Cellular Adsorption Function of the Sialoglycoprotein of

JOURNAL OF VIROLOGY, Apr. 1975, p. 882-893 Copyright ( 1975 American Society for Microbiology Vol. 15, No. 4 Printed in U.S.A. Cellular Adsorption Function of the Sialoglycoprotein of Vesicular Stomatitis Virus and Its Neuraminic Acid ROBERT H. SCHLOEMER AND ROBERT R. WAGNER* Department of Microbiology, The University of Virginia School of Medicine, Charlottesville, Virginia 2291 Received for publication 9 December 1974 Exposure of vesicular stomatitis (VS) virions to neuraminidase resulted in loss of their ability to agglutinate goose erythrocytes and to attach to L cells concomitant with hydrolysis of sialic acid. These viral adsorptive functions were also destroyed by trypsinization. Sialyl transferase resialylation in vitro of neuraminidase-treated VS virions restored their hemagglutinating and adsorptive functions almost to original levels. Erythrocyte and L cell receptors for attachment of VS virions were blocked by fully sialylated fetuin and by VS viral sialoglycopeptides. Smaller VS viral glycopeptides generated by extensive trypsinization were less effective inhibitors of hemagglutination than were larger glycopeptides; neuraminic acid and neuraminosyl lactose had no capacity to inhibit hemagglutination or adsorption of virus to L cells. These data suggest that cellular receptors for viral adsorption recognize sialoglycopeptides of a certain size. Neuraminidase desialylation did not significantly alter the isoelectric point of VS virions. Cells exposed to DEAE-dextran, trypsin, or neuraminidase showed significantly increased capacity to attach fully sialylated but not desialylated VS virions. Neuraminidase desialylation of L cells, Chinese hamster ovary cells, and Madin-Darby bovine kidney cells resulted in enhanced susceptibility to plaque formation by VS virus. The virion of vesicular stomatitis (VS) virus is composed of a helical ribonucleocapsid surrounded by an envelope with two viral proteins, in addition to lipids and glycolipids which are derived from host cells (15, 19, 27, 28). One of the envelope proteins (G protein) contains carbohydrates and comprises the spikes which protrude from the viral membrane (8, 18, 2, 29). The VS viral glycoprotein may serve as the principal viral attachment site for cell surfaces, since the selective removal of the glycoprotein from virions by proteolytic enzymes results in loss of infectivity (3, 23). We have recently shown that VS virions incubated with neuraminidase exhibit a 99% decrease in infectivity concomitant with selective hydrolysis of sialic acid (23). Resialylation of neuraminidasetreated VS virions results in substantial restoration of their infectivity. This rise in infectivity is 882 accompanied by an increase in the number of neuraminic acid residues covalently attached to the glycoproteins of desialylated virions. Surface sialoglycolipids do not appear to contribute to infectivity of the virion. These data point to the hypothesis that the sialoglycoprotein promotes viral infectivity by enhancing adsorption of virions to host cells. In this report, we present evidence that the sialoglycoprotein of VS virions is the principal attachment site of this virus to cell surfaces and that neuraminic acid is involved in the reactions of VS viral hemagglutination and adsorption to host cells. MATERIALS AND METHODS Chemicals and radiochemicals. Trypsin (3 x crystallized) and soybean trypsin inhibitor were purchased from Worthington Biochemical Corp., Freehold, N.J., and Vibrio cholerae neuraminidase was obtained from Behringwerke, Marburg, West Germany. Sigma Chemical Co., St. Louis, Mo., supplied N-acetyl neuraminic acid (9517i pure) and N-acetyl neuraminosyl lactose. Fetuin, purified by the Spiro method, was obtained from Gibco, Grand Island, N.Y. Ampholine carrier ampholytes were from LKB Products, Bromma, Sweden. [3H]leucine (6 Ci/ mmol) and [3H]tyrosine (44 Ci/mmol) were purchased from Schwarz/Mann, Orangeburg, N.Y. D-[6-3H Iglucosamine hydrochloride (7.3 Ci/mmol) and CMP-[4, 5, 6, 7, 8, 9-'4C]neuraminic acid (221 to 23 mci/mmol) were supplied by New England Nuclear Corp., Boston, Mass.; ["C ]-labeled amino acid hydrolysate (57 mci/matom) was procured from Amersham/Searle, Arlington Heights, Ill. Virus and cell cultures. Cultivation of the Indiana serotype of VS virus has been previously described (23). Cell cultures of BHK-21 cells were infected with plaque-purified VS virus at a multiplicity of -1 PFU/cell, which resulted in yields of only B virions. Downloaded from http://jvi.asm.org/ on October 17, 218 by guest

VOL. 15, 1975 For the production of labeled virus, radioactive isotopes were added along with the viral growth medium after the initial adsorption of virus to cells. Virus that had been released into the medium 18 to 2 h postinfection was purified according to the procedure of Emerson and Wagner (7). Infectivity of B particles was determined by plaque assay on monolayers of L cells and expressed as PFU (26). Estimates of the ratio of physical to infectious particles were made from viral protein determinations. Enzyme treatments. Purified B particles of VS virus were incubated in a reaction mixture which contained, in a final volume of.5 ml, 3 Amol of Tris-hydrochloride buffer (ph 7.) and either 5 Mmol of CaCl2 and trypsin or.5 umol of CaCl2 and V. cholerae neuraminidase (23). After incubation at 37 C for the specified times, the reactions were terminated by centrifugation at 125, x g for 9 min. Separation of enzyme and virus was monitored by assaying for trypsin and neuraminidase activity in the pellet as previously described (23). In some instances, trypsinization of virions was terminated by the addition of soybean trypsin inhibitor (1-fold excess [wt/wt] over trypsin), whereas desialylation was inhibited by 1 mm EDTA (1). Sialylation of VS virions with BHK-cell sialyl transferase was accomplished as described previously (23). Briefly, the standard sialyl transferase reaction mixture contained, in a volume of.2 ml, 2. Mmol of EDTA solution which was adjusted to ph 6.9 with 1 N NaOH,.5 Amol of CMP- [IC ]sialic acid (specific activity, 18 to 2 counts/min per pmol), 5 to 1 Mmol of sodium phosphate buffer (ph 6.9),.1 ml of virus preparation, and 1 to 5 sg of sialyl transferase. The reaction was initiated by the addition of partially purified enzyme, allowed to proceed at 37 C, and terminated by centrifugation through a.5-ml pad of 5% glycerol at 125, x g for 9 min. Activity of the sialyl transferase was monitored by the incorporation of ['4C ]sialic acid into VS virions (23). HA and HI assays. Hemagglutination (HA) and hemagglutination inhibition (HI) titrations were performed according to a modification of the goose erythrocyte technique originally