Inhibition of influenza virus synthesis by 2-thiouracil

VIROLOGY

Inhibition

6, 337-347 (19%)

of Influenza

Virus

Synthesis

HAROLD AMOS’ AND ELIZABETH Department

by 2-Thiouracil

VOLLMAYER

oJ” Bacteriology and Immunology, Harvard School, Boston, Massachusetts

Medical

Accepted May 12, 1958

Some aspects of the inhibition of influenza virus synthesis hy thiouracil has been examined. Uracil has been shown to counteract the inhibitor) action. Incorporation of C?-gnanine and P3*-phosphate into tissue RNA has been shown to increase on infection with influenza virus and to be depressed by thiouracil. Results further indicate that production of noninfectious virus may result from exposure of infected tissues to thiouracil. INTRODUCTION

2-Thiouracil, an analog of urncil, has for some time been viewed as a potential inhibitor of RNA synthesis. Though conclusive evidence for this action has not been presented, studies with tobacco mosaic virus (TMV) have contributed materially to this view. Commoner and Mercer (1951, 1952) first reported the inhibition of TMV synthesis in tobacco leaves treated with thiouracil and were able to counteract this effect with uracil. Bawden and Kassanis (1954) confirmed these results, and Holmes (1955) succeeded in curing with thiouracil plants in which lesions caused by infection with TMV had already developed. Similarly, even before the recent characterization of the nucleic acid of type A influenza virus as exclusively RNA (Miller 1956; Ada and Perry 1954), Hannoun (1953) reported (a) that thiouracil inhibited hemagglutinin production in the allantoic membranes of chick embryos infected with this virus; and (b) that uracil reversed the inhibition. The results of closer examination of the effect of thiouracil on influenza virus synthesis are presented in this paper. 1 This work was supported by a research grant, E-957, of the United Stat,es Public Health Service, by the William F. Milton Fund of Harvard Universitv. and by funds from the Eugene Higgins Trust. 337

338

AMOS

MATERIALS

AND

VOLLMAYER

AND

METHODS

Vrus. The PR8 strain of influenza A virus used in these experiments has been propagated for several years in the allantoic sac of the chick embryo. Minced ch&ioallantoic membrane. Growth of the virus in minced chorioallantoic (CA) membrane from ll- or 12-day chick embryos was effected in roller tubes. The cells were suspended in Hanks’ balanced salt solution (BSS). In this and in all other cases where Hanks’ solution was used, it contained 100 units of penicillin and 100 pg of streptomycin per milliliter unless otherwise specified. The carbon and energy source provided during virus synthesis was either glucose alone (0.1%) or sodium pyruvate (0.1%) plus glucose (0.05 %). The pH was adjusted to 7.6, and sufficient phenol red was added to each tube to permit visual detection of gross changes in pH. De-embryonated eggs. De-embryonated eggs were prepared from 14- to 16-day chick embryos by the method of Bernkopf (1949). The membranes were washed liberally with sterile physiological saline and drained, and to each was added 10 ml of Hanks’ solution with glucose. Additions after sealing the rubber caps with paraffin were made with hypodermic needle and syringe through the rubber cap. Hemagglutinin and infectivity determinations. Hemagglutinin titers were determined with fresh hen cells (0.2.5 %) in Hanks’ solution without glucose. Infectivity titers were est,imated by allantoic inoculation of 8to IO-day embryonated eggs, 3 eggs per dilution; the allantoic fluids were harvested after incubation for 48 hours at 35”; 1: 1, 1: 10, and 1:20 dilutions of each fluid were tested for hemagglutinin. Virus inoculum. Unless otherwise stated a seed inoculum of approximately 1000 egg infectious units (EID,a) of virus was employed per vessel. RNA, isolation, specific activity. RNA was isolated by the method of Schmidt and Thannhauser (1945) with the modification that perchloric acid was substituted for trichloroacetic acid. I .O N KOH digestion was permitted to proceed for 10 hours at 37”, after which DNA and protein were precipitated with perchloric acid. The supcrnatant fluid after centrifugation was neutralized with KOH. The RNA was estimated by ultra violet extinction (~2~~= 100 in a Beckman Model DU spectrophotometer. Samples of 0.1 ml were evaporated to dryness on stainless steel planchets, and the radioactivity was measured in a windowless gas-flow counter.

