysaccharides that efficiently interfered with the HVEM-gD interaction were also found to be efficient inhibitors of HSV-1 replication in the plaque assays. Taken together, our earlier results using an aptamer20 and the results of the current study suggest that the molecular assay based on SPR is suitable for screening a wide range of compounds to identify inhibitors that target the initial phase of HSV infection—the interaction of viral glycoproteins with host cell receptors.



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EXPERIMENTAL SECTION Materials. Recombinant HSV-1 gD was expressed as a C-terminal His tag fusion protein (1-285 amino acids) in the baculovirus system as described previously by Zhang et al.11 HVEM was from Sino Biologicals Inc., China. CM5 chips were purchased from GE Healthcare. 3’-Biotinylated multivalent-oligosaccharides were obtained from GlycoTech (MD, USA). The synthetic biotinylated sulfated saccharides used were 3’ HSO3 LacNAcβ-sp-biotin, 3’ HSO3 Galβ1-3GalNac-α-sp-biotin, 3’ sialyllactose-PAAbiotin, 3’ HSO3 Galβ1-3GluNAcβ and heparan sulfate biotin sodium salt. All binding measurements were performed in HBS-P+ buffer (0.01 M HEPES, 0.15 M NaCl, 0.05% v/v Surfactant P20, pH 7.4).

Evaluation of the ability of synthetic and natural polysaccharides to interfere with the gD-HVEM interaction using SPR. To evaluate the ability of the polysaccharides to interfere with the gD-receptor (HVEM) interaction, we used the above-mentioned SPR-based analysis. Initially, 100 nM HVEM was immobilized on an activated CM5 chip through an amino-coupling reaction as described above. The immobilized surface was washed with binding buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, and 0.005% polysorbate 20) before the HVEM-gD interaction was analyzed. Flow cell 1 was used as a control surface, and it was activated and quenched like the experimental flow cell was except without the addition of the protein (HVEM). To obtain the initial binding response, we injected 100 nM gD protein into flow cells 1 and 2. A response curve (representing the HVEM-gD interaction) was initially observed. To determine the effects of the different sulfated saccharides, both the synthetic and natural polysaccharides, on the HVEM-gD interaction, 100 nM gD protein was incubated with different concentrations of sulfated saccharides at 25°C for 10 min. Then, the complexes were injected into both the experimental flow cell (FC2) and the control flow cell (FC1) for 2 min at a flow rate of 30 µl/min. The dissociation time was set for 2 min. Sensorgrams were corrected for non-specific binding by subtracting the response obtained from the control flow cell (FC1) from that for the HVEM-immobilized flow cell (FC2). Similarly, flow cell 3 and 4 was used as a control surface and experimental flow cell, respectively. The sensor chip was then washed with a buffer solution, followed by 6 ACS Paragon Plus Environment

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0.1% SDS, until the response returned to baseline prior to the next analysis. To determine whether this regeneration treatment affects the binding of HVEM to gD, gD alone was injected at the end of the studies to confirm that HVEM was still able to bind to the gD protein. The reduction in the response (Rmax value) with increasing concentrations of aptamer was used to calculate the EC50 value using GraphPad Prism 5 software, as previously described.20 Evaluation of polysaccharides as anti-HSV-1 agents using plaque assays. Vero (Africa green monkey kidney) cells were grown in minimal essential medium (MEM) containing 5% fetal bovine serum (FBS). HSV-1 (KOS strain) was propagated in Vero cells. To assess the cytotoxic effects of the polysaccharides from S. horneri on Vero cells, sample dilutions were prepared and added to Vero cell monolayers in triplicate. After the cells had been incubated at 37˚C for 72 h, the viable cells were counted using the trypan blue exclusion test. The cytotoxicity was determined by calculating the 50% cytotoxic concentration (CC50) from the concentration-response curves. In the antiviral assay, Vero cells were infected with virus at 0.1 plaque-forming units (PFU) per cell for 1 h at room temperature. The cells were then washed with phosphate-buffered saline and incubated at 37˚C for 24 h. The polysaccharides were added during the infection and throughout the subsequent incubation period. The virus yields were determined by plaque assays on 1 day post-infection using Vero cell monolayers. The 50% inhibitory concentrations (IC50) were obtained from the concentration-response curves. The antiviral activities of the polysaccharides were estimated using the selectivity index calculated from the CC50 and IC50 values. RESULTS Rate constants for gD-HVEM binding. The gD protein of HSV-1 is known to interact efficiently with the cell receptor HVEM. The N-terminal amino acid residues of gD are essential for the interaction with HVEM according to structure-functional analyses (PDB 1JMA).8,9 The stoichiometry for gD-HVEM binding has been determined to be 1:1.8 To evaluate the rate constants for gD-HVEM binding, we used SPR-based analyses using a Biacore T100. Initially, HVEM was immobilized on the sensor surface, 7 ACS Paragon Plus Environment


