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Herpes simplex virus type-1 (HSV-1) causes significant health problems from periodic skin and corneal lesions to encephalitis. It is also considered a cofactor in the development of age-related secondary glaucoma. Inhibition of HSV-1 at the stage of viral entry generates a unique opportunity for preventative and/or therapeutic intervention. Here we provide evidence that a sugar-binding antiviral protein, cyanovirin-N (CV-N), can act as a potent inhibitor of HSV-1 entry into natural target cells. Inhibition of entry was independent of HSV-1 gD receptor usage and it was observed in transformed as well as primary cell cultures. Evidence presented herein suggests that CV-N can not only block virus entry to cells but also, it is capable of significantly inhibiting membrane fusion mediated by HSV glycoproteins. While CV-N treated virions were significantly deficient in entering into cells, HSV-1 glycoproteins-expressing cells pretreated with CV-N demonstrated reduced cell-to-cell fusion and polykaryocytes formation. The observation that CV-N can block both entry as well as membrane fusion suggests a stronger potential for this compound in anti-viral therapy against HSV-1.
Herpes simplex virus type-1 (HSV-1) infection is the most common cause of infectious blindness in developed countries (Liesegang et al., 1989; Liesegang, 2001). Following an initial infection in epithelial cells, HSV establishes latency in the host sensory nerve ganglia (Spear, 1993; Spear and Longnecker, 2003). It has recently been reported that over 90 % of the trigeminal ganglia examined post-mortem in a sampling of the American population contained HSV-1 (Hill et al., 2008). The virus emerges sporadically from latency and causes lesions on mucosal epithelium, skin, and the cornea, among other locations. Prolonged or multiple recurrent episodes of corneal infections can result in vision impairment or blindness, due to the development of herpetic stromal keratitis (HSK) (Kaye et al., 2000). This is typically characterized by inflammation leading to scarring, thinning, and vascularization of the corneal stroma (Eisenberg et al., 1985; Ellison et al., 2003). Patients with corneal HSV infection risk lifelong recurrent corneal disease. HSK accounts for 20–48% of all recurrent ocular HSV infection leading to significant vision loss in many patients (Liesegang et al., 1989; Kaye et al., 2000). HSV infection may also lead to several other diseases including retinitis, meningitis, and encephalitis.
Primary infection begins with the entry of HSV into host cells. It is a complex process that is initiated by specific interaction of viral envelope glycoproteins and host cell surface receptors (Spear, 1993; Spear et al., 2000). Both HSV-1 and HSV-2 use glycoproteins B and C (gB and gC, respectively) to mediate their initial attachment to cell surface heparan sulfate proteoglycans (HSPG) (WuDunn and Spear, 1989; Shieh et al., 1992; Herold et al., 1991). Binding of herpesviruses to HSPG likely precedes a conformational change that brings viral glycoprotein D (gD) to the binding domain of host cell surface gD receptors (Whitbeck et al., 1999; Krummenacher et al., 1998, 1999, 2000). Thereafter, a concerted action involving gD, its receptor, three additional HSV glycoproteins; gB, gH, and gL, and possibly an additional gH co-receptor trigger fusion of the viral envelope with the plasma membrane of host cells (Scnalan et al., 2003; Spear and Longnecker, 2003; Perez-Romero et al., 2005). Subsequently viral capsids and tegument proteins are released into the cytoplasm of the host cell.
