Insensitivity of a Ricin-Resistant Mutant of Chinese Hamster Ovary Cells to Fusion Induced by Newcastle Disease Virus

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1 JOURNAL OF VIROLOGY, Apr. 1979, p X/79/4-69/7$2./ Vol. 3, No. 1 Insensitivity of a Ricin-Resistant Mutant of Chinese Hamster Ovary Cells to Fusion Induced by Newcastle Disease Virus PETER G. POLOS AND WILLIAM R. GALLAHER* Department ofmicrobiology, Louisiana State University Medical Center, New Orleans, Louisiana 7112 Received for publication 4 October 1978 The role of membrane components in the interaction of cells with Newcastle disease virus (NDV) was studied using a ricin-resistant mutant of Chinese hamster ovary cells (CHO-15B), in which there is a deficiency in distal saccharides at the plasma membrane. Compared to the parental wild type, the mutant was shown to be 4- to 1-fold less sensitive to either fusion from without or fusion from within induced by NDV. The mutant and wild type were nearly indistinguishable with respect to other interactions with NDV. Viral attachment was investigated with 125I-labeled NDV and found to be comparable in both lines. Functionally equivalent amounts of hemagglutinin were produced, as measured by the fraction of cells positive for hemadsorption, or by the number of erythrocytes adsorbed per cell. No significant differences in the morphogenesis or yield of progeny virus were seen. The ability of the mutant to produce a fusion factor was measured by transfer of infected cells to uninfected monolayers. Infected CHO-15B cells were capable of inducing fusion normally in the indicator wild-type monolayers, but were incapable of inducing fusion in mutant monolayers. These results suggest that the insensitivity of the CHO-15B mutants to fusion may be due to inhibition of an early virus-cell interaction subsequent to viral attachment, whereas other events in infection appear to be unaffected by the cell surface mutation. Glycoproteins play an important role in several stages of viral infection. The surface protreated cells (3, 25, 32), indicating that some as well to neuraminidase-treated cells as to unteins of enveloped viruses are glycosylated, and variability in receptor structure is tolerable for have been implicated as the causative agents of the initiation of infection as well. cytopathology in several viral systems. In the One method of further defining the requirements for cell fusion and other virus-cell inter- case of paramyxoviruses such as Newcastle disease virus (NDV) hemagglutination and neuraminidase activity, and the ability to induce cell cells in which cell surface oligosaccharides are actions is by studying mutants of mammalian fusion, have been definitively associated with altered. We have examined the induction of two glycoproteins designated HN and F, respectively (27). The production of these glycopro- hamster ovary cells. This cell mutant (desig- fusion by NDV in a mutant line of Chinese teins in functional form late in infection requires nated CHO-15B) had been selected for resistance to the toxicity of the lectin ricin (12). The glycosylation of the polypeptide chains, as demonstrated with inhibitors such as 2-deoxy-D-glucose and tunicamycin (1, 14, 18, 23, 29). Studies acetylglucosaminyl transferase activity (11) nec- resistance was due to a deficiency in the N- with other unrelated enveloped viruses on hostinduced modifications of surface oligosaccharide carbohydrate side chains of glycoproteins which essary for addition of distal saccharides to the sequences seem to indicate that some degree of correspond to the binding sites for ricin. variability in glycosylation is tolerable for complete expression of viral functions (7, 15, 26). are insensitive to fusion induced by NDV at a In this report we show that the mutant cells However, the type and extent of this variability step subsequent to viral attachment. We also and tolerance have never been defined. show that the mutant is capable of supporting It has also long been known that the principal productive infection by NDV and is unaffected cellular receptor for paramyxoviruses is a glycosylated protein containing terminal N-acetyl faces and fusion factor late in infection. in its ability to produce hemadsorbing cell sur- neuraminic acid (NANA) (2). The induction of cell fusion at high multiplicities is inhibited by MATERIALS AND METHODS pretreatment of cells with neuraminidase (17). Cell culture. Parental Chinese hamster ovary cells, However, certain strains of NDV attach almost clone K (hereafter designated CHO-wt), and mutant 69 Downloaded from on January 5, 219 by guest

