Neuroblastoma Cell Fusion by a Temperature-Sensitive Mutant of Vesicular Stomatitis Virust

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1 JOURNAL OF VIROLOGY, June 1979, p X/79/ /08$02.00/0 Vol. 30, No. 3 Neuroblastoma Cell Fusion by a Temperature-Sensitive Mutant of Vesicular Stomatitis Virust JOSEPH V. HUGHES,`* BRUCE J. DILLE,' ROBERTA L. THIMMIG,I TERRY C. JOHNSON,' STANLEY G. RABINOWITZ,2 AND MAURO C. DAL CANTO3 Division ofbiology, Kansas State University, Manhattan Kansas 66506,1 and Departments ofmedicine2 and Pathology (Neuropathology),3 Northwestern University Medical School and Veterans Administration Lakeside Hospital, Chicago, Illinois Received for publication 28 December 1978 A temperature sensitive mutant of vesicular stomatitis virus which does not mature properly when grown at 390C promoted extensive fusion of murine neuroblastoma cells at this nonpermissive temperature. Polykaryocytes apparently formed as a result of fusion from within the cells that requires low doses of infectious virions for its promotion and is dependent on viral protein synthesis. Although 90% of infected N-18 neuroblastoma cells were fused by 15 h after infection, larger polykaryocytes continued to form, leading to an average of 28 nuclei per polykaryocyte as a result of polykaryocytes fusing to each other. Two neuroblastoma cell lines have been observed to undergo fusion, whereas three other cell lines (BHK-21, CHO, and 3T3) were incapable of forming polykaryocytes, suggesting that nervous system-derived cells are particularly susceptible to vesicular stomatitis virus-induced fusion. Although the normal assembly of the protein components of this virus is deficient at 39 C, the G glycoprotein was inserted into the infected cell membranes at this temperature. Two lines of evidence suggest that the expression of G at the cell surface promotes this polykaryocyte formation: (i) inhibition of glycosylation, which may be involved in the migration of the G protein to the cellular plasma membranes, will inhibit the cell fusion reaction; (ii) addition of antiserum, directed toward the purified G glycoprotein, will also inhibit cell fusion. Although a number of RNA and DNA viruses induce polykaryocyte formation (19, 20, 23), relatively few viral systems have been analyzed for the viral components that are essential for cellular fusion. The two most distinctive types of virus-induced fusion have been termed fusion from without (FFWO) and fusion from within (FFWI) as first described by Bratt and Gallaher (2). Virus-induced FFWO requires neither viral replication nor viral protein synthesis because it can be produced by both infectious and inactivated virions. A large multiplicity of infection (MOI) is necessary for FFWO, and fusion occurs a short time after addition of virus. In contrast to FFWO, virus-induced FFWI is associated with intracellular replication of virus in either a fully productive or restricted infectious process. FFWI is characterized by a requirement for infectious virions and for some virus-directed protein synthesis and by optimum fusion at a low MOI. Interestingly, viral glycoproteins appear to be important to polykaryocyte formation both in FFWO, as with the F protein of Sendai virus t Contribution no j, Kansas Agricultural Experiment Station, Kansas State University, Manhattan, KS (8, 18, 26, 27), and in FFWI, as with the B2 glycoprotein of herpes simplex virus (15, 24). In our laboratories we have been investigating the effects of intracerebral inoculation of mice with vesicular stomatitis virus (VSV) temperature-sensitive (ts) mutants, one of which, ts G31 (III), produces a prolonged clinical disease and marked status spongiosus of the central nervous system (4, 5, 21). This unique neuropathological lesion has been primarily associated with "atypical infectious agents" which cause slow viral diseases such as kuru and Creutzfeldt-Jakob disease of humans and scrapie and transmissible mink encephalopathy of animals. In addition, these agents promote polykaryocyte formation both in vivo (1, 12, 13) and in vitro (7, 11). Since the ability of these infectious agents to promote cellular fusion may be related to the spongioform central nervous system lesions, it is quite interesting that the VSV mutant, ts G31 (III), is also able to induce polykaryocyte formation. This ts mutant has been shown to be a member of complementation group III whose members are characterized by a ts M protein (9). In this initial report we will present data concerning the fusion 883 Downloaded from on April 29, 2018 by guest

