THE JOURNAL OF BI~L~CKXL CHEM~~RY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 10, Issue of April 5, pp. 5662-5666, 1990 Printed in U.S.A.

Acid pH-induced Fusion of Cells by Herpes Simplex Virus Glycoproteins gB and gD*

(Received for publication, May 12, 1989)

Martin Butcher, Kanakatte RaviprakashS, and Hara P. GhoshQ From the Department of Biochemistry, McMaster University, Hamilton, Ontario L8N 325, Canada

Enveloped animal viruses enter host cells either by direct fusion at neutral pH or by endocytosis. Herpes simplex virus (HSV) is believed to fuse with the plasma membrane of cells at neutral pH, and the glycoproteins gB and gD have been implicated in virus entry and cell fusion. Using cloned gB or gD genes, we show that cells expressing HSV- 1 glycoproteins gB or gD can undergo fusion to form polykaryons by exposure only to acidic pH. The low pa-induced cell fusion was blocked in the presence of monoclonal antibodies specific to the gly- coproteins. Infection of cells expressing gB or gD gly- coproteins with HSV-1 inhibited the low pa-induced cell fusion. The results suggest that although the gly- coproteins gB and gD possess fusogenic activity at acidic pH, other HSV proteins may regulate it such that in the virus-infected cell, this fusion activity is blocked.

Enveloped animal viruses may enter the host cell either by direct fusion with the plasma membrane or by endocytosis of the virus and subsequent fusion of the viral envelope with the endosomal membranes. In both of these cases, the fusion is mediated by virus-coded envelope glycoproteins. The parain- fluenza subgroup of viruses including Sendai, New Castle disease, simian virus 5, measles, as well as human immuno- deficiency virus can fuse with the plasma membrane at neutral pH during the infection process. Cells infected with these viruses also form multinucleated cells or polykaryons at neu- tral pH as a result of the expression of the viral glycoproteins (l-4). On the other hand, viruses belonging to rhabdovirus subgroup, e.g. vesicular stomatitis (VSV),’ orthomyxo subgroup, e.g. influenza, togavirus subgroup, e.g. Semliki For- est, and retroviruses such as mouse mammary tumor virus do not fuse with the plasma membrane at neutral pH but are endocytosed and transported intracellularly via endosome vesicles. In the acidic environment present there (5), the viral envelope fuses with the endosome membrane and releases the viral nucleocapsid in the cytoplasm. In these cases, although the virus cannot fuse directly to the host cells, in the presence

* This work was supported by the Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “uduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Present address: Dept. of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. _

§ To whom correspondence should be sent: Dept. of Biochemistry, McMaster University, 1200 Main St. West, Hamilton, Ontario L8N 325, Canada. Tel.: 416-525-9140 (ext. 2454); fax: 416-521-0048.

’ The abbreviations used are: VSV, vesicular stomatitis virus; HSV, herpes simplex virus; EBV, Epstein-Barr virus; Hepes, 4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid; Mes, 2-(N-morpho- 1ino)ethanesulfonic acid; m.o.i., multiplicity of infection.

of an acidic medium, virus-cell fusion occurs. In addition, exposing cells infected with this group of viruses to acidic pH causes extensive cell-cell fusion and formation of polykaryons (l-3, 6, 7). The presence of agents such as NH&l or chloro- quine, which raise the pH of endosomes and lysosomes (2, 7), inhibits the replication of these viruses by preventing the fusion and hence uncoating of the nucleocapsid.

Hepes simplex virus type 1 (HSV-l), the prototype of the DNA-containing enveloped viruses belonging to the herpes family, contains at least six glycoproteins (3, 8). The three glycoproteins gB, gD, and gH are essential for virus replication and have been implicated in fusion activity of HSV-1 (3, 9- 17). Studies using electron microscopic analysis of HSV-1 infection of cells suggest that either endocytosis or direct fusion of virions to the cell surface at neutral pH could result in the entry of HSV-1 into host cells (13,18-20). In the case of Epstein-Barr virus (EBV), weak bases such as NH&l or chloroquine retarded the deenvelopment and fusion of the virus inside the endosome of normal B lymphocytes, suggest- ing that EBV enters these cells by endocytosis. However, in transformed B lymphoblastoid cells, EBV entered by direct fusion with the plasma membrane (20). The exact mechanism of entry of herpes viruses may, therefore, be dependent on the virus strain, host cell, and other conditions (3).

