A Role for Herpes Simplex Virus Type 1 Glycoprotein E in Induction of Cell Fusion

Transcription

1 J. gen. Virol. (1989), 70, Printed in Great Britain 2157 Key words: HS V-1/glycoprotein E/fusion A Role for Herpes Simplex Virus Type 1 Glycoprotein E in Induction of Cell Fusion By S. CHATTERJEE,* J. KOGA AND R. J. WHITLEY Department of Pediatrics, University of Alabama at Birmingham, Birmingham, Alabama 35294, U.S.A. (Accepted 10 April 1989) SUMMARY The role of herpes simplex virus type 1 (HSV-1) glycoprotein E (ge) in the induction of multinucleate cell (syncytium) formation was investigated using monoclonal antibodies and age deletion mutant, R7023. We found that monoclonal antibodies directed against ge blocked HSV-l-induced syncytium formation in human ce~ls. R7023 also failed to induce syncytium formation in tissue culture cells. The results indicate that ge, in addition to glycoproteins B, D, H and the gene sequence located between and map units, is involved in cell fusion. Thus, this important biological property appears to be regulated by several HSV-1 gene products. Envelope glycoproteins of a variety of animal viruses are essential for adsorption, penetration and, in some instances, cell-to-cell fusion resulting in the formation of multinucleate cells or syncytia. Viral glycoproteins are transported to the plasma membrane and presumably mediate cell fusion, an event which leads to cell-to-cell spread of infection (Hoggan & Roizman, 1959; Hoggan et al., 1960; Lodmell & Notkins, 1974). The exact role of glycosylation in this important biological process is not well understood. It has been reported that glycosylation of the fusioninducing product synthesized by Mason-Pfizer monkey virus is not required for syncytium formation (Chatterjee et al., 1981). However, with Newcastle disease virus and herpes simplex virus type 1 (HSV-1), the ability to induce multinucleate cells can be prevented if glycosylation is blocked (Gallaher et al., 1973). The specific glycoprotein(s) involved in HSV-l-induced cell fusion is unclear. Although several cell surface glycoproteins, gb, gc, gd, ge, gg, gh and gi, have been identified in HSV-infected cells (Baucke & Spear, 1979; Eberle & Courtney, 1980; Marsden et al., 1984; Roizman et al., 1984; Gompels & Minson, 1986; Longnecker et al., 1987), the exact biological functions of these glycoproteins are not completely understood (Roizman & Batterson, 1985). By employing temperature-sensitive mutants, the ionophore monensin and transfection experiments, several workers have demonstrated that at least one of these glycoproteins, gb, is required for HSV-induced cell fusion but other gene products may be required as well (Manservigi et al., 1977; Kousoulas et al., 1983; All et al., 1987; Cai et al., 1988). In fact, monoclonal antibodies against gd and gh block HSV-induced cell fusion which suggests that gd and gh are also involved in this process (Noble et al., 1983 ; Gompels & Minson, 1986). The specific roles of these glycoproteins in cell-to-cell fusion are, however, not known. In this communication, we report that monoclonal antibodies against ge inhibited cell fusion induced by HSV-1 in human fibroblast cells. In addition, age deletion mutant, R7023, failed to induce multinucleate cell formation in tissue culture. Thus, in addition to other gene products, it appears that ge is involved in cell fusion by HSV-1. Monoclonal antibodies against HSV-1 ge were isolated and characterized as described previously (Koga et al., 1986; Metcalf et al., 1988). Fig. 1 (a) demonstrates the radioimmunoprecipitation (RIP) profile obtained with two different monoclonal antibodies (Fd172 and Fd200) against ge using [35S]methionine-labelled, HSV-l-infected HEp-2 cell lysates. Two SGM