2 884 HUGHES ET AL. of neuroblastoma cells by ts G31 (III) which appears to occur by FFWI and is most likely dependent on the expression of the viral G glycoprotein at the cell surface. MATERIALS AND METHODS Viruses. Indiana strain wild-type VSV and ts G31 were each plaque purified and doubly cloned as previously described (21). Stock virus for these experiments was prepared by infecting BHK-21 cells and was purified by density gradient centrifugation (22). Only the B-type virion was used in these experiments. Cell culture lines. Two murine neuroblastoma cell lines were used in these experiments: N-18, obtained from Brian Spooner (Kansas State University) and grown as described elsewhere (9); and N2a, obtained from the American Type Culture Collection (Rockville, Md.) and cultured as previously described (16). The conditions for the growth of BHK-21 cells have been detailed elsewhere (21). 3T3 and Chinese hamster ovary (CHO) cells were propagated in the same medium as the N2a cells. All cell lines were grown at 37 C in a 5% C02-95% air atmosphere in a waterjacketed incubator. Assay for polykaryocyte formation. For most experiments, N-18 cells (106) were seeded onto glass cover slips (22 by 22 mm) in culture dishes (35 mm) with 2 ml of culture medium and incubated 2 to 3 days until confluent. The appropriate virus, usually at a multiplicity of 10 virions per cell, was allowed to adsorb to the cells for 30 min at 25 C; 2 ml of medium was added, and the cells were then incubated at either 31 or 390C. At 5-h intervals after infection, the cells were fixed by placing the cover slips in acetone-methanol (2:1) at -20 C for 10 min. The nuclei were then stained with Mayer hematoxylin stain, and the cells were counterstained with eosin Y (3). Approximately 1,000 nuclei per cover slip were counted, and their distribution in fused and nonfused cells was determined. From this information, two different indexes of fusion were calculated: the percentage of nuclei present in fused cells and the average number of nuclei per fused cell. In some experiments, D-glucosamine (Sigma Chemical Co., St. Louis, Mo.) was added to 1 or 10 mm in the tissue culture medium after the viral infection. Isolation of viral G glycoprotein. The VSV glycoprotein was isolated from sucrose gradient-purified virus by a modification of the procedures outlined by Kelley et al. (10) and Hale et al. (6). Essentially, the viral envelope proteins were solubilized with non-ionic detergent Nonidet P-40 at a concentration of 1%. The nucleocapsids of the virions were then separated from the membrane proteins G and M by pelleting the nucleocapsid through 20% sucrose at 136,000 x g for 90 min. The viral envelope proteins were then collected from the interface of the 20%o sucrose and the top volume. NaCl was added to 0.4 M, and the envelope proteins were separated on a Sephadex G-75 column (56 by 1.1 cm) with phosphate-buffered saline with 0.4 M NaCl to elute the two proteins. The G glycoprotein (molecular weight a 69,000) eluted in the void volume, whereas the M protein (molecular weight M 29,000) eluted later. Analysis by sodium dodecyl J. VIROL. sulfate-polyacrylamide gel electrophoresis has demonstrated that this procedure provides a complete separation of the G and M viral envelope proteins. Preparation of antibody. An antiserum was prepared against the VSV G glycoprotein which was obtained from the concentrated fractions from the Sephadex G-75 column. Complete Freund adjuvant was added to the glycoprotein antigen, and antiserum was raised in New Zealand white rabbits by subcutaneous injection and with booster injections every 2 weeks. The antiserum was purified by the method of McMillen and Consigli (17). RESULTS Cytopathic effect resulting from infection by WT VSV or ts G31. While studying the growth of the WT VSV and a ts mutant virus, ts G31, we observed that infected neuroblastoma cells underwent a variety of cytopathological alterations, including the formation of polykaryocytes. The N-18 neuroblastoma cells attach, spread out on the surface of the culture flask, and extend neurites which can attain lengths of 5 to 10 times that of the cell perikaryon (Fig. 1). Upon infection with WT VSV (MOI of 10), cells became rounded and many appeared to retract or lose their neurites (Fig. 2). At 24 h postinfection, most cells became unattached from the culture vessel surface and floated in the medium. This cytopathological effect was observed at a variety of temperatures at which the WT virus has been grown (31, 37, or 3900). In contrast to WT VSV, infection with ts G31 (MOI of 10) resulted in quite a different cellular response when the cells were incubated at 390C, the nonpermissive temperature for the replication of this virus (9). N-18 cells infected with ts G31 and incubated at 39 C underwent fusion within 15 h (Fig. 3). The fusion initially produced large polykaryocytes, with almost 10 nuclei per polykaryocyte; but upon further incubation many polykaryocytes appeared to fuse with other polykaryocytes. This eventually resulted in polykaryocytes containing as many as 50 to 100 nuclei. Although the actual site of cellular fusion has not been observed, cells often have been observed with long cytoplasmic bridges between polykaryocytes which resemble the neurite extension (Fig. 3). Incubation of ts G31- infected cells at 310C, the permissive temperature for virus replication, did not fuse the cells, but rather they became rounded in a manner similar to the cytopathological effect produced by WT VSV (Fig. 2). Kinetics of cell fusion. In our initial experiments, we examined the effect of the MOI on the formation of polykaryocytes. The best MOI for fusion was 10 infectious virions per cell, which resulted in fusion of 80 to 90% of the cells, Downloaded from on April 29, 2018 by guest