2 7 POLOS AND GALLAHER cells (CHO-15B) derived from them, were obtained through the courtesy of Kenneth Roozen of the University of Alabama, Birmingham. Cell stocks were maintained as monolayer cultures in 75-cm2 tissue culture flasks (Corning) in medium 199 supplemented with 1% fetal calf serum and 5% tryptose phosphate broth (Difco Laboratories). Cells were subcultured every 4 to 7 days and plated at a density of 1.5 x 16 cells per flask. The mutant cells were confirmed as phenotypically stable by demonstrating differences from the wild type in the dose response of agglutination (13) by lectins, ricin II, and concanavalin A (Sigma Chemical Co.), consistent with the results of Gottlieb et al. (12). Both cell lines were periodically monitored for, and found to be free of, viral and mycoplasmal contamination by transmission electron microscopy. For fusion experiments, cells were plated at a density of 1.5 x 16 to 2. x 16 cells per 21-cm2 dish, and for assays of viral adsorption, at a density of 8. x 15 to 9. x 15 cells per 8-cm2 dish. Secondary cultures of chicken embryo fibroblasts used for viral plaque assays were derived from embryonated eggs supplied by Pan American Hatcheries (Hammond, La.) by methods described previously (2). All media and biochemicals were purchased from Grand Island Biological Co., Grand Island, N.Y., unless otherwise indicated. All cultures were incubated at 37 C in a 5% CO2 atmosphere. Virus strains, purification, and fractionation. Partially purified stocks of NDV strains AV (Australia-Victoria, 1932) and IM (Italy-Milano, 1945) were obtained as described previously (2, 5). For preparation of iodinated virus, stocks were further purified by rate zonal centrifugation as described by Clavell and Bratt (5). Hemagglutination microtitrations (HAU) and plaque assays were performed as described previously (2, 5). Quantitation of fusion, hemadsorption, and virus yield. The techniques of Gallaher and Bratt (8, 9) were used to assay fusion from without, which is induced by strain IM immediately after infection, and fusion from within, which is induced by strain AV later as a result of productive infection. Quantitation of cell fusion was based on the equation: fusion events per 1, cells = (number of nuclei - final cell number/ number of nuclei) x 1,. Analysis of a model for cell populations undergoing fusion, to be presented elsewhere, shows this to be a more linear measure of actual fusion events than the previously described method. Assays for fusion factor by transfer of infected cells onto indicator monolayers of sensitive CHO-wt cells or insensitive CHO-15B cells were performed as described by Gallaher and Bratt (9), including the use of cycloheximide (1,ug/ml) to prevent the synthesis of fusion factor after transfer. For quantitative measurement of hemadsorption, monolayers to which chicken erythrocytes were adsorbed, as previously described (1), were prefixed with 1% glutaraldehyde before fixation and dehydration with 95% methanol. Hemadsorption was quantitated by determining the percentage of cells which adsorbed 2 or more erythrocytes in a population of 2 cells, as well as by enumerating the erythrocytes bound to 12 to 15 individual hemadsorbing cells. The standard deviation (a) from the mean was determined J. VIROL. for both methods of quantitation. The yield of virus released from infected cells was measured as previously described (9). Experiments were separately conducted at the different ph optima previously determined for fusion, hemadsorption, and virus yield, respectively (9). lodination of virus. Infectious IM was purified by rate zonal centrifugation as described above and iodinated by a modification of the method of Morrison (21). Virus (2, HAU/ml) was suspended in 1. ml of phosphate-buffered saline (PBS-A) (6), containing 1' M KI. To this was added, in sequence, 5,ul of PBS-A containing.12 mg of lactoperoxidase (Sigma), 5 Ad of Na2S (.5 M), and 5. pi of PBS-A containing.5 mci of ['25I]Na (Amersham/Searle, Arlington Heights, Ill.). At 2-min intervals, from 1 to 15 min, 15 pl of PBS-A containing 1.3 x 1-6 M H22 was added. Iodination was terminated