Herpes Simplex Virus Type-1 (HSV-1) Entry into Human Mesenchymal Stem Cells Is Heavily Dependent on Heparan Sulfate

Hematopoietic stem cells recipients remain susceptible to opportunistic viral infections including herpes simplex virus type-1 (HSV-1). The purpose of this investigation was to analyze susceptibility of human mesenchymal stem cells (hMSCs) to HSV-1 infection and identify the major entry receptor. Productive virus infection in hMSCs was confirmed by replication and plaque formation assays using a syncytial HSV-1 KOS (804) virus. To examine the significance of entry receptors, RT-PCR and antibody-blocking assays were performed. RT-PCR data showed the expression of gD receptors: nectin-1, 3-O sulfotransferase-3 (3-OST-3), and HVEM. Antibody-blocking assay together with heparinase treatment suggested an important role for HS and 3-O-sulfated heparan sulfate (3-OS HS), but not nectin-1 or HVEM, in mediating HSV-1 entry and spread in hMSCs. Taken together, our results provide strong evidence demonstrating that HSV-1 is capable of infecting hMSCs and HS and 3-OS HS serve as its entry receptors during this process.


Introduction
Herpes simplex virus type-1 (HSV-1) is a ubiquitous pathogen associated with facial cold sores [1,2], but severe complications especially in neonates and immunocompromised patients may result in corneal blindness, retinitis, and encephalitis [3,4]. The virus can also cause severe opportunistic infections in the patients following allogeneic stem cell transplant [5][6][7][8]. Such infections could be donor derived or due to the reactivation of an endogenous latent virus [9]. In addition, the drugresistant HSV-1 is also an emerging concern to promote viral infections after allogeneic stem cell transplantation [10]. It has been proposed that human mesenchymal stem cells (hMSCs) may be susceptible to infection if infused in patients with HSV-1 viremia and may compromise the host's defense against infectious agents [7,11].
Although HSV-1 infections lead to significant morbidity and mortality in transplant recipient [6,10,11], the susceptibility of human mesenchymal stems cells (hMSCs) to HSV-1 entry and key entry mediator that allows HSV-1 infection is poorly understood. In this study, we investigated the HSV-1 infection of hMSCs and the role of HSV-1 entry receptors in this process. The finding that HSV-1 can induce cytopathic effect and productively replicate in hMSCs in high titer provides new information on the susceptible nature of target cell population present within the host. We also demonstrated that HS and modified form of HS (3-Osulfated heparan sulfate; 3-OS HS) is a major determinant of HSV-1 entry and spread into hMSCs.

HSV-1 Entry
Assay. Viral entry assays were based on quantitation of β-galactosidase expressed from the viral genome [27]. Cells (hMSCs, HeLa) were plated at 2 × 10 4 per well in 96-well plates at least 16 h prior to infection. After 6h postinfection, β-galactosidase assays were performed using a soluble substrate o-nitrophenyl-β-D-galactopyranoside (ONPG at 3.0 mg/mL) assay. The enzymatic activity was measured at 410 nm using a microplate reader (BioTek Instruments Inc. EL×808 absorbance microplate reader, Vt, USA). Viral entry was further confirmed by 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) staining. The hMSCs were grown in Lab-Tek chamber slides. After 6 hr of infection with the reporter virus, the cells were washed with PBS and fixed with 2% formaldehyde and 0.2% glutaradehyde at room temperature for 15 min. The cells were then washed with PBS and permeabilized with 2 mM MgCl 2 , 0.01% deoxycholate and 0.02% nonidet NP-40 for 15 min. After rinsing with PBS, 1.5 mL of 1.0 mg/mL X-gal in ferricyanide buffer was added to each well, and the blue color developed in the cells was examined. Microscopy was performed using 40x objective of the inverted microscope (Olympus IX50).