described by Halonen et al. (11). Virions which had been initially grown in the presence of.4% bovine plasma albumin were diluted serially twofold with.4% bovine albumin-borate saline buffer, ph 9. (BABS). HA titrations were performed using.2 ml of virus and an equal volume of a.25% goose erythrocyte suspension buffered in the appropriate phosphate-saline solution (erythrocytes were stored as a 1% suspension in glucose-gelatin-veronal buffer). After the addition of cells, the tubes were placed in an ice slurry (4 C) and incubated for 6 min. The end point was read as the highest dilution of virus which caused complete agglutination of erythrocytes. HA units were recorded as the inverse of the highest dilution that caused complete HA. HI was tested by making twofold serial dilutions of a test compound in BABS. An amount of VS virus equivalent to 2 HA units in.1 ml was added to.1 ml of each dilution of the substance. After 3 min at 4 C, NEURAMINIC ACID IN VS VIRUS ATTACHMENT CO)83.2 ml of a.25% suspension of goose erythrocytes was added to each tube in the appropriate phosphatesaline buffer to achieve a final ph of 6.1. After 1 h of incubation at 4 C, the HA pattern was read; 1 HI unit was recorded as the highest dilution of the test substance which caused complete inhibition of HA. Tests for adsorption. (i) Method 1. Confluent monolayers of L cells in 6-mm plastic dishes (-2 x 16 to 3 x 11 cells) were allowed to equilibrate at either 27 or 4 C for 1 min. The medium was removed by aspiration, and tipped plates were allowed to drain to remove all residual medium. A.2-ml portion of phosphate-buffered saline (PBS) which contained -5 x 11 counts/min of [3H]protein-labeled virus was then added to each plate. After incubation for specified times, the inoculum was removed by aspiration, and the plates were washed three times with PBS. The plates were drained well, and monolayers were removed with a rubber policeman into 3 ml of PBS. The cells were pelleted by centrifugation at 8 x g for 1 min, washed one or more times, and resuspended in.5 ml of PBS. (ii) Method 2. Plates containing monolayers of L cells were drained free of medium, and the cells were washed with PBS. The cells were removed with a rubber policeman and pelleted by centrifugation at 8 x g for 1 min. The cells were then resuspended in PBS (5 x 17 cells/ml) and mixed with purified [3H ]virions which had been diluted in PBS. The cells and virus were kept in suspension with the aid of a magnetic stirring bar, and at specified times.2-ml samples of the virus-cell mixtures were removed and diluted 1-fold with PBS. The cells were pelleted by centrifugation, washed twice, and resuspended in.5 ml of PBS. Cell samples to be assayed for cell-associated virus were precipitated in the presence of 1 Mg of bovine plasma albumin with ice-cold trichloroacetic acid at a final concentration of 2%. After 2 h in the cold, the samples were centrifuged at 8 x g, and the pellet was washed with cold acetone and dissolved in.5 ml of.1 N NaOH by standing at room temperature for at least 3 min. Radioactivity was counted in the presence of 1.5 ml of Nuclear-Chicago solubilizer (1:1) and 1 ml of toluene-based scintillation fluid. Other assays. Protein concentrations were determined according to the procedure of Lowry et al. (17) using crystalline bovine plasma albumin as a standard. Desialylated fetuin was prepared by mild acid hydrolysis or by treatment with neuraminidase (23). Sialic acid was measured by paper chromatography (23) or by the thiobarbituric acid assay of Warren (3). Absorbancies were routinely monitored at 549 and 532 nm, and contributions of lipids absorbing at 532 nm were eliminated as suggested by Warren (3). RESULTS Neuraminic acid and glycoprotein requirement for VS viral HA. The role of neuraminic acid in HA of goose erythrocytes was ascertained from the effects of hydrolysis of sialic acid on VS virions by V. cholerae neuraminidase and by subsequent resialylation of the Downloaded from http://jvi.asm.org/ on October 17, 218 by guest

884 SCHLOEMER AND WAGNER J. VIROL. virions. In addition, the comparative hemagglutinating ability of VS virions subjected to trypsinization, a procedure which results in the selective removal of the glycoprotein (3, 23), was also analyzed. In these experiments, purified virions that had been labeled with [3IH ]glucosamine and ["C ]-labeled amino acids were divided into two equivalent aliquots, one of which was treated with the appropriate 124j enzyme and the other exposed to buffer served as the control. After incubation for 9 min at 37 C, the virus-enzyme mixtures were centrifuged and the resulting pellets were washed, suspended in.4% BABS, and assayed for hemagglutinating activity. Figure 1 reveals that hemagglutinating activ-.~:512- CP 256- E A,27Control Il l 125-64- Neuraminidase 32- I or Trypsin - _ 5.5 5.9 6.3 6.7 7.1 ph FIG. 1. Comparative effects of trypsin and neuraminidase on the activity of VS viral hemagglutinin over a broad ph range. Purified B virions (5 rig) that had been labeled with ["4C]-labeled amino acids and [3Hlglucosanrtine were incubated in.5 ml of a reaction mixture consisting of Tris-hydrochloride (ph 7.) and 1 mm CaCI2 in the presence or absence of.1 mg of trypsin or 5 U of neuraminidase. After incubation at 37 C for 9 min, the supernatant fraction and pellet were partitioned by centrifugation at 125, x g for 9 min. The pellet was washed with 1 mm Tris-hydrochloride (ph 7.), suspended in 2 ml of.4% BABS, and sonicated. Serial twofold dilutions of virions were made using BABS, and HA assays were performed. ity of VS virions could be demonstrated only within a narrow ph range, exhibiting an optimum at ph 6.1. However, when either neuraminic acid or the glycoprotein is enzymatically removed from virions, no HA of goose erythrocytes could be detected. Polyacrylamide gel electrophoresis of the trypsinized virus revealed that only the G proteirf was removed, whereas sialic acid was the only hydrolyzed product of the neuraminidase reaction. Relevant electropherograms and paper chromatograms are shown in a previous report (23). These data indicate that the viral glycoprotein and, more specifically, its neuraminic acid are necessary for hemagglutinating activity. Since earlier studies revealed that infectivity of desialylated virions could be restored to nearly original titers by replacing the sialic acid of the viral glycoprotein (23), we sought to determine if the hemagglutinating activity of neuraminidase-treated virions could likewise be restored. In these experiments untreated virus or virions desialylated with neuraminidase, as described above, were suspended in 1 mm sodium phosphate buffer (ph 6.9) and mixed with the sialyl transferase reaction mixture. As a control, CMP- ["C ]neuraminic