by dilution with 4 volumes of PBS-A containing 1o-4 M KI. Iodinated virus was separated from excess label by centrifugation through a 2% sucrose barrier at 12, x g, followed by resuspension of the virus pellet in PBS-A. No loss of hemagglutinating activity occurred as a result of iodination, and little disruption of viral particles was noted upon analysis by rate zonal centrifugation. Attachment of iodinated NDV. To measure attachment of iodinated virus, the medium was removed from CHO monolayers, the monolayer was washed with PBS-A cooled to 4 C, and the cells were precooled to 4C for 15 min. Dilutions of virus were added in.1 ml of chilled PBS-A (ph 7.4). Virus was allowed to adsorb at 4 C for the time indicated in each experiment. The monolayers were then washed twice with ice-cold PBS containing Mg2e and Ca2' (6), scraped off the plates with a latex rubber policeman, suspended in 1. ml of PBS, and counted in a Biogamma spectrometer. The specificity of attachment was measured by competition of attachment with unlabeled virus. Labeled virus (.5 ml) at 2 HAU/ml and unlabeled virus (.5 ml) at 4 HAU/ml were mixed and added to precooled monolayers as described above. At the time indicated, the monolayers were washed, collected, and counted as described above. RESULTS Insensitivity of CHO-15B cells to NDVinduced cell fusion. The relative sensitivity of CHO-wt and CHO-15B cells to fusion from without induced by strain IM is shown in Fig. 1. Substantial fusion was induced in the CHO-wt cultures, with the maximum at 1, HAU/ml corresponding to an average of 2.1 nuclei per ceil. In contrast, CHO-15B cells were four- to tenfold more resistant to fusion induced by NDV, depending on the concentration of virus. CHO-15B cells were also resistant to fusion from within, induced by strain AV. Figure 2 shows the time course of fusion induced in CHOwt cells, which was comparable to that previously described for other sensitive cell types (3, 4). The fusion of CHO-15B cells was not significantly above background 12 h after infection. In Downloaded from on January 5, 219 by guest

3 VOL. 3, 1979 INSENSITIVITY OF CHO MUTANT TO FUSION 71 V) -J -J c D cl U') >- z w cn z CA, UA _ 1 12 HAU/ml FIG. 1. Fusion from without. Monolayers of CHOwt and CHO-15B cells were precooled, after which.2 ml of NDV-IM was added. After adsorption at 4 C for 1 h, PBS (37 C) was added to each culture, and cultures were incubated at 37 C, after which they were washed, fixed and stained. Symbols:, CHOwt;, CHO-15B. U) -j -L w.) z z C') IL L F- _ i -I-_ TIME AFTER INFECTION (hrs) FIG. 2. Fusion from within. Monolayers of CHOwt and CHO-15B were infected with NDV-AV at a multiplicity of infection of 1. PFU/cell at 37 C. At the indicated time points, monolayers were washed, fixed, and stained. Symbols: *, CHO-wt;, CHO- 15B. other experiments (not shown), this contrast in the induction of fusion from within persisted at 24 h after infection, after which infected cultures of both cell clones deteriorated by cell rounding and detachment from the plate. Incubation of infected cells in medium containing.1 to 1,ug of trypsin per ml, which has been shown to activate the induction of fusion by NDV in other insensitive cell types (22, 27), had no effect on the sensitivity of CHO-15B cells. Attachment ofndv to CHO-wt and CHO- 15B cells. The results above could be readily explained by differences in the efficiency of infection between the wild-type and mutant cells. Preliminary experiments (not shown) indicated that both cell clones removed 6 to 8% of the hemagglutinating activity of purified virus from the medium, at viral concentrations of 2 to 2, HAU/ml. To quantitate viral attachment directly, the adsorption of "2I-labeled NDV was measured. Labeled virus was incubated with monolayers of CHO-wt or CHO-15B cells in the presence or absence of additional homologous interfering virus. At an input multiplicity of 1 PFU/cell (1, cpm/culture), CHO-wt cells bound 55% and CHO-15B cells 77%, respectively, of the input virus (Table 1). The mutant therefore actually bound 46% more virus than did the wild type. Homologous "cold" virus decreased by 5% the attachment of labeled virus to each cell type, indicating the same specificity of attachment for both lines. Nonspecific binding of virus to plastic was not significant. Figure 3 shows that attachment of "251-labeled NDV was linear