Fluorescent
Microscopy of Viral Entry. Cultured monolayers of hMSCs were grown overnight in media containing 10% FBS on chamber slides (Lab-Tek). The cells were either mock infected or infected with green fluorescent protein (GFP)-tagged HSV-1 (VP26-GFP) at 50 pfu/cell in serum-free media OptiMEM (Invitrogen). The GFP was fused with the HSV-1 UL35 open reading frame (ORF) which encodes a 12-kDa capsid protein designated VP26 [34]. VP26 is located on the outer surface of the capsid, specifically on the tips of the hexons that constitute the capsid shell. At 90 min post-infection, cells were fixed and stained with 10 nm rhodamine-conjugated phalloidin for F-actin. Images of immunofluorescent labeled cells were acquired using a Olympus IX50 inverted fluorescence microscope.

Effect of Actin Depolymerizing Agents on HSV-1 Entry in hMSCs.
In order to demonstrate the significance of actin cytoskeleton network during HSV-1 entry into hMSCs, the effects of actin depolymerizing agents on entry of HSV into hMSCs were examined. Monolayer cultures of hMSCs (approximately 10 6 cells) cultured in a 96-well plate were pretreated with the indicated concentrations of agents for 1 hr at room temperature with cytochalasin D (Cyto D) and latrunculin B (Lat B) (Sigma) or mock-treated as a control. Cells were infected with the lacZ + HSV-1 KOS (gL86) at 50 PFU/cell for 6 h at 37 • C. ONPG entry assay was performed to estimate the enzymatic activity at 410 nm by spectrophotometer.
2.6. Kinetics of Viral Plaque Assay. Confluent layers of hMSCs (approximately 10 6 ) in six-well dishes were infected with HSV-1 (804) strain at 0.01 PFU/cell or mock infected for 2 hrs at 37 • C. After removal of inoculum, monolayers were overlaid with DMEM containing 2.5% heat inactivated calf serum and incubated at 37 • C. At different time points: 0, 12, 24, and 36 hr, the cells were fixed by using fixative buffer at room temperature for 20 min, followed by Giemsa staining for 45 min. The cells were again washed five times in PBS, and the numbers of plaques were counted. The images were taken by using an Olympus IX50 inverted fluorescence microscope. Virus replication was further examined by quantitative plaque assay. A monolayer of cultured hMSCs (approximately 4 × 10 6 cells per 25-mL flask) was infected (MOI, 0.01) with HSV-1(KOS) or mock infected with PBS alone for 2 h at 37 • C. Vero cells mock infected of infected with the HSV-1 KOS under similar conditions were used as negative and positive controls, respectively. After removal of the inoculum, monolayers were overlaid with DMEM containing 2.5% heat-inactivated calf serum and incubated at 37 • C until the time of harvest (12 to 48 h). Infectious virus titers were determined on Vero cells cultured in triplicate by using an overlay of medium containing methylcellulose. In order to block secondary plaque formation, human immunoglobulin G (IgG; Sigma) was added to the inoculum. The cells were washed with PBS buffer, fixed in alcohol, and stained with Giemsa stain. Infectivity was recorded as the number of PFU. Virus attachment was determined by using Olympus IX50 inverted fluorescence microscope.

Antibody
Blocking Assay. Antibody blocking assay was performed as previously described [36]. hMSCs plated in 96-well plate were preincubated at room temperature with twofold dilutions of Anti-HVEM, anti-nectin-1 antibody (PRR1), and a control antibody (α-TGFbR II) for 90 min. Cells were then challenged with identical doses of HSV-1 (gL86) at 5 × 10 5 pfu per well at 37 • C. After 6 hours, the cells were washed twice with PBS and treated for 1 min with 0.1 M citrate buffer (pH 3.0). The substrate, ImmunoPure ONPG was prepared in PBS buffer with nonionic detergent, and βgalactosidase activity was read at 410 nm. The experiment was repeated three times with similar results.

Heparinase Treatment.
Cultured hMSCs plated on 96well plates were washed with Mg 2+ -and Ca 2+ -free PBS and incubated at room temperature for 90 min with heparinase I (1.5 U mL −1 ; Sigma) or PBS alone and incubated at room temperature. The cells were then washed with PBS and used for HSV-1 entry [33].

