VIROLOGY
63, 191-200 (1975)
Two Different
Mechanisms of Enveloped
of the Inhibition Viruses
C. SCHOLTISSEK, Institut
by Glucosamine
R. ROTT, AND H.-D. KLENK
fiir Virologie, Justus Liebig-Uniuersitiit, Accepted
of the Multiplication
September
Giessen, Germany
3, 1974
In Earle’s medium containing 10 mA4glucose, glucosamine inhibits the multiplication of fowl plague virus (FPV) and Semliki Forest virus (SFV) by interfering with the synthesis of viral glycoproteins, while the synthesis of viral RNA and carbohydrate-free proteins is affected only slightly or not at all. If glucose is replaced by fructose, glucosamine inhibits the synthesis of viral RNA and in this way interferes with the production of all viral proteins. Protein and RNA synthesis of non-infected cells are not significantly impaired under these conditions. The effect on viral RNA synthesis can be readily counteracted by uridine. The inhibition of viral glycoprotein synthesis cannot be reversed at all by uridine and that of viral carbohydrate-free proteins only to a very limited extent. In fructose-containing medium, glucosamine depletes the cells of UTP in such a way that the UTP pool may become rate limiting for viral RNA synthesis but not for cellular RNA synthesis. The multiplication of Newcastle disease virus (NDV) and vesicular stomatitis virus (VW) is inhibited by glucosamine only in fructose-containing medium but not in glucose-containing medium.
containing either glucose, pyruvate or fructose as an energy source. It will be shown Recently it has been shown that glucosathat in glucose-containing medium the mine as well as 2-deoxy-D-glucose inhibits the multiplication of various enveloped effect of the amino sugar is only on the animal viruses by interference with the synthesis of glycoproteins of FPV and SFV leaving synthesis of viral RNA and protein synthesis of viral glycoproteins (Kilbourne, 1959; Kaluza et al., 1972; Gandhi et al., almost intact, while in pyruvate- and fruc1972; Klenk et al., 1972; Courtney et al., tose-containing media the effect is on the 1973; Kaluza et al., 1973; Scholtissek et al., synthesis of viral RNA and of all viral 1974; Klenk et al., 1974). Because 10 mM proteins. Under these latter conditions proglucosamine in glucose-containing medium tein and RNA synthesis in non-infected reduces the UTP pool of the host cell by a cells is almost normal. factor of about 8 (Scholtissek, 1971, 1973), MATERIALS AND METHODS it was suggested that the amino sugar might interfere with the activation of other Virus strains. Fowl plague virus (FPV), sugars via depletion of the UTP pool strain “Restock”; Semliki Forest virus (Klenk et al., 1972). In most of these (SFV), strain “Osterrieth”; vesicular stostudies, medium containing glucose has matitis virus (VSV), strain “Indiana”; and been used; glucose competes with the up- Newcastle disease virus (NDV), strain take and activation of the sugar derivatives “Italien”, were used. FPV and NDV were mentioned. grown by serial passage in embryonated In this paper we compare the effect of eggs, while SFV and VSV were propagated glucosamine on virus multiplication with in primary chick embryo cells at a multiits effect on the UTP pool by using media plicity below one. INTRODUCTION
Copyright 0 1975 by AcademicPress,Inc. All rights of reproduction in any form reserved.
191
192
SCHOLTISSEK,
Tissue cultures. Primary chick embryo cells prepared from 11-day-old embryos were used either 24, 48 or 72 hr after seeding at a cell density of either 6 x lo6 cells in Petri dishes of 5 cm in diameter or 2 x lo7 cells in Petri dishes of 9 cm in diameter. In order to start each experiment with the same number of cells for the 24-hr cultures, 3 x 10’ cells/ml were seeded; for the 46 or 72-hr cultures, 1.5 x lo6 cells/ml were seeded. The cells were infected with a multiplicity of lo-50 PFU/cell. After infection the cells were overlayed with 2 or 6 ml, respectively, of Earle’s medium without glucose. The various sugars or pyruvate as listed in the tables and figures were added to a final concentration of 10 mM. In most experiments, especially when pyruvate medium was investigated, sodium bicarbonate was replaced by Hepes buffer, pH 7.4 (Good et al., 1966). Under these conditions CO, was omitted from the incubator. This became necessary when it was found that pyruvate was toxic for virus multiplication at slightly alkaline pH. Otherwise virus yields were not influenced by the different buffer systems. Virus quantitation. Plaque assays and hemagglutination (HA) titration were carried out according to standard procedures (Zimmermann and Schafer, 1960). Neuraminidase activity. Enzymatic activity was determined as before (Drzeniek et al., 1966) using bovine sialolactose as substrate. The amount of neuraminidase which liberated 1 micromole of sialic acid from the substrate per min at 37” was defined as one enzyme unit. Labeling of protein and RNA. Labeling was done by incubating cells with the respective tritium-labeled precursors. At the time indicated, the radioactivity was determined in RNA, in protein, and in the trichloroacetic acid (TCA)-soluble pool as described recently (Scholtissek, 1971). Polyacrylamide gel electrophoresis. The cells (6 x lop/gel), labeled with a mixture of tritiated amino acids (50 PCilml) from 4 to 5 hr after infection with FPV, were homogenized by sonication. Samples of 0.1 ml were used for gel electrophoresis. Proteins were dissociated with sodium dodecyl
ROTT
AND KLENK
sulfate and mercaptoethanol and separated by electrophoresis on 10%acrylamide gels (Llmmli, 1970). The slicing and processing of gels for the determination of radioactivity by liquid scintillation has been reported previously (Klenk et al., 1970). The determination of viral RNA polymerase. Viral RNA polymerase was deter-
mined as described previously (Scholtissek, 1969). A cytoplasmic fraction prepared 5 hr after infection with FPV was incubated at 32” for different lengths of time with [3H]GTP plus cofactors. The radioactivity was determined in the TCA precipitate. The synthesis
of viral
RNA
in vivo.
Synthesis was followed by incorporation of the labeled nucleoside at various times after infection. For SFV 2 pg./ml of actinomycin D was added immediately after infection and [3H]adenosine was added 3 hr after infection. At different times thereafter cultures were washed three times with phosphate-buffered saline. Radioactivity was determined in the TCA-soluble pool and in RNA (Scholtissek, 1971). For FPV, t3H]uridine was added to the cultures as indicated. At the end of the pulse total RNA was extracted from three cultures by phenol plus 1% sodium dodecyl sulfate at room temperature and was dissolved in 2 ml of 5 mM Tris-HCl buffer, pH 8, containing 1 mM EDTA. Aliquots of 0.1 ml were used for the determination of total radioactivity and for the hybridization procedure after heating for 10 min at 100” and rapid cooling to 0”. Nonlabeled virion RNA was isolated from purified virus particles and was dissolved in 2 x SSC (0.3 M NaCl, 0.03 A4 sodium citrate) to give a final concentration of 0.75 mg/ml RNA. Either 10 or 25 ~1of virion RNA were mixed with the aliquots for hybridization at 65” overnight. Nonlabeled complementary RNA was prepared by incubating a microsomal fraction isolated 5 hr after infection of 50 tissue cultures with FPV in the presence of all four nucleoside triphosphates and 4 mM MgCl, in Tris-HCl buffer, pH 8, at 36” for 20 min. Thereafter the microsomes were centrifuged at 100,000 g at 4” for 90 min. The RNA was extracted
GLUCOSAMINE
193
AND VIRUS MULTIPLICATION
from the supernatant by phenol and was taken up in 2 x SSC to give a concentration of 4.0-4.5 mg RNA/ml. Most of this RNA is microsomal RNA. The actual amount of complementary RNA is not known. Traces of virion RNA in this preparation were bound to complementary RNA forming double-stranded RNA by short heating to 80” and slow cooling to room temperature. Either 50 or 100 ~1 of this RNA solution were mixed with aliquots for hybridization in order to determine the relative amount of labeled virion RNA. Each batch of the respective nonlabeled viral RNA was titrated against viral RNA labeled in uivo from 4 to 5 hr after infection using the same hybridization technique. As a rule, saturation was obtained either with 5 ~1 of nonlabeled virion RNA or 25 ~1 of complementary RNA, respectively. Corresponding saturation curves have been published (Scholtissek and Rott, 1970). The values in Table 2 represent average values of two determinations using the two different concentrations of nonlabeled viral RNA. The highest deviation from each average value was 5%. A more detailed description of the hybridization procedure has been described (Scholtissek and Rott, 1970). Materials. Labeled uridine, [5-3H]uridine, (29 Ci/mmole); [3H]adenosine, generally labeled (0.5 Wmmole); and the tritiated amino acid mixture (TRK 440) were purchased from the Radiochemical Centre, Amersham, England. [8-‘H]GTP (0.80 Ci/mmole) was obtained from Schwarz/Mann, Orangeburg, NY. Dglucosamine-HCl and actinomycin D were obtained from Serva, Heidelberg, Germany. RESULTS
Effect of Glucosamine on the UTP Pool Using Different Media and Cells of Different Age It has been suggested recently (Klenk et al., 1972) that the depletion of the UTP pool by glucosamine might be the fundamental mechanism by which its inhibitory effect is exerted on the production of severa1 enveloped viruses. It is known that the
extent of the effect of glucosamine on the UTP pool depends on the age of the cell (Scholtissek, 1971). In order to find the optimal conditions, this effect was tested at various times after seeding of the cells. In addition, glucose was replaced in the culture medium by fructose, because glucose competes with the uptake of glucosamine. As shown in Table 1, glucosamine enhances the uptake of labeled uridine into noninfected cells (total TCA extract). This can be explained by the finding that the UTP pool regulates the uptake of uridine by negative feedback (Scholtissek, TABLE
1
EFFECT OF GLUCOSAMINE ON THE LABELING OF DIFFERENT COMPOUNDS BY [3H]U~~~~~~ IN MEDIA CONTAINING EITHER GLUCOSE OR FRUCTOSE
Age of cells”
Medium (10mM)
Glucosamine (miW
dpm x 10’ in To;?