Recent studies with cloned cDNAs of the envelope glyco- protein G of VSV (21), HA glycoprotein of influenza virus (22), and E1 and Ez glycoproteins of Semliki Forest virus (23) showed that the expressed glycoproteins could induce cell fusion at low pH in the absence of other viral gene products. Expression of cloned cDNA of the paramyxovirus simian virus 5 fusion protein F in mammalian cells, however, resulted in cell fusion and syncytia formation at neutral pH (24). In an effort to study the mechanism of cell fusion induced by HSV glycoproteins, we have expressed cloned genes of HSV-1 gly- coproteins gB and gD in mammalian cells. We reported pre- viously that gB glycoprotein expressed in COS cells could induce cell fusion at acidic pH (25). In this paper we show that both gB and gD glycoproteins can induce cell fusion at low pH. The cell fusion was blocked with specific monoclonal antibodies. Infection of cells expressing gB or gD glycopro- teins with HSV-1, however, inhibited the low pH-induced cell fusion. These results suggest that endocytosis may also play a role in HSV-1 entry; however, the low pH-induced cell fusion mediated by gB and gD glycoproteins may be regulated by the expression of other viral gene products.

MATERIALS AND METHODS

Construction of Expression Plasmids-The construction of plasmid p9gB containing gB-1 gene inserted into the COS cell expression vector p91023 (27) has already been described (25). The plasmid p9gD was cloned by inserting the 1.65-kilobase HindIII-BstEII fragment containing the complete coding sequence of gD-1 (26) into the unique EcoRI cloning site of p91023B (27).

Expression of Cloned Genes-Transient expression of cloned gB-1

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or gD-1 genes from the plasmids p9gB or p9gD was carried out in COS cells as described earlier (25). The [ “Slmethionine-labeled gly- coproteins were identified by sodium dodecyl sulfate-polyacrylamide gel analysis (25).

Indirect Immunojluorescence-The intracellular and cell surface localizations of the expressed glycoproteins were examined by indirect immunofluorescence as described earlier (25). Monoclonal antibodies 3s (specific for gB-1) and 4s (specific for gD-1 glycoproteins of HSV- 1) (30) were obtained from Dr. M. Zweig of the National Cancer Institute, and anti-HSV-1 antibody was obtained from Dako Labo- ratories, Los Angeles, CA. A rabbit polyclonal anti-gB antibody was raised by inoculating rabbits with a recombinant adenovirus contain- ing HSV-1 gB gene (42). A mixture of two anti-gD monoclonal antibodies LP2 (43) and DLll (44) obtained from Dr. David Johnson, McMaster IJniversity, was also used for immunofluorescence studies.

Cell Fusion-The fusogenic activity of the expressed glycoproteins was monitored by exposing the cells to pH 5.0-6.5 for a very short time and observing the polykaryon formation (6, 25, 28). Typically, monolayers of cells were washed with phosphate-buffered saline at 37 “C and then exposed to the low pH fusion medium (1.8 mM NaH?PO,, 8.4 mM NalHPOI, 10 mM Hepes, 10 mM Mes, and 2.5 mM NaCl, pH adjusted at 5.0-7.0) (21) for 60 s. The medium was washed off with phosphate-buffered saline, and the cells were incubated in the presence of regular medium for 2.5 h. The cells were reexposed to the low pH fusion medium for another 60 s. Washed cells were incubated in the presence of regular medium for a further period of 4 h. The cells were fixed with methanol and stained with Giemsa. Plates were examined under phase contrast, and polykaryons con- taining more than 5 nuclei were counted.

The specificity of cell fusion induced by glycoproteins gB and gD was examined by using monoclonal antibodies 3S and 4S specific for gB-1 and gD-1 glycoproteins, respectively (30). COS cells transfected with p9gB or p9gD plasmids were treated with the antibodies as described by Noble et al. (11). The cells were then exposed to the fusion medium. After washing, cells were incubated for 2.5 h with regular medium containing the antibody. Following exposure to the fusion medium, cells were further incubated for 4 h in the presence of regular medium containing the antibody. The cells were fixed, stained, and examined for polykaryon formation as described earlier.