acid was omitted from the reaction mixture to prevent sialylation. After incubation for 3 h at 37 C, the virions were pelleted by centrifugation through a.5-ml pad of 5% glycerol. This procedure permitted the separation of virions from an inhibitor of VS viral HA present in the sialyl transferase reaction mixture. Infectivity and HA titers of neuraminidasetreated virions and sialylated virus are shown in Table 1. Removal of neuraminic acid from virions resulted in >99.9% decrease in HA titers; resialylation of these virions restored hemagglutinating activity to at least 5% of untreated virus. Infectivity of resialylated virions was restored to a comparable degree. Under conditions in which 4 molecules of sialic acid were incorporated per virion of native VS virus, HA and infectivity levels were not altered. The fact that both infectivity and HA titers are affected to about the same degree by desialylation and resialylation suggests that neuraminic acid is involved only in the binding of virus to cells and not in any other step of the infective process. Role of neuraminic acid in adsorption. As a complement to the HA studies, an assay for adsorption of VS virus employing monolayers of L cells was devised to determine the function of sialoglycoprotein in host cell-virus interaction. Preliminary experiments indicated that there are approximately 13 viral receptor sites per Downloaded from http://jvi.asm.org/ on October 17, 218 by guest

VOL. 15, 1975 TABLE 1. Effect of sialylation of untreated and desialylated VS-B virions on HA and infectivity NEURAMINIC ACID IN VS VIRUS ATTACHMENT Infectivityc Treatmenta HA, (UIO.2 ml) (PFU/ml) None 1,24 4 x 19 Sialylation alone 1,24 4.5 x 19 Neuraminidase alone <2 2.1 x 17 Neuraminidase, then 512 1.7 x 19 sialylation apurified VS virions that had been labeled with [3HJamino acids were desialylated by neuraminidase (5 U). After incubation for 9 min at 37 C, the virions were pelleted by centrifugation at 125, x g for 9 min, washed, and suspended in 1 mm sodium phosphate buffer (ph 6.9). An equal amount of B virions was processed in an identical manner, except that the incubation mixture lacked neuraminidase. Both untreated virions and desialylated virus were sialylated by CMP-sialic acid in the sialyl transferase reaction mixture for 3 h at 37 C. The reaction mixtures were then centrifuged at 125, x g for 9 min through a.5-ml pad of 5% glycerol. The pellets were suspended in.3 ml of Tris-hydrochloride buffer (ph 7.), sonicated, and assayed for hemagglutinating activity and infectivity.,'ha assays were carried out at a final ph of 6.1. c Plaque assays were performed on monolayers of L cells. cell (R. H. Schloemer, unpublished data). Therefore, adsorption studies were performed using input multiplicities of 7 to 1 virus particles per cell to allow for optimal virus-cell interaction. VS virions labeled with [3H ]amino acids which had been desialylated, sialylated, or left untreated were diluted in PBS;.2 ml of this diluted virus (-55, 3H counts/min) was allowed to adsorb to each plate of L cells for various time intervals. As described in Materials and Methods, virus attachment is determined by measuring the amount of 3H radioactivity associated with cells and is expressed as percent of applied virus. Figure 2 shows that attachment of VS virus to L cells is a relatively slow and inefficient process; only 35 to 4% of the applied virus adsorbs after a 2-h period at 4 C. The rate of attachment of VS virions is constant for the first 3 min and then decreases rapidly after 45 min, with very little virus being adsorbed after 1 h. Similar results were obtained when attachment assays were conducted at 27 C, indicating that the adsorption of VS virus to L cells is not a temperature-dependent process (unpublished data). Neuraminidase treatment of VS virions substantially reduces the efficiency of attachment; the small amount of desialylated virions which does attach probably reflects background levels of radioactivity or nonspecific adsorption. Sialylation of virions which were incubated with neuraminidase resulted in considerable restoration of the ability of these virions to bind to L cells (78% of control). Sialylation of native VS virions not previously treated with neuraminidase did not appreciably alter the rate or amount of virus which adsorbed to L cells. Markedly diminished adsorption after removal of the VS viral glycoprotein by trypsin confirms t3 < ) / 3 z 36-18- 12) - L 12-6-)68 885 5 115 3 45 6 12 Minutes of Incubation FIG. 2. Adsorption to L cells of neuraminidasetreated, trypsinized, and sialylated VS virions. Aliquots of purified VS virions (25 Mg) that had been labeled with [3Hjamino acids were incubated at 37 C for 9 min in.5 ml of a reaction mixture consisting of Tris-hydrochloride buffer (ph 7.) and 1 mm CaCI, with either 5 U of neuraminidase or 5 ;ig of trypsin or buffer. The virus-enzyme mixtures were centrifuged at 125, x g for 9 min. Untreated, neuraminidase-treated, and trypsinized virions were sialylated exactly as described in Table 1, except that the trypsinized virions were incubated in a sialyl transferase reaction mixture which lacked CMP- [14C]sialic acid. After incubation, the reaction mixtures were centrifuged through a.5-mi pad of 5%o glycerol; the pellets were resuspended and diluted in PBS. Approximately 5, [3Hlprotein counts/min of virus in.2 ml of PBS were plated on monolayers of L cells (virus-cell ratio of 7), and virions were allowed to adsorb for the specified times at 4 C. Unadsorbed virus was removed and the cells were washed with cold PBS. Cell associated SH radioactivity was counted in the trichloroacetic acid pellet. Symbols:, untreated control virions; A, sialylated control virions; x, neuraminidase desialylated virions;, neuraminidase-treated, resialylated virions;, trypsinized virions. Downloaded from http://jvi.asm.org/ on October 17, 218 by guest

