The previous studies have shown that primary cultures of corneal fibroblasts (CF) expressing the human encoded form of 3-OST-3 receptor leads to a change in the actin cytoskeleton during HSV-1 entry [30Clement C, Tiwari V, Scanlon PM, Vali-Nagy T, Yue BYJT, Shukla D. A novel for phagocytosis-like uptake in herpes simplex virus entry J Cell Biol 2006; 174: 1009-21., 31Tiwari V, Clement C, Xu D, et al. Role for 3-O-sulfated heparan sulfate as a receptor for herpes simplex virus type-1 entry into primary human corneal fibroblasts J Virol 2006; 80: 8970-0.]. Therefore, we examined whether cytoskeletal changes played any significant role in HSV-1 entry into CHO-K1 cells expressing ZF encoded 3-OST-3 isoform. To address this, a high resolution scanning electron microscopy (SEM) was performed. Wild-type HSV-1 (KOS) was added to the cells plated at a low population density for 30 min at 37^oC. SEM images demonstrated that the infected cells expressing ZF encoded 3-OST-3 had numerous virions attached to the cell body while many others were also attached to filopodia-like projections present on the plasma membrane of ZF-3-OST-3 cell (Fig. 1A, panel c). Similar projections were also noticed in human 3-OST-3 isoform expressing CHO-K1 cells (Fig. 1A, panel b). CHO-K1 cells without 3-OST-s expression showed little filopodia induction (Fig. 1A, panel a). The quantification on the number of filopodia formed in ZF-3-OST-3 cells was found to be comparable to the human 3-OST-3 expressing CHO-K1 cells (Fig. 1B). The above data indicates a role of ZF 3-OST-3 in filopodia induction during HSV-1 entry.

Fig. (1) Role of filopodia during HSV-1 entry into Chinese hamster ovary (CHO-K1) cells expressing Zebrafish (ZF) encoded 3- OST-3 receptor. (A) Scanning electron microscopy (SEM) performed on HSV-1 (KOS) infected glycoprotein D (gD) receptor negative (wild-type CHO-K1 cells; panel a) and gD-receptor positive cells (panel b and panel c). CHO-K1 cells expressing human 3-OST-3 (H-3- OST-3), and zebrafish (ZF) encoded 3-OST-3 (ZF-3-OST-3) were grown at a confluence of 40% in a chamber slides (Lab-Tek chamber slide) and were exposed to HSV-1 (25 pfu/cell for 45 min at 37°C). The infected cells were fixed with 2% formaldehyde/4% glutaraldehyde in PBS before SEM. Highlighted regions showing virions bound to wild-type CHO-K1 cells (panel a*) were unable to induce filopodia (red arrow indicates single Filopodia in panel a), while large number of filopodia (panels b* and c*) were observed in human and ZF encoded 3- OST-3 expressing CHO-K1 cells. The arrow indicates presence of virus on the induced filopodia. (B) Determination of percentage of filopodia scored from sampled groups of 100 single cells or clusters (with 5-20 cells) in wild-type CHO-K1 cells, and CHO-K1 cells expressing human and ZF 3-OST-3 receptor in triplicate experiments, 5 μm length of a protrusion and at least 10% of the cell surface covered with 25 or more protrusions is scored positive. ** P< 0.05, one way ANOVA.

Fig. (2) HSV-1 interaction with zebrafish (ZF) encoding 3-OST-3 leads to enhanced filopodia induction. (A) Scanning electron microscopy (SEM) performed on heparan sulfate (HS) negative (CHO-745 cells; panel a) and HS positive (wild-type CHO-K1; panel b and ZF expressing 3-OST-3; panel c) cells in presence of HSV-1 (25 pfu/cell) for 45 min at 37°C. The regions highlighted for filopodia in panels a, b and c has been inverted as a*, b*, and c*. Highlighted regions showing virions bound to CHO-745 cells (panel a*) were unable to induce filopodia, while small number of filopodia were observed in virions bound CHO-K1 cells (panels b*). The maximum number of filopodia were observed in ZF encoded 3-OST-3 expressing CHO-K1 cells (panel c*). (B) Determination of percentage of filopodia scored in HSV-1 (25 pfu/cell for 45 min at 37°C) treated CHO-745, CHO-K1 and CHO-ZF-3-OST-3 is presented. Sampled groups of 50 single cells or clusters (with 5-20 cells) in triplicate experiments, 5 µm lengths of a protrusion and at least 10% of the cell surface covered with 25 or more protrusions is scored positive. ** P< 0.05, one way ANOVA.

