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Inhibitors of IMP dehydrogenase prevent sindbis virus replication and reduce GTP levels in Aedes albopictus cells
VIROLOGY
110,
Inhibitors
281-291
(1981)
of IMP Dehydrogenase and Reduce GTP Levels FRANK
Prevent Sindbis Virus Replication in Aedes albopictus Cells
MALINOSKI
Department of Microbiology, Rutgers Medical
AND VICTOR
College of Medicine School, Piscataway, Accepted
October
STOLLAR’
and Dentistry of New New Jemey 08854
Jersey,
30, 1980
We have investigated the mechanism by which ribavirin (Rbv), mycophenolic acid, and 2-amino-1,3,4-thiadiazole inhibit the replication of Sindbis virus in Aedes albopictus cells. In each case there was a good correlation between inhibition of virus replication and a reduction @O-90%) in the level of cellular GTP. The antiviral effects of all three compounds could be reversed by (1) equimolar amounts of xanthosine but not by guanosine, (2) actinomycin D (0.2 pg/ml), and (3) n-amanitin (10 pg/ml) in o-amanitin-sensitive cells but not in o-amanitinresistant cells. In the case of actinomycin D the reversal of the antiviral effects was correlated with a restoration of the GTP pool to near normal levels and a decrease in the amount of phosphorylated Rbv in acid-soluble cell extracts. The role of the phosphorylated forms of Rbv in inhibiting virus replication and the relationship to cellular RNA synthesis are discussed. INTRODUCTION
Ribavirin (1-P-D-ribofuranosyl-1,2,4-triazole-3-carboxamide; Rbv) is a synthetic guanosine analog which inhibits the replication of a wide range of RNA and DNA viruses (Sidwell et al., 1979). Concerning its mechanism of action, evidence has been presented showing (1) that the monophosphate of Rbv inhibits inosine 5’-monophosphate dehydrogenase, the first enzyme specific for the de novo synthesis of GMP (Streeter et al., 1973) and (2) that in murine lymphoma cells treated with Rbv there is a marked reduction in the intracellular level of GTP (Lowe et al., 197’7). It seems reasonable in turn that the decreased level of GTP can explain the depression of nucleic acid synthesis caused by Rbv (Sarver and Stollar, 1978; Malinoski and Stollar, 1980). However, it has also been proposed that the triphosphate of Rbv (which can be detected within 30 min of treatment with Rbv, see below) is the active form of the drug and that it can inhibit either viral RNA polymerase (Eriksson et al., 1977) or viral capping enzymes 1 Author to whom requests for reprints should be addressed. 281
(Goswami et aZ., 1979). Thus, there are at least three possible explanations for the antiviral action of ribavirin: (1) reduction in the amount of intracellular GTP, (2) inhibition by Rbv triphosphate of viral RNA polymerase, or (3) inhibition by Rbv triphosphate of the RNA capping enzyme, guanyltransferase. Earlier work from this laboratory has shown that Rbv inhibits Sindbis virus (SV) replication in Aedes albopictus mosquito cells (Sarver and Stollar, 1978). We also found that in SV-infected A. albopictus cells, Rbv prevents viral nucleic acid synthesis and that inhibition of both viral RNA synthesis and of virus replication can be reversed by actinomycin (Malinoski and Stollar, 1980). We have now extended our studies by testing (1) the effect of Rbv on another enveloped RNA virus, vesicular stomatitis virus, (2) the antiviral effects of two other inhibitors of inosine monophosphate dehydrogenase (IMPDH), mycophenolic acid (Smith et al., 1974) and 2-amino-1,3,4-thiadiazole (Nelson et al., 1977), and (3) the effects of these inhibitors on the intracellular nucleotide pools of mosquito cells. 0042~6322/81/060281-11$02.00/O Copyright All rights
0 1981 by Academic Press, Inc. of reproduction in any form reserved.
282
MALINOSKI
Our results indicate that mycophenolic acid (MPA) and 2-amino-1,3,4-thiadiazole (TDA), like Rbv, prevent virus replication in A. albopictus cells. Under the various experimental conditions examined and with each of the three inhibitors of IMP dehydrogenase, we found a good correlation between inhibition of virus replication and a decrease in cellular GTP levels. MATERIALS
AND
METHODS
Cells, media, and viruses. Primary chick embryo fibroblast cultures were prepared as described elsewhere (Stollar et al., 1976). Aedes albopictus mosquito cells, clone LT C-7 (Sarver and Stollar, 1978), and the a-amanitin-resistant A. albopictus cell line, Ama(Mento and Stollar, 19’79), were grown at 28” in E medium (Sarver and Stollar, 1978) supplemented with heatinactivated (56”, 30 min) fetal calf serum. Cells were counted using a Coulter counter, Model ZBI. Plaque-purified stocks of Sindbis virus (SV) and vesicular stomatitis virus, Indiana serotype, (VW), were prepared as described elsewhere (Stollar et al., 1972; Shenk and Stollar, 1972; Gillies and Stollar, 1980). Cells were infected with virus at an input multiplicity of approximately 10 PFU/ cell. After adsorption (45 min, 34.5”, 5% CO,) the inoculum was removed, the monolayers were washed with phosphate-buffered saline (PBS), and the cultures were fed with E medium containing 10% fetal calf serum (E-10). Infected cells were incubated at 34.5” unless otherwise stated. Virus titers were measured by plaque assay at 34.5” (Shenk et al., 1974) on chick embryo fibroblasts in the case of SV or on BHK cells in the case of VSV. Analysis of acid-soluble nucleotides. A. albopictus cells were seeded into 60-mm tissue culture dishes at low densities (5 x lo5 cells per dish) in medium E-10 containing 5 to 10 PCi 32P04/m1. When cells reached confluency (after approximately four population doublings or about 3 to 4 days after seeding) cultures were treated with a specific inhibitor of IMP dehydrogenase for the indicated period of time and then analyzed as outlined below.
AND
STOLLAR
Acid-soluble material was extracted from cell cultures following the procedure of Hershfield and Seegmiller (1977). Cells (57 x 106) were scraped from the dishes, pelleted, rinsed twice with cold PBS, and then resuspended in 200 ~1 PBS lacking calcium and magnesium. Perchloric acid, 4 M (20 pl), was added and samples were kept at 4” for 15 to 30 min. Acid-insoluble material was removed by centrifugation (1800 g, 15 min, 4”) and the supernatant was brought to pH 7 with 4 M KOH. Insoluble KClO, was removed by another centrifugation as above and the supernatant acid-soluble extract was collected. Samples were analyzed by ascending chromatography on polyethyleneimine-cellulose (PEI) thin-layer plates (Brinkman) which had been prerun for 3-4 hr in distilled water. The solvent was either 0.85 M KH2POI, pH 3.5 (System A) (Cashel et al., 1969), or 1.2 M LiCl (System B) (Miller et al., 1977). The PEI plates were then air-dried and exposed to Kodak XR-5 film at room temperature. In some experiments, for a more quantitative analysis, sample strips were cut into 5-mm segments which were then placed in scintillation fluid (Formula 949, New England Nuclear) and counted. Chemicals and isotopes. [32P]Orthophosphate (carrier free), [5-3H]ribavirin (15 &i/ mmol), and unlabeled ribavirin were all purchased from ICN Pharmaceuticals. 2-Amino1,3,4-thiadiazole (NCS 4728) was supplied by the National Cancer Institute and mycophenolic acid was the generous gift of Dr. K. F. Kosh, Lilly Research Laboratories. Actinomycin D was purchased from Schwarzl Mann and a-amanitin from Boehringer. RESULTS
Effect of Different Inosine Monophosphate Dehydrogenase Inhibitors on VimLs Replication
Although ribavirin, mycophenolic acid, and 2-amino-1,3,4-thiadiazole all inhibit IMPDH, their structures and perhaps also their specific mechanisms of action are quite different. Rbv is a guanosine analog which, after phosphorylation to the monophosphate, inhibits IMPDH by feedback inhibition. MPA has a similar effect on IMPDH
ANTIVIRAL
EFFECTS
but little is known about the metabolism of this drug in cells. TDA is thought to be converted to a mononucleotide which behaves like a competitive inhibitor of IMPDH with respect to IMP (Nelson et al., 1977). Figure 1 shows the dose-response curve for the inhibition of SV replication in A. albopictus cells treated with Rbv, MPA, or TDA. A loo-fold inhibition of virus yield was obtained with 3 x 10e4M Rbv, 8 x lo-” M MPA, and 8 x 10e3 M TDA. As seen in Table 1, TDA and MPA reduced the yield of both infectious SV and VSV by loo-fold or more, an effect comparable to that seen with Rbv at 6 x low4 M. Lower concentrations of TDA (l-3 x 1O-3 ln) which did not affect the yields of SV, did prevent virus-induced cytopathic effect in A. albopictus cells. Reversal of the Antiviral MPA, and TDA
Effects of Rbv,
Cline et al. (1969) and Streeter et al. (1973) showed that the inhibitory effect of MPA and Rbv on measles virus replication in BSC-1 cells and Vero cells, respectively, could be reversed by the addition of guanosine or xanthosine, but not inosine. Presumably, in these cells the drug-induced block in the conversion of inosine 5’-monophosphate (IMP) to xanthosine 5’-monophosphate can be circumvented by meta-
OF IMPDH INHIBITORS
283
TABLE THE EFFECT OF DIFFERENT ON THE REPLICATION OF VESICULAR STOMATITIS VIRUS
1 IMPDH INHIBITORS SINDBIS VIRUS AND IN A. albopictus CELLS Yield of virus at 24 hr after infection (PFU/ml)
IMPDH inhibitor
added
sv 3.1 x 109
None Ribavirin (6 x lo-’ M) Mycophenolic acid (1.5 x lo-5M) 2-Amino-1,3,4-thiadiazoie (1 x lo-‘M)
3.4 x 10’ 5.0
x 106
1.5 x 10’
vsv 3.0 6.5
x 109 x lo6
8.0 x 106 3.2 x 10’
Note. A. albopictus cells were infected with SV or VSV at an input multiplicity of 10 PFUkell. After 45 min for virus adsorption at 34.5”, cells were washed with PBS and refed with E-10 medium, after which the IMPDH inhibitors were added to the indicated concentrations. Twenty-four hours after infection samples of medium were harvested and assayed for infectious virus. The titer of infectious virus in the medium at 2 hr after infection, a measure of desorbed virus, was 4 to 5 x 10” PFU/ml for both viruses.