886 SCHLOEMER AND WAGNER J. VIROL. the role of the glycoprotein in viral adsorption to cell surfaces. These data indicate that neuraminic acid mediates a critical event in the initiation of viral infection. Inhibition of HA by neuraminic acid-containing compounds. If neuraminic acid does constitute the site on VS virus which recognizes cell receptors, then compounds containing neuraminic acid should be able to compete with VS virus for those receptor sites located on the cell surface. To examine this possibility, several neuraminic acid-containing substances were tested for their capacity to inhibit agglutination of goose erythrocytes by VS virus. Table 2 shows that neither N-acetyl neuraminic acid nor the trisaccharide, N-acetyl neuraminosyl lactose, was effective in inhibiting HA of goose erythrocytes by VS virus. However, fetuin, a glycoprotein which contains abundant sialic acid, was found to prevent HA, but did not itself hemagglutinate goose erythrocytes. Hydrolysis of sialic acid residues of fetuin by mild acid treatment or by neuraminidase treatment reversed this inhibitory effect, a finding which indicates that sialic acid residues of fetuin are responsible for the inhibition of VS viral HA. Neither neuraminic acid nor N-acetyl neuraminosyl lactose altered infectivity titers of VS virus. Infectivity assays employing cells which were incubated with fetuin or desialylated fetuin could not be conducted, since both compounds affected the viability of L cells. Inhibition of viral attachment by fetuin. It seemed important to determine whether fetuin inhibits HA by VS virus by reacting with the surface receptor of erythrocytes or by interacting with the virions. To determine if fetuin does bind to erythrocytes, 2 ml of a 5% suspension of goose erythrocytes was mixed with 1 mg of either fetuin or desialylated fetuin, or with PBS. After incubation for 3 min at 4 C, with occasional shaking to prevent settling of erythrocytes, the mixtures were centrifuged at 8 x g for 15 min at 4 C, washed, suspended in glucose-veronal-gelatin buffer, and used in the TABLE 2. Inhibition of HA by sialic acid-containing compoundsa Test substance (1 mg/ml) HI (U) N-acetyl neuraminic acid... <2 N-acetyl neuraminosyl lactose...... <2 Fetuin... 32 Desialylated fetuin... <2 a The various substances were dissolved in distilled water, and.1 ml of each compound was tested for inhibition of HA. determination of HA titers of untreated VS virions. HA titers of 2,48 U were obtained, employing erythrocytes which had been incubated with desialylated fetuin or with PBS. Incubation of erythrocytes with native fetuin reduced the amount of VS virus able to adsorb to HA titers of' 256. These data suggest that fetuin inhibits HA by binding to erythrocytes and not to virus. To demonstrate this reaction of fetuin with cell receptors more conclusively, attachment of' VS virus to monolayers of L cells which had been preincubated with fetuin or desialylated fetuin was examined. The results shown in Fig. 3 confirm the fact that fetuin competes with VS virus for cellular receptor sites. Virus attached poorly to cells which were preincubated with fetuin. However, if the cells were incubated with desialylated fetuin, no inhibition of attachment of VS virus occurred. These data indicate that fetuin probably inhibits VS viral adsorption by competing for cellular receptor sites which appear to recognize neuraminic acid residues of fetuin. Therefore, N-acetyl neuraminic acid can compete for VS viral receptors, but only when presented as a sialoglycoprotein. cn L. 4- V 3- a) 4 2- ~ -~I. ) 1- U) ~ -------- x,x lb 15 3 45 Minutes of Incubation 6 FIG. 3. Inhibition by fetuin of VS virus adsorption to L cells. A series of plates containing monolayers of L cells were incubated for 15 min at 4 C with 5 mg of either fetuin (x) or desialylated fetuin () or with PBS (). The plates were then washed extensively with PBS and drained well. Two hundred microliters of VS virions that had been labeled with [3Hlamino acids was added (-25, counts/min per ml) to each plate, dispersed by rocking, and allowed to adsorb at 4 C for the times specified. Cells were scraped from the plates, washed with PBS, and precipitated with cold 2% trichloroacetic acid. Cell-associated 3H radioactivity was counted by scintillation spectrophotometry. Downloaded from http://jvi.asm.org/ on October 17, 218 by guest

VOL. 15, 1975 Isoelectric focusing of desialylated VS virus. If the function of VS viral neuraminic acid in the attachment process is solely to maintain the proper charge environment necessary for virus-cell interaction, then removal of sialic acid from virions should result in the formation of a more positively charged VS virus with a less favorable ionic environment for attachment of VS virions to cells. Therefore, we sought to determine by isoelectric focusing the degree to which sialic acids contribute to the negative charge of VS virions. VS virions labeled with [3H ]amino acids were incubated with neuraminidase. After centrifugation to terminate the reaction, the virions were washed and resuspended in distilled water. Thiobarbituric acid analysis showed no detectable sialic acid in the neuraminidase-treated virions; these desialylated VS virions possessed reduced levels of infectivity, no hemagglutinating activity, and decreased efficiency of attachment to L cells as compared to untreated virions. The neuraminidase-treated [3H Ivirions were mixed with [TC ]protein-labeled fully sialylated VS virions and subjected to isoelectric focusing. Figure 4 indicates that the VS virion carries a negative charge with an isoelectric point of 4. and that treatment with neuraminidase does not alter the pl of the virus. Similar results were obtained by isoelectric focusing in ampholine I. 'O C E cn C I () NEURAMINIC ACID IN VS VIRUS ATTACHMENT gradients of ph 3 to 6. No changes in migration of desialylated virus through an electrophoretic field at neutral ph was observed. These data imply that neuraminic acid of VS virions contributes very little to the negative charge of VS virions. Adsorption of VS virus in the presence of DEAE-dextran. Another approach used to elucidate the mechanism by which neuraminic acid enhances adsorption of VS virions to cells was to use DEAE-dextran in an attempt to increase the attachment of virus to cells. Positively charged DEAE-dextran has been shown to increase the adsorption of VS viral nucleocapsids (4) and of VS virions (9) to cells, presumably by influencing the ionic environment existing between the cell and virus. Since desialylation does not result in a detectable change in the negative charge of the virus (see Fig. 4), we sought to determine whether the presence of DEAE-dextran could result in increased efficiency of attachment of desialylated virus. Therefore, [3H]amino acid-labeled VS virus, incubated in the presence or absence of neuraminidase, was diluted with either PBS alone or PBS containing DEAE-dextran and then tested in adsorption assays. The presence of DEAEdextran enhanced the attachment of VS virions to cells by twofold, suggesting that a more positively charged microenvironment results in Fraction FIG. 4. Isoelectric focusing of neuraminidase-treated VS virus. Three hundred micrograms of purified VS-B virions that had been labeled with [3H]amino acids was incubated at 37 C for 4 h with 1 U of neuraminidase. After incubation, the virions were pelleted by centrifugation at 125, x g for 9 min, washed, resuspended in.4 ml of distilled water, and mixed with [14CJprotein-labeled VS virions which were treated in an identical manner, except that neuraminidase was omitted from the