1 Department of Ophthalmology and Visual Sciences, College of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA
2 Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA
3 Divison of Medicinal Chemistry and Natural Products, University of North Carolina, Chapel Hill, NC 27599, USA
Correspondence
Deepak Shukla
dshukla{at}uic.edu
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ABSTRACT |
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MAIN TEXT |
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During entry, penetration proceeds by fusion of the viral envelope with the cell membrane. The current model of HSV entry suggests that viral envelope glycoprotein gC or glycoprotein gB makes the initial contact with HS (Herold et al., 1991; Shieh et al., 1992
; WuDunn & Spear, 1989
). After the attachment, interaction of gD with one of its receptors, in association with three other glycoproteins (gB, gH and gL), triggers the fusion between the viral envelope and cell membrane leading to penetration (Spear & Longnecker, 2003;
Yoon et al., 2003
). Several cellular receptors for HSV-1 gD are known. They belong to three different classes that include a member of the TNF-receptor family named HVEM (HveA) (Montgomery et al., 1996
) and two members of the immunoglobulin superfamily, designated nectin-1 or HveC (Cocchi et al., 2000
; Geraghty et al., 1998
) and nectin-2 or HveB (Warner et al., 1998
). Unique sites within HS chains, generated by 3-OSTs, give rise to the newest family of gD receptors, the 3-OS HS (Shukla et al., 1999
; Xia et al., 2002
). The 3-OSTs act to modify HS late in its biosynthesis (reviewed by Lindahl et al., 1998
), and each isoform recognizes, as substrate, glucosamine residues in regions of the HS chain having specific, but different, prior modifications, including epimerization and sulfation at other positions (Liu et al., 1999
; Xia et al., 2002
). Thus, each 3-OST can generate potentially unique protein-binding sites within HS. To date, six different isoforms of 3-OSTs (3-OST-1, 3-OST-2, 3-OST-3A, 3-OST-3B, 3-OST-4 and 3-OST-5) are known. All, except 3-OST-1, generate HSV-1 entry receptors (Shukla & Spear, 2001
; Xia et al., 2002
). Interestingly, only 3-OST-3A and 3-OST-3B generate structurally identical gD receptors (and hence are used interchangeably throughout this study). The gD receptors generated by other isoforms are very similar, but likely not identical, in structure (Liu & Thorp, 2002
; Shukla et al., 1999
; Shukla & Spear, 2001
; Xia et al., 2002
). 3-OST-1 generates binding sites for antithrombin (Liu et al., 1999
; Shworak et al., 1999
) but fails to produce a receptor that binds to HSV-1 gD (Shukla et al., 1999
). 3-OSTs (one or more) are expressed in human and mouse tissues relevant to HSV-1 infection examined thus far (Mochizuki et al., 2003
; Shworak et al., 1999
; Xia et al., 2002
).
Multinucleated giant cells (polykaryocytes) resulting from virus-induced cell fusion are a hallmark of HSV-1 infection. Recently it was demonstrated that gB, gD, gH, gL (Browne et al., 2001; Muggeridge, 2000
; Turner et al., 1998
), and expression of HVEM or nectin-1, are required for cell fusion (Pertel et al., 2001
). Interestingly, using cell lines deficient in glycosaminoglycan (GAG) biosynthesis but expressing gD protein receptors, it was also demonstrated that cell fusion did not require HS (Browne et al., 2001
; Pertel et al., 2001
). In contrast, the requirement for HS during cell fusion was previously suggested by showing that the syncytial mutant HSV-1 (MP) could induce the fusion of wild-type Chinese hamster ovary (CHO) cells but not of mutant cells defective for HS or GAG biosynthesis (Shieh & Spear, 1994
).