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and different concentrations of purified gD protein were flown over the HVEM surface, during which time, sensorgrams were collected. For this analysis, we used single-cycle kinetics, which allowed us to continuously inject increasing concentrations of HSV-1 gD (20, 40, 80, 160, and 320 nM) and collect the responses without regenerating the surface of the sensor chip (Fig. 1). Single-cycle kinetics experiments have been found to be superior to multi-injection analyses.31,32 The rate of association was measured from the forward reaction, and the dissociation rate was measured from the reverse reaction. The sensorgram data were fitted to a 1:1 binding model. The kinetics studies performed using SPR revealed that gD binds to HVEM with association (kon) and dissociation constants (koff) of 6.0±0.1 x 104 (M-1 s-1) and 3.0±0.1 x 10-3 (s-1), respectively. The equilibrium dissociation constant (KD) for the above complex was 50 (±6) nM. Heparan sulfate interferes with the gD-HVEM interaction in a molecular assay based on SPR. The binding of heparan sulfate to the gD has been reported previously6,27 and has been suggested based on the crystal structure of the HVEM-gD complex. Heparan sulfate potentially interacts with the N-terminal end of gD.8 Because of this interaction, it is speculated that heparan sulfate interferes with the gD-HVEM interaction in a competitive manner by blocking the N-terminal end of the gD protein. This hypothesis has not previously been evaluated directly. To test this hypothesis, we performed a competitive assay using our previously described molecular assay based on SPR. In this assay, HVEM was immobilized on the sensor chip (CM5), and 100 nM HSV-1 gD was passed over the immobilized surface in the same flow cell (Fig. 2a). Upon the binding of gD to HVEM, a response signal was observed (~70 response units). If heparan sulfate competes with the gD-HVEM interaction, we assumed that the signal for this response would decrease. The analyses revealed when the heparan sulfate concentration increased (0.3 – 5.0 µM), the signal for the binding of gD to HVEM decreased in a concentration-dependent manner (Fig. 2a). All of the obtained response signals were corrected by subtracting the heparan sulfate response on the control surface from the HVEM response. The resulting number of response units for each heparan sulfate concentration was used to estimate the concentration of heparan sulfate required for 50% inhibition, and this concentration was estimated to be in the 8 ACS Paragon Plus Environment