RNA
UTP/ UDPsugar
extract Glucose
0 0.08 1 10
404 426 613 784
57 60 92 150
1.88 1.79 1.00 0.68
0 0.08 l 10
405 527 853 882
60 75 125 150
2.42 1.30 0.58 0.44
0 0.08 1 10
498 540 840 1042
70 76 115 146
1.63 1.52 0.79 0.48
0 0.08 1 10
470 784 1084 760
61 103 140 184
1.85 0.85 0.54 0.25
24 hr Fructose
Glucose
48 hr Fructose
’ Chick embryo cells were used either 24 hr or 48 hr after seeding. Earle’s medium without glucose was supplemented with the corresponding sugar as listed in the Table. After 90-min incubation at 37”, IO&i of [BH]uridine were added; 40 min later the cells were processed and the neutralized TCA extracts were analyzed on Dowex columns (Hurlbert et al., 1954; Scholtissek, 1971). The material of two cultures was pooled. The radioactivity in UMP and UDP was negligible.
194
SCHOLTISSEK,
1972). Because of the facilitated uptake of the label and the relative decrease of the UTP pool caused by glucosamine, the specific radioactivity of UTP increases (Scholtissek, 1971). Therefore the incorporation of labeled uridine into RNA is significantly enhanced (Table 1). The relative rise in the radioactivity of UDP-N-acetylglucosamine indicates that most of the UTP is used for the synthesis of this activated amino sugar derivative. Comparing 24-hr-old cells with those of 48 hr of age it can be seen that these effects are expressed more extensively in the older cells. Recently it has been shown that in medium containing 10 n&f glucose, 10 mA4 glucosamine reduces the UTP pool by a factor of about 8 using cells 48 hr after seeding (Scholtissek, 1971, 1973). In fructose-containing medium one needs only about one-tenth of the concentration of glucosamine in order to get the same effect on the UTP pool as compared to glucose-containing medium. In pyruvate-containing medium one observes an intermediate effect (not shown).
ROTI AND KLENK
012
5
10
012 5 x)0125 mM Glucosamlne
II
FIG. 1. Dose response of glucosamine on the multiplication of FPV in different media. Chick embryo cells were infected with FPV 48 hr after seeding. Concentrations of glucosamine as listed on the abscissas were added to the media containing either fructose (A), glucose (0) or pyruvate (0) immediately after infection. Eight hr later the various cell-bound virus activities were determined. There was no effect of glucosamine on virus release.
Effect of Glucosamine Concentration on the Multiplication of Enveloped Viruses in Different Media and on Cells of Different Age
Since the sugar used as energy source for the growth of the cells plays an important role on the effect of glucosamine on the UTP pool, sugar-free Earle’s medium supplemented with 10 mmoles of glucose, pyruvate or fructose was investigated to measure the effect of the amino sugar on multiplication of enveloped viruses. As shown for FPV in Fig. 1 the effect of glucosamine on the yield of infectious virus, on hemagglutinin (HA), and on neuraminidase is highest in fructose-containing medium and lowest in glucose-containing medium. This effect can be reversed completely at least up to 4 hr after infection by replacement with medium without glucosamine (not shown here). Corresponding results have been obtained with SFVinfected chick embryo cells. As shown in Fig. 2 the multiplication of NDV and VSV can be inhibited by glucosamine if fructose-containing medium is
0
2
L
68x)021
6
8
10
mM Glucosamlne
FIG. 2. Dose response of glucosamine on the multiplications of NDV (left) and VSV (right) in different media. Chick embryo cells were infected with either NDV or VSV 48 hr after seeding. Doses of glucosamine as listed on the abscissas were added immediately after infection. Eight hours later either the HA-titer (NDV) or the p.f.u. (VSV) was determined. 0, glucose-containing medium; A, fructose-containing medium.
used. In glucose-containing medium there is no measurable inhibition which is in accord with recent observations (Kaluza et al., 1972). Since in uninfected cells the effect of glucosamine on the UTP pool depends on the age of the cells used (Table l), it was of interest to see whether such a dependence also exists in the case of virus multiplication. There were some fluctuations in the sensitivity of virus multiplication to gluco-
GLUCOSAMINE
AND
VIRUS
195
MULTIPLICATION
samine when different batches of cells were strated in Table 1 (see also Scholtissek, used at different times after seeding. It 1971, 1973) RNA synthesis cannot be meawas, however, a constant finding that cells sured quantitatively by incorporation of infected 24 hr after seeding were much less sensitive than cells infected 48 hr after seeding. If cells were investigated even later glucosamine was again less effective. The results of a typical experiment on the inhibition of the multiplication of FPV and SFV by glucosamine using cells of the same batch at different times after seeding can be seen in Fig. 3. The transport and/or enzyme systems involved in glucosamine metabolism seem to work optimally in cells 48 hr after seeding. Therefore in all further mM Glucosomlne experiments cells of this age were used. Effect of Glucosamine on Cellular Viral RNA Synthesis
and
As shown in Table 2 glucosamine markedly increases the incorporation of labeled uridine into total RNA. Since the amino sugar affects the UTP pool as demon-
FIG. 3. Role of the age of the cultures on virus yield. Dose response of glucosamine. Chick embryo cells were infected either with FPV (left) or with SFV (right) at different times after seeding. Different doses of glucosamine (abscissa) were added to the fructosecontaining medium immediately after infection. Eight hr later the cell-bound activities were determined. 0, 24-hr-old cells; 0, 48hr-old cells; A, 72-hr-old cells.
TABLE EFFECT OF GLUCOSAMINE
Glucosamine (mm
ExperimentR
Glucose 1
3
Pulse from . . till . hours after infection
SYNTHESIS
in Vivo
Total RNA (dpm x 103)
Virion RNA (% oftotal)
Complementary RNA (% of total)
0 5 0 5 5
3-4 3-4 3-4 3-4 3-4
24 62 27 92 84
23.3 21.8 21.0 6.7 1.5
4.6 8.8 4.7 4.1 5.2
Fructose
0 5 0 5 5*
2-3 2-3 4-5 4-5 4-5
64 136 50 109 59
14.0 0.4 29.0 4.6 8.5
9.4 1.3 7.6 4.6 4.5
Fructose
0 5 0 5
3-3.5 3-3.5 3-4.33 3-4.33
28 74 94 187
30.0 5.2 37.0 3.0
5.8 4.3 10.6 3.0
Fructose Pyruvate
2
2
ON FPV-RNA
DIf not otherwise stated, 5 mM glucosamine was added immediately after infection with FPV into Earle’s medium containing Hepes buffer and 10 mM of the energy source listed. Pulses with [sH]uridine, 25 pCi/culture, were applied as listed in the Table. Each value represents the average of three cultures. The percentage of FPV-RNA was determined by hybridization with a surplus of either nonlabeled virion RNA or complementary RNA. Before calculation the corresponding values obtained from noninfected controls were substracted (Scholtissek and Rott, 19703. b Glucosamine was added 2.7 hr after infection.