with viral concentration over the range of 1. to 1 PFU/cell for both cell types. The slightly greater binding of virus to the mutant cells was significant and was reproducible over the entire range of multiplicities of infection shown. Efficiency of infection, hemadsorption, and virus yield. We also assessed the efficiency of plating of strain AV on CHO-wt and CHO- 15B cells by infecting cells at a multiplicity of TABLE 1. Attachment of ini-labeled NDV-IM to CHO celw' 25I.iabeiled Cell t ypendv-im Interfering bound NDV-IM (cpm x % type Wild 1-3) CHO-wt None 55 ± HAU 26 ± CHO-15B None 77 ± HAU 34 ± No cells None 6 ±.4 a NDV-IM was labeled with '25I to 1. x 14 cpm/ HAU and adsorbed to monolayers of CHO-wt and CHO-15B cells at 4 C for 45 min at a multiplicity of 1 PFU/cell, or 1. x 1' cpm per monolayer. After being washed with PBS at 4 C, cells were scraped into vials for determination of radioactivity in a Biogamma spectrometer. Downloaded from on January 5, 219 by guest

4 72 POLOS AND GALLAHER approximately 1. PFU/cell and determining the proportion of cells in cultures of each type which showed positive hemadsorption at 13 h after infection. Table 2 shows that 52.1 and 49.6% of the CHO-wt and CHO-15B cells, respectively, absorbed two or more erythrocytes, which is in reasonable accord with the 62.5% expected from the Poisson distribution at this multiplicity of infection. Therefore, there was no significant difference in the efficiency of plating virus on the mutant cells. There was reproducibly a slightly higher background of hemadsorption with the mutant cell controls than with the wild type, i.e., 1.2 erythrocytes adsorbed to 1% of the cells. But this background was 5-fold lower than the hemadsorption with infected cells and did not affect the significance of the results. Table 2 also shows that the mutant cells were unaffected in their capacity to produce hemad- 2 U) -j -j ~ 5) '...I VIRUS CONCENTRATION (PFU/CELL) FIG. 3. Adsorption of labeled virus. Monolayers of CHO-wt and CHO-15B cells were washed with cold PBS. '25I-labeled NDV-IM was adsorbed for 1 h. The monolayers were then washed, and their radioactivity was counted. Symbols:, CHO-wt;, CHO-15B. TABLE 2. Cell type Hemadsorption to cells infected with NDV-A Va Hemadsorption p Erythrocytes/cell positive CHO-wt 52.1 ±.53b 11.9 ±.6 CHO-15B 49.6 ± ±.6 a Monolayer cultures of each cell type were infected at a multiplicity of infection of 1. PFU/cell and incubated at 41 C for 13 h. The infected monolayers were then chilled, and hemadsorption was assayed. Hemadsorption to control cells, or to infected cells before 6 h postinfection, never exceeded.8 erythrocytes bound to 1.2% of CHO-wt cells, and 1.2 erythrocytes bound to 1.7% of CHO-15B cells. b Each value indicates mean of at least 12 determinations ± standard deviation. sorbing cell surfaces late in infection, since the average number of erythrocytes adsorbed per infected CHO-15B cell did not differ significantly from the number adsorbed to CHO-wt cells. The relative capacity of each cell clone to produce progeny virus is shown in Fig. 4. The yield of virus was comparable for both CHO-wt and CHO-15B cells, and similar to that previously determined for infection of chicken embryo cells by NDV-AV under similar conditions (9). Upon observation by electron microscopy, the budding of viral particles and the appearance of inclusions were morphologically similar in both wild-type and mutant cells (not shown). Synthesis of fusion factor in infected cells. The apparent resistance of the CHO-15B cells to fusion from within could be due either to an insensitivity to the fusion factor or to failure to produce fusion factor late in infection. To distinguish between these two possibilities, we transferred infected CHO-15B cells onto indicator monolayers of CHO-wt cells. The extent of fusion induced in the sensitive monolayer cultures of CHO-wt cells was equivalent for wildtype or mutant infected cells (Table 3). When infected CHO-wt cells were plated onto CHO- 15B cells as indicators, the fusion induced was fourfold lower than that when CHO-wt cells were used as indicator cells. When infected CHO-15B cells were plated onto homologous CHO-15B cells, no fusion at all was observed. These data indicate that CHO-15B cells are capable of producing fusion factor, but are relatively insensitive to the active factor produced by either wild-type or mutant cells. ia- I.X TM) AFTE UWCTION (h,) J. VIROL. FIG. 4. Virus production. Monolayers of CHO-wt and CHO-15B cells were infected with NDV-AV at a multiplicity of infection of 1 PFU/cell at 37 C. At the times after infection indicated, a portion of the medium was taken and assayed for released virus by plaque formation on chicken embryo cells. Symbols: *, CHO-wt;, CHO-15B. Downloaded from on January 5, 219 by guest