2 2158 Short communication I (a) (b) ~5 --II6K -l16k (a) 1 2 (b) (c) K -92K [ --66K -66K --45K -45K - '' gd--, r :: r Fig..1 Fig. 2 Fig. 1. Radioimmunoprecipitation pattern of HSV-1 ge with monoctonal antibodies. (a) HEp-2 cells (5 X 106 cells/60 mm Petri dish) infected with HSV-I strain F (m.o.i. 5) were labelled with [3 S S]methionine (1020 Ci/mmol; Amersham) and then immunoprecipitated with culture supernatants (50 ~tl) as described previously (Koga et al., 1986). Lane 1, immunoprecipitation with supernatant from hybridoma Fd200; lane 2, Fd 172. (b) The experimental procedure was same as in (a), except that the cells were labelled with [3H]glucosamine (40 Ci/mmol; ICN Pharmaceuticals) and immunoprecipitated with clone Fd200. The ge precursor is pge. Fig. 2. Specificity of the monoclonal antibodies against ge. F- and R7023-infected cell lysates were processed for immunoblotting as described previously (Chatterjee et al., 1985) using monoclonal antibodies against gb, gd and ge. Lanes 1, 3 and 5, F-infected cell lysates; lanes 2, 4 and 6, R7023- infected cell lysates. Blot (a) was reacted with monoclonal antibody against ge; blot (b) was reacted with monoclonal antibody against gb, and blot (c) with monoclonal antibody against gd. distinct bands corresponding to the Mr of mature ge (80K) and its precursor pge (69K) were identified. The positions of these two bands were identical when immunoprecipitation profiles obtained with other anti-ge monoclonal antibodies (provided by Dr L. Pereira) were compared. In order to confirm that these monoclonal antibodies were directed against glycosylated polypeptides, a RIP with [3H]glucosamine-labelled, HSV-l-infected HEp-2 cell lysates was performed. Two distinct bands at the same positions as observed with [3SS]methionine-labelled cell lysates indicated that the monoclonal antibodies were directed against two glycosylated polypeptides of 80K and 69K (Fig. 1 b). The specificity of these monoclonal antibodies was confirmed by using R7023, provided by Dr B. Roizman (Longnecker & Roizman, 1986). African green monkey kidney (B-SC-1) cells were infected with either R7023 or strain F of HSV-

3 Short communication 2159 Table 1. Anti-fusion activity of monoclonal antibodies against HSV-1 glycoproteins Clone no. Glycoprotein Inhibition (~) Fe65 Fd79 gb gb Fd Fd48 gd gd Fd172 Fd200 ge ge Fd8 gc 17.0 Table 2. Ability ofr7023 and strain F to induce syncytium formation in B-SC-1 cells Syncytium-forming Experiment no. Virus units/ml 1 F R x l0 s < F R x 10 s <5 x Infected cell lysates were processed for immunoblotting as previously described (Chatterjee et al., 1985). The resulting blots were reacted with monoclonal antibodies against ge and, finally, with 125i_labelled Protein A. As shown in Fig. 2 (a), the specificity of the monoclonal antibodies for ge was confirmed. It should be noted that R7023 also lacks the a sequences located at the junction between the L and S components, an origin of viral DNA synthesis and the ~47 gene, although ge is the only glycoprotein deleted (Longnecker & Roizman, 1986). To demonstrate the presence of other major glycoproteins in the mutant-infected cells, identical blots were reacted with monoclonal antibodies against gb and gd and with 125I-labelled Protein A; this clearly showed the presence of gb and gd in cells infected with R7023 (Fig. 2b and c). None of these anti-ge monoclonal antibodies had significant neutralizing activity in the absence of complement. Clones Fd172 and Fd200 showed complement-dependent neutralizing activities, with titres of 500 and 300 respectively (Metcalf et al., 1988). Nevertheless, these antibodies blocked the multinucleate cell formation induced by HSV-1 as described below. In brief, human foreskin fibroblast cells, grown in a 96-well microtitre plate were adsorbed with different concentrations (m.o.i. of 5, 1, 0-5 and 0.1) of the MP strain of HSV-1 for 1 h. Ninety min after this initial adsorption period, ascites fluids (approx. 50 ~tg of mouse IgG/ml) were added to the infected cells which were then observed at 16 h post-infection after staining with May-Griinwald-Giemsa as previously described (Chatterjee & Hunter, 1979), for their ability to block cell fusion. The number of syncytia per well in each group was determined and the percentage inhibition of syncytium formation was calculated. The result of this experiment (Table 1) indicated that these monoclonal antibodies prevented the ability of HSV-1 to cause fusion in human cells. A threefold dilution of the antibodies showed a proportionate increase in syncytium formation (data not shown). Almost identical results were obtained with the anti-ge monoclonal antibodies provided by Dr L. Pereira (data not shown). In addition to these anti-ge monoclonal antibodies, some of the anti-gb and anti-gd monoclonal antibodies inhibited HSV-l-induced syncytium formation in parallel experiments (Table 1). However, monoclonal antibodies against gc (clone Fd8) did not significantly block multinucleate cell formation which ruled out the possibility of the presence of a common fusion-inhibitory (toxic) factor in these monoclonal antibody preparations. In order to confirm fusion inhibition by monoclonal antibodies, R7023 was studied for the induction of cell-to-cell fusion. B-SC-1 cells were infected with serial dilutions (m.o.i. of 10, 1.0, 0-1 and 0-01) of strain F or R7023. We used monkey kidney cells because the mutant grew well in this cell line but did not in human fibroblast cells. Cells were stained 16 h post-infection as before, and the number of syncytium-forming units/ml was determined. This mutant, unlike strain F, did not induce any fusion of monkey cells in tissue culture (Table 2). Furthermore, to