196
SCHOLTISSEK,
[SH]uridine unless corresponding corrections on pool size etc. are made. Incorporation of [‘Hladenosine can be used, however, to study the effect of glucosamine on RNA synthesis because glucosamine has neither a significant effect on the uptake of adenosine (Fig. 4A and 4C) nor on the pool size of ATP (Scholtissek, 1971, 1972). As shown in Fig. 5 glucosamine has no significant effect on RNA synthesis of noninfected cells regardless of the sugar present in the medium. For comparison, the effect of glucosamine on the incorporation of [3H]leucine has been measured (Fig. 5). There is also no significant effect of the amino sugar on protein synthesis. It has been shown recently that glucosamine has either no or only a slight effect on viral RNA synthesis if the infected cells are
FIG. 4. Effect of glucosamine on SFV-RNA synthesis in uiuo and its reversal by uridine. Chick embryo cells were infected with SFV 48 hr after seeding. Two mM glucosamine and/or 1 mM uridine were added either to glucose-containing medium (left) or to fructose-containing medium (right) immediately after infection. All tissue cultures received 2 pg/ml actinomycin D. Three hr after infection, pulses with 5 pCi adenosine per culture were started. Radioactivity was determined in the TCA extract (upper panels) as well as in RNA (lower panels). From the latter the corresponding values obtained with noninfected controls (between 5 and 10% of those of infected cells, depending on the pulse length) were subtracted. Open symbols, without glucosamine; closed symbols, with 2 mM glucosamine; circles, without uridine; triangles, with 1 mA4 uridine.
RO’IT AND KLENK
/, 0
12
//I,, 3
L 5 01 mM Glucosomlne
, , j 2
3
L
5
FIG. 5. Effect of glucosamine on cellular RNA and protein synthesis using different media. Chick embryo cells were incubated 43 hr after seeding with different doses of glucosamine (abscissa) using medium containing either glucose (0, n ), pyruvate (0, l ), or fructose (A, A). Four hr later pulses were started with either 2 &i adenosine (15 min) or I .2 &i leucine containing 2 pg nonlabeled leucine (30 min)/culture. Open symbols, radioactivity in RNA (left) or protein (right); closed symbols, radioactivity in the TCAsoluble pool.
incubated in glucose-containing medium (Kaluza et al., 1972). These results have been confirmed for SFV and FPV as shown in Fig. 4B and Table 2. In Fig. 4 the effect of glucosamine on SFV-RNA synthesis in chick embryo cells is described. In this sytem [‘Hladenosine has been used as label and actinomycin D has been employed for the specific suppression of cellular RNA synthesis. As shown in the upper panel of Fig. 4 the amino sugar has no significant effect on the uptake of [SH]adenosine into the cell. In the lower panels the incorporation into viral RNA is shown. As can be seen on the right side (Fig. 4D) there is a reduction of about 80% in viral RNA synthesis when 2 mM glucosamine was added to fructose-containing medium. There is no such effect in glucose-containing medium (Fig. 4B). The effect of glucosamine on FPV-RNA synthesis has been analyzed using the technique of specific hybridization of [3H]uridine-labeled virus-specific RNA with a surplus of either nonlabeled virion or complementary RNA (Scholtissek and Rott, 1970). In this case [3H]adenosine is not a suitable label for the hybridization procedure because there is a high background after RNA digestion due to nondi-
GLUCOSAMINE
197
AND VIRUS MULTIPLICATION
gestable oligo(A) stretches. As shown in Table 2 in fructose- or pyruvate-containing medium there is a 70-97s inhibition of virion RNA synthesis. The effect on complementary RNA is less pronounced. The time after infection at which the pulse is started and the pulse length might influence the yield of labeled virus-specific RNA and is probably responsible for the somewhat variable effect of the amino sugar, especially concerning the complementary RNA. Glucosamine is effective even late after infection when the viral RNA polymerase is already present (Scholtissek and Rott, 1969). The results obtained so far are compatible with the concept that the inhibition of viral RNA synthesis by glucosamine is due to a depletion of the UTP pool. If so, it should be possible to counteract this effect by uridine. This hypothesis has been tested by the experiments included in Fig. 4. Uridine at the concentration investigated interferes somewhat with the uptake of [‘Hladenosine (upper panel) (Plagemann, 1971). There is a corresponding decrease in the incorporation of labeled adenosine into viral RNA. If uridine is present in the medium, glucosamine has almost no effect on the incorporation of labeled adenosine into viral RNA. This indicates that uridine is able to reverse the effect of the amino sugar on viral RNA synthesis. In Table 3 it is shown that the reversal is specific for uridine, because guanosine or cytidine is ineffective. When higher doses of glucosamine were investigated, the reversal by uridine was less pronounced.
TABLE 3 REVERSAL OF THE GLUCOSAMINE EFFECT ON SYNTHESIS BY NUCLEOSIDES~
Glucosamine bM)
Uridine Guanosine Cytidine
2 2 2 2 2
Uridine Guanosine Cytidine Uridine”
0
dpm x 10’
N;;leoeoe
0 0 0
-
SFV-RNA
RNA
TCA extract
178
2,560 1,720 1,610 2,040
180 170
180 16 76
9 9 44
2,400 1,745 1,860 2,140 1,800
0 Chick embryo cells were infected by SFV 48 hr after seeding. Immediately after infection 2 mM glucosamine and 2 &ml actinomycin were added as listed in the table. One mM of the corresponding nucleosides was added either immediately after infection or 2 hr later. A pulse of 30 min length with 5 &i/culture of [sH]adenosine was started 3 hr after infection. Further details see Fig. 4. bAdded 2 hr after infection.
by the amino sugar as shown by the polyacrylamide gel patterns presented in Fig. 6. The data demonstrate that the inhibition is dose-dependent. In pyruvate-containing media corresponding results were obtained (not shown here). The inhibitory effect of glucosamine on protein synthesis as detected by polyacrylamide gel electrophoresis could not be counteracted significantly by uridine except for a small effect on the NP and M proteins (Fig. 6C). The effect of glucosamine on the induction of RNA-dependent RNA polymerase Effect of Glucosamine on the Synthesis of of FPV has also been tested. As shown in FPV Proteins and Glycoproteins Using Table 4, in glucose-containing medium Different Media glucosamine has no effect on the induction Recently it has been shown that glucosa- of this virus-specific enzyme which is in mine has a specific inhibitory effect on the accordance with earlier observations (Kasynthesis of viral glycoproteins. Under luza et al., 1972). In fructose- or pyruvatecontaining medium, however, the induccomparable conditions carbohydrate-free proteins were produced in almost normal tion of the enzyme is highly suppressed by yields. In all these studied glucose was glucosamine as indicated by a lower incorpresent in the medium (Kaluza et al., 1972; poration of [3H]GTP into RNA during a 15 Gandhi et al., 1972; Klenk et al., 1972). In min pulse in uitro. This is also shown in fructose-containing medium, however, the Fig. 7, where the time course of the in vitro synthesis of all FPV-proteins is inhibited incorporation of [3H]GTP into RNA has
198
SCHOLTISSEK,
ROTT AND KLENK
is due to an impairment of viral glycoprotein synthesis (Kaluza et al., 1972; Gandhi et al., 1972; Klenk et al., 1972). The data reported now demonstrate the different effect of glucosamine when glucose is replaced by fructose or pyruvate in the culture medium. Under these conditions the multiplication of NDV and VW as well as SFV and FPV is inhibited. The data indicate that the effect is due to an inhibition TABLE 4 EFFECT OF GLUCOSAMINE ON THE INDUCTION OF FPV-RNA POLYMERASEIN DIFFERENT MEDIAN
61
Medium (10 mM)
Glucosamine (mM)
dpm in RNA
HA titer
Glucose
0 5 0 5 0 2 5
22,600 21,600 30,000 8,200 30,000 14,600 4,700
256 8 512 16 512 128 1
1
Frocton
number
FIG. 6. Effect of glucosamine on the synthesis of FPV proteins in fructose-containing medium. Chick embryo cells were infected with FPV 48 hr after seeding. Glucosamine in various concentrations was added immediately after infection into the medium containing fructose. A, no glucosamine; B, 2 mM glucosamine; C, 5 mM glucosamine (O&l. To one infected culture 2 mM uridine plus 5 mM glucosamine were added (crosses in 60. Four hr after infection a pulse with a mixture of tritium-labeled amino acids was started. One hr later samples were prepared for gel electrophoresis. The nomenclature for the various viral polypeptides was adapted from Kilbourne et al. (1972). HA, hemagglutinin precursor; NP, nucleocapsid protein; HA,, large subunit of hemagglutinin; HA,, small subunit of hemagglutinin; M, matrix protein.