bolic derivatives of xanthosine and guanosine. Table 2 illustrates that in A. albopictus cells, equimolar amounts of xanthosine reversed the inhibition of SV replication by Rbv, whereas guanosine did not. However, it should be noted that in these cells guanosine by itself was inhibitory (Stollar and TABLE THE EFFECT THE ANTIVIRAL @US CELLS
2
OF XANTHOSINE AND GUANOSINE ON ACTIVITY OF RIBAVIRIN IN A. albo-
Yield of Sindbis virus (PFU/ml) Ribonucleoside added FIG. 1. Inhibition by different IMP dehydrogenase inhibitors of Sindbis virus replication in A. albopictus cells. A. albopictus cells were infected with SV at an input multiplicity of 10 PFUkell. After a 45min adsorption period, the virus inoculum was removed and the cells were refed with E-10 medium. The inhibitors were added at varying concentrations and cells were incubated at 34.5”. Samples of the medium were harvested at 16 hr postinfection and assayed for infectious virus.
None Xanthosine Guanosine
Without Rbv
With Rbv
x 108 1.1 x 109 2.1 x 10’
3.5 x 106 9.0 x 10” 1.6 x 10’
7.4
Note. A. albopictus cells were infected with SV as described in Table 1. Rbv, xanthosine, and guanosine were each added to a final concentration of 6 x 10ml M. After 16 hr, samples of the medium were harvested and assayed for infectious virus.
MALINOSKI
284
Malinoski, submitted for publication). The effects of xanthosine and guanosine on MPA- and TDA-treated infected cells were similar to those just described for Rbvtreated cells (data not shown). Since TDA is an analog of nicotinamide and its antitumor effects can be reversed by equimolar amounts of nicotinamide (Nelson et al., 19’7’7), we tested the effect of nicotinamide on the antiviral activity of TDA in A. albopictus cells. As indicated in Table 3, nicotinamide (1 x 10e2 M) completely reversed the inhibition of SV replication caused by TDA, but had no effect on the Rbv- or MPA-treated cultures. Actinomycin (AMD), which reverses the antiviral activity of Rbv in A. albopictus cells (Malinoski and Stollar, 1980) was equally effective in reversing the antiviral effects of MPA and TDA (Table 4). In contrast to AMD, which inhibits DNAdependent RNA synthesis by intercalation in the DNA helix (Goldberg et al., 1962), a-amanitin inhibits RNA transcription by binding to RNA polymerase II (Lindell et al., 1970). As seen in Table 5, in (Yamanitin-sensitive A. albopictus cells, (LT C-7), cu-amanitin reversed the inhibition by ribavirin as effectively as did AMD. In contrast, in Amacells, which are resistant to
TABLE
Yield of Sindbis virus (PFU/ml)
inhibitor
added
None Ribavirin (6 x 1O-1 M) Mycophenolic acid (1.5 x lo-5M) 2-Amino-1,3,4-thiadiazole (1 x 10-Z M)
STOLLAR TABLE
4
THE EFFECT OF ACTINOMYCIN ON THE ACTIVITY OF RIBAVIRIN, MYCOPHENOLIC
ANTIVIRAL ACID, AND
Z-AMINO-1,3,&THIADIAZOLE Yield of Sindbis virus (PFUlml)
IMPDH
inhibitor
added
Without
None Ribavirin (6 x 1Om1M) Mycophenolic acid (3 x lo-JM) P-Amino-1,3,4-thiadiazole (1 x lo-*M)
With AMD (0.2 pgiml)
AMD
3.0 x 109 3.4 x 10’
1.6 x lo9 2.3 x 108
2.0 x 10’
1.3 x 109
2.4 x 10’
1.5 x 109
Note. A. albopictus cells were infected with SV as described in Table 1. Cultures were refed with E-10 medium, the various
compounds
were
added
to
give
the
final
concen-
trations indicated, and the cells were reincubated at 34.5”. Sixteen hours after infection samples of the medium were harvested and assayed for infectious virus.
a-amanitin and have an altered RNA polymerase II (Mento and Stollar, 1979), the antiviral effect of Rbv was not reversed by a-amanitin although it was by AMD. As both cr-amanitin and AMD can reverse the antiviral activity of Rbv, MPA, and TDA, (reversal by a+amanitin of MPA and TDA not shown) it appears that in order for these inhibitors to exert their antiviral effect, normal cellular RNA synthesis must occur.
3
THE EFFECT OF NICOTINAMIDE ON THE ANTIVIRAL ACTIVITY OF RIBAVIRIN, MYCOPHENOLIC ACID, AND ?&AMINO-1,3,4-THIADIAZOLE
IMPDH
AND
Without nicotinamide
With nicotinamide (1 x lo-*&f)
2.3 ~10~ 1.0 x 10’
2.5 x lo9 1.2 x 10’
5.0 x 10’
6.3 x 10’
1.5 x 10’
2.7
x 109
Note. A. albopictus cells were infected with SV as described in Table 1. Cultures were refed with medium E-10, the various compounds were added to the concentrations indicated, and the cells were reincubated at 34.5”. Sixteen hours after infection samples of the medium were harvested and assayed for infectious virus.
Phosphorylation
of Ribavirin
Since Rbv must be phosphorylated to be active (see above and Willis et al., 1978) and since the antiviral effects of Rbv can be reversed by AMD and a-amanitin, we wished to know if Rbv is phosphorylated normally in the presence of AMD. Uninfected A. aZbopictus cells were treated with [3H]ribavirin for 4 hr and an acid-soluble extract of the cells was prepared as described under Materials and Methods. Chromatography of the extract (Fig. 2) on PEI-cellulose (System B) revealed a small peak of radioactivity with an R,of 0.26, a minor peak with an Rfof 0.50, a major peak with an Rf of 0.77, and a shoulder on the last peak with an R,of 0.86. Since [3H]ribavirin, chromatographed in a separate channel, had an Rf of 0.86, we consider the shoulder on Peak 3 to be r3H]-
ANTIVIRAL
EFFECTS
OF TABLE
IMPDH
INHIBITORS
285
5
THE EFFECT OF WAMANITIN ON THE ANTIVIRAL ACTIVITY OF RIBAVIRIN IN o-AMANITIN-SENSITIVE AND a-AMANITIN-RESISTANT A. albopictus CELLS Yield of Sindbis virus (PFU/ml) Ama(resistant to cY-amanitin)
LT C-7
Ribavirin (6 x 10-‘&f)
a-Amanitin (10 &ml)
Actinomycin (0.2 pg/ml)
-
-
-
+ + +
+ + -
+ +
(sensitive to cu-amanitin) 1.9 x x x x x x
2.2 5.7 7.2 8.2 6.2
109 107 10” 10” 10” 10”
1.0 x 10” 1.0 x 10” 1.2 x 108 2.0 x 106 1.5 x 10” 1.2 x 108
Note. A. albopictus cells (clones LT C-7 and Ama-18) were infected with SV as described in Table 1. Cultures were refed with E-10 medium, the various compounds were added to give the final concentrations indicated, and the cells were reincubated at 34.5”. Sixteen hours after infection samples of the medium were harvested and assayed for infectious virus.
ribavirin. In this system, ribonucleoside triphosphates migrate very slowly (Ris of 0.18 to 0.30), ribonucleoside diphosphates somewhat more rapidly (R;s of 0.35 to 0.60), and monophosphates still more quickly (R;s of 0.50 to 0.88, see markers at the top of Fig. 2). Thus, from Fig. 2, we have tentatively identified peaks 1, 2, and 3 as the triphosphate, the diphosphate, and monophosphate forms, respectively, of Rbv. In this same system, Miller et al. (1977) obtained Rf values of 0.11, 0.33, and 0.53 for chemically synthesized triphosphate, diphosphate, and monophosphate forms, respectively, of Rbv. Miller et al. also reported an Rfof 0.88 for Rbv. When cultures were treated with AMD (0.4 pg/ml) the cells took up 30% less [3Hlribavirin (as measured by total acid-soluble counts) than control cultures and, as seen in Fig. 2, there were marked reductions (50% or more) in the amounts of both Rbv monophosphate and triphosphate. The amount of unphosphorylated Rbv was unchanged. When cells were labeled for up to 12 hr with [3H]ribavirin, less than 1% of the total radioactivity was found in acidinsoluble material. Thus, at least in the case of AMD, the reversal of the Rbv effect might be partially explained by an inhibition of phosphorylation.
o-e
no AMD
12
4
800 t : 600
400
200
DISTANCE
6
8
10
MIGRATED
12
14 (cm)
FIG. 2. Thin-layer chromatography of acid-soluble extracts prepared from [3H]ribavirin-labeled A. albopi&s cells. Monolayers of A. albopictus cells (5 x lo6 tells/60-mm petri dish) were treated with [3Hlribavirin (10 /.&i/ml) for 4 hr at 34.5” with or without AMD ‘0.4 pg/ml). Acid-soluble extracts were prepared as described under Materials and Methods. Ten microliters of the extract was spotted on PEI-cellulose and chromatographed in 1.2 M LiCl (System B) at room temperature. The chromatogram was cut into 5-mm segments which were then immersed in 5 ml of Formula 949 and counted in a scintillation counter. Standards were located by uv light absorbance.