Fig. (3) Actin depolymerizers block HSV-1 entry into zebrafish (ZF) 3-OST-3 expressing CHO-K1 cells. (A, B) Cultured monolayers of cultured human (H) and zebrafish (ZF) encoded 3-OST-3 CHO-K1 cells were pre-treated with the indicated concentrations of the actin depolymerizing agent Cytochalisin D (Cyto D; panel A) and Lanticulin B (Lat B; panel B) before exposed to β-galactosidase expressing HSV-1 gL86 (25 pfu/cell) virus. Cells treated with 1 x PBS treated cells were used as a control. Viral entry was quantitated 6 h after infection at 410 nm using a spectrophotometer. The experiment was repeated three times with similar results. (C) Visualization on the inhibition of filopodia via scanning electron microscopy (SEM) on HSV-1 infected (25 pfu/cell for 45 min at 37°C) ZF-3-OST-3 expressing CHO-K1 cells in absence (panel a) and presence (panel c) of Cyto D at 0.5 µg/ml. The regions in panel a (filopodia induction) and panel c (reduced filopodia) highlighted are shown in panel b and d. (D) Determination of percentage of filopodia scored in ZF-3-OST-3 infected cells in absence and presence of Cyto D. Sampled groups of 50 single cells or clusters (with 5-20 cells) in a triplicate experiment, 5 µm length of a protrusion and at least 10% of the cell surface covered with 25 or more protrusions is scored positive. ** P< 0.05, one way ANOVA.

Fig. (4) Actin depolymerizer cytochalasin D negatively affects HSV-1 glycoprotein mediated cell fusion with zebrafish (ZF) encoded- 3-OST-3 receptor. In this experiment target CHO-K1 cells expressing either human 3-OST-3 or ZF encoded 3-OST-3 along with luciferase gene were pre-incubated with Cyto D (0.5 µg/ml) or with 1 x PBS for 45 min at room temperature before co-culturing with effector cells expressing four essential HSV-1 glycoproteins (gB, gD, gH-gL) or control plasmid (pCDNA3.1) along with T7 Polymerase in 1:1 ratio for 24 hrs. The graph shows the quantitative cell fusion in presence (line bar) and absence of Cyto D (plain bar). The black bar shown is a negative control. Data represent the means standard deviations of results in triplicate wells from a representative experiment. ** P< 0.05, one way ANOVA.

Because HSV-1 was able to induce filopodia both in wild-type CHO-K1 cells and CHO-K1 cells expressing ZF 3-OST-3, we next evaluated whether changes in actin cytoskeleton were dependent on HS. In order to generate direct and visual evidence of filopodia induction during HSV-1 entry we used SEM. In this experiment HSV-1 (KOS) was used to infect pCDNA3.1 expressing CHO-K1 cells and ZF-3-OST-3 expressing CHO-K1 cells and the above infections was compared for filopodia induction to CHO cells that are deficient in HS (CHO-745). As shown in Fig. (2A, panel a) HSV-1 infected CHO-745 (HS deficient) cells failed to produce significant amount of filopodia compared to the HS expressing wild-type CHO-K1 cells (Fig 2A, panel b). In parallel, pronounced filopodia were observed in CHO-K1 cells expressing ZF encoded 3-OST-3 isoform (Fig. 2A, panel c). Quantification of percentage of cells forming filopodia was highest in ZF-3-OST-3 cells (Fig. 2B). These results indicate that filopodia induction during HSV-1 entry is HS dependent; Furthermore, the presence of 3-OST-3-modified receptor significantly enhances filopodia induction to facilitate HSV-1 entry into ZF expressing 3-OST-3 CHO-K1 cells.

To further demonstrate the significance of actin network in HSV-1 entry, both ZF and human 3-OST-3 receptor expressing CHO-K1 cells were pre-treated with inhibitors of actin polymerization such as cytochalasin D (Cyto D) and lantriculin B (Lat B) [32Gottlieb TA, Ivanov IE, Adesnik M, Sabatini DD. Actin microfilaments play a critical role in endocytosis at the apical but not the basolateral surface of polarized epithelial cells J Cell Biol 1993; 120: 695-710.-34Schafer DA. Regulating actin dynamics at membranes a focus on dynamin Traffic 2004; 5: 463-9.] or mock treated before infecting with Lac Z encoded β-galactosidase expressing HSV-1. It was postulated that pre-treatment would have negative effect on entry provided the actin-based protrusions (such as filopodia) played a role in the virus attachment as well as entry in to cells. As shown in Fig. (3A, B), both of the actin depolymerizers blocked HSV-1 entry in a dosage dependent manner. To generate further visual evidence, SEM was performed in HSV-1 infected ZF-3-OST-3 expressing CHO-K1 cells in presence and absence of Cyto D. Corresponding effects on the inhibition on the number of filopodia and HSV-1 entry was noticed when cells were pre-incubated with Cyto-D (Fig. 3C, D). The quantitation of the above-mentioned data suggests that significant changes in the cytoskeleton occur during initial phase of HSV-1 infection in ZF encoded 3-OST-3 receptor. A similar increase in cellular projections was previously reported with other HSV-1 receptors including nectin-1 [35Tiwari V, Oh MJ, Kovacs M, Shukla SY, Valyi-Nagy T, Shukla D. Role for nectin-1 in herpes simplex virus 1 entry and spread in human retinal pigment epithelial cells FEBS J 2008; 275: 5272-85.].