INHIBITION

OF

VIRUS

TABLE EFFECT OF THIOURACIL VIRUS IN MINCED

Expt. no. 1

1

ON HEMAOGLUTININ PRODUCTION BY PR8 INFLUENZA CA MEMBRANE AND IN DE-EMBRYONATED EGGS

system Minced

CA membrane

Thiouracil Gus/ml) 100 100 200 200

2

Minced

CA membrane

60 60 106 100

3

4

5

Minced

CA membrane

De-embryonated

De-embryonated

339

SYNTHESIS

-

32 32 4 <2 <2 <2 32 64 2 8 <2 <2

60 60 100 100 200 200

64 128 16 64 4 8 <2 <2

60 60 120 120 180 180

> 2560 > 2560 640 320 640 1280 80 160

egg

egg

He~~agrgl$in” Hemagglutinin” e tissue

10 10 20 20 60 60

640 320 160 160 80 20
80

160 40 20 20
340

AMOS

AND

TABLE Expt. no.

6

VOLLMAYER

l-Conlinued T~himx;il m

system

De-embryonated

egg

Hemagglutinina (Per mu

20 20 60 60 120 120

320 320 80
Hemagglutinid’ tissue

80 40 40 10
n Hemagglutinin expressed as the reciprocal of the highest dilution of fluid showing agglutination with 0.25yo hen cells. Fluids harvested after 48 hours’ incubation at 37”. b Tissue ground with Alundum, treated with RDE 2 hours-suspended in 10 ml of Hanks’ solution.

Chemicals. Both uracil and thiouracil were obtained from Nutritional Biochemicals Corporation, PS2 phosphate from the Atomic Energy Commission, and guanine-8-CL4 from Tracerlab, Inc. EXPERIMENTAL

E$ect of Thiouracil

on Influenxa

RESULTS

Virus

Undiluted PR8 virus (log. EID60 = 7.3) incubated at 37” with thiouracil (1 .O mg per milliliter or less) suffered no reduction of hemagglutinating activity or infectiousness for eggs on short incubation (3-5 hours); exposure to thiouracil for as long as 24 hours resulted in no more rapid deterioration of infectivity than in untreated controls. Inhibition

of Hemagglutinin

Productivn by Thiouracil

The addition of thiouracil to minced CA membrane or to de-embryonated eggs at the time of infection resulted in reduced production of virus as shown by determinations of hemagglutinin in the fluid phase at 48 hours (Table 1). Attempts to liberate virus from the tissues by grinding followed by treatment with receptor-destroying enzyme (RDE) were unsuccessful. The concentration of thiouracil required to suppress completely hemagglutinin synthesis varied considerably from one experiment to another and was consistently lower in the de-embryonated egg than with minced tissue in the roller tube. Thiouracil added to infected de-embryonated eggs as late as 16 hours

INHIBITION

OF

VIRUS

TABLE

341

SYNTHESIS

2

EFFECT OF TIME OF ADDITION OF THIOURACIL ON HEMAGGLUTININ IN DE-EMBRYONATED EGGS Thiouracil wdml

Expt. no.

1

2

De-embryonated

De-embryonated

egg

-

egg

120 120 120 120 120 120 120 120 120 120 -

Time of addition after virus (hours)

0 0 2 2 10 10 16 16 24 24 -

120 120 120 120 120 120 120 120 120 120

0 0 2 2 10 10 16 16 24 24

PRODUCTION

HemagglutininQ 24 hours

8 16 <2 <2 <2 <2 <2 <2 <2 <2 8 8 16 16 <2 <2 <2 <2 <2 <2 4 <2 8 32

48 hours

1280 640
a Hemagglutinin expressed as the reciprocal of the highest dilution of fluid showing agglutination with 0.25% hen cells. 24.Hour fluid harvest was made by withdrawing 1 .O ml from the de-embryonated egg with needle and syringe. b Virtually no detectable hemagglutinin could be released by grinding the tissue at 48 hours.

after infection (Table 2) still had some influence on virus hemagglutinin production, while its addition at 24 hours did not affect the yield. Incubation of uninfected tissues (48 hours, 37”) in concentrations of thiouracil as high as 300 pg per milliliter could not be shown to affect adversely their capacity of extension on glass or the Qo, with glucose as substrate.