The gD receptors include cell-surface molecules derived from three structurally unrelated families. These include a member of the tumor necrosis factor (TNF) receptor family, two members of the nectin family of receptors, and the product of certain 3-OSTs, 3-O-sulfated heparan sulfate (3-OS HS) (Spear 1993; Spear and Longnecker, 2003). Herpesvirus entry mediator (HVEM or TNFRSF14) principally mediates entry of HSV-1 and HSV-2 (Montgomery et al., 1996; Marsters et al., 1997; Kwon et al., 1997) into human T lymphocytes and is expressed in many fetal and adult human tissues including the lung, liver, kidney, and lymphoid tissues (Montgomery et al., 1996) and human trabecular meshwork (Tiwari et al., 2007). Nectin-1 and nectin-2, also known as herpesvirus entry proteins C and B (HveC and HveB), respectively, belong to the immunoglobulin superfamily (Cocchi et al., 1998; Milne et al., 2001; Shukla et al., 2000). Both nectin-1 and nectin-2 mediate entry of HSV-1 and HSV-2, but only nectin-1 mediates bovineherpesvirus-1 (BHV-1) entry (Martinez and Spear, 2002; Warner et al., 1998). HSV-1entry mediating activity of nectin-2 is limited to some mutant strains only (Warner at al., 1998; Lopez et al., 2000). Nectin-1 is extensively expressed in human cells of epithelial and neuronal and ocular origin (Richart et al., 2003; Tiwari et al., 2008), while nectin-2 is widely expressed in many human tissues, but with only limited expression in neuronal cells and keratinocytes. The non-protein receptor, 3-OS HS, is expressed in multiple human cell lines (e.g. neuronal and endothelial cells) and mediates entry of HSV-1, but not HSV-2 (Shukla at al., 1999; Shukla and Spear, 2001; Tiwari et al., 2004; Tiwari et al., 2006; Tiwari et al., 2007).
Recently a novel 11-kb antiviral protein cyanovirin-N (CV-N) originally isolated from the cyanobacterium Nostoc ellipsosporum was shown to have potent anti-human immunodeficiency virus (HIV) activity. Its mechanism of action is based on the specific targeting of high mannose oligosaccharides oligomannose-8 (Man-8) and oligomannose-9 (Man-9) on the HIV envelope glycoproteins gp120 and gp41 (O‘Keefe et al., 2000; Bolmstedt et al., 2001; Shenoy et al., 2001). Similar oligosaccharides are known to be present on other viruses, including Ebola, Influenza, and Hepatitis C viruses (O’Keefe et al., 2003; Barrientos et al., 2003). Previous efforts to determine the efficacy of CV-N’s inhibition of HSV-1 entry into target cells have yielded conflicting results (Boyd et al., 1997; O’Keefe et al., 2003). Here, we demonstrate that CV-N significantly inhibits HSV-1 entry into natural target cells of human ocular origin at non-cytotoxic nanomolar concentrations. In addition, we show that CV-N also impairs the viral glycoprotein induced cell-to-cell fusion. These data demonstrate that targeting the HSV-1 envelope glycoproteins is a new and promising approach in the development of antiviral therapies to herpes simplex virus infection.
Wild-type CHO-K1 cells were grown in Ham’s F12 (Invitrogen Corp, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS), while African green monkey kidney (Vero) cells were grown in Dulbecco’s Modified Eagles Medium (DMEM) (Invitrogen Corp.) supplemented with 5% FBS. Cultures of HeLa and RPE cells were grown in L-glutamine containing DMEM (Invitrogen Corp.) supplemented with 10% FBS. As previously described, cultures of human corneal fibroblasts (CF) were derived from the stroma of corneal tissues obtained from the Illinois Eye Bank, Chicago, IL, using institution approved protocol and culture conditions in accordance with the Declaration of Helsinki). CF from the 4th passage was used for the study was kindly provided by Dr. Yue (University of Illinois at Chicago). Recombinant β-galactosidase-expressing HSV-1(KOS) gL86 were used (Montgomery et al., 1996). P.G. Spear (Northwestern University) provided wild-type CHO-K1 cells. GFP expressing HSV-1 (K26GFP) was provided by P. Desai (Johns Hopkins University, Baltimore). The viral stocks were propagated at low multiplicity of infection (MOI) in complementing cell lines, titered on Vero cells and stored at −80°C. Cyanovirin-N (CV-N) used in this study was generous gift of Dr. T. Mori (National Cancer Institute, Bethesda, Maryland).