The aim of this study was to determine whether the gD-binding form of HS, 3-OS HS, has a specific role in HSV-1-induced cell fusion, or not; and to dissect further the virus and cellular requirements of 3-OS HS-mediated cell fusion. For that purpose, we decided to focus on three well-studied 3-OSTs: 3-OST-1, 3-OST-3 and 3-OST-5. While HS modified by the latter two mediate HSV-1 entry, 3-OST-1 failed to generate HSV-1 entry receptor (Shukla et al., 1999; Xia et al., 2002
). In order to quantify HSV-1 glycoproteins and 3-OS HS-induced cell fusion, a luciferase reporter gene activation assay was used (Pertel et al., 2001
). Wild-type CHO-K1 cells express cell surface HS but lack functional gD receptors, including 3-OS HS (Shukla et al., 1999
). As a result, they are resistant to both HSV entry and virus-induced cell fusion (Montgomery et al., 1996
; Shieh et al., 1992
). In our experiments, the CHO-K1 cells 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 the firefly luciferase gene under the T7 promoter (all plasmids described by Pertel et al., 2001
). The other CHO-K1 cell population or target cells were co-transfected with 3-OST-expression plasmid (either 3-OST-1, 3-OST-3B or 3-OST-5) or HVEM plasmid (pBEC10) (Montgomery et al., 1996
; Shukla et al., 1999
; Xia et al., 2002
) and pCAGT7, which expresses T7 RNA polymerase using chicken actin promoter and CMV enhancer (Pertel et al., 2001
). The effector cells expressing pT7EMCLuc and pCDNA3 (devoid of any glycoproteins) and the target cells expressing 3-OST-5 with T7 RNA polymerase were taken as the control. All cells were grown in six-well dishes containing F-12 Ham medium (Gibco) with 10 % fetal bovine serum. At 18 h post-transfection, both the effector and the target cells were mixed together (1 : 1) and co-cultivated. The activation of the reporter luciferase gene as a measure of cell fusion was examined after 24 h. As shown in Fig. 1
(A), CHO-K1 cells (control) in the absence of HSV-1 glycoproteins failed to fuse despite the presence of 3-OST-5 in the target cell, implying an essential role for glycoproteins during cell fusion. Interestingly, cells expressing 3-OST-3B and 3-OST-5 (but not 3-OST-1) induced cell fusion provided all four glycoprotein were co-expressed in the effector cells (Fig. 1A
). The demonstration that 3-OST-1 expression did not result in fusion was very much in line with the fact that 3-OST-1-modified HS fails to bind HSV-1 gD, which is in turn required for the induction of fusion (Pertel et al., 2001
; Shukla et al., 1999
). Interestingly, cell fusion mediated by nectin-1 was higher than that mediated by 3-OST-3 and/or 3-OST-5 (Fig. 1A
). This was not unexpected since it correlates well with higher entry signals reported with nectin-1 compared to 3-OST-3-expressing CHO-K1 cells (Shukla et al., 1999
). One possibility for the observed higher entry and fusion with nectin-1 is enhanced receptor expression; higher than physiological levels of receptors are produced when nectin-1 is overexpressed in CHO-K1 cells. However, since 3-OST-3 and 3-OST-5 are HS-modifying enzymes, their activity is dependent on the availability of specific substrate sites within existing HS chains (Shukla et al., 1999
; Xia et al., 2002
). As shown in Fig. 1(B)
, a cell surface enzyme linked immunosorbent assay (cell-ELISA) did not detect any enhancement of cell surface HS levels by 3-OST-3 and/or 3-OST-5 overexpression in CHO-K1 cells compared to the wild-type cells. A CHO-K1 mutant cell line (pgsA-745) that does not express any cell surface HS was used as the negative control. Thus, overexpression of 3-OST-3 and/or 3-OST-5 does not enhance cell surface HS population, which, in turn, could partly be responsible for the lower HSV-1 entry and fusion compared to nectin-1 (Shukla et al., 1999
).
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HSV-infected cells fuse to form multinucleated polykaryocytes. To verify that 3-OS HS-mediated fusion also results in polykaryocyte formation, we either mock transfected CHO-K1 cells with empty vector (pCAGGS), 3-OST3B-expression plasmid alone, or in combination with 3-OST-5-expression plasmid, as target cells. These cells were then mixed and co-cultivated with the effector cells expressing the four glycoproteins for 24 h and then stained with Giemsa solution. The polykaryocytes observed with cells co-expressing 3-OST-3B and 3-OST-5 were larger and more numerous than cells expressing 3-OST-3B alone (data not shown). This result, taken together with the data shown in Fig. 1(A), suggests that the co-expression of 3-OST-5 and 3-OST-3B enhances both fusion and polykaryocyte formation.