3 VOL. 30, 1979 CELL FUSION BY A VSV MUTANT 885 Downloaded from FIG. 1. Phase-contrast microscopy of uninfected N-18 neuroblastoma cells. These neuroblastoma cells were plated and grown at 37 C for 3 days (x800). whereas either higher or lower doses resulted in significantly less fusion (with approximately 35% of the cells fused at an MOI of either 1 or 100). Using an MOI of 10, we have quantitated the kinetics of cell fusion by determining both the number of cells that uadergo fusion and the average number of nuclei present per polykaryocyte. The kinetics of fusion was linear to 15 h when 90% of the cells were fused; however, by quantitating the average number of nuclei per polykaryocyte, it was clear that fusion continued for at least 40 h (Fig. 4), since the average number of nuclei per polykaryocyte continued to increase from 10 at 15 h to almost 30 at 40 h. Apparently polykaryocytes fused together, resulting in larger polykaryocytes, many of which contained 50 or more nuclei. N-18 cells infected with ts G31 were simultaneously fixed with 2% glutaraldehyde and 2% osmium tetroxide and postfixed with osmium. Thin sections examined by electron microscopy confirmed the multinucleate nature of the polykaryocytes (Fig. 5). Fusion of other cell lines. Five different cell lines have been examined for their ability to undergo polykaryocyte formation after infection with ts G31 at 39 C (Table 1). Only those cell lines derived from the nervous system have proven capable of undergoing fusion. It is not clear at this time why N2a cells, another neuroblastoma cell line, did not promote fusion as readily as the N-18 line, although the neurite extensions of the N2a cells are less elaborate when this line is grown in complete medium. Three other cell lines, including BHK-21 and CHO cells, both commonly used for growing stocks of VSV, did not show any polykaryocyte formation upon infection with ts G31 when incubated at 39 C. All of the cell lines were able to replicate the ts G31 virus at 310C and underwent the normal cytopathic effect characterized by cell rounding at the permissive temperature. Evidence for fusion from within. As described in the introduction, there are two basic types of virus-induced cellular fusion, FFWO and FFWI. The kinetics of fusion (Fig. 4) were consistent with a mechanism involving synthesis on April 29, 2018 by guest

4 886 HUGHES ET AL. of viral components within the cell as the process of initial fusion appeared to proceed in a linear fashion up to 15 h postinfection (whereas FFWO normally occurs quite soon after addition of the virus). In addition, the low viral doses (MOI of 10) that promote cellular fusion were most consistent with FFWI. When the ts G31 virus was exposed to UV light, reducing the infectivity by 5 to 6 logs, the ability of the virus to promote cellular fusion was lost (Table 2). The fusion reaction was also dependent on protein synthesis since the addition of cycloheximide to the infected cells completely abolished the capacity for fusion. The protein synthesis is presumably of viral origin, since the addition of actinomycin D to infected cells did not affect the initial amount of fusion to any great degree (Table 2). Our data are thus consistent with the idea that a viral component, which is responsible for fu- V :;:,*..;,,... J. VIROL. sion, must be synthesized intracellularly and that polykaryocyte formation occurs within a few hours after infection. Importance of the G glycoprotein in the formation of polykaryocytes. A previous study (9) indicated that the assembly of ts G31 is disrupted at the nonpermissive temperature, with most of the M and N proteins remaining in the cytosol rather than associating with the cell membranes, while the G glycoprotein, however, accumulates in the cell membranes. Since we felt that the association of G with the cell membranes may be responsible for the initiation of fusion, we have begun to study the role this protein plays in cellular fusion. Our initial experiments have examined the effect of inhibiting the processing of the G glycoprotein on cell fusion. Since the glycosylation of G is involved in the migration of G to the plasma membranes Downloaded from on April 29, 2018 by guest ANo -i 49;,e) FIG. 2. Phase-contrast microscopy ofn-18 cells infected with WT VSV. N-18 cells weregrown to confluency as in Fig. 1, infected with WT VSVat an MOI of 10, and incubated at 39 C. Most of the cells are rounded and have lost their neurite extensions by 20 h after infection. These same morphological changes are seen with WT VSV-infected cells incubated at 31 C or with N-18 cells infected with ts G31 and incubated at 31 C (x800).