Viral entry assays were based on quantitation of β-galactosidase expressed from the viral genome in which β-galactosidase expression is inducible by HSV infection (Montgomery et al., 1996). Cells were transiently transfected in 6-well tissue culture dishes, using Lipofectamine 2000 with plasmids expressing HSV-1 entry receptors (necitn-1, HVEM and 3-OST-3 expression plasmids) at 1.5 µg per well in 1 ml. At 24 hr post-transfection, cells were re-plated in 96-well tissue culture dishes (2 × 10 4 cells per well) at least 16 hr prior to infection. Cells were washed and exposed to serially diluted pre-incubated virus with CV-N or 1 × PBS at two fold dilutions in 50 µl of phosphate-buffered saline (PBS) containing 0.1% glucose (G) and 1% heat inactivated calf serum (CS) for 6 hr at 37°C before solubilization in 100µl of PBS containing 0.5% NP-40 and the β-galactosidase substrate, o-nitro-phenyl-β-D-galactopyranoside (ONPG; ImmunoPure, PIERCE, Rockford, IL, 3 mg/ml). The enzymatic activity was monitored at 410 nm by spectrophotometry (Molecular Devices spectra MAX 190, Sunnyvale, CA) at several time points after the addition of ONPG in order to define the interval over which the generation of the product was linear with time. In parallel experiment the cells were preincubated with CV-N for 90 min before the HSV-1 infection. Inhibitory effect of CV-N on HSV-1 entry in cells were also confirmed by 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) staining. The cells were grown in Lab-Tek chamber slides (Nunc, Inc., Naperville, IL). After 6 hr of infection with reporter virus treated with CV-N or left untreated with1 × PBS, 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 MgCl2, 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 20 × objective of the inverted microscope (Zeiss, Axiovert 100M). The slide book version 3.0 (imaging software) was used for images. All experiments were repeated a minimum of three times unless otherwise noted.
Purified GFP-expressing HSV-1 (K26 GFP) were pre-incubated with CV-N or with 1 × PBS for 90 min at room temperature were used to the gD receptor expressing CHO-K1 cells or naturally susceptible cells (HeLa, Vero and human CF) grown in Microtest 96-well assay plates (BD Falcon). All cells were incubated at 4°C for 1 hr, washed five times to remove unbound virus, and finally replace with warm medium for further incubation. Viral binding measured as relative fluorescence units (RFU) per treatment were determined by using GENios Pro plate reader (TECAN) at 480-nm excitation and 520-nm emission spectrum. Measurements of 4 replicates of CV-N treated and untreated samples were performed. Data were expressed as mean ± standard deviation (SD).
In this experiment, the CHO-K1 cells (grown in F-12 Ham, Invitrogen) designated “effector” cells were co-transfected with plasmids expressing four HSV-1(KOS) glycoproteins, pPEP98 (gB), pPEP99 (gD), pPEP100 (gH) and pPEP101 (gL), along with the plasmid pT7EMCLuc that expresses firefly luciferase gene under the T7 promoter (Pertel et al., 2001). Wild-type CHO-K1 cells express cell surface HS but lack functional gD receptors, therefore transiently transfected with plasmids expressing entry receptors nectin-1 (pBG38) , HVEM (pBec10) and/or 3-OST-3 (pDS43) (Shukla et al., 1999; Pertel et al., 2001). Wild type CHO-K1 cultured cells expressing HSV-1 entry receptors or naturally susceptible cells (HeLa, Vero and human CF) considered as “target cells” were co-transfected with pCAGT7 plasmid that expresses T7 RNA polymerase using chicken actin promoter and CMV enhancer (Tiwari et al., 2007). CV-N untreated effector cells expressing pT7EMCLuc and HSV-1 essential glycoproteins and the target cells expressing gD receptors transfected with T7 RNA polymerase were used as the positive control. CV-N treated effectors cells were used for the test. For fusion, at 18 hr post transfection, the target and the effector cells were mixed together (1:1 ratio) and co-cultivated in a 24 well- tissue culture plates (Nunc, Inc.). The activation of the reporter luciferase gene as a measure of cell fusion was determined using reporter lysis assay (Promega) at 24 hr post mixing.