MALINOSKI
286
d
J -rr
AND STOLLAR
this system monophosphates migrate with inorganic phosphate and TTP is not resolved from UTP. In extracts of cells treated with ribavirin (6 x 10e4 M) two major changes were seen. First, there was a decrease in the amount of radiolabeled GTP (the numbers below the GTP spot indicate the counts per minute when this spot was cut out and counted). Second, there was a new spot on the chromatogram which migrated ahead of CTP. When this 32P-labeled spot was eluted from the PEI-cellulose with 2.16 M triethylammonium carbonate (Volckaert et al., 1976) and then chromatographed using System B, it had an Rf of 0.20 (Fig. 4), a value close to that of Peak 1 (Rbv triphosphate) in Fig. 2. As shown in Fig. 5, chromatography using System A of an extract from cells labeled with [3H]ribavirin revealed a peak of radioactivity with the same Rf as the new
FIG. 3. Thin-layer chromatography of acid-soluble extracts prepared from 32P-labeled A. albopictus cells treated with ribavirin. A. albopictus cells were grown for ‘72 to 96 hr at 28” to confluent monolayers in medium containing 5 @Zi 32P04/ml. Rbv (6.4 x 10m4M) was added, the cells were incubated at 34.5”, and acidsoluble extracts were prepared at the times indicated as described under Materials and Methods. Five microliters of each extract was spotted on PEI-cellulose and chromatographed in 0.85 M KH,PO, (PH 3.5) (System A). The chromatogram was dried and exposed to X-ray film as described under Materials and Methods. The locations of the nucleotide standards were determined by uv light absorbance. The location of PO, was determined by chromatography of our 3zP0, stock. The numbers below the GTP spots indicate the counts per minute in each spot.
Effect of IMPDH Inhibitors on Intracellular Nucleotide Pools of A. albopictus Cells A. albopictus cells were grown in the presence of 32P-inorganic phosphate for 72 to 96 hr and then treated with Rbv, MPA, or TDA. At various times after the addition of the drugs, extracts were prepared and analyzed by thin-layer chromatography using System A, as described under Materials and Methods. In untreated cells (Fig. 3) six wellseparated spots were resolved which had R;s corresponding to NAD, free inorganic phosphate, UTP, CTP, ATP, and GTP. In
FIG. 4. Thin-layer chromatography of the new 32Plabeled spot from extracts of ribavirin-treated A. albopicks cells. The new 32P-labeled spot seen in Rbvtreated cells (Fig. 3) was eluted from the PEI-cellulose with 2.16 M triethylammonium carbonate and chromatographed using System B on PEI-cellulose. Lane A represents 1 ~1 of the eluent and Lane B, 5 ~1.
ANTIVIRAL
EFFECTS 1
GTP
1
ATP c I
UTP I
FRONl ‘1
300 300
-
ORIGIN
v
OF IMPDH INHIBITORS
Figure 3 also shows that the effect of Rbv on the GTP pool is rapid since within 2 hr of treatment the GTP pool was reduced by 80%. In another experiment (not shown) a 30% reduction in GTP was observed within 30 min by which time the ribavirin triphosphate could also be seen. The reduction of the GTP pool by Rbv was observed in SVinfected cells as well as in uninfected cells (not shown). Mycophenolic acid and 2-amino-1,3,4thiadiazole had effects on GTP levels similar to those of Rbv. Figure 6 illustrates that extracts of cultures treated for 4 hr with 1.2 x 10e5 and 6.0 x low5 M MPA contained 48 and 70%, respectively, less GTP M
loo100
11’1
012
DISTANCE
MIGRATED
287
M M
2341
(cm)
FIG. 5. Thin-layer chromatography of acid-soluble extracts prepared from [3H]ribavirin-labeled A. albop&us cells. Monolayers of A. albopictus cells (5 x lo6 cells) were treated with [5-3H]ribavirin (10 &i/ml) for 12 hr and acid-soluble extracts were prepared as described under Materials and Methods. Ten microliters of the extract was spotted on PEI-cellulose and chromatographed using System A. The chromatogram was then cut into 5-mm segments, which were immersed in 5 ml of Formula 949 and counted. The location of standards was determined by uv light absorbance.
spot seen in Fig. 3. (Free Rbv migrates near the solvent front in this system.) We have also demonstrated that when the new spot (32P-labeled) was eluted from a thin-layer chromatogram like that shown in Fig. 3 and analyzed by high-performance liquid chromatography (a strong anionexchange column and gradient elution were used) it eluted in the ribonucleotide triphosphate region and had a retention time very close to that of ATP (not shown). Using a very similar hplc system, Zimmerman and Deeprose (1978) observed a derivative of ribavirin, formed by human blood cells, which had very similar chromatographic properties and concluded that it was the triphosphate of ribavirin. These results, taken together, strongly suggest that the new spot seen in Fig. 3 is ribavirin triphosphate.
EJ
ORIGIN
FIG. 6. Thin-layer chromatography of acid-soluble extracts prepared from 32P-labeled A. albopictus cells treated with mycophenolic acid. A. albopictus cells were grown in the presence of 32POaas described under Materials and Methods. MPA was added to a fmal concentration of 1.2 or 6 x 10m5M and acid-soluble extracts were prepared 4 hr later, as described under Materials and Methods. Five microliters of each extract was spotted on PEI-cellulose and chromatographed using System A, after which the chromatogram was dried and exposed to X-ray film. The locations of the nucleotide standards and PO, were determined as described for Fig. 3. Abbreviations are AMD for actinomycin D, and MPA for mycophenolic acid. The numbers below the GTP spots indicate the counts per minute in each spot.
288
MALINOSKI
AND STOLLAR
than untreated cultures; similarly, TDA (1 x lop2 M) reduced the GTP level to 20% of normal by 6 hr after treatment (Fig. ‘7). Although no new spots were seen in these chromatograms, it is still possible that phosphorylated forms of these compounds were present but were not resolved from other nucleotides or free inorganic phosphate seen in cell extracts (Fig. 3). Since actinomycin reversed the antiviral effect of each of the IMP dehydrogenase inhibitors and inhibited the phosphorylation of Rbv, we next tested whether AMID was able to reverse the reduction in the GTP pool brought about by RBV, MPA, and TDA. Figure 8 shows that in cells treated with Rbv and AMD the level of GTP is close to that in untreated cells and much higher
FIG. 8. Thin-layer chromatography of acid-soluble extracts prepared from 3ZPOFlabeled A. a1bopictu.s cells treated with ribavirin and actinomycin. A. albopictus cells were grown in the presence of 32P04 as described in Fig. 3. Rbv (6.4 x 10e4 M) and AMD (0.2 pg/ml) were added, as indicated, and the cells were incubated at 34.5”. Twelve hours later, an acid-soluble extract of each sample was prepared as described under Materials and Methods. Five microliters of each extract was spotted on PEI-cellulose and chromate graphed using System A, after which the chromatogram was dried and exposed to X-ray film. The locations of the nucleotide standards and ‘*PO, were determined as described for Fig. 3. The numbers below the GTP spots indicate the counts per minute in each spot.
YI FIG. 7. Thin-layer chromatography of acid-soluble extracts prepared from 32P04-labeled A. albopictus cells treated with Z-amino-1,3,4-thiadiazole (TDA). A. albopictus cells were grown in the presence of 32P0, as described for Fig. 3. TDA was added to a final concentration of 1 x lo-* Y. Acid-soluble extracts were prepared as described under Materials and Methods. Five microliters of each extract was spotted on PEI-cellulose and chromatographed using System A, after which the chromatogram was dried and exposed to X-ray film. The location of nucleotide standards and PO, were determined as described in Fig. 3. The numbers below the GTP spots indicate the counts per minute in each spot.