4 2160 Short communication (a) 1 c 2, (b) C All B D D Fig. 3. Autoradiogram of 12SI-labelled Protein A binding assay to demonstrate the expression ofgb, gd and ge on the surface of R7023- and F-infected B-SC-1 cells. African monkey kidney cells were infected with different concentrations of R7023 (a) or F (b) and then processed for the protein A binding assay as described previously (Chatterjee et al., 1981) except that the ceils were not fixed. (a) The cells were reacted with monoclonal antibodies against gb (rows A and B), gd (rows C and D) and ge (rows E and F). Lane 1, ceils infected with 5 p.f.u./cell; lane 2, cells infected with I p.f.u./cell; lane C, control uninfected cells. (b) The ceils were reacted with monoclonal antibodies against ge (rows A and B), gb (rows C and D) and gd (rows E and F). Lane 1, cells infected with 1 p.f.u./cell; lane 2, cells infected with 0-5 p.f.u./cell; lane C, control uninfected cells. demonstrate that the absence of multinucleate cell formation induced by R7023 was not related to gb and gd expression on the cell surface, the following experiment was performed. Monkey kidney cells were infected with R7023 or F and processed for solid-phase radioimmunoassay 16 h post-infection using monoclonal antibodies against gb, gd and ge. The procedure for this assay was adapted from Chatterjee et al. (1981) without fixing the cells. The result of this experiment (Fig. 3 a) demonstrated the presence of only gb and gd on the surface of the mutantinfected cells whereas the surface of F-infected cells expressed gb, gd and ge as expected (Fig. 3 b). Thus, in spite of the presence of gb and gd on the surface of R7023-infected cells, this mutant did not induce syncytium formation. The inability of R7023 to induce cell fusion was not due to a reduced quantity of progeny virions, as the amount of virus released from the mutantinfected cells was almost the same as the amount of input virions (data not shown). Although several major species of HSV-1-specified glycoproteins, designated gb, gc, gd, ge, gg, gh and gi, have been identified, their specific biological functions have not been precisely determined; gb, gd and gh have been shown to be important for cell fusion activity (see above for references). In this report, we demonstrated that, in addition to these three, ge is involved in HSV-l-induced multinucleate cell formation. We found that monoclonal antibodies directed against ge prevented HSV-l-mediated syncytium formation in human fibroblast cells. Furthermore, the ge deletion mutant R7023 failed to induce multinucleate cell formation in tissue culture, suggesting that ge is involved in cell fusion. Since previous reports have indicated