342

AMOS

Antagonism of Thiouracil

AND

VOLLMAYER

by Uracil

Uracil added at the same time as thiouracil or within 10 hours can counteract the inhibitory effect of thiouracil (Tables 3 and 4). Adenine, guanine, or cytosine at the same concentrations as uracil exerted no such effect. The required concentration of uracil was found to be lower than that of the thiouracil, but a detailed study of this relationship was not attempted. TABLE SUPPRESSION

OF

Expt. no.

1

THIOURACIL

3

INHIBITION OF BY URACIL

System

Minced CA membrane

2

Minced CA membrane

3

De-embryonated egg

ThiouraciP (fig/mU

HEMAGGLUTININ

UracP Gg/ml)

300 300 300 300 300 300 300 300 300 300 300 300 120 120 120 120 120 120

-

PRODUCTION

Hemagglutininb (per ml)

32 64 <2 <2 32 32 64 32 16 64 4 <2 64 32 32 16 1280 640
a Uracil and thiouracil added at time of infection. ) Hemagglutinin expressed as the reciprocal of the highest dilution of fluid showing agglutination with 0.25% hen cells. Fluids harvested after 48 hours’ incubation at 37”.

INHIBITION

OF

VIRUS

TABLE EFFECT OF TIME OF ADDITION System Minced

343

SYNTHESIS

4

ON URACIL REVERSAL OF THIOURACIL Thiouracil” (300wdmu

INHIBITION

Time (hours) of Hemagglutinin in fluid” addition uracilb (200M/ml) Expt. 1 Expt. 2

CA membrane + + + + + + + + + + + + + +

0 0 4 4 8 8 10 10 12 12 15 15

64 128 <2 <2 64 32 64 16 64 64 16 16 <2 <2 <2 <2

32 32 <2 <2 16 64 32 32 16 32 8 8 <2 <2 <2 <2

a Thiouracil added at time of infection. * Uracil added after infection as indicat,ed. c In all cases hemagglutin measurements were made at 48 hours.

Incorporation

of P32 and C14-Guanine into RNA

Experiments were conducted to determine whether the presence of thiouracil altered the incorporation into tissue RNA of P32phosphate and of CY4-guanine. Intact allantoic membranes (four per flask) were shaken gently in 250-ml Erlenmeyer flasks for 24 to 48 hours at 37”. The RNA was isolated and its specific activity determined as described in the section on Materials and Methods. To show significant differences in P3* incorporation, it was necessary to incubate the membranes before infection for 12 to 18 hours in a medium “free” of phosphate. This medium, consisting of Hanks’ solution without phosphate and supplemented with KC1 and 0.005 M tris(hydroxymethyl)aminomethane, was changed twice during the period of ‘Lpreincubation” before the addition of standard Hanks’ solution with P3*. Under such conditions it was possible to show (Table 5) that infection increased the incorporation of phosphorus into the tissue RNA and that thiouracil lowered the incorporation into the RNA of both infected

344

AMOS

AND

VOLLMAYER

TABLE INCORPORATION

OF

P32 INTO

RNA

5

OF

NONINFECTED, CA TISSUEQ

URACIL-TREATED Expt. no. 1

Influenza virus

Ttyril

m

INFECTED,

AND

Hemagglutinin* (per ml)

cpm/mg RNA

-

-

-

2160

1000 EIDsa 1000 EIDS,, -

270 270

64 <2 -

3532 1218 1500

-

-

3017 5312 1710 2022

2

1000 EIDso 1000 EIDb,, -

32 <2

240 240

THIO-

a Initial Pa2 = 0.5 microcurie per flask; fluid volume per flask 70.0 ml of BSS with 0.1% glucose. * Hemagglutinin expressed as reciprocal of the highest dilution of fluid showing agglutination. Fluid harvested after 36 hours’ incubation at 37”. TABLE INCORPORATION

Thiouracil GdmU

Expt. no.

