JOURNAL OF VIROLOGY, June 1976, p. 819-823 Copyright © 1976 American Society for Microbiology
Vol. 18, No. 3 Printed in U.S.A.
Fluorosugars Inhibit Biological Properties of Different Enveloped Viruses MICHAEL F. G. SCHMIDT,* RALPH T. SCHWARZ, AND HANNS LUDWIG Institut far Virologie, Justus Liebig-Universitat, Giessen, Germany Received for publication 10 December 1975
Both 2-deoxy-2-fluoro-D-glucose and 2-deoxy-2-fluoro-D-mannose were found to be potent inhibitors of the synthesis of infectious Semliki forest and fowl plague virus in chicken embryo cells and also of pseudorabies virus grown in rabbit kidney cells. It was found that the pseudorabies virus-mediated cell fusion and the synthesis of functional hemagglutinin of fowl plague virus were blocked. In all cases the 2-deoxy-2-fluoro-r-mannose-caused inhibition was stronger than the 2-deoxy-2-fluoro-D-glucose- or 2-deoxy-D-glucose-mediated blocks. Studies on the virus-specified proteins from Semiliki forest virus-infected cells grown in the presence of the inhibitors show that the target of the fluorosugar action, parallel to the well-studied effects of 2-deoxy-n-glucose, is the glycoprotein biosynthesis.
It has been shown many times that 2-deoxyD-glucose (2-dGlc), an analogue of either glucose or mannose, inhibits the formation of infectious enveloped viruses (for review see reference 13). Biochemical studies have demonstrated that 2-dGlc impairs the formation of functional influenza hemagglutinin (HA) by specifically blocking the glycosylation process (9, 14). Investigations on the metabolism of 2-dGlc in the infected host cell and the interference of the sugar analogue with physiological precursors for glycosylation (12, 14; M. F. G. Schmidt, Ph.D. thesis, Universitat, Giessen, 1975) suggest that 2-dGlc acts mainly as an antimetabolite for mannose rather than for glucose. It should be possible to test this hypothesis, eliminating side effects in one or another direction, by using more specific analogues substituted at carbon 2 in the manno- or gluco-configuration. We therefore compared 2-deoxy-2-fluoro-Dglucose (FG) and 2-deoxy-2-fluoro-D-mannose (FM) for their antiviral activity in different virus-host cell systems, expecting FM to be more effective. Only few data are known about the biological properties of halogen-substituted sugars. FG is able to selectively inhibit glucan formation in yeast, but nothing is known on the specificity of this inhibition related to the fluorine configuration because with FM a similar effect can be observed (4). Furthermore, some of the deoxyfluoro-D-hexopyranoses substituted at carbon atom 2 were shown to interfere with the growth of mouse lymphoma cells, but they did not show any antitumor activity as 6-deoxy-6fluoro-D-glucose did (3). Biochemical studies
showed that fluorine-substituted sugars can be phosphorylated by hexokinase, but 2-chloro-nglucose is a weak substrate and 2-chloro-Dmannose is not a substrate (2). Coe (5) has observed a slight inhibition of glycolysis in ascites tumor cells by 2-deoxy-2-fluoro-D-glucose. We report here the antiviral activity of two fluorosugars that were expected to carry some specificity as analogues for glucose or for mannose. MATERIALS AND METHODS
Virus and cells. The Rostock strain of fowl plague virus (FPV) and the Osterrieth strain of Semliki forest virus (SFV) were grown in monolayers of either primary or secondary chicken embryo fibroblasts produced by a standard procedure (18). The DEK strain of pseudorabies (PsR) virus was grown in primary rabbit kidney (RK) cells prepared from 3to 4-week-old rabbits as described earlier (11). Since the two fluorosugars were available only in minute amounts, most of the inhibitory studies were performed in tissue culture microtest plates (Cooke Microtiter from Greiner, M 220-29 ART, Nurtingen, Germany). For preparing cell cultures, 100 ul of cells in suspension (106 to 3 x 106 cells/ml) was seeded per well of the microplate and incubated at 37 C until complete monolayers had formed. Before infection with SFV, FPV, or PsR virus (multiplicity of infection 50 to 100 PFU/cell), the cells were washed with prewarmed phosphate-buffered saline (PBS). Sixty minutes postinfection (p.i.) the inoculum was withdrawn. After three successive washes with prewarmed phosphate-buffered saline, the cells were flooded with 100 ul of pyruvate containing Earle medium buffered with HEPES (N-2-hydroxymethyl-piperazine-N'-2'-ethanesulfonic acid) (12, 15) and containing the indicated concentrations of 819
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SCHMIDT, SCHWARZ, AND LUDWIG