Surface Immunofluorescence Assay.
To detect the binding of HSV-1 to the surface of hMSCs, cultured monolayers of cells (approximately 10 6 ) were grown overnight on glass bottom culture dishes (Mat Tek Corp.). The cells were then treated with heparinases I (1.5 U mL −1 ) or with PBS buffer for 45 min, exposed to GFP-tagged HSV-1 K26GFP for 30 min at 4 • C, and fixed with acetone. The cells were then stained with phalloidin (F-actin) and mounted on glass slides before examination under an Olympus IX50 inverted fluorescence microscope.

Glycoprotein D (gD)-Fc Binding Assay.
Cultured hMSCs were plated overnight into 96-well dishes (approximately 4 × 10 4 cells/well). They were treated with heparinases I or with PBS buffer as described above for 2 h. This was followed by incubation with gD-Fc (1 μg/mL) for 1 h and fixation at room temperature with 2% formaldehyde and 0.02% glutaraldehyde for 15 min. The cells were washed with PBS containing 3% BSA (Sigma) and then incubated at room temperature sequentially with a biotinylated secondary antibody against rabbit IgG (Sigma) at a 1 : 500 dilution in PBS-3% BSA for 45 min and AMDEX streptavidin-conjugated horseradish peroxidase (Amersham) at a 1 : 20,000 dilution for 30 min. Following washing in PBS, the cells were incubated with 50 μL 3,3 ,5,5 -tetramethylbenzidine (Sigma) in 50 mM phosphate-citrate buffer. Products were quantitated by use of a microplate reader (BioTek Instruments Inc. EL×808 absorbance microplate reader, Vt, USA) to measure optical density at 650 nm (OD 650 ).

HSV-1 Glycoprotein-Induced Cell Fusion Assay.
In this experiment, the CHO-K1 cells designated effector cells were cotransfected with plasmids expressing four HSV-1(KOS) glycoproteins: pPEP98 (gB), pPEP99 (gD), pPEP100 (gH), and pPEP101 (gL), along with plasmid pT7EMCLuc, which expresses the firefly luciferase gene under the control of the T7 promoter [22]. Cultured hMSCs considered target cells were cotransfected with pCalifGT7, which expresses T7 RNA polymerase with the chicken actin promoter and the CMV enhancer [18]. Effector cells expressing pT7EMCLuc and pCDNA3 (devoid of any glycoproteins) and target hMSCs transfected with T7 RNA polymerase alone were used as negative controls. In either case, the total amount of DNA used for transfection was kept constant. After 16 h, target and effector cells were mixed in a 1 : 1 ratio and then replated in 24-well dishes. Activation of the reporter luciferase gene, as a measurement of cell fusion, was examined by reporter lysis assay (Promega) at 24 h postmixing by using a Sirius luminometer (Berthold detection systems). Cell fusion was also visualized for multinucleated giant cells or polykaryocytes by microscopy following Giemsa (Fluka) staining at 24 h postmixing. Photographs of representative cells were imaged using an Olympus IX50 inverted microscope. dilutions of recombinant HSV-1(KOS) gL86 [27], which expresses β-galactosidase upon entry into cells. HeLa cells that are naturally susceptible to HSV-1 entry were used as a positive control. Entry of HSV-1 was measured after 6 h of viral infection. As shown in Figure 1(a), similar to HeLa cells, HSV-1 entered hMSCs in a dose-dependent manner. By contrast, CHO-K1 cells without any receptor remained resistant to HSV-1 entry. Further confirmation of HSV-1 entry was established using an X-gal assay. No β-gal activity was observed in mock infected hMSCs (Figure 1(b), panel A), while a dosage of virus sufficient to infect 100% of hMSCs at 10 PFU/cell (Figure 1(b), panel (B)) also led to a nearly complete infection of hMSCs. In order to obtain more direct and visual evidence of HSV-1 entry, GFP-tagged HSV-1(K26GFP) [34] was used to infect cultured hMSCs, and fluorescence microscopy was used to visualize the virions inside the hMSCs stained with rhodamine phalloidin, which specifically labels F-actin (Figure 1(c), panel (B)). The GFP colocalization with F-actin was seen all over the cell (Figure 1(c), panel (B)). This result correlated with the observed susceptibility of hMSCs to HSV-1 entry during X-gal assay. actin cytoskeleton [36][37][38]. We therefore decided to examine whether cytoskeleton changes played any significant role in HSV-1 entry into hMSCs. To address this issue, fluorescent microscopy of hMSCs exposed to GFP-tagged HSV-1 virions at 30 min postinfection was performed. Quite noticeably, some GFP-tagged virus particles were found to attach at filopodia-like structures in rhodamine-phalloidin stained hMSCs (Figures 2(a) and 2(b)). In order to demonstrate the significance of actin network and filopodia during HSV-1 entry into hMSCs, we used the actin inhibitors latrunculin B (Lat B) or cytochalasin D (Cyto D). Both agents prevent cytoskeleton changes by blocking actin polymerization [39,40]. As shown in Figures 2(c) and 2(d), pretreatment of hMSCs with Lat B and Cyto D resulted in significant inhibition of HSV-1 entry, suggesting that cytoskeleton modification may be needed during the initial phase of HSV-1 infection.