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sub-micromolar range (Fig. 2b). In this analysis, it was also clear that heparan sulfate did not bind to HVEM (Fig. 2a; gray line). These analyses clearly show that heparan sulfate binds to the gD protein of HSV-1 and interferes with the interaction of gD with the host cell receptor HVEM. Next, shorter synthetic sulfated saccharides (HSO3 LacNAcβ-sp-biotin, HSO3 Galβ1-3GalNac-α-sp-biotin, sialyllactose-PAA-biotin, and HSO3 Galβ1-3GluNAcβ) that are available commercially (GlycoTech) were analyzed for their ability to interfere with the gD-HVEM interaction. Our SPR-based analyses showed that all four synthetic sulfated saccharides failed to interfere with this interaction (Supplementary Fig. S-1). Chemical characterization of polysaccharides from S. horneri. Previously, one of us showed that the sulfated polysaccharides (fucoidans) isolated from the sporophyll of Undaria pinnatifida (Mekuba) were able to inhibit efficiently the infection of HSV-1 and HSV-2 with host cells.28,29 Similarly, polysaccharides isolated from S. horneri were also found to exhibit anti-HSV-1 activity.30 In the present study, 14 polysaccharides were isolated from the hot water extract of S. horneri and fractionated. Each fraction containing a polysaccharide was detected as a single peak when analyzed with a TSK-gel GMPW XL column and as a single band by electrophoresis (data not shown). These results revealed that the obtained polysaccharides were homogeneous in terms of the molecular size and charge distribution. Their apparent molecular weights were estimated to range from 43 kDa to 1070 kDa (Table 1). Fucose was the sole or predominant component sugar of these polysaccharides. These results indicate that the polysaccharides were fucan sulfates (fucoidans), with sulfate contents ranging from 14.7 to 42.9% and uronic acid contents ranging from 0.1 to 8.5%, as shown in Table 1. Screening polysaccharides used to identify those that interfere with the gDHVEM interaction using the molecular assay based on SPR. To determine which of the above sulfated polysaccharides isolated from S. horneri can interfere with the HSV1 gD-HVEM interaction, we used our molecular assay based on SPR. Initially, HVEM was immobilized on the activated CM5 chip, and 100 nM gD protein was mixed with different concentrations of the polysaccharides (followed by incubation at room 9 ACS Paragon Plus Environment


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temperature for 30 min to reach equilibrium). The resulting complex injected into the flow cells positioned over the blank CM5 surface (flow cell 1) and the HVEMimmobilized surface (flow cell 2). If any polysaccharides in these fractions interfered with the gD-HVEM interaction, we assumed that the signal for the corresponding response would decrease. As predicted, when the concentrations of some polysaccharides from S. horneri were increased, the signal intensity for the binding of gD to HVEM decreased in a concentration-dependent manner (Fig. 3a-n). All fourteen polysaccharides showed varying levels of interference. Among the 14 polysaccharides, 6 (S3-B2-b, S3-P-1, S3-P-2, S3-B2-a, S3-P-3, and S3-P-4) were found to interfere efficiently the gD-HVEM interaction. Of these six polysaccharides, the polysaccharides S3-B2-b and S3-P-3 were found to have lowest molecular weight (17.1 kDa) and higher sulfate content (30.9), respectively. Three polysaccharides (S2-P-1, S2-P-2, and S2-C1b1) were found to have a moderate interference effect, and five polysaccharides (S2C1-a1, SB-1, SB-2, SB-3, and SD-1) had a weak interference effect (Fig. 3a-n). Using the response signals, we estimated the effectiveness of each of these compounds and classified them into three groups: efficient, moderate and weak interfering compounds (Table 2). These analyses indicated that many sulfated polysaccharides isolated from S. horneri appear to interfere with the gD-HVEM interaction. Anti-HSV-1 activities of the polysaccharides isolated from S. horneri. To determine whether the above analyses are in-line with the anti-viral activity, we performed plaque assays. All of the polysaccharides were evaluated to determine their inhibitory activity against HSV-1 replication in vitro. These polysaccharides showed low toxicities against Vero cells, with CC50 values ranging from 1100 to 4700 µg/ml (Table 3). Their IC50 values, which were much lower than their CC50 values, ranged from 0.22 to 7.2 µg/ml except those of four polysaccharides (SB-1, SB-2, SB-3 and SD-1). The selectivity index (CC50/IC50) of acyclovir, an anti-herpetic agent, was shown to be approximately 950 in a parallel experiment (data not shown), and thus, the resulting selectivity indices indicated that all tested polysaccharides except SB-1, SB-2, SB-3, and SD-1 had highly potent anti-HSV-1 activities (Table 3).