inhibitor at 37 C for 8 (SFV and FPV) or 18 h (PsR virus). Infectivity, hemagglutination, and fusion. Before harvesting the cultures, the fusion events in the herpesvirus-infected cultures were counted with the aid of a graded plastic dish in an inverted microscope and expressed as the percentage of multinucleated cells (11). Virus-infected cells were frozen and thawed three times, and cleared from debris by centrifugation. Aliquots of the cell-free supernatants were tested for infectivity in the standard plaque assay on primary chicken embryo cells. The HA titrations were done by the micro- or macromethod, using either 20 or 100 ,kl of cell-free supernatant of FPV-infected chicken embryo cells (6). Radioactive labeling. Proteins of SFV-infected cells were labeled by adding either 5 ,Ci of 14Clabeled protein hydrolysate or of a mixture of 16.7 ,kCi each of tritiated L-valine, L-tyrosine, and Lleucine in a total volume of 2 ml to the growth medium which contained 10 mM glucose. Labeling was started at 3.5 h after infection and the cells were processed at 5.5 h p.i. Polyacrylamide gel electrophoresis. Labeled cells were washed four times with PBS and homogenized by short sonic treatment, and the same volume of 2 x concentrated sample buffer (10) was added. Samples were then heated for 10 min at 100 C and stored for electrophoresis at -30 C. Samples of a maximal 50 Al were mixed with a drop of bromophenol blue in glycerol and layered on top of the gels in the same buffer. Gels were 5 mm in diameter and 9.5 cm long (8.75% acrylamide, 0.2% methylenebisacrylamide) with a 2-cm long stacking gel (2% acrylamide, 0.07% methylenebisacrylamide). An electric current of 2 mA per gel was applied until the dye just passed the stacking gel and then was raised to 3 mA/gel. After electrophoresis the gels were frozen, cut into 1-mm slices, and prepared for liquid scintillation counting as described by Klenk et al. (8). The nomenclature of the polypeptides of SFV-infected cells was adapted from Simons et al. (16). Chemicals and isotopes. Reagents for polyacrylamide gels, 2-dGlc (reagent grade) and HEPES were obtained from Serva, Heidelberg, Germany. U-
'4C-labeled protein hydrolysate, L-[4,5-3H]leucine (55.5 Ci/mmol), L-[3,5-3H]tyrosine (49 Ci/mmol), and L-[2,3-3H]valine (39 Ci/mmol) were purchased
from Amersham Buchler GmbH, Braunschweig,
Germany. FG and FM were a kind gift from J. H. Westwood, Chester Beatty Research Institute, London.
RESULTS Effect of fluorosugars on infected cells and virus infectivity. As demonstrated for 2-dGlc in a number of virus-host cell systems (13), we find that the fluorosugars inhibit the biosynthesis of infectious virus. As Fig. IA shows, the production of SFV in chicken embryo cells is already blocked at 0.01 to 0.02 mM concen-
trations of FM in the medium, but inhibition by the corresponding analogue of D-glucose can be obtained only at somewhat higher concentrations.
In experiments with FPV using different concentrations of fluorosugars, the formation of infectious virus is only a little lower when FMinduced inhibition is compared with the inhibition by FG (Fig. IB). Despite the low sensitivity of the HA test, the production of HA as a parameter for the formation of functional glycoprotein in FPV-infected chicken embryo cells also reflects the different efficiency in the action of the inhibitors. As was to be expected according to the results given in Fig. 1B, FM affects the HA activity always somewhat stronger than 2-dGlc and FG (not shown in figures). It was of interest whether the same differences in the action of the inhibitors would also occur in a totally different virus-host system. PsR virus-infected RK cells offer the possibility of evaluating not only the infectivity of this virus but also the additional parameter cell fusion. It was found that PsR virus-mediated cell fusion was completely blocked with FM at slightly lower concentrations as with 2-dGlc, whereas FG was less effective on fusion activity at the same concentrations (Table 1). Treatment of uninfected control RK cells under similar conditions did not reveal any morphological alterations, even at 1 mM fluorosugar concentration. The measurement of infectious PsR virus essentially parallels the results of the fusion studies. No infectious virus is formed at concentrations of 0.2 to 0.5 mM FM or 2-dGlc, in contrast to FG (Fig. 1C), which only at higher concentrations inhibits multiplication of PsR virus over 90%. To minimize the possibility that the virus inhibition by the different sugar analogues is due to an irreversible nonspecific inhibition of the host cell metabolism, noninfected cells were pretreated with an inhibitory concentration of fluorosugar in the medium. After 4 h the cells were washed and then infected with SFV or FPV. In both cases the yields of infectious virus were essentially the same as in the nontreated controls (not shown in figures). Reversal of the fluorosugar-mediated inhibition by addition of exogenous hexoses. Whether the addition of physiological hexoses to the culture medium containing the inhibitors could restore the yields of infectious SFV as it is known for mannose in the case of 2-dGlc inhibition has been tested (7). Glucose and mannose interfere with the action of the inhibitor (Fig. 2). In the presence of increasing amounts of mannose both fluorosugars were considerably
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LL LA
D U.