Pyruvate
Fructose
“Cytoplasmic fractions were prepared 5 hr after infection. Aliquots of 0.5 ml were incubated at 32” with 0.5 ml [3H]GTP plus cofactors for 15 min. The protein concentration of the cytoplasm was 1.4 mg/
been studied. When low doses of glucosamine were employed, the inhibition of the enzyme activity could be released by uridine. This was not possible at higher conMowtes centrations of the inhibitor. Corresponding FIG. 7. Effect of glucosamine on the induction of results have been obtained in two other FPV-RNA polymerase. Reversal by uridine. For each experiments. DISCUSSION
Glucosamine interferes with the multiplication of several enveloped viruses (FPV and SFV), while others such as WV and NDV are not influenced by this amino sugar. In these studies medium containing glucose has been used, and it can be shown that the inhibition of virus multiplication
dose, 4 cultures were infected with FPV 48 hr after seeding. Glucosamine and uridine were added to the fructose medium immediately after infection. Five hr later cytoplasmic extracts (1.2 mg/ml protein) were prepared, 0.5 ml aliquots were mixed with [sH]GTP plus cofactors and were incubated at 32” for different lengths of time. 0, no glucosamine and no uridine; 0, 1 mM glucosamine; A, 3 mM glucosamine; 0, = 1 mM glucosamine plus 1 mM uridine; A, = 3 mM glucosamine plus 2 mM uridine.
GLUCOSAMINE
AND VIRUS MULTIPLICATION
of viral RNA synthesis. As a consequence the formation of all viral proteins is inhibited. It can be assumed that this inhibition of viral RNA synthesis is caused by a depletion of the UTP pool because the glucosamine effect on the UTP pool is about 10 times stronger in fructose- or pyruvate-containing medium than in glucose-containing medium. This concept is further supported by the finding that the block in viral RNA synthesis can be released by uridine, but not by other nucleosides. It is not yet clear why the reversal of the block in viral RNA synthesis is not paralleled by a corresponding reversal of the block in protein synthesis, This phenomenon is even less understandable in the light of the finding that under comparable conditions in noninfected cells protein synthesis is not affected (Scholtissek, unpublished results). It is interesting that conditions which almost completely abolish viral RNA synthesis do not significantly impair RNA and protein synthesis in noninfected cells. This suggests that either the viral RNA polymerase tested has a lower affinity for UTP than the cellular enzyme or that two more or less independent UTP pools exist in the host cell. One of these pools would be used for viral RNA synthesis and is emptied easily by glucosamine, v$hile the other pool would be used only for cellular RNA synthesis and is less affected by the amino sugar. Different UTP pools or different affinities of RNA polymerase could also explain the higher sensitivity of FPV virion RNA synthesis for glucosamine as compared to complementary RNA synthesis.
199
oviruses. Biochim. Biophys. Acta 128,547-558. GANDHI, S. S., STANLEY,P., TAYLOR, S. M., and WHITE, D. 0. (1972). Inhibition of influenza virus glycopmtein synthesis by sugars. Microbios 5, 41-50.
GOOD,N. E., WINGET,C. D., WINTER,W., CONOLLY,T. N., ISAWA,S., and SINGH,R. M. M. (1966). Hydrogen ion buffers for biological research. Biochemistry 5,467-477.
HURLBERT,R. B., SCHMITZ,H., BRUMM, A. F., and POTTER,V. R. (1954). Nucleotide metabolism. II. Chromatographic separation of acid-soluble nucleotides. J. Biol. Chem. 209, 23-39. KALUZA, G., SCHOLTISSEK, C., and Roar, R. (1972). Inhibition of the multiplication of enveloped RNA viruses by glucosamine and 2-deoxy-n-glucose. J. Gen. Viral. 14, 251-259.
KALUZA,G., SCHMIDT,M. F. G., and SCHOLTISSEK, C. (1973). Effect of 2-deoxy-n-glucose on the multiplication of Semliki Forest virus and the reversal of the block by mannose. Virology 54, 179-189. KILBOURNE,E. D. (1959). Inhibition of influenza virus multiplication with a glucose antimetabolite (2-deoxy-o-glucose). Nature (London) 183,271-272. KILBOURNE,E. D., CHOPPIN,P. W., SCHULZE,I. T., SCHOLTISSEK, C., and BUCHER,D. L. (1972). Influenza virus polypeptides and antigens. J. Infect. Dis. 125, 447-455.
KLENK, H.-D., CALIGUIRI,L. A., and CHOPPIN,P. W. (1970). The proteins of parainfluenza virus SV 5. II. The carbohydrate content and glycoproteins of the virion. Virology 42, 473-481. KLENK, H.-D., SCHOLTISSEK, C., and ROIT, R. (1972). Inhibition of glycoprotein biosynthesis of influenza virus by D-glUCOSaXIh? and 2-deoxy-o-glucose. Virology 49, 723-734. KLENK, H.-D., WBLLERT,W., SCHOLTISSEK, C., and Rorr, R. (1974). Association of influenza virus proteins with cytoplasmic fractions. Virology 57, 28-41. LAMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227,680-685. PLAGEMANN, P. G. W. (1971). Nucleoside transport by ACKNOWLEDGMENTS Novikoff rat hepatoma cells growing in suspension culture. Specificity and mechanism of transport We thank Miss M. Orlich, Mrs. B. Homann, and reactions and relationship to nucleoside incorporaMiss E. Otto for their excellent technical assistance. tion into nucleic acids. Biochim. Biophys. Acta 233, This work was supported by the Sonderforschungs688-701. bereich 47 (Virologie). SCHOLTISSEK, C. (1969). Synthesis in vitro of RNA REFERENCES complementary to parental viral RNA by RNA COURTNEY,R. J., STEINER, S. M., and BENYESH- polymerase induced by influenza virus. Biochim. MELNICK,M. (1973). Effect of 2-deoxy-n-glucose on Biophys. Acta 179, 389-397. herpes simplex virus replication. Virology 52, SCHOLTISSEK, C. (1971). Detection of an unstable RNA 447-455. in chick fibroblasts after reduction of the UTP-pool DRZENIEK, R., SETO, J. T., and Roar, R. (1966). by glucosamine. Eur. J. Biochem. 24, 358-365. Characterization of neuraminidase from myx- SCHOLTISSEK, C. (1972). Influence of glucosamine on
200
SCHOLTISSEK,
the uptake of nucleosides by chick fibroblasts and on the incorporation into RNA. Biochim. Biophys. Acta 277, 459-465. SCHOLTISSEK, C. (1973). Glycoprotein synthesis of enveloped viruses. Aduan. Biosciences 11,193-203. SCHOLTISSEK, C., and Row, R. (1969). Ribonucleic acid nucleotidyl transferase induced in chick fibroblasts after infection with an influenza virus. J. Gen. Virol. 4, 125-137. SCHOLTISSEK, C., and ROTT, R. (1970). Synthesis in
RO’IT AND KLENK vivo of influenza virus plus and minus strand RNA and its preferential inhibition by antibiotics. Virology 40,989-996. SCHOLTISSEK, C., ROIT, R., HAU, G., and KALUZA, G. (1974). Inhibition of the multiplication of vesicular stomatitis and Newcastle disease virus by 2-deoxyD-glUC0.W. J. Viral. 13, 1166-1193. ZIMMERMANN, T., and SCH~EX., W. (1960). Effect of p-fluorophenylalanine on fowl plague virus multiplication. Virology 11, 676-698.