5 Short communication 2161 that gb, gd and gh are involved in cell fusion, it is likely that this property is regulated by several distinct gene products of HSV-1. The biological importance of ge is also supported by recent observations from this laboratory that anti-ge monoclonal antibodies prevent stromal keratitis and protect mice against encephalitis (Metcalf et al., 1988). During past years, a great deal of effort has been devoted to the study of the role of glycoproteins in HSV-l-induced cell fusion. The mechanism of HSV-l-induced multinucleate cell formation might be analogous to that proposed for Sendai virus-induced cell fusion. The fusion process in the latter case consists of two steps, a binding step mediated by HN glycoproteins, and the fusion step mediated by F glycoproteins (Choppin et al., 1981). It should be noted that ge is known to be responsible for the induction of Fc-binding activity on virusinfected cells (Baucke & Spear, 1979). Since HSV-1 glycoproteins B, D, E and H might play a role in cell fusion, it will be of interest to pinpoint the specific polypeptide(s) involved in the binding activity and in the actual fusion event. However, at this time the possibility of involvement of other gene products in this biological process can not be ruled out. In fact it has been reported that the gene sequence located between and map units of HSV-1 (KOS strain) is also associated with cell fusion (Debroy et al., 1985). We thank Dr L. Pereira and Dr B. Roizman for providing us with anti-ge monoclonal antibodies and R7023, respectively. We also thank Sherlyn Burks for her help in preparation of this manuscript. This work was supported by NIH grants AI-25120, AI and by a grant from the Japan Chemical Research Company, Kobe, Japan. REFERENCES ALX, ~. A., BUTCHER, M. & GHOSH, H. P. (1987). Expression and nuclear envelope localization of biologically active fusion glycoprotein gb of herpes simplex virus in mammalian cells using cloned DNA. Proceedings of the National Academy of Scienees, U.S.A. 84, BAUCKE, R. B. & SPEAR, P. t3. (1979). Membrane proteins specified by herpes simplex virus. V. Identification of an Fc-binding glycoprotein. Journal of Virology 32, CAt, W., Or0, B. ~ PERSON, S. (1988). Role of glyeoprotein B of herpes simplex virus type 1 in viral entry and cell fusion. Journal of Virology 62, CHATTERJEE, S. & HUNTER, E. (1979). The characterization of Mason-Pfizer monkey virus-induced cell fusion. Virology 95, CHATTERJEE, S., BRAOAC, J. & HUNTER, E. (1981). Effect of tunicamycin on cell fusion induced by Mason-Pfizer monkey virus. Journal of Virology 38, CHATTERJEE, S., HUNTER, E. & WHITLEY, R. (1985). Effect of cloned human interferons on protein synthesis and morphogenesis of herpes simplex virus. Journal of Virology 56, cnoppin, P. w., RICHARt~SON, C. D., MERZ, D. C., HALL, W. W. & SCh'EID, A. (1981). The functions and inhibition ofthe membrane glycoproteins of paramyxoviruses and myxoviruses and the role of the measles virus M protein in subacute sclerosing panencephalitis. Journal of Infectious Diseases 143, DEBROY, C., PEDERSON, N. & PERSON, S. (1985). Nucleotide sequence of a herpes simplex virus type 1 gene that causes cell fusion. Virology 145, EBERLE, R. & COtmTNEy, R, J. (1980). ga and gb glycoproteins of herpes simplex virus type 1 : two forms of a single polypeptide. Journal of Virology 36, t3allaher, W. R., LEVlTAN, D. B. & SLOUGH, H. A. (1973). Effect of 2-deoxy-D-glucose on cell fusion induced by Newcastle disease and herpes simplex viruses. Virology 55, COMPELS, U. & MINSON, A. (1986). The properties and sequence of glycoprotein H of herpes simplex virus type 1. Virology 153, HOOCAN, M. D. & ROIZMAN, B. (1959). The isolation and properties of a variant of herpes simplex producing multinucleated giant cells in monolayer cultures in the presence of antibody. American Journal of Hygiene 70, HOGGAN, M. D., ROIZMAN, B. & TURNER, T. B. (1960). The effect of the temperature of incubation on the spread of herpes simplex virus in an immune environment in cell culture. Journal of Immunology 84, KOOA, J., CHATTERJEE, S. & WmTLEY, R. J. (1986). Studies on herpes simplex virus type 1 glycoproteins using monoclonal antibodies. Virology 151, KOUSOOLAS, K. G., BZXK, o. J. & PERSON, S. (1983). Effect of the ionophore monensin on herpes simplex virus type 1- induced cell fusion, glycoprotein synthesis and virion infectivity. Intervirology 20, LODMELL, D. L. & NOTKINS, ~,. L. (1974). CeUular immunity to herpes simplex virus mediated by interferon. Journal of Experimental Medicine 140, LONGNECKER, R. & ROIZMAN, B. (1986). Generation of an inverting herpes simplex virus 1 mutant lacking the L-S junction a sequences, an origin of DNA synthesis, and several genes including those specifying glycoprotein E and the 0t47 gene. Journal of Virology 58,

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