LL-
vl) j
L-
cr
1,o Concentration of Inhibitor [mM]
FIG. 1. Effect of FG (M), FM (-), and 2-dGlc (A) on the production of infectious SFV (A), FPV (B), and PsR (C). SFV and FPV were grown in chicken embryo cells and PsR virus was grown in RK cells using pyruvate-containing medium in both cases. The inhibitors were added 1 h p.i. and the concentrations of infectious virus were determined with the plaque assay on chicken embryo cells 8 h p.i. for FPV and SFV and 18 h p.i. for PsR virus.
less effective, whereas glucose did not restore the growth of infectious SFV to the same extent even when counteracting the inhibition by FG. This observation would suggest that, as in the case of 2-dGlc-inhibition of virus multiplication, FG and FM both act on the mannose pathway. Further support for this hypothesis will be given later. Synthesis of viral proteins in the presence of fluorosugars. Since it was not clear whether
the fluorosugars would primarily effect glycosylation like 2-dGlc or would block cell metabolism in general, the synthesis of SFV-specific proteins in chicken embryo cells was studied. As shown in Fig. 3 both fluorosugars have a distinct effect only on the virus-specific glycoproteins. The precursor molecule NSP 68, which only appears in the infected cells, but not in the virus particle (16), migrates faster in the gels, when the cells are grown in the presence
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SCHMIDT, SCHWARZ, AND LUDWIG
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antagonist for mannose and not so much for glucose as suggested by many studies in animal cells (7, 12, 17). Therefore one should expect by FG FM 2-dGlc Concn using more specific analogues to obtain sup(MM) porting evidence for this hypothesis. As we 0 0 0 o have shown, FM compared with FG was indeed 0 20 0 0.01 10 50 the more potent inhibitor in all experiments. 5 0.05 30 100 50 0.1 Our results were obtained by using enveloped 70 100 90 0.5 viruses from different taxonomic groups as 90 100 100 1.0 SFV, FPV, and PsR virus, as well as different a PsR virus-infected RK cells were treated with host cell systems (chicken embryo cells and RK different concentrations of the indicated substances. cells). The percentage of inhibition of syncytia formation The observation that FG also results in an (numbers) was evaluated 18 h p.i. inhibition of virus multiplication and the fact that mannose can reverse the effect of both FG and FM are strong indications that the cellular TABLE 1. Inhibition of herpesvirus-induced cell fusion by sugar analoguesa
_
_
-Control titer
A
1ou E _
-
75
15
ose . ~~~~~~~Gluc
,.
I NSP 68-
on?
Mannose
"I
10-
, Glucose 10' A 5'
QOS
02 05 01 Concentration of MGnnose
10 or
Glucose
X
-2
25
IE
x
mM
FIG. 2. Dose response of glucose and mannose on SFV infectivity in the presence of 0.2 mM FG (a) or FM (a). In both cases the indicated concentrations (abscissa) of competing physiological sugars and the inhibitors were applied at 1 h after infection. The plaque assay was performed after an 8-h incubation.
NSP68
EjFl.E2
C
of one of the fluorosugars, which is compatible with a lower molecular weight. In the case of the viral glycoproteins El and E2, which are not resolved in these gels (-49,000 and -51,000 molecular weight), both inhibitors lead to the occurrence of a new product which migrates only slightly faster in the gels than El and E2. Equimolar concentrations of FM (Fig. 3B) Fractions cause a stronger reduction of the molecular FIG. 3. Polyacrylamide gel electrophoresis of weight of NSP 68 than does FG (Fig. 3A). This SFV-infected chicken embryo cells labeled with 14C fluorosugar-induced modification of virus-spe- protein hydrolysate in the presence of FG (A) or of cific glycoproteins of SFV-infected cells was FM (B). The sugar analogues were applied 1 h p.i. found to be dose dependent for both inhibitors with SFV at 5 mM concentration in Earle medium (not shown in figures). In all cases there was no containing 10 mM glucose. Labeling was from 3.5 to change detected in the mobility of the core pro- 5.5 h p.i. Samples were subjected to co-electrophoresis tein (-35,000 molecular weight) which does not with SFV-infected cells labeled with a mixture of tritiated amino acids. Abbreviations: NSP 68, noncontain any sugar. structural protein (-68,000 molecular weight); E, + E2, the two envelope glycoproteins (-49,000 and DISCUSSION -51,000 molecular weights, respectively) which are 2-dGlc, which is an analogue for glucose as not resolved in the gels used; C, core protein well as for mannose, acts in vivo mainly as an (-35,000 molecular weight).