In order to verify the glycoprotein requirement for fusion mediated by 3-OS HS, we transfected CHO-K1 cells with various combinations of HSV-1 glycoproteins and performed the fusion assay using CHO-K1 with 3-OST-3B as target cells. As shown in Table 1, 3-OS HS-mediated fusion required co-expression of all four glycoproteins, gB, gD, gH and gL, which were also reported to be essential for fusion via HVEM and nectin-1 (Pertel et al., 2001
; Turner et al., 1998
). An identical glycoprotein requirement was also observed with target cells expressing 3-OST-5. Therefore it is possible that all three gD receptors (3-OS HS, HVEM and nectin-1) mediate cell fusion via a common mechanism. Interestingly, the three receptors are already known to bind gD with almost identical affinities (Spear et al., 2000
).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Browne, H., Bruun, B. & Minson, T. (2001). Plasma membrane requirements for cell fusion induced by herpes simplex virus type 1 glycoproteins gB, gD, gH and gL. J Gen Virol 82, 14191422.
Chen, J., Duncan, M. B., Carrick, K., Pope, R. M. & Liu, J. (2003). Biosynthesis of 3-O-sulfated heparan sulfate: unique substrate specificity of heparan sulfate 3-O-sulfotransferase isoform 5. Glycobiology 13, 785794.
Cocchi, F., Menotti, L., Mirandola, P., Lopez, M. & Campadelli-Fiume, G. (1998). The ectodomain of a novel member of the immunoglobulin subfamily related to the poliovirus receptor has the attributes of a bona fide receptor for herpes simplex virus types 1 and 2 in human cells. J Virol 72, 999210002.
Cocchi, F., Menotti, L., Dubreuil, P., Lopez, M. & Campadelli-Fiume, G. (2000). Cell-to-cell spread of wild-type herpes simplex virus type-1, but not of syncytial strains, is mediated by the immunoglobulin-like receptors that mediate virion entry, nectin1 (PRR1/HveC/HIgR) and nectin2 (PRR2/HveB). J Virol 74, 39093917.
Ernst, S., Langer, R., Cooney, C. L. & Sasisekharan, R. (1995). Enzymatic degradation of glycosaminoglycans. Crit Rev Biochem Mol Biol 30, 387444.[Abstract]
Geraghty, R. J., Krunnenacher, C., Cohen, G. H., Eisenberg, R. J. & Spear, P. G. (1998). Entry of alphaherpesviruses mediated by poliovirus receptor-related protein 1 and poliovirus receptor. Science 280, 16181620.
Herold, B. C., WuDunn, D., Soltys, N. & Spear, P. G. (1991). Glycoprotein C of herpes simplex virus type 1 plays a principle role in the adsorption of virus to cells and in infectivity. J Virol 65, 10901098.[Medline]
Lindahl, U., Kusche-Gullberg, M. & Kjellen, L. (1998). Regulated diversity of heparan sulfate. J Biol Chem 273, 2497924982.
Liu, J. & Thorp, S. C. (2002). Cell surface heparan sulfate and the roles in assisting viral infections. Med Res Rev 22, 125.[CrossRef][Medline]
Liu, J., Shriver, Z., Blaiklock, P., Yoshida, K., Sasisekharan, R. & Rosenberg, R. D. (1999). Heparan sulfate D-glucosaminyl 3-O-sulfotransferase-3A sulfates N-unsubstituted glucosamine residues. J Biol Chem 274, 3815538162.
Mochizuki, H., Yoshida, K., Gotoh, M. & 9 other authors (2003). Characterization of a heparan sulfate 3-O-sulfotransferase-5, an enzyme synthesizing a tetrasulfated disaccharide. J Biol Chem 278, 2678026787.