After establishing the role of ZF encoded 3-OST-3 in filopodia induction during HSV-1 entry, we next examined whether ZF 3-OST-3 mediated cell-to-cell fusion is also an actin network dependent process. CHO-K1 cells were used that are resistant to virus-induced cell fusion due to the absence of a gD receptor [28Tiwari V, Clement C, Duncan MB, Chen J, Liu J, Shukla D. A role for 3-O-sulfated heparan sulfate in cell fusion induced by herpes simplex virus type 1 J Gen Virol 2004; 85: 805-9.]. A previously described luciferase reporter gene assay was performed to quantify the induced cell fusion between 3-OS HS cells modified by ZF encoded 3-OST-3 and HSV-1 glycoproteins in presence and absence of Cyto D [28Tiwari V, Clement C, Duncan MB, Chen J, Liu J, Shukla D. A role for 3-O-sulfated heparan sulfate in cell fusion induced by herpes simplex virus type 1 J Gen Virol 2004; 85: 805-9.]. The ‘‘effector’’ CHO-K1 cells were transiently transfected with each of four glycoprotein plasmids: pPEP98 (gB), pPEP99 (gD) pPEP100 (gH), and pPEP101 (gL), as well as the plasmid pT7EMCLuc that expresses a luciferase reporter gene [29Pertel P, Fridberg A, Parish M, Spear PG. Cell fusion induced by herpes simplex virus glycoproteins gB gD and gH-gL requires a gD receptor but not necessarily heparan sulfate Virology 2001; 279: 313-24.]. The ‘‘target’’ cells were transfected with a 3-OST plasmid expressing ZF encoded 3-OST-3 and the plasmid pCAGT7, which expresses T7 RNA polymerase to induce expression of the luciferase gene. For a negative control, cells were transfected with T7 RNA polymerase and control vector pCDNA3.1. The cells expressing human 3-OST-3 and T7RNA polymerase served as a positive control. As shown in Fig. (4) a slightly higher amount of fusion occurred in human 3-OST-3 isoform, while ZF encoded 3-OST-3 expressing cells also showed fusion (grey bar) compared to negative control cells without 3-OST-3 expression (black bar). Interestingly significant inhibition in cell-to-cell fusion was observed in presence of Cyto D (grey bars with black lines). These results reinforce our finding that changes in actin cytoskeleton play a critical role during HSV-1 entry and spread and cells expressing ZF 3-OST-3 facilitate filopodia induction.

The Zebrafish encoded 3-OST-3 gene was cloned into pDream2.1 plasmid vector (Genscript), while the Human 3-OST-3 expressing plasmid (pDS43) was provided by Dr. Shukla (University of Illinois at Chicago) [11Shukla D, Liu J, Blaiklock P, et al. A novel role for 3-O-sulfated heparan sulfate in herpes simplex virus 1 entry Cell 1999; 99: 13-22.]. The HSV-1 (KOS) glycoprotein expressing plasmids used were pPEP98 (gB), pPEP99 (gD), pPEP100 (gH), and pPEP101 (gL) [28Tiwari V, Clement C, Duncan MB, Chen J, Liu J, Shukla D. A role for 3-O-sulfated heparan sulfate in cell fusion induced by herpes simplex virus type 1 J Gen Virol 2004; 85: 805-9.]. Other plasmids used in this study include pCAGT7 (T7 RNA polymerase), and pT7EMCLuc (luciferase gene) for the luciferase assay [29Pertel P, Fridberg A, Parish M, Spear PG. Cell fusion induced by herpes simplex virus glycoproteins gB gD and gH-gL requires a gD receptor but not necessarily heparan sulfate Virology 2001; 279: 313-24.], and a control empty vector pCDNA3.1 from Invitrogen (Carlsbad, CA, USA).

Wild-type Chinese Hamster ovary-K1 (CHO-K1) and heparan sulfate defective cells (CHO-745) were kindly provided by P.G. Spear (Northwestern University, Chicago). Both CHO-K1 and CHO-745 cells were grown in Ham’s F-12 medium (Gibco/BRL, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS), and penicillin and streptomycin (Gibco/BRL). The β-galactosidase expressing recombinant HSV-1 (KOS) gL86 was provided by P.G. Spear (Northwestern University, Chicago).