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enzymes like hexokinase (2) or nucleotidyltransferases are not able to recognize the applied fluorosugar exclusively as the appropriate analogue of one hexose only. Biochemical studies on the metabolism of FM and FG give evidence that FG and also FM can form both the guanosine 5'-diphosphate and the uridine 5'diphosphate nucleotide sugar derivatives (manuscript in preparation). Further experiments using radioactively labeled fluorosugars will clairfy this point and will give additional information on the metabolic fate of these two analogues. These studies will also answer the question whether the fluorosugars can be incorporated into macromolecular material. There is no doubt that the fluorosugars, like 2-dGlc, predominantly effect glycosylation of viral proteins. This interpretation is strongly supported by the observation that under our conditions viral protein synthesis is only little impaired and by the finding that there are no drastic changes in the energy charge indices as measured according to Atkinson (1) (unpublished data). The exact site of the block, however, is not known. It can be placed at sites of synthesis of precursor molecules for glycosylation (e.g., direction of glucose-6-phosphate- or mannose-6-phosphate-isomerase or the generation of monosaccharides via gluconeogenesis when pyruvate is used as carbon source) or directly on the level of the glycosyl transferases, which is the case for the 2-dGlc-caused inhibition of glycosylation (Schmidt, Ph.D. thesis, 1975). ACKNOWLEDGMENTS We thank U. Elsasser, I. Weibel, and J. Weiel for excellent technical assistance. We are grateful to C. Scholtissek and R. Rott for continuous advice and support and P Biely, Bratislava, CSSR, for helpful discussion. We are greatly indebted to J. H. Westwood and A. B. Foster for providing 2-deoxy-2-fluoro-D-glucose and 2-deoxy2-fluoro-D-mannose. This work was supported by the Sonderforschungsbereich 47 (Virologie).
3.
4.
5.
6.
7.
8.
9.
10. 11.
12.
13. 14. 15.
16. 17.
LITERATURE CITED 1. Atkinson, D. E. 1968. The energy charge of the adenylate pool as a regulatory parameter. Interaction with feedback modifiers. Biochemistry 7:4030-4034. 2. Bessell, E. M., A. B. Foster, and J. H. Westwood. 1972.
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The use of deoxyfluoro-D-glucopyranoses and related compounds in a study of yeast hexokinase specificity. Biochem. J. 128:199-204. Bessell, E. M., V. D. Courtenay, A. B. Foster, M. Jones, and J. H. Westwood. 1973. Some 'in vivo' and 'in vitro' antitumour effects of the deoxyfluoro-Dgluco-pyranoses. Eur. J. Cancer 9:463-470. Biely, P., J. Kovarik, and S. Bauer. 1973. Lysis of Sacchaomyces cerevisiae with 2-deoxy-2-fluoro-Dglucose, an inhibitor of the cell wall glucan synthesis. J. Bacteriol. 115:1108-1120. Coe, E. L. 1972. Inhibition of glycolysis in ascites tumor cells preincubated with 2-deoxy-2-fluoro-D-glucose. Biochim. Biophys. Acta 264:319-327. Davenport, R. M., R. Rott, and W. Schafer. 1960. Physical and biological properties of influenza virus components obtained after ether treatment. J. Exp. Med. 112:765-782. Kaluza, G., M. F. G. Schmidt, and C. Scholtissek. 1973. Effect of 2-deoxy-D-glucose on the multiplication of Semliki Forest virus and the reversal of the block by mannose. Virology 54:179-189. Klenk, H.-D., L. A. Caliguiri, and P. W. Choppin. 1970. The proteins of parinfluenza virus SV5. II. The carbohydrate content and glycoproteins of the virion. Virology 42:473-481. Kienk, H.-D., C. Scholtissek, and R. Rott. 1972. Inhibition of glycoprotein biosynthesis of influenze virus by D-glucosamine and 2-deoxy-D-glucose. Virology 49:723-734. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. Ludwig, H., H. Becht, and R. Rott. 1974. Inhibition of herpesvirus-induced cell fusion by Concanavalin A, antisera, and 2-deoxy-D-glucose. J. Virol. 14:307-314. Schmidt, M. F. G., R. T. Schwarz, and C. Scholtissek. 1974. Nucleoside diphosphate derivatives of 2-deoxy1-glucose in animal cells. Eur. J. Biochem. 49:237247. Scholtissek, C. 1975. Inhibition of the multiplication of enveloped viruses by glucose derivatives. Curr. Top. Microbiol. Immunol. 70:101-119. Schwarz, R. T., and H.-D. Klenk. 1974. Inhibition of glycosylation of the influenza virus hemagglutinin. J. Virol. 14:1023-1034. Shipman, C. 1969. Evaluation of 4-(2-hydroxyethyl)-1piperazine-ethane-sulfonic acid (HEPES) as tissue culture buffer. Proc. Soc. Exp. Biol. Med. 130:305310. Simons, K., S. Keranen, and L. Karikinen. 1973. Identification of a precursor of the Semliki Forest virus membrane proteins. FEBS Lett. 29:87-91. Steiner, S., R. J. Courtney, and J. L. Melnick. 1973. Incorporation of 2-deoxy-D-glucose into glycoproteins of normal and Simian virus 40-transformed cells. Cancer Res. 33:2402-2407. Youngner, J. S. 1954. Monolayer tissue cultures. I. Preparation and standardization of suspensions of trypsin-dispersed-monkey-kidney cells. Proc. Soc. Exp. Biol. 85:202-207.