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DISCUSSION The entry of herpes simplex virus into host cells involves a complex series of events that require concerted inputs from multiple glycoproteins of HSV. Among these glycoproteins, the gD protein of HSV-1 and HSV-2 plays an important role as a sensing molecule that detects specific receptors on the host cell and facilitates membrane fusion by coordinating with other HSV glycoproteins. Compounds that interfere with the sensing of host cell receptors by viral glycoproteins are attractive and useful targets to combat enveloped viruses such as HSV. Previously, two sulfated polysaccharides (heparan sulfate and chondroitin) from animal sources and naturally occurring sulfated polysaccharides isolated from marine algae and other sources have been shown to exhibit anti-HSV activity.33 Although these sulfated polysaccharides are efficient inhibitors, the molecular basis for their inhibition needs further studies. To evaluate these sulfated polysaccharides and other inhibitory compounds, different bioassays based on intact HSV or recombinant HSV virions have been reported.14-18 Previously, it was suggested that sulfated polysaccharides block the initial interactions with host cell receptors by binding to the gD protein of HSV.30 In the present study, we used our previously developed molecular assay (based on SPR) to determine the molecular basis of the inhibitory activity of sulfated polysaccharides. First, we evaluated the HVEM-gD interaction using SPR-based analyses and found that this interaction is similar to other protein-protein interactions. HVEM and the HSV-1 gD protein were found to interact efficiently, with an apparent equilibrium dissociation constant of KD ~50 nM. This KD value is in accordance with prior SPR measurements (37 nM).34 Once this interaction had been studied, we then evaluated the ability of heparan sulfate to interfere with the HVEM-gD interaction. For this analysis, we incubated gD with increasing concentrations of heparan sulfate from 0.3 µM to 5 µM and found that with increasing heparan sulfate concentrations, the binding of gD to its cognate receptor, HVEM, decreased. These analyses showed that the effector concentration (EC50) of heparan sulfate for 50% inhibition was 2.1 µM for interfering with the HVEM-gD interaction. These studies revealed that heparan sulfate binds to gD and is able to block the site that interacts with the HVEM receptor. The crystal structure of 11 ACS Paragon Plus Environment


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the gD-HVEM complex was solved using ammonium sulfate as a precipitant. Based on the sulfate atom's position in the complex, it was suggested that heparan sulfate is equivalent to the anionic atoms in the complex structure. Taken together, the above results and the results of previous studies suggest that heparan sulfate binds to gD along the axis of the sulfate atoms.27 Similarly, we evaluated four shorter sulfated saccharides and found that they failed to interfere with the HVEM-gD interaction. Earlier studies indicated that the antiviral activities of polysaccharides isolated from different sea algae appear to depend on the molecular weight and sulfate content. In general, polysaccharides that have higher MWs up to 100 kDa, and sulfate contents above the 20% possess anti-viral activity.36 Additionally, a higher uronic acid content and a lower sulfate content in the polysaccharides were found to inversely affect the antiviral activity.36 In the current study, we isolated polysaccharides from S. horneri. The molecular weights and sulfate and uronic acid contents of these polysaccharides, were determined. To evaluate whether these isolated polysaccharides can interfere with the HVEM-gD interaction, we used our previously developed molecular assay based on SPR. Of the fourteen natural polysaccharides that we tested, six efficiently (S3-B2-b, S3-P-1, S3-P-2, S3-B2-a, S3-P-3, and S3-P-4), three moderately (S2-P-1, S2-P-2, and S2-C1-b1) and five weakly (S2-C1-a1, SB-1, SB-2, SB-3, and SD-1) interfered with the HVEM-gD interaction (Table 2). All of these polysaccharides were evaluated further, using plaque assays to determine whether these polysaccharides possess antiviral activity. Five polysaccharides (S3-P-1, S3-P-2, S3-B2-a, S3-P-3, and S3-P-4) exhibited high antiviral activity (IC50 between 0.34 and 0.74 µg/ml), five exhibited moderate antiviral activity (S3-B2-b, S2-P-1, S2-P-2, S2-C1-b1, and S2-C1-a1; IC50 values between 2.1 and 7.2 µg/ml), and four exhibited weak antiviral activity (SB-1, SB-2, SB-3, and SD-1; IC50 values between 58 and 200 µg/ml) (Table 3). Interestingly, all of these polysaccharides had high molecular weights (except S3-B2-b) and sulfate contents (>20%) and low uronic acid contents. The polysaccharides that failed to interfere with the HVEM-gD interaction generally had lower molecular weights and higher uronic acid and sulfate contents. These results are in agreement with earlier observations reviewed in Ghosh et al.33