In this experiment CHO-K1 effector cells were co-transfected with plasmids expressing four HSV-1(KOS) glycoproteins (gB, gD, gH-gL) along with the plasmid pT7EMCLuc that expresses firefly luciferase gene under the T7 promoter plus pDSRed-N1 plasmid (BD Falcon) constructs. The target CHO-K1 cells expressing gD receptor (3-OST-3 modified 3OS HS) were co-transfected with pCAGT7 plasmid that expresses T7 RNA polymerase using chicken actin promoter and CMV enhancer plus green fluorescent expression plasmid. During co-transfection effector and target cells were balanced with empty vector plasmid pCDNA3.1 to keep equal amount of DNA in both cell-types. Before co-culture, effector cells were pre-incubated with 10 nM CV-N or 1× PBS for 90 min. Then both populations of effector and target cells were cultured in 1:1 ratio for 24 hrs. The cells were then fixed and mounted in Vectorshield mounting medium (Vector Laboratories, Inc. Burlingame, CA). Leica confocal microscope SP2 was used at 40 × magnification. A group of multinucleate cells (8–10 joint cells) were scored positive for polykaryocytes formation.
To determine the effect of CV-N on HSV-1 entry, we first tested the ability of HSV-1, in presence and absence of CV-N, to infect CHO-K1 cells expressing gD receptors. HSV-1 entry into cell was determined by using β-galactosidase expressing HSV-1 reporter virus (gL86). As shown in Fig. 1 HSV-1 pre-incubated with CV-N significantly blocked viral entry in a dose dependent manner in CHO-K1 cells expressing gD receptors (nectin-1, HVEM and 3-OST-3 modified 3OS HS). The blocking activity of CV-N was seen at low nano-molar concentrations and clearly, entry was inhibited irrespective of the gD receptors used. The inhibition seen was not due to the ability of CV-N to block reporter assay since strong enzyme activity was observed in CV-N treated cells transfected with a β-galactosidase expression plasmid (data not shown) (Montgomery et al., 1996).Taken together, the results indicated that the role of CV-N in HSV-1 entry blocking is not receptor specific phenomenon.
Next, to confirm blocking activity of CV-N on HSV-1 entry, we used natural target cells. We used HeLa and primary cultures of human corneal fibroblasts (CF). Human CF is a natural target cell line that has been shown previously to express 3-O sulfated heparan sulfate as a receptor (Tiwari et al., 2007; Tiwari et al., 2008). As shown in Fig. 2 (panel A and panel D) HSV-1 virions pre-treated with CV-N (50 nM) showed significant reduction of entry in both HeLa and CF. These results were further confirmed by X-gal assay. As demonstrated in panel C and F (Fig 2.) the HSV-1 treatment with CV-N significantly reduced the number of blue cells in both HeLa and CF cells (panels C and F). While corresponding untreated virus were able to infect all the cells as 100% cells turned blue (panels B and E). Taken together, the results indicated the role of CV-N in HSV-1 entry blocking is also observed in natural target cells including primary cells cultured from the human cornea.
Subsequently, we asked whether the inhibitory activity of CV-N on HSV-1 entry occurs at the cellular receptor level or it could be attributed to viral glycoproteins. To answer this question, instead of preincubating HSV-1 with CV-N, we first preincubated cells with CN-V for 90 min and then infected target cells (HeLa, Vero, RPE and CF) with 25 pfu/cell of HSV-1(KOS). As shown in Fig. 3 pre-incubation of the cells with CV-N had relatively minor effects on HSV-1 entry, suggesting that anti-HSV-1 activity of CV-N is likely exerted on glycoproteins expressed on viral envelopes.
Because CV-N blocked HSV-1 entry, we next tested its ability to affect viral binding to the cells. To determine the difference between CV-N treated versus untreated virus on attachment or binding we used GFP-expressing HSV-1 (K26GFP). GFP was fused in frame with the UL35 ORF to generate a VP26-GFP fusion protein in HSV-1 KOS (Desai and Person, 1998). As shown in Fig. 4 the GFP signal on cell surface was significantly weaker when virus was treated with CV-N in both gD receptors-expressing CHO-K1 cells (panel A) and natural target cells (panel B). This data clearly indicated that CV-N significantly affects viral entry at attachment step. In a parallel experiment the post-treatment of CV-N after 45 min of HSV-1 infection had no effect on viral binding or attachment on the cells (data not shown).