Vol. 18, No. 3 Printed in U.S.A.
Fluorosugars Inhibit Biological Properties of Different Enveloped Viruses MICHAEL F. G. SCHMIDT,* RALPH T. SCHWARZ, AND HANNS LUDWIG Institut far Virologie, Justus Liebig-Universitat, Giessen, Germany Received for publication 10 December 1975
Both 2-deoxy-2-fluoro-D-glucose and 2-deoxy-2-fluoro-D-mannose were found to be potent inhibitors of the synthesis of infectious Semliki forest and fowl plague virus in chicken embryo cells and also of pseudorabies virus grown in rabbit kidney cells. It was found that the pseudorabies virus-mediated cell fusion and the synthesis of functional hemagglutinin of fowl plague virus were blocked. In all cases the 2-deoxy-2-fluoro-r-mannose-caused inhibition was stronger than the 2-deoxy-2-fluoro-D-glucose- or 2-deoxy-D-glucose-mediated blocks. Studies on the virus-specified proteins from Semiliki forest virus-infected cells grown in the presence of the inhibitors show that the target of the fluorosugar action, parallel to the well-studied effects of 2-deoxy-n-glucose, is the glycoprotein biosynthesis.
It has been shown many times that 2-deoxyD-glucose (2-dGlc), an analogue of either glucose or mannose, inhibits the formation of infectious enveloped viruses (for review see reference 13). Biochemical studies have demonstrated that 2-dGlc impairs the formation of functional influenza hemagglutinin (HA) by specifically blocking the glycosylation process (9, 14). Investigations on the metabolism of 2-dGlc in the infected host cell and the interference of the sugar analogue with physiological precursors for glycosylation (12, 14; M. F. G. Schmidt, Ph.D. thesis, Universitat, Giessen, 1975) suggest that 2-dGlc acts mainly as an antimetabolite for mannose rather than for glucose. It should be possible to test this hypothesis, eliminating side effects in one or another direction, by using more specific analogues substituted at carbon 2 in the manno- or gluco-configuration. We therefore compared 2-deoxy-2-fluoro-Dglucose (FG) and 2-deoxy-2-fluoro-D-mannose (FM) for their antiviral activity in different virus-host cell systems, expecting FM to be more effective. Only few data are known about the biological properties of halogen-substituted sugars. FG is able to selectively inhibit glucan formation in yeast, but nothing is known on the specificity of this inhibition related to the fluorine configuration because with FM a similar effect can be observed (4). Furthermore, some of the deoxyfluoro-D-hexopyranoses substituted at carbon atom 2 were shown to interfere with the growth of mouse lymphoma cells, but they did not show any antitumor activity as 6-deoxy-6fluoro-D-glucose did (3). Biochemical studies
showed that fluorine-substituted sugars can be phosphorylated by hexokinase, but 2-chloro-nglucose is a weak substrate and 2-chloro-Dmannose is not a substrate (2). Coe (5) has observed a slight inhibition of glycolysis in ascites tumor cells by 2-deoxy-2-fluoro-D-glucose. We report here the antiviral activity of two fluorosugars that were expected to carry some specificity as analogues for glucose or for mannose. MATERIALS AND METHODS
Virus and cells. The Rostock strain of fowl plague virus (FPV) and the Osterrieth strain of Semliki forest virus (SFV) were grown in monolayers of either primary or secondary chicken embryo fibroblasts produced by a standard procedure (18). The DEK strain of pseudorabies (PsR) virus was grown in primary rabbit kidney (RK) cells prepared from 3to 4-week-old rabbits as described earlier (11). Since the two fluorosugars were available only in minute amounts, most of the inhibitory studies were performed in tissue culture microtest plates (Cooke Microtiter from Greiner, M 220-29 ART, Nurtingen, Germany). For preparing cell cultures, 100 ul of cells in suspension (106 to 3 x 106 cells/ml) was seeded per well of the microplate and incubated at 37 C until complete monolayers had formed. Before infection with SFV, FPV, or PsR virus (multiplicity of infection 50 to 100 PFU/cell), the cells were washed with prewarmed phosphate-buffered saline (PBS). Sixty minutes postinfection (p.i.) the inoculum was withdrawn. After three successive washes with prewarmed phosphate-buffered saline, the cells were flooded with 100 ul of pyruvate containing Earle medium buffered with HEPES (N-2-hydroxymethyl-piperazine-N'-2'-ethanesulfonic acid) (12, 15) and containing the indicated concentrations of 819