63, 191-200 (1975)
Two Different
Mechanisms of Enveloped
of the Inhibition Viruses
C. SCHOLTISSEK, Institut
by Glucosamine
R. ROTT, AND H.-D. KLENK
fiir Virologie, Justus Liebig-Uniuersitiit, Accepted
of the Multiplication
September
Giessen, Germany
3, 1974
In Earle’s medium containing 10 mA4glucose, glucosamine inhibits the multiplication of fowl plague virus (FPV) and Semliki Forest virus (SFV) by interfering with the synthesis of viral glycoproteins, while the synthesis of viral RNA and carbohydrate-free proteins is affected only slightly or not at all. If glucose is replaced by fructose, glucosamine inhibits the synthesis of viral RNA and in this way interferes with the production of all viral proteins. Protein and RNA synthesis of non-infected cells are not significantly impaired under these conditions. The effect on viral RNA synthesis can be readily counteracted by uridine. The inhibition of viral glycoprotein synthesis cannot be reversed at all by uridine and that of viral carbohydrate-free proteins only to a very limited extent. In fructose-containing medium, glucosamine depletes the cells of UTP in such a way that the UTP pool may become rate limiting for viral RNA synthesis but not for cellular RNA synthesis. The multiplication of Newcastle disease virus (NDV) and vesicular stomatitis virus (VW) is inhibited by glucosamine only in fructose-containing medium but not in glucose-containing medium.
containing either glucose, pyruvate or fructose as an energy source. It will be shown Recently it has been shown that glucosathat in glucose-containing medium the mine as well as 2-deoxy-D-glucose inhibits the multiplication of various enveloped effect of the amino sugar is only on the animal viruses by interference with the synthesis of glycoproteins of FPV and SFV leaving synthesis of viral RNA and protein synthesis of viral glycoproteins (Kilbourne, 1959; Kaluza et al., 1972; Gandhi et al., almost intact, while in pyruvate- and fruc1972; Klenk et al., 1972; Courtney et al., tose-containing media the effect is on the 1973; Kaluza et al., 1973; Scholtissek et al., synthesis of viral RNA and of all viral 1974; Klenk et al., 1974). Because 10 mM proteins. Under these latter conditions proglucosamine in glucose-containing medium tein and RNA synthesis in non-infected reduces the UTP pool of the host cell by a cells is almost normal. factor of about 8 (Scholtissek, 1971, 1973), MATERIALS AND METHODS it was suggested that the amino sugar might interfere with the activation of other Virus strains. Fowl plague virus (FPV), sugars via depletion of the UTP pool strain “Restock”; Semliki Forest virus (Klenk et al., 1972). In most of these (SFV), strain “Osterrieth”; vesicular stostudies, medium containing glucose has matitis virus (VSV), strain “Indiana”; and been used; glucose competes with the up- Newcastle disease virus (NDV), strain take and activation of the sugar derivatives “Italien”, were used. FPV and NDV were mentioned. grown by serial passage in embryonated In this paper we compare the effect of eggs, while SFV and VSV were propagated glucosamine on virus multiplication with in primary chick embryo cells at a multiits effect on the UTP pool by using media plicity below one. INTRODUCTION
Copyright 0 1975 by AcademicPress,Inc. All rights of reproduction in any form reserved.
191
192
SCHOLTISSEK,
Tissue cultures. Primary chick embryo cells prepared from 11-day-old embryos were used either 24, 48 or 72 hr after seeding at a cell density of either 6 x lo6 cells in Petri dishes of 5 cm in diameter or 2 x lo7 cells in Petri dishes of 9 cm in diameter. In order to start each experiment with the same number of cells for the 24-hr cultures, 3 x 10’ cells/ml were seeded; for the 46 or 72-hr cultures, 1.5 x lo6 cells/ml were seeded. The cells were infected with a multiplicity of lo-50 PFU/cell. After infection the cells were overlayed with 2 or 6 ml, respectively, of Earle’s medium without glucose. The various sugars or pyruvate as listed in the tables and figures were added to a final concentration of 10 mM. In most experiments, especially when pyruvate medium was investigated, sodium bicarbonate was replaced by Hepes buffer, pH 7.4 (Good et al., 1966). Under these conditions CO, was omitted from the incubator. This became necessary when it was found that pyruvate was toxic for virus multiplication at slightly alkaline pH. Otherwise virus yields were not influenced by the different buffer systems. Virus quantitation. Plaque assays and hemagglutination (HA) titration were carried out according to standard procedures (Zimmermann and Schafer, 1960). Neuraminidase activity. Enzymatic activity was determined as before (Drzeniek et al., 1966) using bovine sialolactose as substrate. The amount of neuraminidase which liberated 1 micromole of sialic acid from the substrate per min at 37” was defined as one enzyme unit. Labeling of protein and RNA. Labeling was done by incubating cells with the respective tritium-labeled precursors. At the time indicated, the radioactivity was determined in RNA, in protein, and in the trichloroacetic acid (TCA)-soluble pool as described recently (Scholtissek, 1971). Polyacrylamide gel electrophoresis. The cells (6 x lop/gel), labeled with a mixture of tritiated amino acids (50 PCilml) from 4 to 5 hr after infection with FPV, were homogenized by sonication. Samples of 0.1 ml were used for gel electrophoresis. Proteins were dissociated with sodium dodecyl
ROTT
AND KLENK
sulfate and mercaptoethanol and separated by electrophoresis on 10%acrylamide gels (Llmmli, 1970). The slicing and processing of gels for the determination of radioactivity by liquid scintillation has been reported previously (Klenk et al., 1970). The determination of viral RNA polymerase. Viral RNA polymerase was deter-
mined as described previously (Scholtissek, 1969). A cytoplasmic fraction prepared 5 hr after infection with FPV was incubated at 32” for different lengths of time with [3H]GTP plus cofactors. The radioactivity was determined in the TCA precipitate. The synthesis
of viral
RNA
in vivo.