reaction mixture. The isoelectric point of VS virions was determined using an LKB electrofocusing column (LKB Products, Bromma, Sweden). Linear to 6% sucrose gradients which contained 1% ampholines were generated in a two-channel mixing chamber. After one-fourth of the gradient had been poured, the virus preparation which was free of salts was added to the channel containing 6% sucrose and allowed to mix, and then the remainder of the gradient was formed. The bottom of the gradient was in contact with 1% (vol/vol) sulfuric acid in 6% sucrose while 2% monoethanolamine (vol/vol) was present at the top. Focusing was carried out at 3 V using a current of 3.5 ma; after 6 h this declined to.8 ma and the voltage was increased to 32 V. After isoelectric focusing for 42 h, during which time the temperature was maintained at 4 C, the gradient was fractionated into.9-mi aliquots. Each fraction was analyzed with respect to ph and radioactivity. IN ) -._ 1 cn C -1-8 -6 1 4-2 cl 887 Downloaded from http://jvi.asm.org/ on October 17, 218 by guest

888 SCHLOEMER AND WAGNER J. VIROL. virus-cell surface interaction (Fig. 5). However, DEAE-dextran did not have any effect upon the binding of neuraminidase-treated virus to L cells. Increased attachment of VS virus to desialylated L cells. The above experiments indicate that viral neuraminic acid is essential for efficient attachment of VS virions to cell surfaces. In view of the fact that cellular sialic acid serves as the receptor site for myxo- and paramyxoviruses (6, 1, 24), we investigated the role, if any, of the neuraminic acid present on the surfaces of L cells. In addition, we also examined the susceptibility of the cellular receptor site for VS virus to the proteolytic enzyme, trypsin. Suspensions of 5 x 17 L cells/ml were incubated with either trypsin, neuraminidase, U) I ) a) -_ ' C,) 4- c a) C) CL a) a-) 5 115 Minutes 3, 3 45 6 of Incubation FIG. 5. Influence of DEAE-dextran on the attachment of VS virions and desialylated VS virions to L cells. Equal amounts of [3H]protein-labeled VS virions were incubated for 2 h at 37 C in the presence of Tris-hydrochloride (ph 7.) or 5 Uof neuraminidase. The virions were then centrifuged at 125, x g for 9 min, resuspended in.1 ml of PBS, and sonicated. Desialylated virus and untreated virions were diluted with either PBS alone or PBS containing 1 Ig of DEAE-dextran per ml to achieve a final virus concentration of -25, 3H counts/min per ml. To each of a series of plates of monolayers of L cells,.2 ml of the virus was added and allowed to adsorb at 4 C for the specified times. Cell-associated SH radioactivity was then determined. Symbols: Untreated virus with () and without () DEAE-dextran; desialylated virus with (x) and without (A) DEAE-dextran. or buffer for 1 h. The cells were then centrifuged, washed extensively with PBS, and resuspended in PBS to their original volume. The enzymatic treatments did not influence the gross morphology of these cells. The attachment of [3H ]protein-labeled VS virus to these cells was determined at different times as described in Materials and Methods. The effects of neuraminidase and trypsin on the ability of L cells to absorb VS virus are shown in Fig. 6. Cells that were treated with either trypsin or neuraminidase not only absorb VS virions at a faster rate, but also attach twice as much virus than do untreated L cells. In addition, the initial rate of attachment of VS virions is even greater to cells which were incubated with trypsin than it is to desialylated cells. These results indicate that the cellular receptor site for VS virus is neither sialic acid nor surface glycoproteins accessible to trypsin. Since removal of sialic acid from L cells produces a more positively charged cell surface (25) and results in greater attachment of virions as compared to untreated cells, desialylated L cells were tested in the attachment assay to ascertain if the adsorption of neuraminidasea 64- I 48-32- 5 1 15 3 45 6 Minutes of Incubation FIG. 6. Adsorption of untreated VS virions and desialylated VS virions to L cells treated with trypsin and neuraminidase. Suspensions of 5 x 17 L cells per ml of 1 mm Tris-hydrochloride (ph 7.) were treated with 2 U of neuraminidase or I mg of trypsin for 3 min at 37 C. Subsequently, the cells were washed three times with 1 volumes of PBS and finally resuspended in PBS to their original volume. [3H]protein-labeled VS virions which had been untreated or desialylated with neuraminidase were mixed with the cells so that the ratio of virus particle to cell was -8:1. After incubation at 4 C for the specified times,.2-ml aliquots were removed and assayed for cell-associated 3H radioactivity. Symbols: attachment of untreated VS virus to untreated (O), trypsinized (A), and desialylated cells (); attachment of desialylated virions to untreated (x) and desialylated cells (M). Downloaded from http://jvi.asm.org/ on October 17, 218 by guest

VOL. 15, 1975 NEURAMINIC ACID IN VS VIRUS AYTACHMENT treated virions could likewise by enhanced. VS virions which were treated with neuraminidase adsorb to the same limited extent to desialylated L cells as they do to untreated cells (Fig. 6). This experiment indicates that removal of cell sialic acid does not merely create a more favorable ionic environment for adsorption of VS virus which still requires neuraminic acid for efficient adsorption. Increased plating efficiency of VS virions on monolayers of desialylated cells. Since removal of neuraminic acid from L cells resulted in greater attachment of VS virions, the possibility was examined that infectivity of untreated VS virus could also be increased if plaque assays were performed using monolayers of desialylated cells. Therefore, a series of plates containing either L cells, Madin-Darby bovine kidney cells, or Chinese hamster ovary cells were desialylated and then tested for their susceptibility to plaque formation by untreated VS virions. As a comparison, virus was plated in the presence of DEAE-dextran, which also increases VS viral absorption (see Fig. 5). Results of these experiments on the ability of desialylated cells to support growth of VS virus are shown in Table 3. The use of desialylated monolayers of L cells resulted in a 1% increase in the number of plaques as compared to untreated controls. DEAE-dextran did not in- TABLE 3. Increased plaque formation of VS virus plated on monolayers of desialylated cellsa Cell lineb Treatment Infectivity L None 7.5 x 1' DEAE-dextran 7.6 x 1' Neuraminidase 17.7 x 1' CHO None 19 DEAE-dextran 1.3 x 19 Neuraminidase 3.5 x 19 MDBK None 2.7 x 17 DEAE-dextran 3. x 17 Neuraminidase 4.2 x 17 a Series of plates containing monolayers of cells were incubated at 37 C for 2 h with.5 ml of 1 mm Tris-hydrochloride (ph 7.) and 1 mm CaCl2 in the presence or absence of 5 U of neuraminidase. The plates were washed extensively with PBS to remove neuraminidase. Dilutions of VS virus were then plated on treated or untreated cells, adsorbed for 1 h, and overlaid with agar. Plaques were counted after neutral red staining at 48 h. In those instances where DEAE-dextran was employed, virus was diluted in the presence of 1 ug of DEAE-dextran per ml. Triplicate assays were reproducible with an error of less than 15%. CHO, Chinese hamster ovary; MDBK, Madin- Darby bovine kidney. 