than in cells treated only with ribavirin. In cells treated with AMD alone, there was a considerable increase in the size of the GTP pool. Actinomycin also reversed the reduction in GTP brought about by MPA (Fig. 6) or by TDA (data not shown). Consistent with the results in Fig. 2, AMD prevented the appearance of the new spot, ribavirin triphosphate (Fig. 8). DISCUSSION
At least three different mechanisms have been proposed (see Introduction) which might explain the antiviral effect of Rbv. Depending on the specific mechanism invoked, either Rbv monophosphate or Rbv triphosphate would be the active intracellu-
ANTIVIRAL
EFFECTS
lar form of the drug. Miller et al. (1977) have shown that 8 hr after receiving an oral dose of Rbv, ribavirin, as well as the various phosphorylated derivatives, were found in the liver of rats. Of the total ribavirin in the liver, 50% was unphosphorylated, while 23, 18, and 5% were found as Rbv monophosphate, Rbv diphosphate, and Rbv triphosphate, respectively. In our experiments with [3H]Rbv in A. albopictus cells, only 12% of the total intracellular ribavirin remained unphosphorylated after 4 hr while the major phosphorylated form was Rbv monophosphate. One or two percent was found in the disphosphate form and about 8% in the triphosphate form. Although Rbv triphosphate has been shown to inhibit both influenza virus RNA transcriptase and vaccinia virus guanyltransferase, it is not yet known whether the concentrations of Rbv triphosphate achieved in cells approach the levels needed to inhibit these activities either in viva or in vitro. Similarly, while the inhibition of IMPDH by Rbv monophosphate has been demonstrated, there had been no evidence which correlated the decrease in GMP (or GTP) with an inhibition of virus replication in the same cell system. The one report which described decreased levels of GTP in cells treated with Rbv, did not include any data on antiviral activity in the Rbv-treated cells. We, therefore, sought in our experiments to determine which phosphorylated forms of ribavirin were found in A. albopictus cells and to see whether correlations could be drawn between the antiviral effect of Rbv and specific changes in the intracellular nucleotide pools. In this way, we hoped to find out whether or not the inhibition of virus replication could be explained mainly by an inhibition of cellular IMPDH (and the resultant reduction in GMP and GTP) and to examine possible mechanisms by which the antiviral activity of ribavirin can be reversed. We found that with Rbv as well as with two other inhibitors of IMPDH (MPA and TDA), there was a good correlation in A. albopictus cells between antiviral activity and a reduction in the GTP pool. Our findings on the GTP levels are consistent with those of Lowe et al. (197’7) who reported
OF IMPDH
INHIBITORS
289
that MPA and Rbv decrease the level of GTP by 90% in L15’78Y murine lymphoma cells. Lowe et ~2. also found that on a molar basis MPA was 100 times as effective as Rbv in decreasing GTP levels and inhibiting cell growth. Similarly, we found in our experiments that, as measured by antiviral activity, MPA was 20 times as effective as Rbv. The antiviral effects of Rbv, MPA, and TDA in A. albopictus cells could be reversed in several ways. Although both xanthosine and guanosine have been shown to reverse the action of Rbv and MPA in measles virusinfected Vero cells and BSC-1 cells, respectively, in our studies only xanthosine was effective. The failure of guanosine to reverse the action of ribavirin is likely due to the fact that by itself, guanosine inhibits virus replication in these cells (Stellar and Malinoski, submitted for publication). Two inhibitors of cellular RNA synthesis, actinomycin D and cu-amanitin, were shown to reverse the antiviral action of Rbv, and in the ease of actinomycin, return the GTP to levels similar to those in untreated cells. Although these effects on GTP levels could be attributed to a decreased demand for ribonucleotide triphosphates because of decreased cellular RNA synthesis in the presence of AMD or a-amanitin, it is more difficult to explain why actinomycin prevents the phosphorylation of ribavirin (Figs. 2 and 8). At any rate, the observations that both actinomycin D and cr-amanitin reverse the antiviral action of three different IMPDH inhibitors suggests strongly that in order for the antiviral effects of these compounds to be expressed, normal cellular RNA synthesis must be carried on. Possibly cellular RNA synthesis and certain cell functions are needed for the conversion of these compounds to their active forms. Another point of interest is that the concentration of Rbv necessary to inhibit SV replication in A. albopictus cells (6 x lo-“M) is much higher than that needed for a 50% plaque reduction of four strains of influenza Type A virus on calf kidney cells (4 x lo+ M; Tisdale and Bauer, 1977). Whether this difference relates to the two viruses studied, to different mechanisms of inhibition of the two viruses, to differences between the host cells, or differences in growth conditions of
290
MALINOSKI
AND STOLLAR
the cells is unknown. We are currently exploring the role of the host cell by testing the antiviral effects of Rbv in a variety of different cell types as well as in mutants of A. albopictus cells which have alterations in purine metabolism. ACKNOWLEDGMENTS This investigation was supported by Grant AI-11290 from the National Institute of Allergy and Infectious Diseases, by the United States-Japan Medical Science Program through Public Health Service Grant AI05920, and by the Institutional National Research Service Award CA-09069 from the National Cancer Institute. REFERENCES CASHEL, M., LAZZARINI, R. A., and KALBACHER, B. (1969). An improved method for thin-layer chromatography of nucleotide mixtures containing 32Plabeled orthophosphate. J. Chromatogr. 40, 103109. CLINE, J. C., NELSON, J. D., GERZON, K., WILLIAMS, R. H., and DELONG, D. C. (1969). In vitro antiviral activity of mycophenolic acid and its reversal by guanine-type compounds. Appl. Microbial. 18,14-20. ERIKSSON, B., HELGSTRAND, E., JOHANSSON, N. G., LARSSON, A., MISIORNY, A., NOREN, J. O., PHILIPSON, L., STENBERG, K., STENING, G., STRIDH, S., and OBERG, B. (1977). Inhibition of influenza virus ribonucleic acid polymerase by ribavirin triphosphate. Antimicrob. Agents Chemother. 11,946-951. GILLIES, S., and STOLLAR, V. (1980). The production of high yields of infectious vesicular stomatitis virus in A. albopictus cells and comparisons with replication in BHK-21 cells. Virology 107,509-513. GOLDBERG, I. H., RABINOWITZ, M., and REICH, E. (1962). Basis of actinomycin action, I. DNA binding and inhibition of RNA-polymerase synthetic reactions by actinomycin. Proc. Nat. Acad. Sci. USA 48,2094-2101. GOSWAMI, B. B., BOREK, E., SHARMA, 0. K., FUJITAKI, J., and SMITH, R. A. (1979). The broad spectrum antiviral agent ribavirin inhibits capping of mRNA. Biochem. Biophys. Res. Commun. 89,830836. HERSHFIELD, M. S., and SEEGMILLER, J. E. (1977). Regulation of de novo purine synthesis in human lymphoblasts. Similar rates of de novo synthesis during growth by normal cells and mutants deficient in hypoxanthine-guanine phosphoribosyltransferase activity. J. Biol. Chem. 252, 6002-6010. HERSHFIELD, M. S., SNYDER, F. F., and SEEGMIL-
LER, J. E. (1977). Adenine and adenosine are toxic to human lymphoblast mutants defective in purine salvage enzymes. Science 197, 1284-1287. LINDELL, T. J., WEINBERG, F., MORRIS, P. W., ROEDER, R. G., and RUTTER, W. J. (1970). Specific inhibition of nuclear RNA polymerase II by cy-amanitin. Science 170,447~449. LOWE, J. K., BROX, L., and HENDERSON, J. F. (1977). Consequences of inhibition of guanine nucleotide synthesis by mycophenolic acid and virazole. Cancer Res. 37, 736-743.
MALINOSKI, F., and STOLLAR, V. (1980). Inhibition of Sindbis virus replication in Aedes albopictus cells by virazole (ribavirin) and its reversal by actinomycin: A correction. Virology 102,473-476. MENTO, S. J., and STOLLAR, V. (1979). a-Amanitin resistant RNA polymerase II from Aedes albopictus cell mutants resistant to cY-amanitin. Cell Biol. Znt. Rep.
3, 725-732.
MILLER, J. P., STREETER, D. G., ROBINS, R. K., and SIMON, L. N. (1977). The relationship between the metabolism of ribavirin and its proposed mechanism of action. Ann. N. Y. Acad. Sci. 284,211-229. NELSON, J. A., ROSE, L. M., and BENNETT, L. L., JR. (1977). Mechanism of action of 2-amino-1,3,4-thiadiazole (NSC 4728). Cancer Res. 37, 182-187. SARVER, N., and STOLLAR, V. (1978). Virazole prevents production of Sindbis virus and virus-induced cytopathic effect in Aedes albopictus cells. Virology
91,267-282. SHENK, T. E., and STOLLAR, V. (1972). Viral RNA species in BHK-21 cells infected with Sindbis virus serially passaged at high multiplicity of infection. Biochem. Biophys. Res. Commun. 49, 60-67. SHENK, T. E., KOSHELNYK, K. A., and STOLLAR, V. (1974). Temperature-sensitive virus from Aedes albopictus cells chronically infected with Sindbis virus. J. Viral. 13, 439-447. SIDWELL, R. W., ROBINS, R. K., and HILLYARD, I. W. (1979). Ribavirin: An antiviral agent. Pharmucol. Ther. 6, 123-146.
SMITH,C.M.,FONTINELLE,L.J.,MUZIK,H.,PATERSON, A. R. P., UNGER, H., BROX, L. W., and HENDERSON, J. F. (1974). Inhibitors of inosinate dehydrogenase activity in Ehrlich ascites tumor cells in vitro. Biochem. Pharmacol. 23, 2727-2735. STOLLAR, V., SHENK, T. E., and STOLLAR, B. D. (1972). Double stranded RNA in hamster, chick and mosquito cells infected with Sindbis virus. Virology 47, 122-132. STOLLAR,V., STOLLAR, B. D., Koo, R., HARRAP, K. A., and SCHLESINGER, R. W. (1976). Sialic acid contents of Sindbis virus from vertebrate and mosquito cells. Equivalence of biological and immunological viral properties. Virology 69, 104-115.
ANTIVIRAL
EFFECTS
STREETER, D. G., WITKOWSKI, J. T., KHARE, G. P., SIDWELL, R. W., BAUER, R. J., ROBINS, R. K., and SIMON, L. N. (1973). Mechanism of action of l-pn-ribofuranosyl-1,2,4-triazole-3-carboximide (Virazole), a new broad spectrum antiviral agent. Proc. Nat. Acad. Sci. USA 70, 1174-1178. TISDALE, M., and BAUER, D. J. (1977). The relative potencies of anti-influenza compounds. Ann. N. Y. Acad.
Sci. 284,254-263.
VOLCKAERT, G., MIN Jou, W., and FIERS, W. (1976). Analysis of 32P-labeled bacteriophage MS2 RNA by
OF IMPDH INHIBITORS
291
a mini-fingerprinting procedure. Anal. Biochem. 72, 433-446. WILLIS, R. C., CARSON, D. A., and SEEGMILLER, J. E. (1978). Adenosine kinase initiates the major route of ribavirin activation in a cultured human cell line. Proc.
Nat.
Acad.
Sci.
USA
75, 3042-3044.