5 VOL. 30, 1979 CELL FUSION BY A VSV MUTANT 887 Downloaded from FIG. 3. N-18 cells infected with ts G31 at 39 C. N-18 cells were infected at an MOI of 10 and incubated at 39 C for 24 h after infection, at which time a number ofpolykaryocytes were forming (x800). (14, 25), we have added glucosamine, an inhibitor of glycosylation, to cells infected with ts G31 to prevent the incorporation of G into the cell surface. Either 1 or 10 mm glucosamine inhibited the fusion reaction initially to about 75% (Fig. 6, compared with the controls in Fig. 4). Even upon further incubation up to 40 h postinfection, the fusion reaction appeared inhibited. Since glucosamine could have inhibited protein synthesis (28), we measured the effects of 1 and 10 mm glucosamine on both cellular and virusdirected translation. Although 10 mm reduced cellular and viral protein synthesis by approximately 50% in N-18 cells, the lower dose of 1 mm inhibited neither host cell nor viral protein synthesis. This study indicates that the process of glycosylation is essential for the promotion of fusion, presumably by its importance for the normal migration of the G protein into the cellular plasma membranes. As a more direct assay of the role of the G glycoprotein at the cell surface, we have examined the effects of antiserum specific for G on fusion. For this experiment, N-18 cells were infected with ts G31 and then incubated at 39 C. At 2 h after infection, the specific serum (diluted 1:10) was added to the infected cells. The N-18 cells fused up to 80 ± 5% (for triplicate samples) when no rabbit serum was added. The addition of antiserum from a rabbit immunized with purified G completely inhibited the formation of polykaryocytes (no fusion was observed). Control serum collected from the rabbit before immunization did not inhibit fusion when added to the incubation medium of the infected cells (82 ± 2% of the N-18 cells were fused by 40 h). DISCUSSION The formation of polykaryocytes by ts G31 appeared to be the result of an intracellular defective maturation and assembly of the viral polypeptides. This FFWI was induced by infectious virions, and the kinetics of cellular fusion coincided with the periods of intensive viral protein synthesis (9). It is interesting to note that on April 29, 2018 by guest

6 888 HUGHES ET AL. 0 'a. I U. S.I Zq U "Z 0 z 20Z 20 EzI 10i. Hours After Infection FIG. 4. Kinetics of polykaryocyte formation induced by ts G31 in N-18 cells. N-18 cells were seeded onto glass cover slips, incubated 3 days until confluent, and then infected with ts G31 at an MOI of 10 and incubated at 39 C. At 5-h intervals after infection, the cover slips were fixed and stained and the amount of polykaryocyte formation was determined as described in the text. Two different indices of cellular fusion are presented: (A) The percentage of nuclei present in fused cells, and (B) the average number of nuclei per fused cell. All determinations are the average of triplicate samples ± the range. the cellular fusion occurs at 390C (the nonpermissive temperature for viral replication) so that the production of infectious virions did not appear to be necessary for polykaryocyte formation. The use of the metabolic inhibitors cycloheximide and actinomycin D has demonstrated that some viral protein metabolism was essential for cellular fusion. In fact, our later experiments have suggested that a single VSV protein, the G glycoprotein, may be responsible for this FFWI. Our previous report on ts G31 had demonstrated that, although the other proteins of the virus (in particular, the M protein) appear incapable of undergoing the normal assembly process at the nonpermissive temperature, the newly synthesized G protein is almost totally localized in the membranes of the cell (9). Inhibition of glycosylation with D-glucosamine, which presumably blocks the normal migration of the G glycoprotein to the plasma membrane (14), also blocked the process of cell fusion (Fig. 6). Although glucosamine may have inhibited fusion by other mechanisms (28), a decrease in protein synthesis was not the reason for decreased polykaryocyte formation. Finally, antiserum directed against G completely abolishes the capacity of the cells to form polykaryocytes. These experiments all indicate that the expression of the VSV G glycoprotein at the cell surface was uniquely involved in the formation of polykaryocytes by VSV. Similar viral glycoproteins have been associated with the cellular fusion induced by other viruses, including the F protein in Sendai virus and B2 in herpes simplex virus (15, 24, 26, 27). Presently, the mechanism by which these viral glycoproteins initiate the process of cell fusion is unclear, as well as whether all these viral glycoproteins act in a similar fashion at the cell surface. Our experiments have also demonstrated that the virus-induced