Synthesis was followed by incorporation of the labeled nucleoside at various times after infection. For SFV 2 pg./ml of actinomycin D was added immediately after infection and [3H]adenosine was added 3 hr after infection. At different times thereafter cultures were washed three times with phosphate-buffered saline. Radioactivity was determined in the TCA-soluble pool and in RNA (Scholtissek, 1971). For FPV, t3H]uridine was added to the cultures as indicated. At the end of the pulse total RNA was extracted from three cultures by phenol plus 1% sodium dodecyl sulfate at room temperature and was dissolved in 2 ml of 5 mM Tris-HCl buffer, pH 8, containing 1 mM EDTA. Aliquots of 0.1 ml were used for the determination of total radioactivity and for the hybridization procedure after heating for 10 min at 100” and rapid cooling to 0”. Nonlabeled virion RNA was isolated from purified virus particles and was dissolved in 2 x SSC (0.3 M NaCl, 0.03 A4 sodium citrate) to give a final concentration of 0.75 mg/ml RNA. Either 10 or 25 ~1of virion RNA were mixed with the aliquots for hybridization at 65” overnight. Nonlabeled complementary RNA was prepared by incubating a microsomal fraction isolated 5 hr after infection of 50 tissue cultures with FPV in the presence of all four nucleoside triphosphates and 4 mM MgCl, in Tris-HCl buffer, pH 8, at 36” for 20 min. Thereafter the microsomes were centrifuged at 100,000 g at 4” for 90 min. The RNA was extracted
GLUCOSAMINE
193
AND VIRUS MULTIPLICATION
from the supernatant by phenol and was taken up in 2 x SSC to give a concentration of 4.0-4.5 mg RNA/ml. Most of this RNA is microsomal RNA. The actual amount of complementary RNA is not known. Traces of virion RNA in this preparation were bound to complementary RNA forming double-stranded RNA by short heating to 80” and slow cooling to room temperature. Either 50 or 100 ~1 of this RNA solution were mixed with aliquots for hybridization in order to determine the relative amount of labeled virion RNA. Each batch of the respective nonlabeled viral RNA was titrated against viral RNA labeled in uivo from 4 to 5 hr after infection using the same hybridization technique. As a rule, saturation was obtained either with 5 ~1 of nonlabeled virion RNA or 25 ~1 of complementary RNA, respectively. Corresponding saturation curves have been published (Scholtissek and Rott, 1970). The values in Table 2 represent average values of two determinations using the two different concentrations of nonlabeled viral RNA. The highest deviation from each average value was 5%. A more detailed description of the hybridization procedure has been described (Scholtissek and Rott, 1970). Materials. Labeled uridine, [5-3H]uridine, (29 Ci/mmole); [3H]adenosine, generally labeled (0.5 Wmmole); and the tritiated amino acid mixture (TRK 440) were purchased from the Radiochemical Centre, Amersham, England. [8-‘H]GTP (0.80 Ci/mmole) was obtained from Schwarz/Mann, Orangeburg, NY. Dglucosamine-HCl and actinomycin D were obtained from Serva, Heidelberg, Germany. RESULTS
Effect of Glucosamine on the UTP Pool Using Different Media and Cells of Different Age It has been suggested recently (Klenk et al., 1972) that the depletion of the UTP pool by glucosamine might be the fundamental mechanism by which its inhibitory effect is exerted on the production of severa1 enveloped viruses. It is known that the
extent of the effect of glucosamine on the UTP pool depends on the age of the cell (Scholtissek, 1971). In order to find the optimal conditions, this effect was tested at various times after seeding of the cells. In addition, glucose was replaced in the culture medium by fructose, because glucose competes with the uptake of glucosamine. As shown in Table 1, glucosamine enhances the uptake of labeled uridine into noninfected cells (total TCA extract). This can be explained by the finding that the UTP pool regulates the uptake of uridine by negative feedback (Scholtissek, TABLE
1
EFFECT OF GLUCOSAMINE ON THE LABELING OF DIFFERENT COMPOUNDS BY [3H]U~~~~~~ IN MEDIA CONTAINING EITHER GLUCOSE OR FRUCTOSE
Age of cells”
Medium (10mM)
Glucosamine (miW
dpm x 10’ in To;?
RNA
UTP/ UDPsugar
extract Glucose
0 0.08 1 10
404 426 613 784
57 60 92 150
1.88 1.79 1.00 0.68
0 0.08 l 10
405 527 853 882
60 75 125 150
2.42 1.30 0.58 0.44
0 0.08 1 10
498 540 840 1042
70 76 115 146
1.63 1.52 0.79 0.48
0 0.08 1 10
470 784 1084 760
61 103 140 184
1.85 0.85 0.54 0.25
24 hr Fructose
Glucose
48 hr Fructose
’ Chick embryo cells were used either 24 hr or 48 hr after seeding. Earle’s medium without glucose was supplemented with the corresponding sugar as listed in the Table. After 90-min incubation at 37”, IO&i of [BH]uridine were added; 40 min later the cells were processed and the neutralized TCA extracts were analyzed on Dowex columns (Hurlbert et al., 1954; Scholtissek, 1971). The material of two cultures was pooled. The radioactivity in UMP and UDP was negligible.
194
SCHOLTISSEK,
1972). Because of the facilitated uptake of the label and the relative decrease of the UTP pool caused by glucosamine, the specific radioactivity of UTP increases (Scholtissek, 1971). Therefore the incorporation of labeled uridine into RNA is significantly enhanced (Table 1). The relative rise in the radioactivity of UDP-N-acetylglucosamine indicates that most of the UTP is used for the synthesis of this activated amino sugar derivative. Comparing 24-hr-old cells with those of 48 hr of age it can be seen that these effects are expressed more extensively in the older cells. Recently it has been shown that in medium containing 10 n&f glucose, 10 mA4 glucosamine reduces the UTP pool by a factor of about 8 using cells 48 hr after seeding (Scholtissek, 1971, 1973). In fructose-containing medium one needs only about one-tenth of the concentration of glucosamine in order to get the same effect on the UTP pool as compared to glucose-containing medium. In pyruvate-containing medium one observes an intermediate effect (not shown).
ROTI AND KLENK
012
5
10
012 5 x)0125 mM Glucosamlne
II
FIG. 1. Dose response of glucosamine on the multiplication of FPV in different media. Chick embryo cells were infected with FPV 48 hr after seeding. Concentrations of glucosamine as listed on the abscissas were added to the media containing either fructose (A), glucose (0) or pyruvate (0) immediately after infection. Eight hr later the various cell-bound virus activities were determined. There was no effect of glucosamine on virus release.
Effect of Glucosamine Concentration on the Multiplication of Enveloped Viruses in Different Media and on Cells of Different Age
Since the sugar used as energy source for the growth of the cells plays an important role on the effect of glucosamine on the UTP pool, sugar-free Earle’s medium supplemented with 10 mmoles of glucose, pyruvate or fructose was investigated to measure the effect of the amino sugar on multiplication of enveloped viruses. As shown for FPV in Fig. 1 the effect of glucosamine on the yield of infectious virus, on hemagglutinin (HA), and on neuraminidase is highest in fructose-containing medium and lowest in glucose-containing medium. This effect can be reversed completely at least up to 4 hr after infection by replacement with medium without glucosamine (not shown here). Corresponding results have been obtained with SFVinfected chick embryo cells. As shown in Fig. 2 the multiplication of NDV and VSV can be inhibited by glucosamine if fructose-containing medium is
0
2
L
68x)021
6
8
10
mM Glucosamlne
FIG. 2. Dose response of glucosamine on the multiplications of NDV (left) and VSV (right) in different media. Chick embryo cells were infected with either NDV or VSV 48 hr after seeding. Doses of glucosamine as listed on the abscissas were added immediately after infection. Eight hours later either the HA-titer (NDV) or the p.f.u. (VSV) was determined. 0, glucose-containing medium; A, fructose-containing medium.
used. In glucose-containing medium there is no measurable inhibition which is in accord with recent observations (Kaluza et al., 1972). Since in uninfected cells the effect of glucosamine on the UTP pool depends on the age of the cells used (Table l), it was of interest to see whether such a dependence also exists in the case of virus multiplication. There were some fluctuations in the sensitivity of virus multiplication to gluco-
GLUCOSAMINE
AND
VIRUS
195
MULTIPLICATION
samine when different batches of cells were strated in Table 1 (see also Scholtissek, used at different times after seeding. It 1971, 1973) RNA synthesis cannot be meawas, however, a constant finding that cells sured quantitatively by incorporation of infected 24 hr after seeding were much less sensitive than cells infected 48 hr after seeding. If cells were investigated even later glucosamine was again less effective. The results of a typical experiment on the inhibition of the multiplication of FPV and SFV by glucosamine using cells of the same batch at different times after seeding can be seen in Fig. 3. The transport and/or enzyme systems involved in glucosamine metabolism seem to work optimally in cells 48 hr after seeding. Therefore in all further mM Glucosomlne experiments cells of this age were used. Effect of Glucosamine on Cellular Viral RNA Synthesis
and
As shown in Table 2 glucosamine markedly increases the incorporation of labeled uridine into total RNA. Since the amino sugar affects the UTP pool as demon-
FIG. 3. Role of the age of the cultures on virus yield. Dose response of glucosamine. Chick embryo cells were infected either with FPV (left) or with SFV (right) at different times after seeding. Different doses of glucosamine (abscissa) were added to the fructosecontaining medium immediately after infection. Eight hr later the cell-bound activities were determined. 0, 24-hr-old cells; 0, 48hr-old cells; A, 72-hr-old cells.