ZIMMERMAN, T. P., and DEEPROSE, R. D. (1978). Metabolism of 5amino-I-&~ribofuranosyl-imidazole4-carboxamide and related five-membered heterocycles to 5’-triphosphates in human blood and L5178Y cells. Biochem. Pharmacol. 27, 709-716.
110,
Inhibitors
281-291
(1981)
of IMP Dehydrogenase and Reduce GTP Levels FRANK
Prevent Sindbis Virus Replication in Aedes albopictus Cells
MALINOSKI
Department of Microbiology, Rutgers Medical
AND VICTOR
College of Medicine School, Piscataway, Accepted
October
STOLLAR’
and Dentistry of New New Jemey 08854
Jersey,
30, 1980
We have investigated the mechanism by which ribavirin (Rbv), mycophenolic acid, and 2-amino-1,3,4-thiadiazole inhibit the replication of Sindbis virus in Aedes albopictus cells. In each case there was a good correlation between inhibition of virus replication and a reduction @O-90%) in the level of cellular GTP. The antiviral effects of all three compounds could be reversed by (1) equimolar amounts of xanthosine but not by guanosine, (2) actinomycin D (0.2 pg/ml), and (3) n-amanitin (10 pg/ml) in o-amanitin-sensitive cells but not in o-amanitinresistant cells. In the case of actinomycin D the reversal of the antiviral effects was correlated with a restoration of the GTP pool to near normal levels and a decrease in the amount of phosphorylated Rbv in acid-soluble cell extracts. The role of the phosphorylated forms of Rbv in inhibiting virus replication and the relationship to cellular RNA synthesis are discussed. INTRODUCTION
Ribavirin (1-P-D-ribofuranosyl-1,2,4-triazole-3-carboxamide; Rbv) is a synthetic guanosine analog which inhibits the replication of a wide range of RNA and DNA viruses (Sidwell et al., 1979). Concerning its mechanism of action, evidence has been presented showing (1) that the monophosphate of Rbv inhibits inosine 5’-monophosphate dehydrogenase, the first enzyme specific for the de novo synthesis of GMP (Streeter et al., 1973) and (2) that in murine lymphoma cells treated with Rbv there is a marked reduction in the intracellular level of GTP (Lowe et al., 197’7). It seems reasonable in turn that the decreased level of GTP can explain the depression of nucleic acid synthesis caused by Rbv (Sarver and Stollar, 1978; Malinoski and Stollar, 1980). However, it has also been proposed that the triphosphate of Rbv (which can be detected within 30 min of treatment with Rbv, see below) is the active form of the drug and that it can inhibit either viral RNA polymerase (Eriksson et al., 1977) or viral capping enzymes 1 Author to whom requests for reprints should be addressed. 281
(Goswami et aZ., 1979). Thus, there are at least three possible explanations for the antiviral action of ribavirin: (1) reduction in the amount of intracellular GTP, (2) inhibition by Rbv triphosphate of viral RNA polymerase, or (3) inhibition by Rbv triphosphate of the RNA capping enzyme, guanyltransferase. Earlier work from this laboratory has shown that Rbv inhibits Sindbis virus (SV) replication in Aedes albopictus mosquito cells (Sarver and Stollar, 1978). We also found that in SV-infected A. albopictus cells, Rbv prevents viral nucleic acid synthesis and that inhibition of both viral RNA synthesis and of virus replication can be reversed by actinomycin (Malinoski and Stollar, 1980). We have now extended our studies by testing (1) the effect of Rbv on another enveloped RNA virus, vesicular stomatitis virus, (2) the antiviral effects of two other inhibitors of inosine monophosphate dehydrogenase (IMPDH), mycophenolic acid (Smith et al., 1974) and 2-amino-1,3,4-thiadiazole (Nelson et al., 1977), and (3) the effects of these inhibitors on the intracellular nucleotide pools of mosquito cells. 0042~6322/81/060281-11$02.00/O Copyright All rights
0 1981 by Academic Press, Inc. of reproduction in any form reserved.
282
MALINOSKI
Our results indicate that mycophenolic acid (MPA) and 2-amino-1,3,4-thiadiazole (TDA), like Rbv, prevent virus replication in A. albopictus cells. Under the various experimental conditions examined and with each of the three inhibitors of IMP dehydrogenase, we found a good correlation between inhibition of virus replication and a decrease in cellular GTP levels. MATERIALS
AND
METHODS
Cells, media, and viruses. Primary chick embryo fibroblast cultures were prepared as described elsewhere (Stollar et al., 1976). Aedes albopictus mosquito cells, clone LT C-7 (Sarver and Stollar, 1978), and the a-amanitin-resistant A. albopictus cell line, Ama(Mento and Stollar, 19’79), were grown at 28” in E medium (Sarver and Stollar, 1978) supplemented with heatinactivated (56”, 30 min) fetal calf serum. Cells were counted using a Coulter counter, Model ZBI. Plaque-purified stocks of Sindbis virus (SV) and vesicular stomatitis virus, Indiana serotype, (VW), were prepared as described elsewhere (Stollar et al., 1972; Shenk and Stollar, 1972; Gillies and Stollar, 1980). Cells were infected with virus at an input multiplicity of approximately 10 PFU/ cell. After adsorption (45 min, 34.5”, 5% CO,) the inoculum was removed, the monolayers were washed with phosphate-buffered saline (PBS), and the cultures were fed with E medium containing 10% fetal calf serum (E-10). Infected cells were incubated at 34.5” unless otherwise stated. Virus titers were measured by plaque assay at 34.5” (Shenk et al., 1974) on chick embryo fibroblasts in the case of SV or on BHK cells in the case of VSV. Analysis of acid-soluble nucleotides. A. albopictus cells were seeded into 60-mm tissue culture dishes at low densities (5 x lo5 cells per dish) in medium E-10 containing 5 to 10 PCi 32P04/m1. When cells reached confluency (after approximately four population doublings or about 3 to 4 days after seeding) cultures were treated with a specific inhibitor of IMP dehydrogenase for the indicated period of time and then analyzed as outlined below.
AND
STOLLAR
Acid-soluble material was extracted from cell cultures following the procedure of Hershfield and Seegmiller (1977). Cells (57 x 106) were scraped from the dishes, pelleted, rinsed twice with cold PBS, and then resuspended in 200 ~1 PBS lacking calcium and magnesium. Perchloric acid, 4 M (20 pl), was added and samples were kept at 4” for 15 to 30 min. Acid-insoluble material was removed by centrifugation (1800 g, 15 min, 4”) and the supernatant was brought to pH 7 with 4 M KOH. Insoluble KClO, was removed by another centrifugation as above and the supernatant acid-soluble extract was collected. Samples were analyzed by ascending chromatography on polyethyleneimine-cellulose (PEI) thin-layer plates (Brinkman) which had been prerun for 3-4 hr in distilled water. The solvent was either 0.85 M KH2POI, pH 3.5 (System A) (Cashel et al., 1969), or 1.2 M LiCl (System B) (Miller et al., 1977). The PEI plates were then air-dried and exposed to Kodak XR-5 film at room temperature. In some experiments, for a more quantitative analysis, sample strips were cut into 5-mm segments which were then placed in scintillation fluid (Formula 949, New England Nuclear) and counted. Chemicals and isotopes. [32P]Orthophosphate (carrier free), [5-3H]ribavirin (15 &i/ mmol), and unlabeled ribavirin were all purchased from ICN Pharmaceuticals. 2-Amino1,3,4-thiadiazole (NCS 4728) was supplied by the National Cancer Institute and mycophenolic acid was the generous gift of Dr. K. F. Kosh, Lilly Research Laboratories. Actinomycin D was purchased from Schwarzl Mann and a-amanitin from Boehringer. RESULTS
Effect of Different Inosine Monophosphate Dehydrogenase Inhibitors on VimLs Replication
Although ribavirin, mycophenolic acid, and 2-amino-1,3,4-thiadiazole all inhibit IMPDH, their structures and perhaps also their specific mechanisms of action are quite different. Rbv is a guanosine analog which, after phosphorylation to the monophosphate, inhibits IMPDH by feedback inhibition. MPA has a similar effect on IMPDH
ANTIVIRAL
EFFECTS
but little is known about the metabolism of this drug in cells. TDA is thought to be converted to a mononucleotide which behaves like a competitive inhibitor of IMPDH with respect to IMP (Nelson et al., 1977). Figure 1 shows the dose-response curve for the inhibition of SV replication in A. albopictus cells treated with Rbv, MPA, or TDA. A loo-fold inhibition of virus yield was obtained with 3 x 10e4M Rbv, 8 x lo-” M MPA, and 8 x 10e3 M TDA. As seen in Table 1, TDA and MPA reduced the yield of both infectious SV and VSV by loo-fold or more, an effect comparable to that seen with Rbv at 6 x low4 M. Lower concentrations of TDA (l-3 x 1O-3 ln) which did not affect the yields of SV, did prevent virus-induced cytopathic effect in A. albopictus cells. Reversal of the Antiviral MPA, and TDA
Effects of Rbv,
Cline et al. (1969) and Streeter et al. (1973) showed that the inhibitory effect of MPA and Rbv on measles virus replication in BSC-1 cells and Vero cells, respectively, could be reversed by the addition of guanosine or xanthosine, but not inosine. Presumably, in these cells the drug-induced block in the conversion of inosine 5’-monophosphate (IMP) to xanthosine 5’-monophosphate can be circumvented by meta-
OF IMPDH INHIBITORS
283
TABLE THE EFFECT OF DIFFERENT ON THE REPLICATION OF VESICULAR STOMATITIS VIRUS
1 IMPDH INHIBITORS SINDBIS VIRUS AND IN A. albopictus CELLS Yield of virus at 24 hr after infection (PFU/ml)
IMPDH inhibitor
added
sv 3.1 x 109
None Ribavirin (6 x lo-’ M) Mycophenolic acid (1.5 x lo-5M) 2-Amino-1,3,4-thiadiazoie (1 x lo-‘M)
3.4 x 10’ 5.0
x 106
1.5 x 10’
vsv 3.0 6.5
x 109 x lo6
8.0 x 106 3.2 x 10’
Note. A. albopictus cells were infected with SV or VSV at an input multiplicity of 10 PFUkell. After 45 min for virus adsorption at 34.5”, cells were washed with PBS and refed with E-10 medium, after which the IMPDH inhibitors were added to the indicated concentrations. Twenty-four hours after infection samples of medium were harvested and assayed for infectious virus. The titer of infectious virus in the medium at 2 hr after infection, a measure of desorbed virus, was 4 to 5 x 10” PFU/ml for both viruses.