cellular fusion was highly dependent on the cell type infected (Table 1). Only the two neuroblastoma cell lines underwent polykaryocyte formation, with N-18 cells fusing much more extensively than N2a cells. The only morphological difference between these neuroblastoma cell lines was the greater elaboration and length of the neurite outgrowths of N-18 TABLE 1. Fusion induced in various cell lines by ts G31 at 390C0 Cell line % Nuclei in fused cells N ± 5% N2a 10 ± 1.5% CHO 0 BHK T3 0 a The various cell lines, propagated on glass cover slips were infected when confluent with ts G31 at an MOI = 10 and incubated at 390C. Fusion was determined at 15 h after infection and is presented as the percentage of the nuclei that were present in fused ceuls ± standard deviation. Continued incubation (to 40 h) did not result in greater fusion. TABLE 2. Virus Evidence for fusion from within' Addition to cells J. VIROL. % Nuclei in ts G ± 3.0 ts G31 inactivated 0 with UV light ts G31 +Cyclohexi- 0 mide at 100 isg/ml ts G31 +Actinomycin 67.0 ± 2.0 D at 5,ug/ml N-18 cells were infected with ts G31 at an MOI of 10, or the same amount of virus after UV inactivation, and the cells were incubated at 39 C. Cycloheximide or actinomycin D was added directly after the cells were infected, and fusion was assayed at 15 h after infection. The percentage of the nuclei that were present in fused cells ± standard deviation for triplicate cultures is presented. Downloaded from on April 29, 2018 by guest

7 VOL. 30, 1979 CELL FUSION BY A VSV MUTANT 889 Downloaded from FIG. 5. Electron micrograph of N-18 cells infected with ts G31 at 39 C. N-18 cells were infected at an MOI of 10, incubated at 39 C, and then fixed at 24 h after infection for electron microscopic observation (x3,300). 90 *0.9 t) 70 US L.6 CSO S.5 Z A. S. B / Hours After Infction _ z %. 2b cells. The presence of these neurites may be responsible for the initial fusion reaction and the cytoplasmic bridges between fusing cells (Fig. 3). Alternatively, more subtle differences of the cell surface cytoskeleton architecture or membrane biosynthesis may underlie the differential sensitivity of cell lines to cellular fusion. Polykaryocyte formation which was seen with these neuroblastoma cells may be restricted to nervous system-derived cells infected with some ts VSV mutants and may be only a part of the unique cytopathological effect of ts VSV on FIG. 6. Kinetics ofpolykaryocyte formation in the presence ofglucosamine. N-18 cells were infected with ts G31 as indicated in Fig. 4, except glucosamine was added to the incubation media to either 1 mm (0) or 10 mm (0). The infected cells were then incubated at 39 C, and the amount of fusion was quantitated as described in the text. The determinations are the average of duplicate samples + the range. on April 29, 2018 by guest

8 890 HUGHES ET AL. nervous tissue. ts G31-infected N-18 cells, when incubated at the nonpermissive temperature, also undergo a degeneration of mitochondria and develop a number of large intracellular vacuoles similar to the pathological effects seen in the spinal cord of mice injected with the same virus (4, 5, 21). Perhaps the alteration of the cellular membranes that promotes cellular fusion in tissue culture underlies the rnechanisms associated with these ultrastructural changes that occur in vivo in the mouse spinal cord. More importantly, it may be significant that ts G31 infection results in cellular ultrastructure changes and a status spongiosus that are similar to slow viral infections such as kuru and Creutzfeldt-Jakob diseases (4, 5, 21). Perhaps, the mechanisms that lead to the central nervous system spongioform changes and polykaryocyte formation will prove to have a common basis. ACKNOWLEDGMENTS We thank Sevinc Akinc for her technical assistance. This study was supported by Public Health Service grant NS from the National Institutes of Health, the Kenyon Giese Memorial Grant for Cancer (BC-232) from the American Cancer Society, and MRS7319 from the Veterans Administration. S. G. R. is a Clinical Investigator of the Veterans Administration. LITERATURE CITED 1. Beck, E., I. J. Bak, J. F. Christ, D. C. Gajdusek, C. J. Gibbs, Jr., and R. Hassler Experimental kuru in the spider monkey. Brain 98: Bratt, M. A., and W. R. Gallaher Preliminary analysis of the requirements for fusion from within and fusion from without by Newcastle's Disease Virus. Proc. Natl. Acad. Sci. U. S. A. 64: Clark, G., R. E. Coalson, and R. E. Nordquist Staining procedures used by the Biological Stain Commission, p The Williams and Wilkins Company, Baltimore. 4. Dal Canto, M. C., S. G. Rabinowitz, and T. C. Johnson An ultrastructural study of central nervous system disease produced by wild-type and temperaturesensitive mutants of vesicular stomatitis virus. 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