889 crease plaque formation. The universality of this phenomenom is suggested by the fact that desialylated Madin-Darby bovine kidney and Chinese hamster ovary cells also supported growth of VS virus better than did untreated control cells. Biological activity of VS viral tryptic sialoglycopeptides. Since proteolytic digestion of the VS viral glycoprotein with Pronase has been shown to result in the formation of several classes of glycopeptides (2, 2), we investigated the possibility that mild proteolytic digestion of the intact VS virion could result in the formation of soluble, biologically active glycopeptides. These glycopeptides would not be expected to hemagglutinate goose erythrocytes, since the HA reaction requires bivalent attachment sites, but they might inhibit VS viral HA by competing with intact virus for the receptor sites on the erythrocytes. To this end, intact VS virus, the G protein of which had been labeled with ["C ]-labeled amino acids and [3H ]glucosamine, was subjected to mild digestion with trypsin. After incubation for specified times, soybean trypsin inhibitor was added to terminate the reaction. The supernatant fraction and the pellet were partitioned by centrifugation of the virusenzyme mixture at 125, x g for 9 min. The hemagglutinating ability of trypsinized VS virions and the ability of the supernatant fractions to inhibit HA of goose erythrocytes by untreated VS virus were examined. Figure 7 shows that, upon incubation with trypsin, virions progressively lost the ability to hemagglutinate goose erythrocytes, a fact which confirms the observation that the VS viral glycoprotein is the viral hemagglutinin (J. J. McSharry and P. W. Choppin, Abstr. Annu. Meet. Am. Soc. Microbiol., 1974, p. 266, V395). The supernatant fraction of the trypsin-treated virus mixtures was found to contain a VS viral hemagglutinin inhibitor, the activity of which increased with incubation. Since the control virus supernatant fraction did not exhibit this inhibitory effect, soybean trypsin inhibitor could not be involved in this inhibition of HA by VS virus. Other experiments reveal that boiled trypsin or a combination of trypsin and soybean trypsin inhibitor does not affect HA titers of VS virus. The decline with prolonged trypsinization of HI activity of the supernatant fraction is probably due to degradation of the VS viral G protein to smaller, less active sialoglycopeptides. Polyacrylamide gel electrophoresis of the supernatant fraction which exhibited maximal HI activity did not demonstrate a discrete peak of Downloaded from http://jvi.asm.org/ on October 17, 218 by guest

89 SCHLOEMER AND WAGNER J. VIROL. I c._ %I- - I (I a) ) 'o. Minutes of Incubation with Trypsin FIG. 7. Release of an inhibitor of HA by trypsin treatment of intact VS virions. To aliquots ofpurified VS virions (3 gg) in.3 ml of 1 mm Tris-hydrochloride buffer, ph 7., containing 1 mm CaCl2, 25 gl of buffer containing 5. Mg of trypsin or 25 Ml of buffer was added and incubated at 37 C for the times specified. The reaction was terminated by adding 5 Mg of soybean trypsin inhibitor. The reaction mixtures were then centrifuged at 125, x g for 9 min, and the pellet and supernatant fractions were partitioned. The pellet was dissolved in.3 ml of.4% BABS and assayed for hemagglutinating activity. The supernatant fraction was lyophilized, dissolved in BABS, and used in the HI assays. Symbols: O-, untreated virions; **; trypsinized virions; - -, supernatant of untreated virus; - -, supernatant of trypsinized virus. radioactivity, but the heterogeneous material labeled with both [14C ]-labeled amino acids and [3H ]glucosamine which migrated with the tracking dye undoubtedly represents the small glycopeptides (Fig. 8). Gel electrophoresis of the trypsinized virions revealed that only the G protein was degraded by trypsin (data not shown). Since we have proposed that neuraminic acid of the viral glycoprotein is involved in HA, the small glycopeptides which are inhibitors of HA should contain sialic acid, the removal of which should reverse the inhibitory effect. Therefore, VS virions which had been labeled with [3H]glucosamine and [14C]-labeled amino acid were subjected to mild trypsinization for 45 min, exactly as described in the legend to Fig. 7. After the addition of soybean trypsin inhibitor, the virus-enzyme mixture was centrifuged and the resulting supernatant fraction was divided into two equal parts. One sample was incubated at 37 C for 1 h with 5 U of neuraminidase; the '-._ U) I- second was incubated with buffer. Both samples received.3 umol of EDTA to inhibit neuraminidase activity (1) and then were assayed for their ability to inhibit HA by VS virions. The tryptic glycopeptide which was treated with neuraminidase exhibited no inhibition of HA, whereas the sample which was not desialylated contained a total of 64 HI units. Moreover, paper chromatography of the supernatant sample which was treated with neuraminidase revealed the presence of free neuraminic acid. These data indicate that a soluble glycopeptide which exhibits biological activity can be generated by mild trypsin treatment of VS virions and that the glycopeptide contains neuraminic acid which contributes to HI. DISCUSSION Evidence that the glycoprotein of VS virus is responsible for attachment of the virus to cell surfaces and that neuraminic acid residues of VS virus are involved in the attachment process is demonstrated by the following criteria: (i) removal of the glycoprotein by trypsin or hydrolysis of terminal sialic acid from virions 1 1- DYE L G N NS M b_ - 1 2 3 4 5 6 7 8 + Gel Fraction FIG. 8. Polyacrylamide gel electrophoresis of the soluble tryptic VS viral glycopeptides. Purified VS virions that had been labeled with [3Hlglucosamine and ["Cl-labeled amino acids were subjected to mild trypsin treatment at 37 C for 45 min as described in the legend to Fig. 7. After the addition of soybean trypsin inhibitor, the virus-enzyme mixtures were centrifuged at 125, x g for 9 min. The proteins in the supernatant fraction were precipitated in the presence of 5 Ag of bovine plasma albumin with 1 volume of cold 2% trichloroacetic acid. After cooling on ice, the contents of the tubes were pelleted by centrifugation. The precipitate was washed with cold acetone and dissolved in 1 mm sodium phosphate buffer (ph 7.4), 1% sodium dodecyl sulfate, and 1% 2-mercaptoethanol. Samples were boiled for 2 min and subjected to electrophoresis for 8 h at 5 ma/gel on 7.5% polyacrylamide gels. The arrows mark the positions of the viral proteins as determined from parallel gels. Symbols:, ["4Clamino acid;, [3Hlglucosamine. Downloaded from http://jvi.asm.org/ on October 17, 218 by guest

