Gliotoxin: inhibitor of poliovirus RNA synthesis that blocks the viral RNA polymerase 3Dpol.

JOURNAL OFVIROLOGY,Apr. 1992,p. 1971-1976 0022-538X/92/041971-06$02.00/0

Copyright © 1992,American SocietyforMicrobiology

Gliotoxin: Inhibitor of Poliovirus

RNA

Synthesis

That

Blocks

the Viral RNA Polymerase

3DPo1

PEDRO L.RODRIGUEZAND LUIS CARRASCO*

Centro deBiologiaMolecular, Universidad Aut6noma, CantoBlanco, 28049Madrid, Spain Received23 September1991/Accepted3 December1991

Themodeof action of gliotoxinagainstpoliovirushas beenanalyzedindetail. Thisfungalmetabolite inhibits theappearanceofpoliovirus proteinswhen present from thebeginningofinfection but hasnoeffectonviral

translation when added atlate times. In agreementwith previous findings, this toxin potently inhibited the incorporation of[3Hluridineintopoliovirus RNAsoonafteritsadditiontothe culture medium.Analysisof the synthesis of poliovirus plus-orminus-strandedRNA inthepresenceofgliotoxinsuggests that thiscompound effectively hampered bothprocesses.This resultcontrastswith the mode of action of other inhibitors ofpoliovirus RNAsynthesis, suchasguanidineorflavones, thatselectivelyblockplus-strandedRNAsynthesisand suggests that the target ofgliotoxindiffers from the target ofguanidine, i.e., poliovirus protein2C.Indeed, gliotoxinwas found to be a potent inhibitor ofpoliovirus RNA synthesis in cell-free systems, using membranous crude replication complexes,areaction that isnotblockedby guanidineorRo 09-0179.Moreover,invitroactivityof

thepurified polioviruspolymerase3D°"'wasefficientlyinhibitedby gliotoxin.These resultsindicate that this toxin

actsonthe poliovirus polymerase3D1"',providingthe firstdescriptionofaninhibitor of this viralenzyme.

Picornaviruses are among the simplest and best-known animalviruses in molecularterms(10). Despitetheir simplic-ity,severalaspectsof theirreplicative cycleremainobscure (3, 11, 16, 25). Hopefully, detailed studies of the mode of action of selective inhibitors ofpicornavirus replicationwill provideabetterunderstandingof thoseprocesses. Although the main steps in the replication of poliovirus RNA have been delineated, there are still some reactions that remain puzzling (25). Thus, it is known thatoncethegenomicRNA is free in thecytoplasm, it is first translatedtogiverisetoan array of viral proteins (3, 11, 16). An unknown number of

these viralpolypeptides usetheplus-stranded genomicRNA as a template to make a complementary minus-stranded

RNA(19, 25). Thisnegative-stranded RNA isused, inturn,

as atemplatetosynthesizemorecopiesofpositive-stranded RNA molecules that can fulfill several requirements, i.e., they can be used as mRNAs to make viral proteins or as templates to make more copiesofnegative-stranded RNA, ortheycanbeencapsidatedtogenerate maturevirions(19, 25).

The virus-encoded RNA-dependent RNA polymerase, knownasprotein 3DPo1,is ableto elongate already initiated plus- or minus-stranded RNA molecules (24, 25). It was suggested that thegenome-linkedprotein VPg (viral protein 3B),wasusedas aprimertoinitiatenewchainsof viral RNA (19, 20). However, recent evidence indicates that this

pro-tein iscovalentlylinkedtothe RNAwell after the initiation event (22). Therefore, the exact mechanism of initiation of picornavirus RNA synthesis still remains controversial. Moreover, viral RNA replication complexes are tightly

bound to membranes, and the inhibition of membrane for-mation leadstoimmediatearrestofpoliovirus RNA synthe-sis(8).Perhapsviralprotein2Coritsprecursor2BC playsa

part in thecoupling between the RNAreplication complex and the membranous vesicles that proliferate throughout infection (1). A role for protein 2C in poliovirus RNA replication was suspected on the basis that the selective

*Correspondingauthor.

inhibitor ofpoliovirusRNA synthesis, guanidine, acted on protein 2C (17, 25). Apartfromthatfinding,theexactroleof protein 2C in RNAsynthesisis obscure. It isevenpossible

that other viralproteins,suchas2Bor3A,arealsoinvolved

in the replication of viral genomes (19, 25). Therefore, a

better understanding of the mode ofaction of inhibitors of poliovirus RNAreplicationcanhelptounravel thisprocess.