5 VOL. 3, 1979 TABLE 3. Homologous and heterologous transfer of cell-associated fusion factor' Cell type infected ~Fusion with with NDV-AV Indicator cell type events/1,ooo INSENSITIVITY OF CHO MUTANT TO FUSION ~~~~~~~~cells CHO-wt CHO-wt 384 CHO-15B CHO-wt 372 CHO-wt CHO-15B 95 CHO-15B CHO-15B 5 Monolayer cultures of each cell type were infected with NDV-AV at a multiplicity of infection of 5 PFU/ cell and incubated at 41C for 6 h. The infected cells were then removed from the plates, centrifuged, suspended in minimal essential medium and added to indicator monolayers at a concentration of 1. x 17 cells/ml in the presence of cycloheximide. The indicator monolayers were fixed and stained 4.5 h after the addition of infected cells. DISCUSSION We have shown that a ricin-resistant mutant of Chinese hamster ovary cells is also resistant to both fusion from without and fusion from within induced by NDV. Consistent with these findings was the failure of infected wild-type cells, which are capable of synthesizing fusion factor, to induce fusion when plated on monolayers of mutant cells. The observed resistance was not due to a defect in viral attachment. In fact, attachment of iodinated virus was slightly greater to mutant than to wild-type cells, indicating that receptors in the mutant cells were functionally intact. This is in accord with the fact that the quantity of NANA, a principal component of the receptor for paramyxoviruses, was found to be 72% of that present in the wild type (12). Nor was the resistance due to failure of NDV to infect mutant cells after attachment, since the efficiency of viral plating was equivalent to that found with the wild-type cells. Results of preliminary experiments to be reported subsequently also indicate that CHO-15B cells are resistant to lysis induced by NDV, as measured by release of Na5"CrO4. Our findings on the late events of infection by NDV have shown that, in the mutant cells, each of the membrane functions mediated by viral envelope glycoproteins was unaffected. The production of hemadsorbing cell surfaces was quantitatively equivalent with both mutant and wildtype cells. Likewise, production of fusion factor by infected mutant cells, as measured by the transfer of such cells onto indicator monolayers of fusion-sensitive cells, was also equivalent to that of wild-type cells. The yield of progeny virus from mutant cells was comparable to that obtained from wild-type cells or from chicken embryo cells. We also have not observed any morphological differences between the two cell types in the maturation of viral particles. In other cell systems, insensitivity to paramyxovirus-induced fusion was due to the inability of cells to activate the F glycoprotein by proteolytic cleavage of its precursor, Fo (22, 27). Several results indicate that this mechanism is not likely to play a significant role in the insensitivity of CHO-15B cells to fusion. The mutant cells are insensitive to fusion from without, induced by biologically active NDV grown in eggs, and to fusion induced by infected CHO-wt cells which are capable of inducing fusion from within. Infected mutant cells remain insensitive to fusion from within when incubated in the presence of trypsin at concentrations which activate fusion of other cell types (22, 27). The results of the transferring of infected cells onto indicator monolayers of CHO-wt or CHO-15B cells are also the opposite of those reported for MDBK cells (3), which are defective in cleavage of Fo (27) and incapable of inducing fusion in sensitive indicator cells. Such results are consistent with the previous finding that the CHO-15B mutant did not differ from the wild type in its interactions with either Sindbis or vesicular stomatitis viruses (28). Therefore, there is as yet no indication that the alterations in glycosylation which result from the mutation affect significantly the synthesis or activity of viral glycoproteins. The foregoing