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inhibitor at 37 C for 8 (SFV and FPV) or 18 h (PsR virus). Infectivity, hemagglutination, and fusion. Before harvesting the cultures, the fusion events in the herpesvirus-infected cultures were counted with the aid of a graded plastic dish in an inverted microscope and expressed as the percentage of multinucleated cells (11). Virus-infected cells were frozen and thawed three times, and cleared from debris by centrifugation. Aliquots of the cell-free supernatants were tested for infectivity in the standard plaque assay on primary chicken embryo cells. The HA titrations were done by the micro- or macromethod, using either 20 or 100 ,kl of cell-free supernatant of FPV-infected chicken embryo cells (6). Radioactive labeling. Proteins of SFV-infected cells were labeled by adding either 5 ,Ci of 14Clabeled protein hydrolysate or of a mixture of 16.7 ,kCi each of tritiated L-valine, L-tyrosine, and Lleucine in a total volume of 2 ml to the growth medium which contained 10 mM glucose. Labeling was started at 3.5 h after infection and the cells were processed at 5.5 h p.i. Polyacrylamide gel electrophoresis. Labeled cells were washed four times with PBS and homogenized by short sonic treatment, and the same volume of 2 x concentrated sample buffer (10) was added. Samples were then heated for 10 min at 100 C and stored for electrophoresis at -30 C. Samples of a maximal 50 Al were mixed with a drop of bromophenol blue in glycerol and layered on top of the gels in the same buffer. Gels were 5 mm in diameter and 9.5 cm long (8.75% acrylamide, 0.2% methylenebisacrylamide) with a 2-cm long stacking gel (2% acrylamide, 0.07% methylenebisacrylamide). An electric current of 2 mA per gel was applied until the dye just passed the stacking gel and then was raised to 3 mA/gel. After electrophoresis the gels were frozen, cut into 1-mm slices, and prepared for liquid scintillation counting as described by Klenk et al. (8). The nomenclature of the polypeptides of SFV-infected cells was adapted from Simons et al. (16). Chemicals and isotopes. Reagents for polyacrylamide gels, 2-dGlc (reagent grade) and HEPES were obtained from Serva, Heidelberg, Germany. U-
'4C-labeled protein hydrolysate, L-[4,5-3H]leucine (55.5 Ci/mmol), L-[3,5-3H]tyrosine (49 Ci/mmol), and L-[2,3-3H]valine (39 Ci/mmol) were purchased
from Amersham Buchler GmbH, Braunschweig,
Germany. FG and FM were a kind gift from J. H. Westwood, Chester Beatty Research Institute, London.
RESULTS Effect of fluorosugars on infected cells and virus infectivity. As demonstrated for 2-dGlc in a number of virus-host cell systems (13), we find that the fluorosugars inhibit the biosynthesis of infectious virus. As Fig. IA shows, the production of SFV in chicken embryo cells is already blocked at 0.01 to 0.02 mM concen-
trations of FM in the medium, but inhibition by the corresponding analogue of D-glucose can be obtained only at somewhat higher concentrations.
In experiments with FPV using different concentrations of fluorosugars, the formation of infectious virus is only a little lower when FMinduced inhibition is compared with the inhibition by FG (Fig. IB). Despite the low sensitivity of the HA test, the production of HA as a parameter for the formation of functional glycoprotein in FPV-infected chicken embryo cells also reflects the different efficiency in the action of the inhibitors. As was to be expected according to the results given in Fig. 1B, FM affects the HA activity always somewhat stronger than 2-dGlc and FG (not shown in figures). It was of interest whether the same differences in the action of the inhibitors would also occur in a totally different virus-host system. PsR virus-infected RK cells offer the possibility of evaluating not only the infectivity of this virus but also the additional parameter cell fusion. It was found that PsR virus-mediated cell fusion was completely blocked with FM at slightly lower concentrations as with 2-dGlc, whereas FG was less effective on fusion activity at the same concentrations (Table 1). Treatment of uninfected control RK cells under similar conditions did not reveal any morphological alterations, even at 1 mM fluorosugar concentration. The measurement of infectious PsR virus essentially parallels the results of the fusion studies. No infectious virus is formed at concentrations of 0.2 to 0.5 mM FM or 2-dGlc, in contrast to FG (Fig. 1C), which only at higher concentrations inhibits multiplication of PsR virus over 90%. To minimize the possibility that the virus inhibition by the different sugar analogues is due to an irreversible nonspecific inhibition of the host cell metabolism, noninfected cells were pretreated with an inhibitory concentration of fluorosugar in the medium. After 4 h the cells were washed and then infected with SFV or FPV. In both cases the yields of infectious virus were essentially the same as in the nontreated controls (not shown in figures). Reversal of the fluorosugar-mediated inhibition by addition of exogenous hexoses. Whether the addition of physiological hexoses to the culture medium containing the inhibitors could restore the yields of infectious SFV as it is known for mannose in the case of 2-dGlc inhibition has been tested (7). Glucose and mannose interfere with the action of the inhibitor (Fig. 2). In the presence of increasing amounts of mannose both fluorosugars were considerably
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821
LL LA
D U.