Wild-type CHO-K1, CHO-745 cells and CHO-K1 cells expressing human (H) and zebrafish (ZF) 3-OST-3 receptor were infected with HSV-1 (KOS) at 25 pfu/cell for 45 min at 37^oC in triplicate experiments. In a parallel experiment, the CHO-K1 cells expressing ZF-3-OST-3 were pre-incubated with 1× PBS or with an actin polymerizer cyto-D followed by HSV-1 infection at 25 pfu/cell for 45 min at 37^oC. The cells were then fixed with 2% formaldehyde/4% glutaraldehyde in 1 × phosphate buffer saline (PBS) prior to SEM study. This was followed by fixing cells with 1% osmium tetroxide formaldehyde/glutaraldehyde for 40 minutes. Dehydration was done using 25% ethanol, 50% ethanol, 70% ethanol, 90% ethanol, 95% ethanol, 100% ethanol at five minutes each respectively. 100% ethanol was repeated to ensure dehydration. Cover slips were removed from dishes and mounted on aluminum studs previously cleaned with 100% ethanol. Cover slip edges were painted with colloidal silver for conduction and dried in a Critical Point Dryer (Samdri-780A). Samples were then coated with gold using a Sputter Coater (Hummer VI-A) for 2.5 minutes. Samples were viewed using a Hitachi S-2700 Scanning Electron Microscope (SEM). Images were captured at 1000-5000x using Revolution image capture system and a controller/analysis system for energy dispersive X-ray spectroscopy.

As previously described [22Hubbard S, Darmani NA, Thrush GR, et al. Zebrafish-encoded 3-O-sulfotransferase-3 isoform mediates herpes simplex virus type 1 entry and spread Zebrafish 2010; 7: 181-7.] CHO-K1 cells were grown in 6-well plates to subconfluence and transfected with 2.5 μg of human and or ZF encoded 3-OST isoforms (3-OST-3) or control plasmid (pDream2.1 or pCDNA3.1) using LipofectAMINE (Gibco/BRL) [22Hubbard S, Darmani NA, Thrush GR, et al. Zebrafish-encoded 3-O-sulfotransferase-3 isoform mediates herpes simplex virus type 1 entry and spread Zebrafish 2010; 7: 181-7.]. At 16 h post-transfection, the cells were replated into 96-well dishes for pre-treatment with actin depolymerizers (cyto-D and lant-B at indicated concentrations for 45 min at room temperature) followed by infection with β-galactosidase expressing recombinant HSV-1 gL86 virus. After 6-h post infection, β-galactosidase assay were performed using a soluble substrate o-nitrophenyl-β-D-galactopyranoside (ONPG; ImmunoPure, Pierce). The enzymatic activity was measured at 410 nm using a micro-plate reader [22Hubbard S, Darmani NA, Thrush GR, et al. Zebrafish-encoded 3-O-sulfotransferase-3 isoform mediates herpes simplex virus type 1 entry and spread Zebrafish 2010; 7: 181-7.].

A cell-to-cell fusion assay described previously was used [22Hubbard S, Darmani NA, Thrush GR, et al. Zebrafish-encoded 3-O-sulfotransferase-3 isoform mediates herpes simplex virus type 1 entry and spread Zebrafish 2010; 7: 181-7., 28Tiwari V, Clement C, Duncan MB, Chen J, Liu J, Shukla D. A role for 3-O-sulfated heparan sulfate in cell fusion induced by herpes simplex virus type 1 J Gen Virol 2004; 85: 805-9.]. CHO-K1 cells were grown in 6-well plates to subconfluent levels. The cultured CHO-K1 ‘‘target’’ cells were transfected with plasmids expressing either human or zebrafish (ZF) 3-OST-3 isoform and the luciferase gene. The ‘‘effector’’ or virus-like cells were co-transfected with four HSV-1(KOS) glycoproteins as previously described [28Tiwari V, Clement C, Duncan MB, Chen J, Liu J, Shukla D. A role for 3-O-sulfated heparan sulfate in cell fusion induced by herpes simplex virus type 1 J Gen Virol 2004; 85: 805-9.]. In either case, the total amount of DNA used for transfection was kept constant. For transfection, CHO-K1 cells were grown to 70% confluency in a 6 well dishes. 2.5 µg of plasmid DNA was mixed with 8 µl of Lipofectamine (Gibco/BRL) in to a total volume of 1 ml with serum-free media. After 16 h, target and effector cells were mixed in a 1:1 ratio and then replated in 24-well dishes. In parallel effector cells transfected only with pCDNA3.1 along with T7 RNA polymerase was mixed with the target cells expressing either human or ZF 3-OST-3 isoform was used as a negative control. The activation of the reporter luciferase gene as a measure of cell fusion was examined after 24 h. Luciferase activity was quantified using the luciferase reporter assay system (Promega, Madison, WI).