results with mutant CHO-15B cells are similar to those obtained by Toyama et al. (3), who studied a mutant of KB cells, designated KB-sil, selected by resistance to lysis induced by Sendai virus. As with NDV and CHO-15B cells, KB-sil cells adsorbed normal amounts of Sendai virus, and were capable of producing fusion factor to the same extent as the parental KB cells, but were resistant to virus-induced fusion (3). In light of these results, we propose that the mechanism of resistance to virus-induced fusion seen in CHO-15B cells may be due to inhibition of an early virus-cell interaction subsequent to viral attachment, such as the rate at which the viral envelope becomes fused with the plasma membrane at the inception of viral penetration. This hypothesis is consistent with the results of Toyama et al. (3) and with reports which show that a configurational change in the envelope of Sendai virus is essential for initiation of fusion between the envelope and the plasma membrane (1, 16). Such a membrane rearrangement may be affected, and cell-to-cell fusion either inhibited or enhanced, by the presence on the cell surface of ligands such as lectins or antibodies (1, 24, 31), or by the absence of certain oligosaccharide sequences at the cell surface, as in the CHO-15B cell system. Altematively, CHO-15B may be deficient in sites other than NANA 73 Downloaded from on January 5, 219 by guest

6 74 POLOS AND GALLAHER which react with viral glycoproteins. Such sites have been postulated to account for the attachment of NDV to neuraminidase-treated cells, and as mediators of the tighter binding of virions to the cell surface correlated with the induction of fusion (3, 25, 32). The identity of the oligosaccharide sequences which may modulate sensitivity to paramyxovirus-induced fusion, and whether these are bound to glycoproteins or glycolipids, have not been determined. However, the availability of mutants such as CHO-15B cells should make such determinations feasible. In addition to these effects of surface mutants on the interactions of cells with paramyxoviruses, mutants of L cells have recently been reported which are resistant to Sindbis virus. One mutant does not bind the virus, whereas a second oversialylates surface glycoproteins apparently resulting in defective mutation of viral particles (S. Schlesinger, R. Gibson, R. Leavitt, C. Gottlieb, and S. Kornfeld, Fed. Proc. 37:1772, 1978). Enhancement of fusion induced by herpes simplex virus has been reported for surface mutants of BHK-21 (K. S. Kirwin, M.S. thesis, University of Pennsylvania, Philadelphia 1977) and KB (3) cells. Also, the fusion factor of herpes simplex virus has been found to require terminal fucose for full activity (19). Together with the results on inhibitors of glycosylation, such as 2-deoxy-D-glucose and tunicamycin (1, 14, 18, 23, 29), these various findings indicate that alterations in glycosylation can affect either the synthesis or the activity of viral glycoproteins, as well as the sensitivity of cells to viral action, in ways specific for each viral glycoprotein. ACKNOWLEDGMENTS We are indebted to Charlene Gottlieb and Kenneth Roozen for providing the CHO cell clones, to Terry W. Fenger for iodination of NDV, and to Sylvia Breland and Kay Redmond for technical assistance. We are also grateful to K. Siena Kirwin and to T. Bachi, H. A. Blough, and S. Toyama for making their unpublished findings available. This study was supported by Public Health Service biomedical research grant RR from the National Institutes of Health, by research grant AI-1945 from the National Institute of Allergy and Infectious Diseases, and by a grant from the Edward G. Schlieder Educational Foundation. LITERATURE CITED 1. Bachi, T., G. Eichenberger, and H. P. Hauri Sendai virus hemolysis: influence of lectins and analysis by immune fluorescence. Virology 85: Bratt, M. A., and W. R. Gallaher Preliminary analysis of the requirements for fusion from within and fusion from without by Newcastle disease virus. Proc. Natl. Acad. Sci. U.S.A. 64: Bratt, M. A., and W. R. Gallaher Biological parameters of fusion from within and fusion from without, p In C. F. Fox (ed.), Membrane research. Academic Press Inc., New York. 4. Bratt, M. A., and H. Rubin Specific interference J. VIROL. among strains of Newcastle disease virus. I. 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