Virus Infection of Cells-Nontransfected as well as transfected COS cells were infected with wild-type HSV-1 KOS strain, gB mutant HSV-1, K082 (40), and gD mutant HSV-1, FgD@ (17) virus at a m.o.i. of 5-10. Fusion assay was done as described earlier at 18-24 h postinfection. The K082 virus was obtained from Drs. J. Glorioso and M. Levine of the University of Michigan, Ann Arbor, and was grown in D6 cells (40). FgDp virus grown in VD60 cells (17) was obtained from Dr. D. Johnson of McMaster University.

RESULTS

Cells Fusion by HSVGlycoproteins-Wild-type HSV cannot induce the fusion of cultured cells, but variants known as syn mutants can induce cell fusion and syncytia formation at neutral pH (8). That HSV-1 cannot induce polykaryon for- mation at acid pH was shown by exposing COS cells infected with HSV-1 to medium that has been adjusted to pH 5.0-7.0. No formation of polykaryons under these conditions was observed (data not presented), confirming that wild-type HSV-l-infected cells do not form syncytia at neutral or acidic PH.

We noted previously that COS cells transfected with gB- containing plasmid and expressing gB glycoprotein would form polykaryons when exposed to acid pH (25). Since the essential glycoprotein gD has also been implicated in the HSV entry and cell fusion process (3, 13, 15-17), we also exposed COS cells expressing gD glycoprotein to acidic pH (pH 5.7). As in the case of COS cells expressing gB glycoproteins (Fig. 1, panel b), cells expressing gD also showed formation of polykaryons when exposed to acidic pH (Fig. 1, panel d). The polykaryons contained between 8 and 15 nuclei in the giant cells. No polykaryons were observed when COS cells trans- fected with the gB- or gD-containing plasmid were not treated with the fusion medium at acidic pH. When nontransfected COS cells were exposed to acid pH, only a few polykaryons

FIG. 1. Low pa-induced fusion of cells expressing gB or gD glycoprotein. Cells were exposed to fusion medium at pH 5.7 for 60 s, then incubated in regular medium for 2.5 h, treated again with pH 5.7 fusion medium for 60 s, incubated in regular medium for 4 h, and then fixed, stained, and photographed using a phase-contrast micro- scope equipped with a camera. COS cells were transfected with p9gB (a, b) or p9gD (c, d) for 40 h. Panels a and c show cells treated with fusion medium at pH 7.5, whereas panels b and d show cells exposed to acid pH (pH 5.7) fusion medium. Magnification, X 250.

PH

FIG. 2. pH dependence of cell fusion induced by expression of gB-1 glycoprotein. Cells were twice exposed as described under “Materials and Methods” to fusion medium of varying acidity in the pH range 5.0-7.0. The number of polykaryons containing more than five nuclei in 10 random fields was counted. COS cells were trans- fected with p9gB plasmids (w), p9gD plasmids (w), p9tgB plasmids (M), p91023 plasmids (A-A), and no plasmids (A-A), respectively.

containing two to four nuclei were observed. It has been observed previously that the extent of fusion of

cells varies with the pH of the fusion medium and the virus used (6,21,22,29). We therefore determined the pH optimum of fusion of COS cells transfected with the plasmids p9gB or p9gB. Results presented in Fig. 2 show that COS cells express- ing gB glycoproteins showed a sharp pH optimum of cell fusion at pH 5.7 in comparison with a broader pH dependence of cell fusion induced by VSV G glycoprotein (28). The

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exposure of COS cells, expressing a truncated form of gB (tgB) which lacks the carboxyl-terminal membrane anchor and cytoplasmic domains and therefore is not associated with cellular membranes (25), to medium at pH 5.0-pH 6.5 did not show the formation of any polykaryons. COS cells expressing gD glycoproteins, however, showed a pH optimum of cell fusion at pH 5.5. In order to determine the efficiency of cell fusion by gB or gD proteins, transfected cells were examined by surface immunofluorescence using anti-gB or anti-gD an- tibody with or without exposure to acidic fusion medium. It was observed that typically 40-60s of cells expressing gB or gD glycoproteins formed polykaryons induced by acid pH.