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Of the six polysaccharides identified as efficient interfering compounds (for the HVEM-gD interaction) by the SPR analyses, five exhibited high antiviral activity (all except S3-B2-b). In addition, S2-C1-a1, identified as a weakly interfering compound (for the HVEM-gD interaction) in the SPR analyses, showed moderate antiviral activity. These two polysaccharides showed different degrees of effectiveness in the two different assays, most likely because their mode of action differs from that of the 12 other polysaccharides. Among the 14 polysaccharides analyzed by the SPR and plaque assays, 12 had consistent results between the two techniques, and thus, the two assays yielded consistent predictions of the potential antiviral effectiveness of these 12 compounds. These results indicate that the antiviral activity observed in the plaque assay most likely originated from the interference of the polysaccharides with the interactions between gD and host receptors such as HVEM, as observed in our SPR assay. Similarly, it is possible to explore compounds that interfere with other glycoproteins of HSV-1 intereactions with different receptors of the host cell. Our earlier20 and present studies suggest that our molecular assay based on SPR not only allows the analysis of viral-host protein interactions but also has potential applications in the routine screening of compounds to identify those that interfere with the first step of viral entry, thus facilitating the rapid development of novel antiviral compounds against HSV. Moreover the described method can be automated continuously without significantly damaging the sensing surface for screening number of drug leads. ACKNOWLEDGMENT The authors would like to thank Drs. Emi Suenaga and Hiroshi Mizuno for useful discussion. Supporting Information Experimental details and surface plasmon response curves obtained during the analyses of heparan sulfate and synthetic sulfated saccharides ability to interfere the HVEM-gD interactions. This material is available free of charge via the internet at http://pubs.acs.org.



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REFERENCES (1)

Campadelli-Fiume, G.; Cocchi, F.; Menotti, L.; Lopez, M. Rev. Med. Virol. 2000,

10, 305-319. (2)

Spear, P.G.; Eisenberg, R.J.; Cohen, G.H. Virology 2000, 275,1-8.

(3)

Whitbeck, J.C.; Peng, C.; Lou, H.; Xu, R.; Willis, S.H.; Ponce de Leon,

M.; Peng, T.; Nicola, A.V.; Montgomery, R.I.; Warner, M.S.; Soulika, A.M.; Spruce, L.A.; Moore, W.T.; Lambris, J.D.; Spear, P.G.; Cohen, G.H.; Eisenberg, R.J. J. Virol. 1997, 71, 6083-6093. (4)

Montgomery, R.I.; Warner, M.S.; Lum, B.J.; Spear, P.G. Cell 1996, 87, 427-436.

(5)

Warner, M.S.; Geraghty, R.J.; Martinez, W.M.; Montgomery, R.I.; Whitbeck, J.C.;

Xu, R.; Eisenberg, R.J.; Cohen, G.H.; Spear, P.G. Virology 1998, 246, 179–189. (6)

Shukla, D.; Liu, J.; Blaiklock, P.; Shworak, N.W.; Bai, X.; Esko, J.D.; Cohen,

G.H.; Eisenberg, R.J.; Rosenberg, R.D.; Spear, P.G. Cell 1999, 99, 13–22. (7)

Heldwein, E.E.; Krummenacher, C. Cell Mol. Life Sci. 2008, 65, 1653-1668.