TABLE EFFECT OF GLUCOSAMINE
Glucosamine (mm
ExperimentR
Glucose 1
3
Pulse from . . till . hours after infection
SYNTHESIS
in Vivo
Total RNA (dpm x 103)
Virion RNA (% oftotal)
Complementary RNA (% of total)
0 5 0 5 5
3-4 3-4 3-4 3-4 3-4
24 62 27 92 84
23.3 21.8 21.0 6.7 1.5
4.6 8.8 4.7 4.1 5.2
Fructose
0 5 0 5 5*
2-3 2-3 4-5 4-5 4-5
64 136 50 109 59
14.0 0.4 29.0 4.6 8.5
9.4 1.3 7.6 4.6 4.5
Fructose
0 5 0 5
3-3.5 3-3.5 3-4.33 3-4.33
28 74 94 187
30.0 5.2 37.0 3.0
5.8 4.3 10.6 3.0
Fructose Pyruvate
2
2
ON FPV-RNA
DIf not otherwise stated, 5 mM glucosamine was added immediately after infection with FPV into Earle’s medium containing Hepes buffer and 10 mM of the energy source listed. Pulses with [sH]uridine, 25 pCi/culture, were applied as listed in the Table. Each value represents the average of three cultures. The percentage of FPV-RNA was determined by hybridization with a surplus of either nonlabeled virion RNA or complementary RNA. Before calculation the corresponding values obtained from noninfected controls were substracted (Scholtissek and Rott, 19703. b Glucosamine was added 2.7 hr after infection.
196
SCHOLTISSEK,
[SH]uridine unless corresponding corrections on pool size etc. are made. Incorporation of [‘Hladenosine can be used, however, to study the effect of glucosamine on RNA synthesis because glucosamine has neither a significant effect on the uptake of adenosine (Fig. 4A and 4C) nor on the pool size of ATP (Scholtissek, 1971, 1972). As shown in Fig. 5 glucosamine has no significant effect on RNA synthesis of noninfected cells regardless of the sugar present in the medium. For comparison, the effect of glucosamine on the incorporation of [3H]leucine has been measured (Fig. 5). There is also no significant effect of the amino sugar on protein synthesis. It has been shown recently that glucosamine has either no or only a slight effect on viral RNA synthesis if the infected cells are
FIG. 4. Effect of glucosamine on SFV-RNA synthesis in uiuo and its reversal by uridine. Chick embryo cells were infected with SFV 48 hr after seeding. Two mM glucosamine and/or 1 mM uridine were added either to glucose-containing medium (left) or to fructose-containing medium (right) immediately after infection. All tissue cultures received 2 pg/ml actinomycin D. Three hr after infection, pulses with 5 pCi adenosine per culture were started. Radioactivity was determined in the TCA extract (upper panels) as well as in RNA (lower panels). From the latter the corresponding values obtained with noninfected controls (between 5 and 10% of those of infected cells, depending on the pulse length) were subtracted. Open symbols, without glucosamine; closed symbols, with 2 mM glucosamine; circles, without uridine; triangles, with 1 mA4 uridine.
RO’IT AND KLENK
/, 0
12
//I,, 3
L 5 01 mM Glucosomlne
, , j 2
3
L
5
FIG. 5. Effect of glucosamine on cellular RNA and protein synthesis using different media. Chick embryo cells were incubated 43 hr after seeding with different doses of glucosamine (abscissa) using medium containing either glucose (0, n ), pyruvate (0, l ), or fructose (A, A). Four hr later pulses were started with either 2 &i adenosine (15 min) or I .2 &i leucine containing 2 pg nonlabeled leucine (30 min)/culture. Open symbols, radioactivity in RNA (left) or protein (right); closed symbols, radioactivity in the TCAsoluble pool.
incubated in glucose-containing medium (Kaluza et al., 1972). These results have been confirmed for SFV and FPV as shown in Fig. 4B and Table 2. In Fig. 4 the effect of glucosamine on SFV-RNA synthesis in chick embryo cells is described. In this sytem [‘Hladenosine has been used as label and actinomycin D has been employed for the specific suppression of cellular RNA synthesis. As shown in the upper panel of Fig. 4 the amino sugar has no significant effect on the uptake of [SH]adenosine into the cell. In the lower panels the incorporation into viral RNA is shown. As can be seen on the right side (Fig. 4D) there is a reduction of about 80% in viral RNA synthesis when 2 mM glucosamine was added to fructose-containing medium. There is no such effect in glucose-containing medium (Fig. 4B). The effect of glucosamine on FPV-RNA synthesis has been analyzed using the technique of specific hybridization of [3H]uridine-labeled virus-specific RNA with a surplus of either nonlabeled virion or complementary RNA (Scholtissek and Rott, 1970). In this case [3H]adenosine is not a suitable label for the hybridization procedure because there is a high background after RNA digestion due to nondi-
GLUCOSAMINE
197
AND VIRUS MULTIPLICATION
gestable oligo(A) stretches. As shown in Table 2 in fructose- or pyruvate-containing medium there is a 70-97s inhibition of virion RNA synthesis. The effect on complementary RNA is less pronounced. The time after infection at which the pulse is started and the pulse length might influence the yield of labeled virus-specific RNA and is probably responsible for the somewhat variable effect of the amino sugar, especially concerning the complementary RNA. Glucosamine is effective even late after infection when the viral RNA polymerase is already present (Scholtissek and Rott, 1969). The results obtained so far are compatible with the concept that the inhibition of viral RNA synthesis by glucosamine is due to a depletion of the UTP pool. If so, it should be possible to counteract this effect by uridine. This hypothesis has been tested by the experiments included in Fig. 4. Uridine at the concentration investigated interferes somewhat with the uptake of [‘Hladenosine (upper panel) (Plagemann, 1971). There is a corresponding decrease in the incorporation of labeled adenosine into viral RNA. If uridine is present in the medium, glucosamine has almost no effect on the incorporation of labeled adenosine into viral RNA. This indicates that uridine is able to reverse the effect of the amino sugar on viral RNA synthesis. In Table 3 it is shown that the reversal is specific for uridine, because guanosine or cytidine is ineffective. When higher doses of glucosamine were investigated, the reversal by uridine was less pronounced.
TABLE 3 REVERSAL OF THE GLUCOSAMINE EFFECT ON SYNTHESIS BY NUCLEOSIDES~
Glucosamine bM)
Uridine Guanosine Cytidine
2 2 2 2 2
Uridine Guanosine Cytidine Uridine”
0
dpm x 10’
N;;leoeoe
0 0 0
-
SFV-RNA
RNA
TCA extract
178
2,560 1,720 1,610 2,040
180 170
180 16 76
9 9 44
2,400 1,745 1,860 2,140 1,800
0 Chick embryo cells were infected by SFV 48 hr after seeding. Immediately after infection 2 mM glucosamine and 2 &ml actinomycin were added as listed in the table. One mM of the corresponding nucleosides was added either immediately after infection or 2 hr later. A pulse of 30 min length with 5 &i/culture of [sH]adenosine was started 3 hr after infection. Further details see Fig. 4. bAdded 2 hr after infection.