LL-
vl) j
L-
cr
1,o Concentration of Inhibitor [mM]
FIG. 1. Effect of FG (M), FM (-), and 2-dGlc (A) on the production of infectious SFV (A), FPV (B), and PsR (C). SFV and FPV were grown in chicken embryo cells and PsR virus was grown in RK cells using pyruvate-containing medium in both cases. The inhibitors were added 1 h p.i. and the concentrations of infectious virus were determined with the plaque assay on chicken embryo cells 8 h p.i. for FPV and SFV and 18 h p.i. for PsR virus.
less effective, whereas glucose did not restore the growth of infectious SFV to the same extent even when counteracting the inhibition by FG. This observation would suggest that, as in the case of 2-dGlc-inhibition of virus multiplication, FG and FM both act on the mannose pathway. Further support for this hypothesis will be given later. Synthesis of viral proteins in the presence of fluorosugars. Since it was not clear whether
the fluorosugars would primarily effect glycosylation like 2-dGlc or would block cell metabolism in general, the synthesis of SFV-specific proteins in chicken embryo cells was studied. As shown in Fig. 3 both fluorosugars have a distinct effect only on the virus-specific glycoproteins. The precursor molecule NSP 68, which only appears in the infected cells, but not in the virus particle (16), migrates faster in the gels, when the cells are grown in the presence
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SCHMIDT, SCHWARZ, AND LUDWIG
822
antagonist for mannose and not so much for glucose as suggested by many studies in animal cells (7, 12, 17). Therefore one should expect by FG FM 2-dGlc Concn using more specific analogues to obtain sup(MM) porting evidence for this hypothesis. As we 0 0 0 o have shown, FM compared with FG was indeed 0 20 0 0.01 10 50 the more potent inhibitor in all experiments. 5 0.05 30 100 50 0.1 Our results were obtained by using enveloped 70 100 90 0.5 viruses from different taxonomic groups as 90 100 100 1.0 SFV, FPV, and PsR virus, as well as different a PsR virus-infected RK cells were treated with host cell systems (chicken embryo cells and RK different concentrations of the indicated substances. cells). The percentage of inhibition of syncytia formation The observation that FG also results in an (numbers) was evaluated 18 h p.i. inhibition of virus multiplication and the fact that mannose can reverse the effect of both FG and FM are strong indications that the cellular TABLE 1. Inhibition of herpesvirus-induced cell fusion by sugar analoguesa
_
_
-Control titer
A
1ou E _
-
75
15
ose . ~~~~~~~Gluc
,.
I NSP 68-
on?
Mannose
"I
10-
, Glucose 10' A 5'
QOS
02 05 01 Concentration of MGnnose
10 or
Glucose
X
-2
25
IE
x
mM
FIG. 2. Dose response of glucose and mannose on SFV infectivity in the presence of 0.2 mM FG (a) or FM (a). In both cases the indicated concentrations (abscissa) of competing physiological sugars and the inhibitors were applied at 1 h after infection. The plaque assay was performed after an 8-h incubation.
NSP68
EjFl.E2
C
of one of the fluorosugars, which is compatible with a lower molecular weight. In the case of the viral glycoproteins El and E2, which are not resolved in these gels (-49,000 and -51,000 molecular weight), both inhibitors lead to the occurrence of a new product which migrates only slightly faster in the gels than El and E2. Equimolar concentrations of FM (Fig. 3B) Fractions cause a stronger reduction of the molecular FIG. 3. Polyacrylamide gel electrophoresis of weight of NSP 68 than does FG (Fig. 3A). This SFV-infected chicken embryo cells labeled with 14C fluorosugar-induced modification of virus-spe- protein hydrolysate in the presence of FG (A) or of cific glycoproteins of SFV-infected cells was FM (B). The sugar analogues were applied 1 h p.i. found to be dose dependent for both inhibitors with SFV at 5 mM concentration in Earle medium (not shown in figures). In all cases there was no containing 10 mM glucose. Labeling was from 3.5 to change detected in the mobility of the core pro- 5.5 h p.i. Samples were subjected to co-electrophoresis tein (-35,000 molecular weight) which does not with SFV-infected cells labeled with a mixture of tritiated amino acids. Abbreviations: NSP 68, noncontain any sugar. structural protein (-68,000 molecular weight); E, + E2, the two envelope glycoproteins (-49,000 and DISCUSSION -51,000 molecular weights, respectively) which are 2-dGlc, which is an analogue for glucose as not resolved in the gels used; C, core protein well as for mannose, acts in vivo mainly as an (-35,000 molecular weight).