HSV-1 Replicates in Cultured hMSCs.
Because HSV-1 was able to enter cultured hMSCs, we next evaluated whether this entry led to a productive virus replication. Initially, microscopy was used to obtain visual evidence of HSV-1 replication. Syncytial plaque forming HSV-1 (KOS) 804 virus [41] was used for infecting cultured hMSCs, and the virus was allowed to replicate. Cells were fixed at different time points and Giemsa stained. The cytopathic effect in the form of plaque formation increased significantly overtime in virus-infected hMSCs as seen in Figures 3(a) and 3(b). Furthermore, to assess viral replication, the infectious yields of virus were determined by plaque assays with Vero cells. As shown in Figure 3(c), inoculum harvested from infected hMSCs produced a larger number of plaques overtime. In contrast, CHO-K1 cells infected with identical doses of the same virus failed to produce infectious virions. These results, together with those of the entry assay, show that entry of HSV-1 into cultured hMSCs leads to productive infection.

Expression of HSV-1 gD in Cultured hMSCs Renders
Resistance to HSV-1 Entry. In order to determine if the gD receptors play a role in human hMSCs infection by HSV-1, a gD-mediated interference assay was used [35]. This assay is based on the principle that cells that are normally susceptible to viral entry become resistant upon expression of viral gD because of sequestration of gD receptors by cell-expressed gD [35]. To carry out the assay, hMSCs were transiently transfected with an HSV-1 gD-expressing plasmid (or an equal amount of the empty vector, pCDNA3, as a control), followed by infection with serial dilutions of β-galactosidaseexpressing HSV-1(KOS) gL86. As shown in Figure 4, HSV-1 entry into gD-expressing hMSCs was approximately 2.0-fold lower compared to the pCDNA3 control. Thus, entry into hMSCs is likely mediated by gD receptors expressed on the cell surface in a gD-dependent manner.

Identification of gD Receptors Expressed in Cultured hMSCs.
Reverse transcriptase (RT)-PCR analysis was performed to determine the identity of gD receptors potentially expressed in hMSCs. Gene-specific primers for nectin-1, 3-OST-3 and HVEM were used. As shown in Figure 5(a), products of the expected sizes for cDNAs encoding nectin-1, 3-OST-3 and HVEM were all detected, indicating that all three genes are normally expressed in hMSCs.

Anti-Nectin-1 and Anti-HVEM Antibody Do Not Significantly Affect HSV-1 Entry in hMSCs.
To determine which receptor were important for HSV-1 entry into hMSCs, previously established receptor specific antibodies were used [33,36]. As shown in Figure 5(b), antibody against nectin-1 affected HSV-1 entry by 20%, while HVEM had no significant effect on HSV-1 entry suggesting that neither of these receptors plays a critical role in mediating HSV-1 infection of hMSCs.