bolic derivatives of xanthosine and guanosine. Table 2 illustrates that in A. albopictus cells, equimolar amounts of xanthosine reversed the inhibition of SV replication by Rbv, whereas guanosine did not. However, it should be noted that in these cells guanosine by itself was inhibitory (Stollar and TABLE THE EFFECT THE ANTIVIRAL @US CELLS
2
OF XANTHOSINE AND GUANOSINE ON ACTIVITY OF RIBAVIRIN IN A. albo-
Yield of Sindbis virus (PFU/ml) Ribonucleoside added FIG. 1. Inhibition by different IMP dehydrogenase inhibitors of Sindbis virus replication in A. albopictus cells. A. albopictus cells were infected with SV at an input multiplicity of 10 PFUkell. After a 45min adsorption period, the virus inoculum was removed and the cells were refed with E-10 medium. The inhibitors were added at varying concentrations and cells were incubated at 34.5”. Samples of the medium were harvested at 16 hr postinfection and assayed for infectious virus.
None Xanthosine Guanosine
Without Rbv
With Rbv
x 108 1.1 x 109 2.1 x 10’
3.5 x 106 9.0 x 10” 1.6 x 10’
7.4
Note. A. albopictus cells were infected with SV as described in Table 1. Rbv, xanthosine, and guanosine were each added to a final concentration of 6 x 10ml M. After 16 hr, samples of the medium were harvested and assayed for infectious virus.
MALINOSKI
284
Malinoski, submitted for publication). The effects of xanthosine and guanosine on MPA- and TDA-treated infected cells were similar to those just described for Rbvtreated cells (data not shown). Since TDA is an analog of nicotinamide and its antitumor effects can be reversed by equimolar amounts of nicotinamide (Nelson et al., 19’7’7), we tested the effect of nicotinamide on the antiviral activity of TDA in A. albopictus cells. As indicated in Table 3, nicotinamide (1 x 10e2 M) completely reversed the inhibition of SV replication caused by TDA, but had no effect on the Rbv- or MPA-treated cultures. Actinomycin (AMD), which reverses the antiviral activity of Rbv in A. albopictus cells (Malinoski and Stollar, 1980) was equally effective in reversing the antiviral effects of MPA and TDA (Table 4). In contrast to AMD, which inhibits DNAdependent RNA synthesis by intercalation in the DNA helix (Goldberg et al., 1962), a-amanitin inhibits RNA transcription by binding to RNA polymerase II (Lindell et al., 1970). As seen in Table 5, in (Yamanitin-sensitive A. albopictus cells, (LT C-7), cu-amanitin reversed the inhibition by ribavirin as effectively as did AMD. In contrast, in Amacells, which are resistant to
TABLE
Yield of Sindbis virus (PFU/ml)
inhibitor
added
None Ribavirin (6 x 1O-1 M) Mycophenolic acid (1.5 x lo-5M) 2-Amino-1,3,4-thiadiazole (1 x 10-Z M)
STOLLAR TABLE
4
THE EFFECT OF ACTINOMYCIN ON THE ACTIVITY OF RIBAVIRIN, MYCOPHENOLIC
ANTIVIRAL ACID, AND
Z-AMINO-1,3,&THIADIAZOLE Yield of Sindbis virus (PFUlml)
IMPDH
inhibitor
added
Without
None Ribavirin (6 x 1Om1M) Mycophenolic acid (3 x lo-JM) P-Amino-1,3,4-thiadiazole (1 x lo-*M)
With AMD (0.2 pgiml)
AMD
3.0 x 109 3.4 x 10’
1.6 x lo9 2.3 x 108
2.0 x 10’
1.3 x 109
2.4 x 10’
1.5 x 109
Note. A. albopictus cells were infected with SV as described in Table 1. Cultures were refed with E-10 medium, the various
compounds
were
added
to
give
the
final
concen-
trations indicated, and the cells were reincubated at 34.5”. Sixteen hours after infection samples of the medium were harvested and assayed for infectious virus.
a-amanitin and have an altered RNA polymerase II (Mento and Stollar, 1979), the antiviral effect of Rbv was not reversed by a-amanitin although it was by AMD. As both cr-amanitin and AMD can reverse the antiviral activity of Rbv, MPA, and TDA, (reversal by a+amanitin of MPA and TDA not shown) it appears that in order for these inhibitors to exert their antiviral effect, normal cellular RNA synthesis must occur.
3
THE EFFECT OF NICOTINAMIDE ON THE ANTIVIRAL ACTIVITY OF RIBAVIRIN, MYCOPHENOLIC ACID, AND ?&AMINO-1,3,4-THIADIAZOLE
IMPDH
AND
Without nicotinamide
With nicotinamide (1 x lo-*&f)
2.3 ~10~ 1.0 x 10’
2.5 x lo9 1.2 x 10’
5.0 x 10’
6.3 x 10’
1.5 x 10’
2.7
x 109
Note. A. albopictus cells were infected with SV as described in Table 1. Cultures were refed with medium E-10, the various compounds were added to the concentrations indicated, and the cells were reincubated at 34.5”. Sixteen hours after infection samples of the medium were harvested and assayed for infectious virus.
Phosphorylation
of Ribavirin
Since Rbv must be phosphorylated to be active (see above and Willis et al., 1978) and since the antiviral effects of Rbv can be reversed by AMD and a-amanitin, we wished to know if Rbv is phosphorylated normally in the presence of AMD. Uninfected A. aZbopictus cells were treated with [3H]ribavirin for 4 hr and an acid-soluble extract of the cells was prepared as described under Materials and Methods. Chromatography of the extract (Fig. 2) on PEI-cellulose (System B) revealed a small peak of radioactivity with an R,of 0.26, a minor peak with an Rfof 0.50, a major peak with an Rf of 0.77, and a shoulder on the last peak with an R,of 0.86. Since [3H]ribavirin, chromatographed in a separate channel, had an Rf of 0.86, we consider the shoulder on Peak 3 to be r3H]-
ANTIVIRAL
EFFECTS
OF TABLE
IMPDH
INHIBITORS
285
5
THE EFFECT OF WAMANITIN ON THE ANTIVIRAL ACTIVITY OF RIBAVIRIN IN o-AMANITIN-SENSITIVE AND a-AMANITIN-RESISTANT A. albopictus CELLS Yield of Sindbis virus (PFU/ml) Ama(resistant to cY-amanitin)
LT C-7
Ribavirin (6 x 10-‘&f)
a-Amanitin (10 &ml)
Actinomycin (0.2 pg/ml)
-
-
-
+ + +
+ + -
+ +
(sensitive to cu-amanitin) 1.9 x x x x x x
2.2 5.7 7.2 8.2 6.2
109 107 10” 10” 10” 10”
1.0 x 10” 1.0 x 10” 1.2 x 108 2.0 x 106 1.5 x 10” 1.2 x 108
Note. A. albopictus cells (clones LT C-7 and Ama-18) were infected with SV as described in Table 1. Cultures were refed with E-10 medium, the various compounds were added to give the final concentrations indicated, and the cells were reincubated at 34.5”. Sixteen hours after infection samples of the medium were harvested and assayed for infectious virus.
ribavirin. In this system, ribonucleoside triphosphates migrate very slowly (Ris of 0.18 to 0.30), ribonucleoside diphosphates somewhat more rapidly (R;s of 0.35 to 0.60), and monophosphates still more quickly (R;s of 0.50 to 0.88, see markers at the top of Fig. 2). Thus, from Fig. 2, we have tentatively identified peaks 1, 2, and 3 as the triphosphate, the diphosphate, and monophosphate forms, respectively, of Rbv. In this same system, Miller et al. (1977) obtained Rf values of 0.11, 0.33, and 0.53 for chemically synthesized triphosphate, diphosphate, and monophosphate forms, respectively, of Rbv. Miller et al. also reported an Rfof 0.88 for Rbv. When cultures were treated with AMD (0.4 pg/ml) the cells took up 30% less [3Hlribavirin (as measured by total acid-soluble counts) than control cultures and, as seen in Fig. 2, there were marked reductions (50% or more) in the amounts of both Rbv monophosphate and triphosphate. The amount of unphosphorylated Rbv was unchanged. When cells were labeled for up to 12 hr with [3H]ribavirin, less than 1% of the total radioactivity was found in acidinsoluble material. Thus, at least in the case of AMD, the reversal of the Rbv effect might be partially explained by an inhibition of phosphorylation.
o-e
no AMD
12
4
800 t : 600
400
200
DISTANCE
6
8
10
MIGRATED
12
14 (cm)
FIG. 2. Thin-layer chromatography of acid-soluble extracts prepared from [3H]ribavirin-labeled A. albopi&s cells. Monolayers of A. albopictus cells (5 x lo6 tells/60-mm petri dish) were treated with [3Hlribavirin (10 /.&i/ml) for 4 hr at 34.5” with or without AMD ‘0.4 pg/ml). Acid-soluble extracts were prepared as described under Materials and Methods. Ten microliters of the extract was spotted on PEI-cellulose and chromatographed in 1.2 M LiCl (System B) at room temperature. The chromatogram was cut into 5-mm segments which were then immersed in 5 ml of Formula 949 and counted in a scintillation counter. Standards were located by uv light absorbance.