VOL. 15, 1975 NEURAMINIC ACID IN VS VIRUS ATTACHMENT 891 resulted in a loss in the ability of VS virions to hemagglutinate goose erythrocytes and to attach to mouse fibroblast (L) cells; and (ii) resialylation of virions which had been treated with neuraminidase reconstituted both hemagglutinating activity and attachment of virions to L cells. It is likely that those neuraminic acid residues which contribute to the attachment process are located on the viral glycoprotein, since the soluble tryptic glycopeptides which inhibit HA by VS virions contain neuraminic acid. Moreover, neuraminic acid on the viral glycolipids do not appear to be involved in the infective process (23). The exact function of viral sialic acids in the attachment process is not entirely clear. Neuraminic acid could serve as the viral site for attachment to the cell surface or could be involved in the attachment process simply by maintaining the necessary ionic environment between the viral and cellular receptor sites. Alternatively, negatively charged sialic acid may serve only to maintain the glycoprotein extended in the proper orientation for attachment to cell surface. Electron microscopic study of desialylated VS virions revealed that the glycoprotein spikes remained in the same spatial orientation as they do in untreated controls. No folding of the desialylated spikes was noted, a situation which may be expected to occur if the glycoproteins were maintained in the proper spatial orientation by electrostatic repulsion due to the negatively charged terminal neuraminic acid residues (unpublished data). The hypothesis that neuraminic acid constitutes the viral attachment site for cellular surfaces is not inconsistent with these results. The fact that fetuin reacts with cell surfaces and this inhibits viral HA as well as cell attachment of VS virions, and that removal of neuraminic acid residues from fetuin reverses this inhibition, suggests that receptor sites present on cell surfaces recognize sialic acid residues on sialoglycopeptides. Moreover, soluble viral glycopeptides were shown to contain neuraminic acid and to inhibit HA of goose erythrocytes by VS virions. If viral sialic acid does indeed serve as the specific receptor-recognition component, the peptide moiety could provide a backbone of sufficient length on which neuraminic acid is properly arrayed. Consistent with this is the observation that neither free sialic acid nor N-acetyl neuraminosyl lactose inhibits HA, whereas fetuin and trypsin-generated VS viral glycopeptide of low molecular weight do. However, it cannot be determined whether sialic acid constitutes the entire viral attachment site or just part of it, possibly in conjunction with other carbohydrates or with the peptide moiety of the viral glycoprotein. Both the HA and viral adsorption assays are inadequate in terms of sensitivity to detect greater than 99% loss of activity upon neuraminidase or trypsin treatment. The observation that VS virions incubated with trypsin exhibited a 3-log greater decrease in infectivity than did neuraminidase-treated virus (23) tends to argue against the supposition that sialic acid is the only component of the viral attachment site. The question whether viral neuraminic acid provides the proper ionic environment for attachment of VS virus to cells remains partially unresolved. Although desialylation of VS virions did not alter the isoelectric point of these virions or the electrophoretic mobility of the virions at neutral ph, it remains uncertain whether neuraminic acid contributes to a small extent to the negative charge of VS virus. It has been demonstrated that neuraminidase treatment of purified VS viral glycoprotein altered its isoelectric position (6). Conceivably, the negative charge of VS virus is caused to a small extent by sialic acid, and the removal by desialylation of that portion of the negative charge may not be sufficient to result in a detectable change in the isoelectric point of the virion or in the electrophoretic mobility of the virus. Similar findings that neuraminic acid does not influence the charge of cell membranes have been observed in ascites tumor cells and other mammalian cells (13, 25). If viral sialic acid functions in attachment solely by providing the proper ionic milieu for virus-cell interaction, then any alteration of charge environment which leads to an enhancement of attachment of native VS virions could be expected to increase the adsorption of desialylated virions. However, when desialylated cells or DEAE-dextran were employed in the attachment assays, the adsorption of only untreated virions and not of desialylated virions is increased. This observation tends to suggest that viral neuraminic acid functions in the attachment process by serving as the viral site for attachment to cells, rather than influencing the charge environment. The role of cellular neuraminic acid in viruscell interaction is puzzling. Cellular sialic acid appears to prevent the attachment of VS virus to cells. Since both VS virus and animal cell membranes are negatively charged, the removal of the negatively charged sialic acid from the cell surfaces conceivably produces a more positively charged cell surface microenvironment, thereby promoting greater electrostatic attraction for VS virions. The fact that greater Downloaded from http://jvi.asm.org/ on October 17, 218 by guest