The dependence of the cell fusion on the expression of specific glycoproteins was shown by two approaches. COS cells expressing gB or gD glycoproteins were treated with polyclonal anti-HSV-1 antibody or monoclonal antibodies 3s or 4s specific for gB or gD glycoproteins, respectively (30), before exposure to acidic pH. Pretreatment with polyclonal anti-HSV-1 antiserum blocked the formation of polykaryons in both gB- and gD-expressing COS cells (data not shown). As shown in Fig. 3, cells expressing gB and gD also did not show the formation of any polykaryons when pretreated with monoclonal antibodies 3s and 4s specific for gB (panel 6) and gD (panel f), respectively. The inhibition was specific for gB and gD glycoproteins because fusion was not blocked in the presence of nonspecific monoclonal antibodies to other HSV glycoproteins. In order to establish that the cells in- volved in acid-induced polykaryon formation were indeed expressing gB or gD glycoproteins, we used indirect immu- nofluorescence to detect the presence of glycoproteins in the polykaryons. Cells expressing gB or gD glycoproteins were analyzed for the presence of glycoproteins inside the cells by incubating first with rabbit anti-HSV-1 antibody and then staining the cells with anti-rabbit IgG antibody conjugated with fluorescein. As shown in Fig. 4, the polykaryons formed in the presence of acid pH by COS cells expressing gB (panel d) or gD (panel h) glycoproteins showed strong internal immunofluorescence. The staining was very high around the nuclei in the fused multinucleated cells. Although the cells

FIG. 3. Blocking of cell fusion with specific antibodies to the glycoproteins. COS cells transfected with p9gB or p9gD were treated with rabbit anti-HSV-1 antiserum or with monoclonal anti- bodies 3s and 4S specific to gB and gD, respectively (30). The cells were then exposed to the fusion medium at pH 5.7, and polykaryon formation was determined as described under “Materials and Meth- ods.” Shown here are the results with monoclonal antibodies. Panels a-c and d-/ represent COS cells transfected with p9gB and p9gD plasmids, respectively. Panels a and d, no pretreatment; b and e, pretreatment with 3S anti-gB monoclonal antibody; c and j, pretreat- ment with 4S anti-gD monoclonal antibody, respectively. Magnifica- tion, X 330.

were treated for internal immunofluorescence, staining was also noticed around the periphery of the giant cell. As ex- pected, the extent of surface staining under these conditions was much weaker. COS cells expressing gB (panel b) or gD (panel f) but not exposed to acidic pH did not result in polykaryon formation and showed internal staining only on isolated cells.

Inhibition of Cell Fusion by HSV-1 Infection-It has been suggested previously that the HSV-l-induced cell fusion may be regulated by the expression of a number of viral genes (3, 8, 9, 31, 32). We therefore decided to determine the effect of infection of COS cells expressing gB or gD protein with HSV- 1. Results presented in Fig. 5 show that infection with HSV- 1 resulted in inhibition of low pH-induced cell fusion in the cases of cells expressing gB and gD glycoproteins (panels b and d, respectively). In an effort to quantitate the extent of cell fusion induced by the glycoproteins, we counted the polykaryons formed in COS cells transfected with the HSV gB or gD glycoprotein genes (Table I). The results show that although COS cells transfected with the vector alone showed very little polykaryon formation at neutral or acidic pH, COS cells transfected with gB- or gD-containing plasmids showed an increase of 25 and 16-fold, respectively, in the number of the polykaryons formed after treatments with the acidic fu- sion medium. Infection of the cells expressing gB or gD glycoproteins with HSV-1 at an m.o.i. of 10 followed by treatment with fusion medium at acidic pH resulted in com- plete inhibition of polykaryon formation. These results sug- gest that expression of HSV-1 genes did not allow the cells to fuse under conditions in which noninfected cells expressing gB or gD glycoprotein would fuse.

In order to show that the transfected cells are still express- ing the glycoproteins gB or gD after HSV-1 infection, we used two mutant HSV-1 strains defective either in gB gene (40) or in gD gene (17), respectively. The gB-defective virus (K082) was generated by introducing a chain termination codon into codon 43 of the gB-1 gene (40). The K082 virus could not grow on Vero cells but could grow on a gB-transformed cell line (D6) that provided gB protein (40). The K082 virus grown in D6 cell line contained gB protein in the virion and could infect cells. The infected cells did not synthesize any gB-1 protein because of the defect in the gB gene, but other HSV- specific proteins were synthesized (14, 40). The gD-defective mutant (FgDP) was constructed by replacing gD sequences of HSV-1 with Escherichia coli /3-galactosidase gene (17). The defective virus FgD@, when grown in a cell line (VD60) expressing gD-1 protein, would produce infectious FgD/3 virus. Cells infected with FgD@ virus synthesize HSV-specific pro- teins except gD-1 protein (17).