MALINOSKI
286
d
J -rr
AND STOLLAR
this system monophosphates migrate with inorganic phosphate and TTP is not resolved from UTP. In extracts of cells treated with ribavirin (6 x 10e4 M) two major changes were seen. First, there was a decrease in the amount of radiolabeled GTP (the numbers below the GTP spot indicate the counts per minute when this spot was cut out and counted). Second, there was a new spot on the chromatogram which migrated ahead of CTP. When this 32P-labeled spot was eluted from the PEI-cellulose with 2.16 M triethylammonium carbonate (Volckaert et al., 1976) and then chromatographed using System B, it had an Rf of 0.20 (Fig. 4), a value close to that of Peak 1 (Rbv triphosphate) in Fig. 2. As shown in Fig. 5, chromatography using System A of an extract from cells labeled with [3H]ribavirin revealed a peak of radioactivity with the same Rf as the new
FIG. 3. Thin-layer chromatography of acid-soluble extracts prepared from 32P-labeled A. albopictus cells treated with ribavirin. A. albopictus cells were grown for ‘72 to 96 hr at 28” to confluent monolayers in medium containing 5 @Zi 32P04/ml. Rbv (6.4 x 10m4M) was added, the cells were incubated at 34.5”, and acidsoluble extracts were prepared at the times indicated as described under Materials and Methods. Five microliters of each extract was spotted on PEI-cellulose and chromatographed in 0.85 M KH,PO, (PH 3.5) (System A). The chromatogram was dried and exposed to X-ray film as described under Materials and Methods. The locations of the nucleotide standards were determined by uv light absorbance. The location of PO, was determined by chromatography of our 3zP0, stock. The numbers below the GTP spots indicate the counts per minute in each spot.
Effect of IMPDH Inhibitors on Intracellular Nucleotide Pools of A. albopictus Cells A. albopictus cells were grown in the presence of 32P-inorganic phosphate for 72 to 96 hr and then treated with Rbv, MPA, or TDA. At various times after the addition of the drugs, extracts were prepared and analyzed by thin-layer chromatography using System A, as described under Materials and Methods. In untreated cells (Fig. 3) six wellseparated spots were resolved which had R;s corresponding to NAD, free inorganic phosphate, UTP, CTP, ATP, and GTP. In
FIG. 4. Thin-layer chromatography of the new 32Plabeled spot from extracts of ribavirin-treated A. albopicks cells. The new 32P-labeled spot seen in Rbvtreated cells (Fig. 3) was eluted from the PEI-cellulose with 2.16 M triethylammonium carbonate and chromatographed using System B on PEI-cellulose. Lane A represents 1 ~1 of the eluent and Lane B, 5 ~1.
ANTIVIRAL
EFFECTS 1
GTP
1
ATP c I
UTP I
FRONl ‘1
300 300
-
ORIGIN
v
OF IMPDH INHIBITORS
Figure 3 also shows that the effect of Rbv on the GTP pool is rapid since within 2 hr of treatment the GTP pool was reduced by 80%. In another experiment (not shown) a 30% reduction in GTP was observed within 30 min by which time the ribavirin triphosphate could also be seen. The reduction of the GTP pool by Rbv was observed in SVinfected cells as well as in uninfected cells (not shown). Mycophenolic acid and 2-amino-1,3,4thiadiazole had effects on GTP levels similar to those of Rbv. Figure 6 illustrates that extracts of cultures treated for 4 hr with 1.2 x 10e5 and 6.0 x low5 M MPA contained 48 and 70%, respectively, less GTP M
loo100
11’1
012
DISTANCE
MIGRATED
287
M M
2341
(cm)
FIG. 5. Thin-layer chromatography of acid-soluble extracts prepared from [3H]ribavirin-labeled A. albop&us cells. Monolayers of A. albopictus cells (5 x lo6 cells) were treated with [5-3H]ribavirin (10 &i/ml) for 12 hr and acid-soluble extracts were prepared as described under Materials and Methods. Ten microliters of the extract was spotted on PEI-cellulose and chromatographed using System A. The chromatogram was then cut into 5-mm segments, which were immersed in 5 ml of Formula 949 and counted. The location of standards was determined by uv light absorbance.
spot seen in Fig. 3. (Free Rbv migrates near the solvent front in this system.) We have also demonstrated that when the new spot (32P-labeled) was eluted from a thin-layer chromatogram like that shown in Fig. 3 and analyzed by high-performance liquid chromatography (a strong anionexchange column and gradient elution were used) it eluted in the ribonucleotide triphosphate region and had a retention time very close to that of ATP (not shown). Using a very similar hplc system, Zimmerman and Deeprose (1978) observed a derivative of ribavirin, formed by human blood cells, which had very similar chromatographic properties and concluded that it was the triphosphate of ribavirin. These results, taken together, strongly suggest that the new spot seen in Fig. 3 is ribavirin triphosphate.
EJ
ORIGIN
FIG. 6. Thin-layer chromatography of acid-soluble extracts prepared from 32P-labeled A. albopictus cells treated with mycophenolic acid. A. albopictus cells were grown in the presence of 32POaas described under Materials and Methods. MPA was added to a fmal concentration of 1.2 or 6 x 10m5M and acid-soluble extracts were prepared 4 hr later, as described under Materials and Methods. Five microliters of each extract was spotted on PEI-cellulose and chromatographed using System A, after which the chromatogram was dried and exposed to X-ray film. The locations of the nucleotide standards and PO, were determined as described for Fig. 3. Abbreviations are AMD for actinomycin D, and MPA for mycophenolic acid. The numbers below the GTP spots indicate the counts per minute in each spot.
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than untreated cultures; similarly, TDA (1 x lop2 M) reduced the GTP level to 20% of normal by 6 hr after treatment (Fig. ‘7). Although no new spots were seen in these chromatograms, it is still possible that phosphorylated forms of these compounds were present but were not resolved from other nucleotides or free inorganic phosphate seen in cell extracts (Fig. 3). Since actinomycin reversed the antiviral effect of each of the IMP dehydrogenase inhibitors and inhibited the phosphorylation of Rbv, we next tested whether AMID was able to reverse the reduction in the GTP pool brought about by RBV, MPA, and TDA. Figure 8 shows that in cells treated with Rbv and AMD the level of GTP is close to that in untreated cells and much higher
FIG. 8. Thin-layer chromatography of acid-soluble extracts prepared from 3ZPOFlabeled A. a1bopictu.s cells treated with ribavirin and actinomycin. A. albopictus cells were grown in the presence of 32P04 as described in Fig. 3. Rbv (6.4 x 10e4 M) and AMD (0.2 pg/ml) were added, as indicated, and the cells were incubated at 34.5”. Twelve hours later, an acid-soluble extract of each sample was prepared as described under Materials and Methods. Five microliters of each extract was spotted on PEI-cellulose and chromate graphed using System A, after which the chromatogram was dried and exposed to X-ray film. The locations of the nucleotide standards and ‘*PO, were determined as described for Fig. 3. The numbers below the GTP spots indicate the counts per minute in each spot.
YI FIG. 7. Thin-layer chromatography of acid-soluble extracts prepared from 32P04-labeled A. albopictus cells treated with Z-amino-1,3,4-thiadiazole (TDA). A. albopictus cells were grown in the presence of 32P0, as described for Fig. 3. TDA was added to a final concentration of 1 x lo-* Y. Acid-soluble extracts were prepared as described under Materials and Methods. Five microliters of each extract was spotted on PEI-cellulose and chromatographed using System A, after which the chromatogram was dried and exposed to X-ray film. The location of nucleotide standards and PO, were determined as described in Fig. 3. The numbers below the GTP spots indicate the counts per minute in each spot.