VOL. 18, 1976
FLUOROSUGAR INHIBITION OF ENVELOPED VIRUSES
enzymes like hexokinase (2) or nucleotidyltransferases are not able to recognize the applied fluorosugar exclusively as the appropriate analogue of one hexose only. Biochemical studies on the metabolism of FM and FG give evidence that FG and also FM can form both the guanosine 5'-diphosphate and the uridine 5'diphosphate nucleotide sugar derivatives (manuscript in preparation). Further experiments using radioactively labeled fluorosugars will clairfy this point and will give additional information on the metabolic fate of these two analogues. These studies will also answer the question whether the fluorosugars can be incorporated into macromolecular material. There is no doubt that the fluorosugars, like 2-dGlc, predominantly effect glycosylation of viral proteins. This interpretation is strongly supported by the observation that under our conditions viral protein synthesis is only little impaired and by the finding that there are no drastic changes in the energy charge indices as measured according to Atkinson (1) (unpublished data). The exact site of the block, however, is not known. It can be placed at sites of synthesis of precursor molecules for glycosylation (e.g., direction of glucose-6-phosphate- or mannose-6-phosphate-isomerase or the generation of monosaccharides via gluconeogenesis when pyruvate is used as carbon source) or directly on the level of the glycosyl transferases, which is the case for the 2-dGlc-caused inhibition of glycosylation (Schmidt, Ph.D. thesis, 1975). ACKNOWLEDGMENTS We thank U. Elsasser, I. Weibel, and J. Weiel for excellent technical assistance. We are grateful to C. Scholtissek and R. Rott for continuous advice and support and P Biely, Bratislava, CSSR, for helpful discussion. We are greatly indebted to J. H. Westwood and A. B. Foster for providing 2-deoxy-2-fluoro-D-glucose and 2-deoxy2-fluoro-D-mannose. This work was supported by the Sonderforschungsbereich 47 (Virologie).
3.
4.
5.
6.
7.
8.
9.
10. 11.
12.
13. 14. 15.
16. 17.
LITERATURE CITED 1. Atkinson, D. E. 1968. The energy charge of the adenylate pool as a regulatory parameter. Interaction with feedback modifiers. Biochemistry 7:4030-4034. 2. Bessell, E. M., A. B. Foster, and J. H. Westwood. 1972.
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The use of deoxyfluoro-D-glucopyranoses and related compounds in a study of yeast hexokinase specificity. Biochem. J. 128:199-204. Bessell, E. M., V. D. Courtenay, A. B. Foster, M. Jones, and J. H. Westwood. 1973. Some 'in vivo' and 'in vitro' antitumour effects of the deoxyfluoro-Dgluco-pyranoses. Eur. J. Cancer 9:463-470. Biely, P., J. Kovarik, and S. Bauer. 1973. Lysis of Sacchaomyces cerevisiae with 2-deoxy-2-fluoro-Dglucose, an inhibitor of the cell wall glucan synthesis. J. Bacteriol. 115:1108-1120. Coe, E. L. 1972. Inhibition of glycolysis in ascites tumor cells preincubated with 2-deoxy-2-fluoro-D-glucose. Biochim. Biophys. Acta 264:319-327. Davenport, R. M., R. Rott, and W. Schafer. 1960. Physical and biological properties of influenza virus components obtained after ether treatment. J. Exp. Med. 112:765-782. Kaluza, G., M. F. G. Schmidt, and C. Scholtissek. 1973. Effect of 2-deoxy-D-glucose on the multiplication of Semliki Forest virus and the reversal of the block by mannose. Virology 54:179-189. Klenk, H.-D., L. A. Caliguiri, and P. W. Choppin. 1970. The proteins of parinfluenza virus SV5. II. The carbohydrate content and glycoproteins of the virion. Virology 42:473-481. Kienk, H.-D., C. Scholtissek, and R. Rott. 1972. Inhibition of glycoprotein biosynthesis of influenze virus by D-glucosamine and 2-deoxy-D-glucose. Virology 49:723-734. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. Ludwig, H., H. Becht, and R. Rott. 1974. Inhibition of herpesvirus-induced cell fusion by Concanavalin A, antisera, and 2-deoxy-D-glucose. J. Virol. 14:307-314. Schmidt, M. F. G., R. T. Schwarz, and C. Scholtissek. 1974. Nucleoside diphosphate derivatives of 2-deoxy1-glucose in animal cells. Eur. J. Biochem. 49:237247. Scholtissek, C. 1975. Inhibition of the multiplication of enveloped viruses by glucose derivatives. Curr. Top. Microbiol. Immunol. 70:101-119. Schwarz, R. T., and H.-D. Klenk. 1974. Inhibition of glycosylation of the influenza virus hemagglutinin. J. Virol. 14:1023-1034. Shipman, C. 1969. Evaluation of 4-(2-hydroxyethyl)-1piperazine-ethane-sulfonic acid (HEPES) as tissue culture buffer. Proc. Soc. Exp. Biol. Med. 130:305310. Simons, K., S. Keranen, and L. Karikinen. 1973. Identification of a precursor of the Semliki Forest virus membrane proteins. FEBS Lett. 29:87-91. Steiner, S., R. J. Courtney, and J. L. Melnick. 1973. Incorporation of 2-deoxy-D-glucose into glycoproteins of normal and Simian virus 40-transformed cells. Cancer Res. 33:2402-2407. Youngner, J. S. 1954. Monolayer tissue cultures. I. Preparation and standardization of suspensions of trypsin-dispersed-monkey-kidney cells. Proc. Soc. Exp. Biol. 85:202-207.