COS cells were infected with either K082 or FgD@ viruses, and the infected cells were examined by immunofluorescence for expression of HSV-l-specific proteins as well as for gB or gD proteins. In agreement with the published results (17,40), we also found that cells infected with K082 virus showed no expression of gB protein at the cell surface although all of the cells showed strong immunofluorescence when reacted with anti-HSV-1 antibody or anti-gD antibody. Similarly, COS cells infected with the FgD/3 virus did not show the synthesis any gD-1 protein, although all of the cells were expressing gB-1 protein (data not shown). The expression of gB or gD proteins from COS cells transfected with p9gB or p9gD plas- mids after infection with the mutant K082 and FgBP virus is shown in Fig. 6, u-h. Infection of p9gB-transfected COS cells with gB mutant K082 virus resulted in expression of gD protein in all the cells (panel b), but only isolated cells showed fluorescent staining when treated with anti-gB antibody

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FIG. 1. Immunofluorescent stain- ing of polykaryons induced by acid pH treatment of COS cells trans- fected with p9gB and p9gD. Panels a-t1 and Ph show results with COS cells t ranstected with P9gH and p9gD. respec- tively. I’nncis (I, h. c, and /‘. without PH shock: and c. d, g, and II, with pH shock. I’nrre/s n. c. c, and ,g show Phase-contrast Photographs: b. f, d, and h show immu- nofluorescent staining Lvith anti-HSV-1 antiserum. Magnification. X 1000.

a

FIG. 5. Inhibition of low pa-induced gB and gD glycopro- tein-mediated cell fusion by infection with HSV-1. COS cells were transfected with p9gR (paneI.~ n, h), p9gD fpnnel~ L, d), and the plasmid cector p91OX1 (pcrneLs e, f), respectively. The transfected cells were then infected with KOS stram of HSV-1 vu-us at a m.0.t. of 10. I’nrarls a. c, E, and b, d, fshow COS cells untnf’ected and infected with HSV-1 KOS virus, respectiveI>. Magnif’tcation, X ?A).

(panel a). Similarly, COS cells transfected with p9gD and infected with gD-defective FgD@ virus showed expression of gD only in isolated cells (panel f), whereas the entire mono- layer showed expression of gB glycoprotein (panel e). Results presented in Fig. 6, i-l, show that the acid-induced polykaryon formation by COS cells transfected with p9gB (panel i) or p9gD (panel h) was blocked on infection with HSV-1 mutant

TABLE I

containing no insert or with expression vector containing HSV p!?-1 or gD-1 glycoprotein genes. Cells were exposed to medium containing pH 5.5 buffer at 40 h posttransf’ection as described under “Materials and Methods.” Transf’ected cells were also infected with HSV-1 (KOS strain) at an m.o.i. of 10, and at 18 h Postinfection the cells were esposed to pH .5.7 buffer-containin, u medium. Polykaryons formed were counted as described under “Materials and Methods.” The values show the mean number of’ Polykaryons 2 standard deviation. The results were obtained from two independent sets of’ experiments, and triplicate Plates were used for each set. Only polykaryons containing more than five nuclei were counted.

p9012I 1.5 * 1.0 2.2 f 1.3 1.3 + 1.6 P9XR 4.2 * 1.3 107.8 f 8.9 2.3 * 1.6 p9pD 4.7 + 1.6 -x.2 c 5.4 3.2 5 1.3

viruses K082 (panel j) or FgDp (panel 1), respectively. Taken together, these results suggest that the inhibition of acid- induced cell fusion by gB or gD glycoprotein by infection with HSV-1 could not be due to direct inhibition of glycoprotein synthesis or expression of the glycoprotein on cell surface. Thus, the cell fusion induced by gB and gD glycoproteins may be controlled by some gene products of HSV-1.

It was suggested that the glycoprotein gC-1 might act as a negative regulator of cell fusion and syncytia formation (9). We investigated the effect of co-transfection of gB, gC, and gD proteins on acid-induced cell fusion. When COS cells were co-transfected with gB- and gD-containing plasmids, no in- crease in cell fusion at acidic pH was noted (Fig. 7). Co- transfection of cells with gB- and gC-, or gD- and gc-contain- ing plasmids did not show any inhibition of acid-induced cell fusion.