than in cells treated only with ribavirin. In cells treated with AMD alone, there was a considerable increase in the size of the GTP pool. Actinomycin also reversed the reduction in GTP brought about by MPA (Fig. 6) or by TDA (data not shown). Consistent with the results in Fig. 2, AMD prevented the appearance of the new spot, ribavirin triphosphate (Fig. 8). DISCUSSION
At least three different mechanisms have been proposed (see Introduction) which might explain the antiviral effect of Rbv. Depending on the specific mechanism invoked, either Rbv monophosphate or Rbv triphosphate would be the active intracellu-
ANTIVIRAL
EFFECTS
lar form of the drug. Miller et al. (1977) have shown that 8 hr after receiving an oral dose of Rbv, ribavirin, as well as the various phosphorylated derivatives, were found in the liver of rats. Of the total ribavirin in the liver, 50% was unphosphorylated, while 23, 18, and 5% were found as Rbv monophosphate, Rbv diphosphate, and Rbv triphosphate, respectively. In our experiments with [3H]Rbv in A. albopictus cells, only 12% of the total intracellular ribavirin remained unphosphorylated after 4 hr while the major phosphorylated form was Rbv monophosphate. One or two percent was found in the disphosphate form and about 8% in the triphosphate form. Although Rbv triphosphate has been shown to inhibit both influenza virus RNA transcriptase and vaccinia virus guanyltransferase, it is not yet known whether the concentrations of Rbv triphosphate achieved in cells approach the levels needed to inhibit these activities either in viva or in vitro. Similarly, while the inhibition of IMPDH by Rbv monophosphate has been demonstrated, there had been no evidence which correlated the decrease in GMP (or GTP) with an inhibition of virus replication in the same cell system. The one report which described decreased levels of GTP in cells treated with Rbv, did not include any data on antiviral activity in the Rbv-treated cells. We, therefore, sought in our experiments to determine which phosphorylated forms of ribavirin were found in A. albopictus cells and to see whether correlations could be drawn between the antiviral effect of Rbv and specific changes in the intracellular nucleotide pools. In this way, we hoped to find out whether or not the inhibition of virus replication could be explained mainly by an inhibition of cellular IMPDH (and the resultant reduction in GMP and GTP) and to examine possible mechanisms by which the antiviral activity of ribavirin can be reversed. We found that with Rbv as well as with two other inhibitors of IMPDH (MPA and TDA), there was a good correlation in A. albopictus cells between antiviral activity and a reduction in the GTP pool. Our findings on the GTP levels are consistent with those of Lowe et al. (197’7) who reported
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INHIBITORS
289
that MPA and Rbv decrease the level of GTP by 90% in L15’78Y murine lymphoma cells. Lowe et ~2. also found that on a molar basis MPA was 100 times as effective as Rbv in decreasing GTP levels and inhibiting cell growth. Similarly, we found in our experiments that, as measured by antiviral activity, MPA was 20 times as effective as Rbv. The antiviral effects of Rbv, MPA, and TDA in A. albopictus cells could be reversed in several ways. Although both xanthosine and guanosine have been shown to reverse the action of Rbv and MPA in measles virusinfected Vero cells and BSC-1 cells, respectively, in our studies only xanthosine was effective. The failure of guanosine to reverse the action of ribavirin is likely due to the fact that by itself, guanosine inhibits virus replication in these cells (Stellar and Malinoski, submitted for publication). Two inhibitors of cellular RNA synthesis, actinomycin D and cu-amanitin, were shown to reverse the antiviral action of Rbv, and in the ease of actinomycin, return the GTP to levels similar to those in untreated cells. Although these effects on GTP levels could be attributed to a decreased demand for ribonucleotide triphosphates because of decreased cellular RNA synthesis in the presence of AMD or a-amanitin, it is more difficult to explain why actinomycin prevents the phosphorylation of ribavirin (Figs. 2 and 8). At any rate, the observations that both actinomycin D and cr-amanitin reverse the antiviral action of three different IMPDH inhibitors suggests strongly that in order for the antiviral effects of these compounds to be expressed, normal cellular RNA synthesis must be carried on. Possibly cellular RNA synthesis and certain cell functions are needed for the conversion of these compounds to their active forms. Another point of interest is that the concentration of Rbv necessary to inhibit SV replication in A. albopictus cells (6 x lo-“M) is much higher than that needed for a 50% plaque reduction of four strains of influenza Type A virus on calf kidney cells (4 x lo+ M; Tisdale and Bauer, 1977). Whether this difference relates to the two viruses studied, to different mechanisms of inhibition of the two viruses, to differences between the host cells, or differences in growth conditions of
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the cells is unknown. We are currently exploring the role of the host cell by testing the antiviral effects of Rbv in a variety of different cell types as well as in mutants of A. albopictus cells which have alterations in purine metabolism. ACKNOWLEDGMENTS This investigation was supported by Grant AI-11290 from the National Institute of Allergy and Infectious Diseases, by the United States-Japan Medical Science Program through Public Health Service Grant AI05920, and by the Institutional National Research Service Award CA-09069 from the National Cancer Institute. REFERENCES CASHEL, M., LAZZARINI, R. A., and KALBACHER, B. (1969). An improved method for thin-layer chromatography of nucleotide mixtures containing 32Plabeled orthophosphate. J. Chromatogr. 40, 103109. CLINE, J. C., NELSON, J. D., GERZON, K., WILLIAMS, R. H., and DELONG, D. C. (1969). In vitro antiviral activity of mycophenolic acid and its reversal by guanine-type compounds. Appl. Microbial. 18,14-20. ERIKSSON, B., HELGSTRAND, E., JOHANSSON, N. G., LARSSON, A., MISIORNY, A., NOREN, J. O., PHILIPSON, L., STENBERG, K., STENING, G., STRIDH, S., and OBERG, B. (1977). Inhibition of influenza virus ribonucleic acid polymerase by ribavirin triphosphate. Antimicrob. Agents Chemother. 11,946-951. GILLIES, S., and STOLLAR, V. (1980). The production of high yields of infectious vesicular stomatitis virus in A. albopictus cells and comparisons with replication in BHK-21 cells. Virology 107,509-513. GOLDBERG, I. H., RABINOWITZ, M., and REICH, E. (1962). Basis of actinomycin action, I. DNA binding and inhibition of RNA-polymerase synthetic reactions by actinomycin. Proc. Nat. Acad. Sci. USA 48,2094-2101. GOSWAMI, B. B., BOREK, E., SHARMA, 0. K., FUJITAKI, J., and SMITH, R. A. (1979). The broad spectrum antiviral agent ribavirin inhibits capping of mRNA. Biochem. Biophys. Res. Commun. 89,830836. HERSHFIELD, M. S., and SEEGMILLER, J. E. (1977). Regulation of de novo purine synthesis in human lymphoblasts. Similar rates of de novo synthesis during growth by normal cells and mutants deficient in hypoxanthine-guanine phosphoribosyltransferase activity. J. Biol. Chem. 252, 6002-6010. HERSHFIELD, M. S., SNYDER, F. F., and SEEGMIL-
LER, J. E. (1977). Adenine and adenosine are toxic to human lymphoblast mutants defective in purine salvage enzymes. Science 197, 1284-1287. LINDELL, T. J., WEINBERG, F., MORRIS, P. W., ROEDER, R. G., and RUTTER, W. J. (1970). Specific inhibition of nuclear RNA polymerase II by cy-amanitin. Science 170,447~449. LOWE, J. K., BROX, L., and HENDERSON, J. F. (1977). Consequences of inhibition of guanine nucleotide synthesis by mycophenolic acid and virazole. Cancer Res. 37, 736-743.
MALINOSKI, F., and STOLLAR, V. (1980). Inhibition of Sindbis virus replication in Aedes albopictus cells by virazole (ribavirin) and its reversal by actinomycin: A correction. Virology 102,473-476. MENTO, S. J., and STOLLAR, V. (1979). a-Amanitin resistant RNA polymerase II from Aedes albopictus cell mutants resistant to cY-amanitin. Cell Biol. Znt. Rep.
3, 725-732.
MILLER, J. P., STREETER, D. G., ROBINS, R. K., and SIMON, L. N. (1977). The relationship between the metabolism of ribavirin and its proposed mechanism of action. Ann. N. Y. Acad. Sci. 284,211-229. NELSON, J. A., ROSE, L. M., and BENNETT, L. L., JR. (1977). Mechanism of action of 2-amino-1,3,4-thiadiazole (NSC 4728). Cancer Res. 37, 182-187. SARVER, N., and STOLLAR, V. (1978). Virazole prevents production of Sindbis virus and virus-induced cytopathic effect in Aedes albopictus cells. Virology
91,267-282. SHENK, T. E., and STOLLAR, V. (1972). Viral RNA species in BHK-21 cells infected with Sindbis virus serially passaged at high multiplicity of infection. Biochem. Biophys. Res. Commun. 49, 60-67. SHENK, T. E., KOSHELNYK, K. A., and STOLLAR, V. (1974). Temperature-sensitive virus from Aedes albopictus cells chronically infected with Sindbis virus. J. Viral. 13, 439-447. SIDWELL, R. W., ROBINS, R. K., and HILLYARD, I. W. (1979). Ribavirin: An antiviral agent. Pharmucol. Ther. 6, 123-146.
SMITH,C.M.,FONTINELLE,L.J.,MUZIK,H.,PATERSON, A. R. P., UNGER, H., BROX, L. W., and HENDERSON, J. F. (1974). Inhibitors of inosinate dehydrogenase activity in Ehrlich ascites tumor cells in vitro. Biochem. Pharmacol. 23, 2727-2735. STOLLAR, V., SHENK, T. E., and STOLLAR, B. D. (1972). Double stranded RNA in hamster, chick and mosquito cells infected with Sindbis virus. Virology 47, 122-132. STOLLAR,V., STOLLAR, B. D., Koo, R., HARRAP, K. A., and SCHLESINGER, R. W. (1976). Sialic acid contents of Sindbis virus from vertebrate and mosquito cells. Equivalence of biological and immunological viral properties. Virology 69, 104-115.
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EFFECTS
STREETER, D. G., WITKOWSKI, J. T., KHARE, G. P., SIDWELL, R. W., BAUER, R. J., ROBINS, R. K., and SIMON, L. N. (1973). Mechanism of action of l-pn-ribofuranosyl-1,2,4-triazole-3-carboximide (Virazole), a new broad spectrum antiviral agent. Proc. Nat. Acad. Sci. USA 70, 1174-1178. TISDALE, M., and BAUER, D. J. (1977). The relative potencies of anti-influenza compounds. Ann. N. Y. Acad.
Sci. 284,254-263.
VOLCKAERT, G., MIN Jou, W., and FIERS, W. (1976). Analysis of 32P-labeled bacteriophage MS2 RNA by
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a mini-fingerprinting procedure. Anal. Biochem. 72, 433-446. WILLIS, R. C., CARSON, D. A., and SEEGMILLER, J. E. (1978). Adenosine kinase initiates the major route of ribavirin activation in a cultured human cell line. Proc.
Nat.
Acad.
Sci.
USA
75, 3042-3044.
ZIMMERMAN, T. P., and DEEPROSE, R. D. (1978). Metabolism of 5amino-I-&~ribofuranosyl-imidazole4-carboxamide and related five-membered heterocycles to 5’-triphosphates in human blood and L5178Y cells. Biochem. Pharmacol. 27, 709-716.