Montgomery, R. I., Warner, M. S., Lum, B. J. & Spear, P. G. (1996). Herpes simplex virus-1 entry into cells mediated by a novel member of the TNF/NGF receptor family. Cell 87, 427436.[Medline]
Muggeridge, M. I. (2000). Characterization of cellcell fusion mediated by herpes simplex virus 2 glycoproteins gB, gD, gH and gL in transfected cells. J Gen Virol 81, 20172027.
Pertel, P., Fridberg, A., Parish, M. & Spear, P. G. (2001). Cell fusion induced by herpes simplex virus glycoproteins gB, gD, and gHgL requires a gD receptor but not necessarily heparan sulfate. Virology 279, 313324.[CrossRef][Medline]
Rosenberg, R. D., Shworak, N. W., Liu, J., Schwartz, J. J. & Zhang, L. (1997). Heparan sulfate proteoglycans of the cardiovascular system. Specific structures emerge but how is synthesis regulated? J Clin Invest 99, 20622070.
Shieh, M. T. & Spear, P. G. (1994). Herpesvirus-induced cell fusion that is dependent on cell surface heparan sulfate or soluble heparin. J Virol 68, 12241228.[Abstract]
Shieh, M. T., WuDunn, D., Montgomery, R. I., Esko, J. D. & Spear, P. G. (1992). Cell surface receptors for herpes simplex virus are heparan sulfate proteoglycans. J Cell Biol 116, 12731281.[Abstract]
Shukla, D. & Spear, P. G. (2001). Herpesviruses and heparan sulfate: an intimate relationship in aid of viral entry. J Clin Invest 108, 503510.
Shukla, D., Liu, J., Blaiklock, P. & 7 other authors (1999). A novel role for 3-O-sulfated heparan sulfate in herpes simplex virus 1 entry. Cell 99, 1322.[Medline]
Shworak, N. W., Liu, J., Petros, L. M., Schwartz, J. J., Zhang, L., Kobayashi, M., Copeland, N.G., Jenkins, N. A. & Rosenberg, R. D. (1999). Multiple isoforms of heparan sulfate D-glucosaminyl 3-O-sulfotransferase. Isolation, characterization, and expression of human cDNAs and identification of distinct genomic loci. J Biol Chem 274, 51705184.
Spear, P. G., Longnecker, R. (2003). Herpesvirus entry: an update. J Virol 77, 1017910185.
Spear, P. G., Eisenberg, R. & Cohen, G. (2000). Three classes of cell surface receptors for alphaherpesvirus entry. Virology 275, 18.[CrossRef][Medline]
Turner, A., Bruun, B., Minson, T. & Browne, H. (1998). Glycoprotein gB, gD, and gHgL of herpes simplex virus type 1 are necessary and sufficient to mediate membrane fusion in a Cos cell transfection system. J Virol 72, 873875.
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. (1998). A cell surface protein with herpesvirus entry activity (HveB) confers susceptibility to infection by mutants of herpes simplex virus type 1, herpes simplex virus type 2, and pseudorabies virus. Virology 246, 179189.[CrossRef][Medline]
WuDunn, D. & Spear, P. G. (1989). Initial interaction of herpes simplex virus with cells is binding to heparan sulfate. J Virol 63, 5258.[Medline]
Yoon, M., Zago, A., Shukla, D. & Spear, P. G. (2003). Mutations in the N termini of herpes simplex virus type 1 and 2 gDs alter functional interactions with the entry/fusion receptors HVEM, nectin-2, and 3-O-sulfated heparan sulfate but not with nectin-1. J Virol 77, 92219231.
Xia, G., Chen, J., Tiwari, V., Ju, W., Li, J.-P., Malmström, A., Shukla, D. & Liu, J. (2002). Heparan sulfate 3-O-sulfotransferase isoform 5 generates both an antithrombin-binding site and an entry receptor for herpes simplex virus, type 1. J Biol Chem 277, 3791237919.
Received 9 September 2003;
accepted 7 January 2004.