1

2

6

OF CWGUANINE INTO RNA THIOURACIL-TREATED

1000 EIDSo 1000 EIDso -

240 240

OF NONINFECTED,

Hemagglutinin’ (per ml)

1000 EII1ho

240 240

AND

cpm/mg RNA

128 <2 -

600 1232 416 373

64 <2

312 592 247 214

1000 EIDso

INFECTED,

CA TISSUEQ

a Guanine-Cl4 initially 0.2 microcurie per flask (1 mg guanine). Fluid volume per flask 70.0 ml of BSS with 0.1% glucose. * Hemagglutinin expressed as reciprocal of the highest dilution of fluid showing agglutination. Fluid harvested after 36 hours’ incubation at 37”.

and noninfected tissues. Experiments on the incorporation of CY4-guanine yielded similar results (Table 6). In the latter experiments it was not necessary to “preincubate” the tissue in a medium free of phosphate. Virus production under the conditions employed for these incorporation studies was relatively poor, as can be deduced from measurements

INHIBITION

of hemagglutinin estimated.

OF VIRUS

in the fluid phase. The infectivity

Zffect of l’hiouracil Virus

345

SYNTHESIS

upon the Proportion

of the fluids was not

of Infectious and Noninfectious

The influence of thiouracil upon the relative amount of infectious virus in fluids from de-embryonated eggs was examined. For this experiment concentrations of thiouracil were favored that were insufficient to reduce too sharply the hemagglutinin titer of the experimental fluids. A seed inoculum was used throughout and the fluids were harvested after incubation for 48 hours at 37”. Results of three representative experiments from a series of eight are presented in Table 7. It would appear that under the influence of thiouracil in low concentrations, a smaller proportion of the virus synthesized is infectious. TABLE EFFECT

Ihpt.

OF

THIOURACIL

no.”

Log EIDao/ml

1

7

ON THE PROPORTION NONINFECTIOUS VIRUS

OF INFECTIOUS

Log HA titer/ml

2.2 2.2 1.6

20 20

6.7 6.3 4.2 3.7 4.5 4.0

20 20

7.7 6.9 5.6 5.5 7.1 7.3 5.4 6.0 5.1 5.5

10 10

2

3 10

10 20 20

Log ratio -AFAE

4.5 4.1 2.6

1.0

1.8

1.9 1.9

2.6 2.1

3.1 2.8 3.4 3.1

4.6 4.1 2.2 2.4

2.5 2.8 2.5 2.5 2.8 2.5

4.6 4.5 2.9 3.5 2.3 3.0

u In all three experiments the system was de-embryonated B Each value represents a single de-embryonated egg.

TO

egg.