DISCUSSION

The resu1t.s presented in this paper show that HSV-1 gly- coproteins gB and gD, when expressed in mammalian cells in the absence of any other HSV-1 gene products, can induce cell fusion under acidic conditions. Electron microscopic stud- ies have shown that HSV could enter cells either by a phag- ocytic process (18) or by direct fusion with plasma membrane (19). Recent electron microscopic analysis of cells exposed to infectious or neutralized HSV-1 also showed that it entered by direct fusion at the plasma membrane (13). Agents such

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FIG. 6. Inhibition of gB and gD glycoprotein-induced cell fusion by infection with HSV-1 mutants de- fective in gB and gD expression. Panels a-h, expression of viral proteins by transfected cells infected with mutant viruses. COS cells were transfected with p9gB plasmid and infected with K082 virus (panels a-d) or with p9gD plasmid and infected with FgDB virus (panels e- h), respectively. Panels (I and e, stained with anti-gB antibody; panels b and f, stained with anti-gD antibody. Panels c, d, g, and h show phase-contrast photo- micrographs corresponding to panels a, b, e, and f, respectively. Magnification, x 750. Punels i-I, inhibition of low pH- induced cell fusion. COS cells were transfected with p9gB (panels i, j) and p9gD (panels k, 1) plasmids, respectively. Panels j and 1 show transfected cells in- fected with K082 and FgDP viruses, re- spectively. Magnification, x 330.

Cell Fusion Induced by HS V Glycoprotein

FIG. I. Acid-induced cell fusion after co-transfection of COS cells with pSgB, pSgD, and p9gC plas- mids. Panels a, e, cells co-transfected with p9gB and p9gD; panels b, f, cells co- transfected with p9gB and p9gC; panels c, g, cells co-transfected with p9gD and p9gC; panels d, h, cells transfected with p9gC plasmids, respectively. Panels u-d show cells treated with fusion at pH 7.5; panels e-h show cells exposed to acid pH (pH 5.7) fusion medium. Magnification, x 250.

as NH&l, which raised the pH of the endosomes or lysosomes, had no effect on HSV-1 replication, suggesting that HSV infection may not occur via endocytosis (33). In contrast, it was shown that HSV-1 can enter by endocytosis into BJ cells constitutively expressing gD glycoprotein (34). However, in this case, the infection was abortive, and the virus was be- lieved to be degraded inside the cell. That herpes viruses can infect cells by endocytosis was clearly shown in the case of EBV infecting normal B lymphocytes (20). As with other virus systems using the endocytic pathway, agents like NH&l and chloroquine inhibited viral deenvelopment and intracel- lular fusion and thus blocked virus infection. The entry of EBV into B lymphocytes therefore required an acid pH- induced fusion step. In contrast, if transformed Raji lympho- blastoid cells were used as host, EBV entered by direct fusion with the plasma membrane (20). That the same virus could

use either pathway of entry into cells was also observed in the case of WV, which normally enter the cell by endocytosis and involve low pH-induced cell fusion (6,21), but ts mutants of VSV defective in the matrix protein M could induce fusion at neutral pH at the nonpermissive temperature (35).

In the case of COS cells, immunofluorescence shows that only about 5-10s of the cells express gB and gD glycoproteins at the cell surface. The presence of acidic pH must thus bring about some change in the conformation of the glycoproteins which results in fusion of the surrounding cells to form polykaryons. Immunofluorescence of the fused nuclei shows the presence of gB or gD glycoproteins along the profile of the polykaryon and the perinuclear region. It appears that after exposure to low pH, cells surrounding the cell expressing gB or gD glycoprotein are fused together, and the glycopro- teins synthesized are associated with the fused polykaryons

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(Fig. 4). It may also be pointed out that the observed low pH- induced fusion is dependent on the incorporation of the glycoproteins in the cellular membranes, as a mutant gB glycoprotein (tgB) that lacked the membrane anchor domain failed to induce any cell fusion. However, cells expressing mutant gB glycoproteins containing shortened cytoplasmic tail or lacking part of the hydrophobic sequence of 69 amino acids near the carboxyl terminus underwent fusion when exposed to low pH medium,2 suggesting that the extracyto- plasmic domain of gB may be involved in the fusion activity. It was shown recently that mutations within the extracyto- plasmic region of gB decreased its fusogenic activity (14).