892 SCHLOEMER AND WAGNER J. VIROL. amounts of VS virus attach to cells treated with neuraminidase than to untreated cells may indicate that cellular sialic acid either masks additional receptor sites for the virus or prevents the formation of strong electrostatic bonds, in which case VS virus could more readily desorb. No attempts have yet been made to determine desorption of VS virions. Sindbis virus adsorbs to cells in two ways, a loose attachment and tight binding, both of which are dependent upon ionic conditions (22). Conceivably, cellular neuraminic acid could dictate the type of binding for VS virus. Trypsin treatment of L cells, in addition to enhancing the amount of VS virions which attach to the cells, causes a more rapid attachment of virions, a situation comparable to that which occurs with adenovirus (21). Possibly, the cellular surface glycoproteins sterically hinder the attachment of VS virions. The reason for increased plating efficiency on desialylated cells is not clear, but is not necessarily due to increased attachment of VS virus since no increase in the number of plaques occur if VS virions are plated in the presence of DEAE-dextran, which also increases viral attachment. The identity of the cellular receptor site for VS virus is unknown, except that neuraminic acid and that portion of the surface glycoproteins which is removed by trypsin are not involved in the attachment of VS virus. Since VS virus has an exceptionally wide host range, the receptor site is probably a common constituent of all cell membranes. It is possible that the cellular receptor site could be a membrane protein or even a lipid. Haywood (12) has demonstrated that Sendai virions can adsorb to and fuse with liposomes. Experiments are being conducted in this laboratory to identify the nature of the cellular receptor site for VS virus. The requirement for viral neuraminic acid for attachment of VS virions contrasts sharply with that of other enveloped viruses. The paramyxoviruses and influenza virus, which possess neuraminidase activity and lack neuraminic acid, recognize cellular sialic acid as their receptor site (1, 16, 24). In sharp contrast to VS virus, removal of cellular neuraminic acid prevents the binding of influenza virus to cells. Even more striking is the apparent lack of function of neuraminic acid in another envelope virus, Semliki Forest virus. Desialylation of Semliki Forest virus was not shown to result in a loss of infectivity or HA, but did apparently increase the isoelectric point of the virus (14). ACKNOWLEDGMENTS This research was supported by grant GB-35257-X from the National Science Foundation, by grant VC-88 from the American Cancer Society, and by Public Health Service grant AI-11112 from the National Institute of Allergy and Infectious Diseases. We thank Ruth Snyder for technical assistance and W. Steinberg for assistance with the isoelectric focusing apparatus. LITERATURE CITED 1. Ada, G. L., E. L. French, and P. E. Lind. 1961. Purification and properties of neuraminidase from Vibrio cholerae. J. Gen. Microbiol. 24:49-421. 2. Burge, B. W., and A. S. Huang. 197. Comparison of membrane protein glycopeptides of Sindbis virus and vesicular stomatitis virus. J. Virol. 6:176-182. 3. Cartwright, B., C. J. Smale, and F. Brown. 1969. Surface structure of vesicular stomatitis virus. J. Gen. Virol. 5:1-1. 4. Cartwright, B., C. J. Smale, and F. Brown. 197. Dissection of vesicular stomatitis virus into the infective ribonucleoprotein and immunizing components. J. Gen. Virol. 7:19-32. 5. Dales, S. 1973. Early events in cell-animal virus interactions. Bacteriol. Rev. 37:13-135. 6. Dietzschold, B., L. G. Schneider, and J. H. Cox. 1974. Serological characterization of the three major proteins of vesicular stomatitis virus. J. Virol. 14:1-7. 7. Emerson, S. U., and R. R. Wagner. 1972. Dissociation and reconstitution of the transcriptase and template activities of vesicular stomatitis B and T virions. J. Virol. 1:297-39. 8. Etchison, J. R., and J. J. Holland. 1974. Carbohydrate composition of the membrane glycoprotein of vesicular stomatitis virus. Virology 6:217-229. 9. Flamand, A., and D. H. L. Bishop. 1973. Primary in vivo transcription of vesicular stomatitis virus and temperature-sensitive mutants of five vesicular stomatitis virus complementation groups. J. Virol. 12:1238-1252. 1. Gottschalk, A. 1957. Neuraminidase: the specific enzyme of influenza virus and Vibrio cholerae. Biochim. Biophys. Acta 23:645-646. 11. Halonen, P. E., F. A. Murphy, B. N. Fields, and D. R. Reese. 1968. Hemagglutinin of rabies and some other bullet-shaped viruses. Proc. Soc. Exp. Biol. Med. 127:137-142. 12. Haywood, A. M. 1974. Characteristics of Sendai virus receptors in a model membrane. J. Mol. Biol. 83:427-436. 13. Kajima, K., and A. Maekawa. 197. Difference in electrokinetic charge of cells between two cell types of ascites hepatoma after removal of sialic acid. Cancer Res. 3:2858-2862. 14. Kennedy, S. I. T. 1974. The effect of enzymes on structural and biological properties of Semliki Forest virus. J. Gen. Virol. 23:129-143. 15. Klenk, H-D., and P. W. Choppin. 1971. Glycolipid content of vesicular stomatitis virus grown in baby hamster kidney cells. J. Virol. 7:416-417. 16. Klenk, H-D., R. W. Compans, and P. W. Choppin. 197. An electron microscope study of the presence or absence of neuraminic acid in enveloped viruses. Virology 42:1158-1162. 17. Lowry,. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 18. McSharry, J. J., R. W. Compans, and P. W. Choppin. 1971. Proteins of vesicular stomatitis virus and of phenotypically mixed vesicular stomatitis virus-simian virus 5 virions. J. Virol. 8:722-729. 19. McSharry, J. J., and R. R. Wagner. 1971. Lipid composition of purified vesicular stomatitis virus. J. Virol. 7:59-7. 2. McSharry, J. J., and R. R. Wagner. 1971. Carbohydrate composition of vesicular stomatitis virus. J. Virol. 7:412-415. Downloaded from http://jvi.asm.org/ on October 17, 218 by guest

VOL. 15, 1975 NEURAMINIC ACID IN VS VIRUS ATTACHMENT 893 21. Philipson, L., K. Lonberg-Holm, and U. Petterson. 1968. Virus-receptor interaction in an adenovirus system. J. Virol. 2:164-175. 22. Pierce, J. S., E. G. Strauss, and J. H. Strauss. 1974. Effect of ionic strength on the binding of Sindbis virus to chick cells. J. Virol. 13:13-136. 23. Schloemer, R. H., and R. R. Wagner. 1974. Sialoglycoprotein of vesicular stomatitis virus: role of the neuraminic acid in infection. J. Virol. 14:27-281. 24. Schulze, I. T. 1973. Structure of the influenza virion, p. 1-56. In M. A. Lauffer, F. B. Bang, K. Maramorosch, and K. M. Smith (ed.), Advances in virus research, vol. 18. Academic Press Inc., New York. 25. Simon-Reuss, I., G. M. W. Cook, G. V. F. Seaman, and D. H. Heard. 1964. Electrophoretic studies on some types of mammalian tissue cell. Cancer Res. 24:238-243. 26. Wagner, R. R., A. H. Levy, R. M. Snyder, G. A. Ratcliff, Jr., and D. F. Hyatt. 1963. Biologic properties of two plaque variants of vesicular stomatitis virus (Indiana serotype). J. Immunol. 91:112-122. 27. Wagner, R. R., L. Prevec, F. Brown, D. F. Summers, F. Sokol, and R. MacLeod. 1972. Classification of rhabdovirus proteins: a proposal. J. Virol. 1:1228-123. 28. Wagner, R. R., T. C. Schnaitman, R. M. Snyder, and C. A. Schnaitman. 1969. Protein composition of the structural components of vesicular stomatitis virus. J. Virol. 3:611-618. 29. Wagner, R. R., R. M. Snyder, and S. Yamazaki. 197. Proteins of vesicular stomatitis virus: kinetics and cellular sites of synthesis. J. Virol. 5:548-558. 3. Warren, L. 1959. The thiobarbituric acid assay of sialic acids. J. Biol. Chem. 234:1971-1975. Downloaded from http://jvi.asm.org/ on October 17, 218 by guest