Significance of HS and 3-OST-3 as the Mediator of HSV-1 Entry and gD Binding to Cultured hMSCs.
Expression of 3-OST-3 raised the possibility that 3-OS HS could be a mediator of HSV-1 entry into hMSCs. Since cells that use 3-OS HS as the major entry receptor become significantly more resistant to HSV-1 upon removal of HS from the surface [37,38], we examined the effect of HS-degrading enzyme heparinase-I (1.5 U mL −1 ) on HSV-1K26GFP binding and entry into hMSCs. This enzyme is known to cleave off both heparin and HS chains containing 1-4 linkages between glucosamine and O-sulfated iduronic acid residues [42]. As shown in Figure 5(c), heparinase-treated hMSCs (H+) were resistant to HSV-1 binding (panel B) compared to the mock-treated cells (H-, panel A). Similar resistance to HSV-1 KOS gL86 entry was also observed as a result of heparinase treatment ( Figure 5(d)). Next, we tested whether a soluble recombinant form of HSV-1 gD or gD-Fc [29,35] would bind cultured hMSCs and whether this binding could be adversely affected by removal of HS from the surface using cell enzyme-linked immunosorbent assay [26,28,33]. Cells either treated with heparinases or mock-treated were allowed to bind equal amount of gD, and binding was detected with a spectrophotometer. Figure 5(e) shows that gD binding to mock-treated hMSCs was not affected; however, the binding of gD to heparinase-treated hMSCs was reduced by about 75%. This finding strongly supports that gDbinding receptors were specifically removed upon heparinase treatment in hMSCs, indicating that HS and 3-OST-3 play critical roles in mediating HSV-1 entry through gD binding to cultured hMSCs.

Requirement of 3-OS HS for HSV-1 Glycoprotein-Induced
Cell-to-Cell Fusion of Cultured hMSCs. To further verify that 3-OS HS is indeed the mediator of the membrane fusion that occurs during HSV-1 entry into hMSCs, we used a quantitative and efficient cell-to-cell fusion assay [14,22]. It has been shown that, similar to the membrane fusion that occurs during entry, cells expressing HSV-1 glycoproteins gB, gD, gH, and gL fuse with cells expressing gD receptors [18], and thus cell-to-cell fusion mimics the minimum requirement for entry. First, we studied the significance of HS in the fusion of hMSCs with HSV-1 glycoproteins expressing effector CHO-K1 cells. HS dependence is a way to determine whether 3-OS HS is the principal mediator of membrane fusion since, unlike nectin-1 and HVEM, which do not require HS for membrane fusion, 3-OS HS-mediated cell fusion is highly dependent on the expression of HS on the cell surface [22]. As expected, fusion of hMSCs with effector cells expressing the HSV-1 glycoproteins was significantly reduced upon heparinase treatment (Figure 6(a)), and a similar result was seen with CHO-K1 expressing 3-OST-3 (data not shown), suggesting that expression of HS and 3-OS HS in hMSCs are required for effective fusion with glycoprotein expressing-CHO-K1 cells. In a separate set of experiments, heparinase treatment also reduced multinucleated giant cell formation or polykaryons in hMSCs cocultured with effector CHO-K1 cells expressing HSV-1 glycoproteins (Figures 6(b) and 6(c)). Taken together, our results demonstrate that HS and 3-OS HS are required for HSV-1 glycoprotein-induced cell to cell fusion in cultured hMSCs, further implying their significant role in mediating HSV-1 entry and spread into hMSCs.