(8)

Carfi, A.; Willis, S.H.; Whitbeck, J.C.; Krummenacher, C.; Cohen, G.H; Eisenberg,

R.J.; Wiley, D.C. Mol. Cell 2001, 8, 169-179. (9)

Krummenacher, C.; Supekar, V.M.; Whitbeck, J.C.; Lazear, E.; Connolly, S.A.;

Eisenberg, R.J.; Cohen, G.H.; Wiley, D.C.; Carfi, A. EMBO J. 2005, 24, 4144-4153. (10) Di Giovine, P.; Settembre, E.C.; Bhargava, A.K.; Luftig, M.A.; Lou, H.; Cohen, G.H.; Eisenberg, R.J.; Krummenacher, C.; Carfi, A. PLOS Pathog. 2002, 7, e1002277. (11) Zhang, N.; Yan, J.; Lu, G.; Guo, Z.; Fan, Z.; Wang, J.; Shi, Y.; Qi, J.; Gao, G.F.; Nat. Commun. 2011, 2, 577.


Page 15 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(12) Connolly, S.A.; Landsburg, D.J.; Carfi, A.; Wiley, D.C.; Eisenberg, R.J.; Cohen, G.H. J. Virol. 2002, 76, 10894–10904. (13) Connolly, S.A.; Landsburg, D.J.; Carfi, A.; Wiley, D.C.; Cohen, G.H.; Eisenberg, R.J. J. Virol. 2003, 77, 8127–8140. (14) Dicker, I.B.; Blasecki, J.W.; Seetharam, S. Antiviral Res. 1995,28,213-224. (15) Wang, Y.C.; Kao, C.L.; Liu, W.T.; Sun, J.R.; Tai, Y.E.; Kung, S.H. J. Med. Virol. 2002, 68, 599-605. (16) Stránská, R.; Schuurman, R.; Scholl, D.R.; Jollick, J.A.; Shaw, C.J.; Loef, C.; Polman, M.; van Loon, A.M. Antimicrob. Agents Chemother. 2004,48,2331-2333. (17) Tanaka, M.; Kodaira, H.; Nishiyama, Y.; Sata, T.; Kawaguchi, Y. Microbes Infect. 2004, 6, 485-493. (18) Taylor, T.J.; Knipe, D.M. Methods Mol. Biol. 2009, 515, 239-248. (19) Zhu, Q.C.; Wang, Y.; Peng, T. J. Biomol. Screen. 2010,15, 1016-1020. (20) Gopinath, S.C.B.; Hayashi, K.; Kumar, P.K.R. J. Virol. 2012, 86, 6732-6744. (21) Nahmias, A.J.; Kibrick, S. J. Bacteriol. 1964, 87, 1060–1066. (22) WuDunn, D.; Spear, P.G. J. Virol. 1989, 63, 52–58. (23) Antoine, T.E.; Park, P.J.; Shukla, D. Rev. Med. Virol. 2013, 23, 194–208. (24) Xia, G.; Chen, J.; Tiwari, V.; Ju, W.; Li, J-P.; Malmström, A.; Shukla, D.; Liu, J. J. Biol. Chem. 2002, 277, 37912–37919. (25) Xu, D.; Tiwari, V.; Xia, G.; Clement, C.; Shukla, D.; Liu, J. Biochem. J. 2005, 385, 451–459. (26) O’Donnell, C.D.; Tiwari, V.; Oh, M.J.; Shukla, D. Virology 2006, 346, 452–459.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(27) Liu, R.J.; Raska,

J.; Shriver,

Z.; Pope,

C.S.; Yoshida,

K.;