by the amino sugar as shown by the polyacrylamide gel patterns presented in Fig. 6. The data demonstrate that the inhibition is dose-dependent. In pyruvate-containing media corresponding results were obtained (not shown here). The inhibitory effect of glucosamine on protein synthesis as detected by polyacrylamide gel electrophoresis could not be counteracted significantly by uridine except for a small effect on the NP and M proteins (Fig. 6C). The effect of glucosamine on the induction of RNA-dependent RNA polymerase Effect of Glucosamine on the Synthesis of of FPV has also been tested. As shown in FPV Proteins and Glycoproteins Using Table 4, in glucose-containing medium Different Media glucosamine has no effect on the induction Recently it has been shown that glucosa- of this virus-specific enzyme which is in mine has a specific inhibitory effect on the accordance with earlier observations (Kasynthesis of viral glycoproteins. Under luza et al., 1972). In fructose- or pyruvatecontaining medium, however, the induccomparable conditions carbohydrate-free proteins were produced in almost normal tion of the enzyme is highly suppressed by yields. In all these studied glucose was glucosamine as indicated by a lower incorpresent in the medium (Kaluza et al., 1972; poration of [3H]GTP into RNA during a 15 Gandhi et al., 1972; Klenk et al., 1972). In min pulse in uitro. This is also shown in fructose-containing medium, however, the Fig. 7, where the time course of the in vitro synthesis of all FPV-proteins is inhibited incorporation of [3H]GTP into RNA has
198
SCHOLTISSEK,
ROTT AND KLENK
is due to an impairment of viral glycoprotein synthesis (Kaluza et al., 1972; Gandhi et al., 1972; Klenk et al., 1972). The data reported now demonstrate the different effect of glucosamine when glucose is replaced by fructose or pyruvate in the culture medium. Under these conditions the multiplication of NDV and VW as well as SFV and FPV is inhibited. The data indicate that the effect is due to an inhibition TABLE 4 EFFECT OF GLUCOSAMINE ON THE INDUCTION OF FPV-RNA POLYMERASEIN DIFFERENT MEDIAN
61
Medium (10 mM)
Glucosamine (mM)
dpm in RNA
HA titer
Glucose
0 5 0 5 0 2 5
22,600 21,600 30,000 8,200 30,000 14,600 4,700
256 8 512 16 512 128 1
1
Frocton
number
FIG. 6. Effect of glucosamine on the synthesis of FPV proteins in fructose-containing medium. Chick embryo cells were infected with FPV 48 hr after seeding. Glucosamine in various concentrations was added immediately after infection into the medium containing fructose. A, no glucosamine; B, 2 mM glucosamine; C, 5 mM glucosamine (O&l. To one infected culture 2 mM uridine plus 5 mM glucosamine were added (crosses in 60. Four hr after infection a pulse with a mixture of tritium-labeled amino acids was started. One hr later samples were prepared for gel electrophoresis. The nomenclature for the various viral polypeptides was adapted from Kilbourne et al. (1972). HA, hemagglutinin precursor; NP, nucleocapsid protein; HA,, large subunit of hemagglutinin; HA,, small subunit of hemagglutinin; M, matrix protein.
Pyruvate
Fructose
“Cytoplasmic fractions were prepared 5 hr after infection. Aliquots of 0.5 ml were incubated at 32” with 0.5 ml [3H]GTP plus cofactors for 15 min. The protein concentration of the cytoplasm was 1.4 mg/
been studied. When low doses of glucosamine were employed, the inhibition of the enzyme activity could be released by uridine. This was not possible at higher conMowtes centrations of the inhibitor. Corresponding FIG. 7. Effect of glucosamine on the induction of results have been obtained in two other FPV-RNA polymerase. Reversal by uridine. For each experiments. DISCUSSION
Glucosamine interferes with the multiplication of several enveloped viruses (FPV and SFV), while others such as WV and NDV are not influenced by this amino sugar. In these studies medium containing glucose has been used, and it can be shown that the inhibition of virus multiplication
dose, 4 cultures were infected with FPV 48 hr after seeding. Glucosamine and uridine were added to the fructose medium immediately after infection. Five hr later cytoplasmic extracts (1.2 mg/ml protein) were prepared, 0.5 ml aliquots were mixed with [sH]GTP plus cofactors and were incubated at 32” for different lengths of time. 0, no glucosamine and no uridine; 0, 1 mM glucosamine; A, 3 mM glucosamine; 0, = 1 mM glucosamine plus 1 mM uridine; A, = 3 mM glucosamine plus 2 mM uridine.
GLUCOSAMINE
AND VIRUS MULTIPLICATION
of viral RNA synthesis. As a consequence the formation of all viral proteins is inhibited. It can be assumed that this inhibition of viral RNA synthesis is caused by a depletion of the UTP pool because the glucosamine effect on the UTP pool is about 10 times stronger in fructose- or pyruvate-containing medium than in glucose-containing medium. This concept is further supported by the finding that the block in viral RNA synthesis can be released by uridine, but not by other nucleosides. It is not yet clear why the reversal of the block in viral RNA synthesis is not paralleled by a corresponding reversal of the block in protein synthesis, This phenomenon is even less understandable in the light of the finding that under comparable conditions in noninfected cells protein synthesis is not affected (Scholtissek, unpublished results). It is interesting that conditions which almost completely abolish viral RNA synthesis do not significantly impair RNA and protein synthesis in noninfected cells. This suggests that either the viral RNA polymerase tested has a lower affinity for UTP than the cellular enzyme or that two more or less independent UTP pools exist in the host cell. One of these pools would be used for viral RNA synthesis and is emptied easily by glucosamine, v$hile the other pool would be used only for cellular RNA synthesis and is less affected by the amino sugar. Different UTP pools or different affinities of RNA polymerase could also explain the higher sensitivity of FPV virion RNA synthesis for glucosamine as compared to complementary RNA synthesis.
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oviruses. Biochim. Biophys. Acta 128,547-558. GANDHI, S. S., STANLEY,P., TAYLOR, S. M., and WHITE, D. 0. (1972). Inhibition of influenza virus glycopmtein synthesis by sugars. Microbios 5, 41-50.
GOOD,N. E., WINGET,C. D., WINTER,W., CONOLLY,T. N., ISAWA,S., and SINGH,R. M. M. (1966). Hydrogen ion buffers for biological research. Biochemistry 5,467-477.
HURLBERT,R. B., SCHMITZ,H., BRUMM, A. F., and POTTER,V. R. (1954). Nucleotide metabolism. II. Chromatographic separation of acid-soluble nucleotides. J. Biol. Chem. 209, 23-39. KALUZA, G., SCHOLTISSEK, C., and Roar, R. (1972). Inhibition of the multiplication of enveloped RNA viruses by glucosamine and 2-deoxy-n-glucose. J. Gen. Viral. 14, 251-259.
KALUZA,G., SCHMIDT,M. F. G., and SCHOLTISSEK, C. (1973). Effect of 2-deoxy-n-glucose on the multiplication of Semliki Forest virus and the reversal of the block by mannose. Virology 54, 179-189. KILBOURNE,E. D. (1959). Inhibition of influenza virus multiplication with a glucose antimetabolite (2-deoxy-o-glucose). Nature (London) 183,271-272. KILBOURNE,E. D., CHOPPIN,P. W., SCHULZE,I. T., SCHOLTISSEK, C., and BUCHER,D. L. (1972). Influenza virus polypeptides and antigens. J. Infect. Dis. 125, 447-455.
KLENK, H.-D., CALIGUIRI,L. A., and CHOPPIN,P. W. (1970). The proteins of parainfluenza virus SV 5. II. The carbohydrate content and glycoproteins of the virion. Virology 42, 473-481. KLENK, H.-D., SCHOLTISSEK, C., and ROIT, R. (1972). Inhibition of glycoprotein biosynthesis of influenza virus by D-glUCOSaXIh? and 2-deoxy-o-glucose. Virology 49, 723-734. KLENK, H.-D., WBLLERT,W., SCHOLTISSEK, C., and Rorr, R. (1974). Association of influenza virus proteins with cytoplasmic fractions. Virology 57, 28-41. LAMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227,680-685. PLAGEMANN, P. G. W. (1971). Nucleoside transport by ACKNOWLEDGMENTS Novikoff rat hepatoma cells growing in suspension culture. Specificity and mechanism of transport We thank Miss M. Orlich, Mrs. B. Homann, and reactions and relationship to nucleoside incorporaMiss E. Otto for their excellent technical assistance. tion into nucleic acids. Biochim. Biophys. Acta 233, This work was supported by the Sonderforschungs688-701. bereich 47 (Virologie). SCHOLTISSEK, C. (1969). Synthesis in vitro of RNA REFERENCES complementary to parental viral RNA by RNA COURTNEY,R. J., STEINER, S. M., and BENYESH- polymerase induced by influenza virus. Biochim. MELNICK,M. (1973). Effect of 2-deoxy-n-glucose on Biophys. Acta 179, 389-397. herpes simplex virus replication. Virology 52, SCHOLTISSEK, C. (1971). Detection of an unstable RNA 447-455. in chick fibroblasts after reduction of the UTP-pool DRZENIEK, R., SETO, J. T., and Roar, R. (1966). by glucosamine. Eur. J. Biochem. 24, 358-365. Characterization of neuraminidase from myx- SCHOLTISSEK, C. (1972). Influence of glucosamine on
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