Discussion
HSV-1 infections remain a significant problem for allogeneic stem cell recipients. Understanding viral entry into hMSCs O or the same RNA that was used in generating cDNA templates was used as PCR template to control for potential genomic DNA contamination. The products were separated by electrophoresis on an agarose gel. (b) HSV-1 entry into hMSCs was not significantly affected by anti-nectin-1 or anti-HVEM antibody treatment. Cells were preincubated with anti-nectin-1 and anti-HVEM antibodies (1 : 10 dilution) followed by infection with reporter HSV-1 (KOS) gL86 virus. β-galactosidase activity was recorded 6 h later to determine HSV-1 entry. The experiment was repeated three times with similar results. (c) Visualization of heparinase treatment on HSV-1 binding into hMSCs. Cultured hMSCs were mock-treated with PBS (H−) or heparinase I (1.5 U mL −1 ) (H+), followed by exposure to GFP-tagged HSV-1 K26GFP virus at 50 pfu/cell for 30 min at 4 • C. Cells were then stained with rhodamine phalloidin that recognizes F-actin (red). Only mock-treated but not heparinase-treated cells showed HSV-1 GFP binding, which appeared yellow when its green fluorescence overlaps with red fluorescence. Virus attachment was determined by using Olympus IX50 inverted fluorescence microscope. (d) Effect of heparinase treatment on HSV-1 entry into hMSCs. Cells were treated with heparinase I (1.5 U mL −1 ) (white bar) or mock-treated with PBS alone (black bar), followed by exposure to HSV-1 KOS gL86 at 50 pfu/cell. Viral entry was quantitated 6 hr later by ONPG assay. Viral entry was reduced more than 3 folds in cells pretreated with heparinase compared to mock-treated cells ( * * P < .01, t test). Data shown are the means of triplicate measures. (e) Effect of heparinase treatment on HSV-1 gD binding to hMSCs. The cells were either pretreated with a mixture of heparinase-I (treated at 1.5 U mL −1 ; white bar) or mocktreated (untreated; black bar), followed by exposure to a soluble recombinant form of HSV-1 gD (gD:Fc). Binding of gD:Fc was detected by using HRP-tagged antibody against IgG:Fc and subsequent HRP enzyme detection. Asterisks indicate significant difference from the control treatment ( * * P < .01, t test). Error bars represent standard deviation (SD). in a healthy donor or in a recipient host is important for two reasons. First, productive viral infection into hMSCs may trigger immune response during or after stem cell transplant and, second, the infected population of hMSCs may become large reservoir of virions which may constantly expand both immune response and the viral transmission to the localized cells or tissues or may find the new host cells and tissues in the recipient. Therefore, viral receptor on these cells could determine the type of the infection, nature of immune response, and viral transmission. We cultured hMSCs derived from the adipose tissue of healthy donors to develop an in vitro model for studies of HSV-1 entry and replication and for identification of cellular mediators of entry. Although all the three HSV-1 gD-receptors (nectin-1, 3-OST-3 and HVEM) were detected, however, antibodies against nectin-1 and HVEM failed to block significantly HSV-1 entry (Figures 5(a) and 5(b)). Therefore, our focus shifted to 3-OS HS as a potential entry receptor. Adding further strength to this possibility, heparinase enzymes completely abolished both HSV-1 attachment (Figure 5 and entry into cultured hMSCs ( Figure 5(d)). In addition, the gD binding exhibited sensitivity to enzyme treatment ( Figure 5(e)) and the effect of heparinase to viral spread during cell to cell fusion assay was also significantly inhibited in cultured hMSCs ( Figure 6). Similar effect of heparinase treatment on HSV-1 entry and fusion has been shown in primary cultures of corneal fibroblasts obtained from human eye donors or in the cells exclusively expressing 3-OST isoforms [28,29,33]. Taken together, we speculate one interesting possibility that although all the three known gDreceptors are expressed in hMSCs but HSV-1 preferentially uses HS/3-OS-HS to gain entry could be related for two main reasons. First HS and 3-OS HS being larger in size are widely distributed and expressed all over the surface of the cells.
Second they are also easily accessible to HSV-1 unlike nectin-1 which is localized in cell junctions. This is also possible that nectin-1 and HVEM receptors are involved in other complex interactions that do not support HSV-1 entry into hMSCs. In summary, we conclude HS and 3-OS HS play a key role during HSV-1 infection into hMSCs. The finding provides new information on susceptible nature of hMSCs population within the host cell and contributes the broader significance of HS and 3-OS HS receptor in HSV-1 infection. Because HS also play a key role in signaling and immune activation [43], it is possible that they might play a dual role allowing both HSV-1 infection and facilitating the inflammation to further damage hMSCs. This unique feature of HS/3-OS HS in hMSCs can be exploited to develop the agent that blocks HSV-1 gD interaction with 3-OS HS receptor to suppress both viral entry/spread and immune response. Finally, hMSCs have potential to differentiate into many other celltypes including neurons. Interestingly, HS is known to mediate the proliferation and differentiation in mesenchymal stem cells [44]. Therefore it will be interesting to evaluate the expression pattern of gD-receptors in differentiated stem cells including neuronal stem cells where HSV-1 can easily infect and establish latency. Similarly the strategic development to isolate population of stem cell lines that does not support HSV-1 infection may further suppress the HSV-1-induced morbidity and mortality and bring enhanced success of stem cell transplantation.