R.M.; Thorp,

Page 16 of 25

S.C.; Duncan,

Eisenberg,

R.J.; Cohen,

M.B.; Copeland, G.; Linhardt,

R.J.; Sasisekharan, R. J. Biol. Chem. 2002, 277, 33456-33467. (28) Lee, J.B.; Hayashi, K.; Hashimoto, M.; Nakano, T.; Hayashi, T. Chem. Pharm. Bull. 2004, 52,1091–1094. (29) Hayashi, K.; Nakano, T.; Hashimoto, M.; Kanekiyo, K.; Hayashi, T. J. Int. Immunopharm. 2008, 8, 109-116. (30) Srisomporn, P.; Hayashi, K.; Lee, J-B.; Sankawa, U.; Hayashi, T. Chem. Pharm. Bull. 2001, 49, 484-485 (31) Karlsson, R.; Katsamba, P.S.; Nordin, H.; Pol, E.; Myszka, D.G. Anal. Biochem. 2006, 349, 136-147. (32) Suenaga, E.; Mizuno, H.; Kumar, P.K.R. Biosens. Bioelectron. 2012, 32, 195-201. (33) Ghosh, T.; Chattopadhyay, K.; Marschall, M.; Karmakar, P.; Mandal, P.; Ray, B. Glycobiology 2009,19, 2-15. (34) Rux, A.J.; Willis, S.H; Nicola, A.V.; Hou, W.; Peng, C.; Lou, H.; Cohen, G.H.; Eisenberg, R.J. J.Vitol. 1998, 72, 7091-098. (35) Witvrouw, M.; De Clercq, E. Gen. Pharmacol. 1997, 29, 497-511. (36) Hemmingson, J.A.; Falshaw, R.; Furneaux, R.H.; Thompson, K.J. J. Appl. Phycol. 2006,18, 185–193.


Page 17 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 1. Chemical characterization of polysaccharides isolated from S. horneri

MW Polysaccharide

(x 104)

S3-B2-b

17.1

S3-P-1

Neutral sugar (mol %)

Uronic acid (%)

Sulfate

Protein

(%)

(%)

Fuc *(Gal)**

1.0

23.4

0.0

107.0

Fuc (Gal)

3.2

20.3

2.3

S3-P-2

71.1

Fuc (Gal)

1.8

n.d.

S3-B2-a

48.5

Fuc (Gal)

0.1

22.4

0.0

S3-P-3

84.3

Fuc

1.0

30.9

3.3

S3-P4

39.3

Fuc

2.2

21.3

1.1

S2-P-1

43.9

Fuc (Gal)

1.5

16.4

2.8

S2-P-2

38.5

Fuc (Gal)

1.4

17.1

1.5

S2-c1-b1

79.3

Fuc (Gal)

4.2

18.9

0.0

S2-c1-a1

104.0

Fuc (Gal)

1.4

14.7

0.0

SB-1

17.3

Fuc (Gal)

5.7

22.0

1.7

SB-2

9.2

Fuc (Xyl)

8.5

15.4

2.5

SB-3

4.3

Fuc (Gal)

6.8

19.5

2.4

SD-1

4.7

Fuc (Xyl)

4.3

42.9

0.7

n.d.: not determined. * Fuc: fucose; Gal: galactose; Xyl: xylose. ** (trace)


2.1


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 25

Table 2. Ability of the polysaccharides isolated from S. horneri to interfere with the gD-HVEM interaction as determine by the SPR-based assay Polysaccharide

Level of inhibition (EC50)

S3-B2-b

70 ± 10

S3-P-1

50 ± 7

S3-P-2

50 ± 10

S3-B2-a

95 ± 16

S3-P-3

75 ± 15

S3-P-4

80 ± 12

S2-P-1

310 ± 25

S2-P-2

310 ± 15

S2-C1-b1

140 ± 27

S2-C1-a1

140 ± 14

SB-1

>5000

SB-2

3000 ± 130

SB-3

>5000

SD-1

>5000

Efficient (EC50): ~90 nM; Moderate (EC50): 140-310 nM; Weak (EC50): > 3.0 µM


Page 19 of 25

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Table 3. Anti-viral activities of the polysaccharides isolated from S. horneri Cytotoxicity Antiviral activity Selectivity index Polysaccharide (CC50, µg/ml) (IC50, µg/ml) (CC50/IC50) S3-B2-b

3000

2.1

1400

S3-P-1

4500

0.34

13000

S3-P-2

4600

0.74

6200

S3-B2-a

4700

0.54

8700

S3-P-3

4200

0.22

19000

S3-P-4

4600

0.36

13000

S2-P-1

1100

2.5

440

S2-P-2

4600

4.6

1000

S2-C1-b1

4500

7.2

630

S2-C1-a1

4600

4.4

1050

SB-1

4300

>200

200

200

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