Finally, we tested the role of CV-N during HSV-1 glycoproteins mediated cell-to-cell fusion. Cell-to-cell fusion has been used to demonstrate the viral and cellular requirements during virus-cell interactions and also as means of viral spread (Pertel et al., 2001). We sought to determine whether CV-N interaction with HSV-1 envelope glycoproteins essential for viral entry may also affect cell-to-cell fusion. Surprisingly, effector cells expressing HSV-1 glycoproteins treated with CV-N impaired the cell-to-cell fusion in both CHO-K1 cells expressing specific gD receptors or naturally susceptible cells (Fig 5). This result was further confirmed during fluorescent -labeled cell fusion assay, where HSV-1 glycoprotein expressing effector cells co-transfected with pDSRed N1 fluorescent plasmid incubated with CV-N for 90 min failed to fuse with GFP-expressing CHO-3OST-3 target cells. In contrast, the control CV-N untreated effector red-cells fused (yellow color) with green target cells (Fig. 6). This response was further observed when polykaryocytes formation was estimated. Again CV-N treated effector cells failed to form polykaryons when co-cultured with target cells, while in control untreated effector cells efficiently showed larger number of polykaryons. Inhibitory fusion activity CV-N has been previously reported for human Herpesvirus 6 and measles virus (Dey et al., 2000).
Viral entry into cell is the first critical step required for the onset of disease (Sieczkari and Whittaker, 2005). Hence viral entry into cell provides a unique opportunity to study virus cell interactions in detail and find novel ways to block viral cell interactions for therapeutic interventions (Dimitrov, 2004). Here we demonstrated that cyanobacterial protein cyanovirin-N (CV-N) affects both HSV-1 entry and viral glycoprotein mediated cell-to-cell fusion using in vitro cell culture models. The anti-HSV-1 activity of CV-N was not limited to any particular gD receptors. Our results showed that HSV-1 entry was significantly blocked in CHO-K1 cells expressing either protein receptors (nectin-1 and or HVEM) or a sugar receptor (3-OST-3 modified 3OS HS). Similar blocking was also observed in a natural target CF cells isolated from the human cornea which expresses 3OS HS as the prime gD receptor (Tiwari et al., 2006). The blocking of CV-N was more pronounced with pre-treatment of HSV-1 virions compared to pre-treatment of host cells. This result suggested that CV-N inhibition resulted predominantly from CV-N-virions interactions. Similar conclusions have been made in previous reports with HIV, Hepatitis C and Ebola virus entry (Boyd et al., 1997; Helle et al., 2006; Dey et al., 2000; Barrientos et al., 2003). It has been proposed that potent antiviral property of CV-N stems from the fact that it is sugar binding protein. In case of HIV, CV-N binds to envelope glycoprotein gp120 and gp41 that are rich in high-mannose oligosaccharides structures Man-8 and Man-9 (Boyd et al., 1997). Similar oligosaccharides are known to present on other viruses including Influenza virus, flaviviruses and herpesviruses (Cohen et al., 1983). Importantly, CV-N activity against HSV-1 was at low nanomolar concentrations. It remains to be investigated whether CV-N blocks viral attachment. Similarly, CV-N treatment may affect cell-to-cell fusion at the level of cell binding.
The observation that CV-N affected cell-to-cell fusion and polykaryocytes formation, it is likely that CV-N may have more pronounced effects in blocking HSV-1 infection in vivo as well, especially since it seems to affect the membrane fusion phenomena. The latter is required for both virus entry and cell-to-cell spread (Pertel et al., 2001). Therefore, CV-N becomes very relevant in the development of new preventative therapeutics. Recently, in vivo efficacy of CV-N gel in vaginal model of female macaques (Macaca fascicularis) was demonstrated without any cytotoxic or clinical adverse effect (Tsai et al., 2004).
The potent inhibitory activity of CV-N against HSV-1 and multiple other enveloped (Balzarini, 2007; Buffa et al., 2009) suggests an important possibility that seemingly unrelated viruses may share something in common that can be used for the development of “broad-spectrum” anti-viral. Our work with HSV-1 provide a new template for future investigation if CV-N has similar ability to block on genital herpes (HSV-2) virus entry in vaginal cell culture model.
This investigation was supported by NIH RO1 grants Al057860 (DS) and a grant from the Glaucoma Foundation (DS). The authors wish to thank confocal imaging facility in the Department of Ophthalmology at University of Illinois.
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