The existence of syn mutants that allow extensive fusion of HSV-l-infected cells at neutral pH suggests the involve- ment of HSV-1 genes other than the three glycoproteins gB, gD, and gH in the process of cell-cell fusion (3, 8). The syn gene had been cloned and sequenced (31,32), but the predicted syn gene product glycoprotein has not yet been detected or identified. Defect in the syn gene usually resulted in increased cell fusion, and thus this syn gene product could be a negative effector of cell fusion. To determine if gene products of HSV- 1 could block cell fusion induced by gB or gD glycoproteins, we infected COS cells expressing gB or gD with HSV-1. The results show that infection with HSV-1 blocked cell fusions induced by both of the glycoproteins. It seems that cells expressing gB or gD glycoproteins could no longer recruit for fusion the surrounding cells infected with HSV-1. That the inhibition of acid-induced cell fusion by gB or gD glycoprotein by infection with HSV-1 was not due to direct inhibition of gB or gD synthesis by the transfected cells or expression of the glycoproteins on the cell surface was shown by the ob- served inhibition of cell fusion by HSV-1 mutants lacking gB or gD glycoprotein genes. The sequence of HSV-1 gene has now been determined (4I), and 70 virus-coded protein se- quences are predicted. Not all of the virus-coded proteins have yet been identified, and the biological roles of only a number of these proteins are currently known.

Recently, spontaneous pH-independent fusion of a baby hamster kidney cell line constitutively expressing gD glyco- protein was reported (32). The fusogenic effect was enhanced by exposure to polyethylene glycol. However, only cell lines expressing low amounts of gD glycoprotein showed fusogenic activity. It was also reported that baby hamster kidney cell lines constitutively expressing gB glycoprotein showed mark- edly decreased cell fusion, and exposure to polyethylene glycol did not enhance the fusogenic activity. The gD- and gB- expressing baby hamster kidney cell lines did not show any cell fusion by exposure to acid pH (32). In contrast, our studies show that COS cells transiently expressing gB and gD glyco- proteins can be induced to fuse by exposure to low pH. The observed discrepancy could be due to (a) the different cell lines used; (b) the amount of the glycoprotein expressed in the cells; and (c) the presence of surrounding cells not ex- pressing gB or gD glycoproteins. In fact, the route of entry of EBV into the host was shown to depend on the nature of the cell line used (20). Also, baby hamster kidney cell lines ex- pressing higher amounts of gD failed to fuse; only cell lines expressing lower amounts of gD can fuse (32). It was reported previously that increased cell fusion occurred when cells were infected at a lower m.o.i. than at higher m.o.i. (37) or when infected cells were mixed with noninfected cells (38). The observed interference of HSV infection by cells expressing gD glycoprotein (32, 39) was proposed to be due to saturation of a gD-recognizing cellular membrane component essential for cell-virus or cell-cell fusion by the expressed glycoprotein. In

* K. Raviprakash, L. Rasile, and H. P. Ghosh, unpublished results.

the case of COS cells expressing gB or gD glycoprotein tran- siently, the neighboring cells not expressing the viral glyco- protein would allow necessary interactions between gB or gD glycoproteins and the cell surface components essential for virus-cell or cell-cell fusion. The fusogenic activity of gB or gD glycoprotein may normally be mediated at neutral pH by interaction with other HSV proteins, and this process may be controlled by the syn gene products. In the absence of any viral proteins, the acidic pH may bring changes in the confor- mation of the glycoproteins, which can then interact with the uninfected cell membranes to induce cell-cell fusion.

Acknowledgments-We thank Dr. Roger Watson for HSV-1 gD clone; Dr. Randy Kaufman for p91023 expression vector; Dr. Jack Rose for pSVGL; Dr. David Johnson for the gD mutant (FgDP) HSV virus, the anti-gD monoclonal antibodies, and the recombinant ade- novirus containing gB-1 gene; Drs. Joseph Glorioso and Myron Le- vine for the gB mutant (K082) HSV virus and D6 cell line; Dr. Martin Zweig for monoclonal antibodies to gB, gC, and gD proteins: and Bonnie Murphy for typing the manuscript.

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M Butcher, K Raviprakash and H P GhoshAcid pH-induced fusion of cells by herpes simplex virus glycoproteins gB an gD.

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