346

AMOS

AND

VOLLMAYER

DISCUSSION

Hannoun (1953) previously reported that thiouracil inhibited influenza virus production in the chick embryo; he demonstrated further that uracil could counteract this inhibitory action. The results reported in this paper confirm these effects with minced CA membrane tissue and in the de-embryonated egg. Some inhibition of hemagglutinin formation was observed even when thiouracil was added as late as 16 hours after infection. That is to say, the amount of hemagglutinin found in the fluid and tissue after 48 hours of incubation was very much reduced when thiouracil was added at 16 hours or before. However, thiouracil added at 24 hours did not appear to affect either the synthesis or release of virus during the next 24 hours. The effect of uracil in reversing the thiouracil inhibition was observed when the uracil was added up to 10 hours after infection. This antagonistic effect of uracil suggests that thiouracil may interfere with RNA synthesis. Further support for this hypothesis is to be found in the finding that thiouracil depresses the incorporation of P32 and of C4-guanine into tissue RNA. Increased turnover or synthesis of RNA in cells infected with influenza virus has been suggested from P32measurements by Cohn (1952) and by Womack and Kass (1953). In both of these studies small but consistent increases in specific activity were demonstrated in infected tissues. By preincubating the tissues in a medium without phosphate before infection, we have been able to demonstrate a greater effect of infection upon the incorporation of phosphorus into the tissue RNA. Apparently more readily utilizable phosphate reserves in the cells mask the increased RNA turnover in infected tissues not subject to such preincubation. Probably the most interesting finding in these experiments is the reduced relative production of infectious virus in the presence of thiouracil. The analyses of Ada (1957) and Schafer (1957) indicate that some varieties of “incomplete” virus contain less RNA than fully infectious virus. It would be of interest to examine thiouracil-grown virus for its RNA content and for the extent of thiouracil substitution into viral RNA. Comparable studies have already been accomplished with TMV; from its RNA thiouridylic acid has been isolated and characterized (Jeener and Roseels, 1953; Matthews, 1956; Mandel et al., 1957). REFERENCES ADA, G. L. (1957). Ribonucleic posium. The Nature of Viruses,

acid in influenza virus. C&a Foundation pp. 104115. Little, Brown, Boston.

Sym-

INHIBITION

OF VIRUS

SYNTHESIS

347

G. L., and PERRY, B. T. (1954). The nucleic acid content of influenza virus. Australian J. Exptl. Biol. Med. Sci. 32, 453-468. BAWDEN, F. C., and KASSANIS, B. (1954). Some effects of thiouracil on virusinfected plants. J. Gen. Micro&ok 10, 160-173. BERNKOPF, H. (1949). Cultivation of influenza virus in the chorio-allantoic membrane of de-embryonated eggs. Proc. Sot. Exptl. Biol. Med. 72, 680-682. COHN, Z. A. (1952). Quantitative distribution of phosphorus in chorioallantoic membrane as affected by infection with influenza virus. Proc. Sot. Exptl. Biol. Med. 79, 566-568. COMMONER, B., ~~~MERCER, F. L. (1951). Inhibition of the biosynthesis of tobacco mosaic virus by thiouracil. Nature 168, 113-114. COMMONER, B., and MERCER, F. L. (1952). The effect of thiouracil on the rate of tobacco mosaic virus biosynthesis. Arch. Biochem. Biophys. 36, 278-289. HANNOUN, C. (1953). Action des analogues puriques et pyrimidiques sm- la multiplication des virus de la grippe dans l’embryon de poulet. Atti congr. interna. microbial. 6th Congr. Rome, Italy 1965 1, 431-432. HOLMES, F. 0. (1955). Preventive and curative effects of thiouracil treatment,s in mosaic-hypersensitive tobacco. Virology 1, 1-9. JEENER, R., and ROSEELS, J. (1953). Incorporation of 2-thiouracil S3S in the ribose nucleic acid of tobacco mosaic virus. Biochem. et Biophys. Acta 11, 438. MANDEL, H. G., MARKHAM, R., and MATTHEWS, R. E. F. (1957). The distribution of thiouracil in nucleic acid of tobacco mosaic virus. Biochem. et Biophys. Acta 24, 205-206. MATTHEWS, R. E. F. (1956). Thiouracil in tobacco mosaic virus. Biochim. et Biophys. Acta 19, 559. MILLER, H. K. (1956). The nucleic acid content of influenza virus. Virology 2, 312-320. SCHHFER, W. (1957). Units isolated after splitting fowl plague virus. Ciba Foundation Symposium. l’he Nature of Viruses, pp. 91-103. Little, Brown, Boston. SCHMIDT, G., and THANNHAUSER, S. T. (1945). A method for the determination of desoxyribonucleic acid, ribonucleic acid, and phosphoproteins in animal tissues. J. Biol. Chem. 161, 83-89. WOMACK, C. It., and KAS~, E. H. (1953). Influenza virus in allantoic sac tissue. J. Zmmunol. 71, 152-167. ADA,