In addition to guanidine, which has been the classic inhibitor ofpoliovirusRNAsynthesis (21),otheragentshave been reported to selectively block the step of viral RNA replication. Thus, some natural flavones, such as 3-meth-ylquercetin (4, 5),orRo 09-0179 (7, 9),selectively block the synthesis of plus-stranded RNA without inhibiting minus-strandedRNAsynthesis at concentrations 1,000-fold lower than those forguanidine (7). 2-(a-Hydroxybenzyl)-benzimid-azole (HBB) (6), penicillamine (14), and gliotoxin (15) also block viral RNAreplication, but the detailedmechanism of action of thoseagentsis stillunknown. High concentrations ofpenicillamine (above 7 mM)orHBB (above 0.1 mM)are requiredtoblockpoliovirus (6, 14). Gliotoxin andanumber

ofrelated compounds,either from naturalsources or chem-icallysynthesized, arethemostpotent agentsthat interfere with poliovirus RNA synthesis (15, 23). We undertook an

analysis of the mode of action ofthis toxin, andthe results presented suggest that it differs from the effect of known poliovirus RNA inhibitors, such as guanidine and the fla-vones.

MATERIALS ANDMETHODS

Cells and viruses. HeLa cells were grown in Dulbecco

modified Eagle's medium supplemented with 10% newborn calfserum (GIBCO, Grand Island, N.Y.) andincubated at

37°C ina5% CO2 atmosphere. Poliovirus type 1 (Mahoney strain) (American Type Culture Collection) was grown in

HeLa cells. Cell culture and virus titration were as

previ-ouslydescribed(7).

Compounds. 4'-5-Dihydroxy-3-3',7-trimethoxyflavone

(Ro-1971

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1972 RODRIGUEZ AND CARRASCO

+

POLIOVIRUS

1 2345 6 7

-.M-- --2C

-.

1 2 345 6 7

B

+ POLIOVIRUS MocKinf ected cells

0 h.p.i. 4 h.p.i. 0 h.pi

addption

00.3061.5 3 0 030.61.53 00.30.61.53 GTX (pM)

Pi-

II

rn=_

4- 44

iim1 g.t"_

1-0

*0_ _s t a"_{IC=_}

as

C am*"_am

" At NM

li~ ~~, _ _

-Act'in

1C-_a

FIG. 1. Effect of gliotoxinontranslation. (A) Timecourseof the proteins synthesized in poliovirus-infected cells minus (-)orplus(+)

1.5 ,uM gliotoxin (GTX) fromzerotime. HeLa cellsgrownin 24-well plateswereinfected with poliovirusatanMOIof50PFUpercell. After

a60-min adsorption period, cellswerelabeledeveryhour with 20 ,uCi of [35S]methionineperml for 1 h. Proteinswereanalyzedon a15%

polyacrylamide-SDS gel asindicated in Materials and Methods. (B) Analysis of the proteins synthesized in poliovirus-infected cells or

mock-infectedcells between4to5hp.i. Different concentrations of GTXwereaddedat0or4h p.i.

09-0179)waskindly provided byH. Ishitsuka and K.Yokose, Nippon Roche Research Center (Kamamura, Japan). Glio-toxin, guanidine, and dactinomycin were purchased from Sigma Chemical Co., St. Louis, Mo.

Virus infection ofcell monolayers. HeLa cells were

in-fected with poliovirus (-1 h) in Dulbecco modified Eagle's medium supplemented with 2% newborn calfserum. After incubation at 37°C for 1 h, unattached viruswas removed and fresh mediumwas added(zero hours).

Polyacrylamide gel analysisofproteins.At the times indi-cated,cellswereincubated in methionine-free medium in the

presence of 20 ,uCi of [35SJmethionine (1,450 Ci/mmol; Amersham) perml. One hour later the radioactive medium

was removed, the cell monolayer was washed with phos-phate-buffered saline, and cellswere collected in 200

RI

of buffer containing 62 mM Tris-HCI (pH 6.8), 2% sodium dodecyl sulfate (SDS), 0.1 M dithiothreitol, 17% glycerol, and 0.0024%bromophenolblue. Eachsamplewassonicated

to reduce viscosity and heated at 90°C for 5 min. Five microliterswasappliedtoa15%polyacrylamide gelandrun overnightat 100 V. Fluorographyof thegelwascarriedout with 2,5-diphenyloxazole-dimethyl sulfoxide (20% wt/vol).

Estimation of uridine incorporation in RNA. HeLa cells

grown in 24-well plates were infected with poliovirus at a multiplicityof infection (MOI)of 50 PFUpercell. Dactino-mycin (5

jig/ml)

wasadded atzero hours. Everyhour after infection, 10 ,uCi of [3H]uridine (25 to 30 Ci/mmol; Amer-sham)permlwasadded. Thelabelingmediumwasremoved

after 1h, and the cellmonolayerwastreated with 0.5 ml of

5% trichloroacetic acid, washed twice with ethanol, dried under an infrared lamp, and dissolved in 200 ,u of 0.1 N NaOH-1% SDS. Samplesof 150plwerecounted inaliquid

scintillation spectrophotometer.

Synthesis of labelled RNA probe. The poliovirus

strand-specificRNAprobewasgenerated byinvitrotranscription ofafragmentofpoliovirus cDNA encompassing nucleotides 2099to4600, subcloned inaBluescriptvector (Stratagene). The poliovirus cDNAsequence was flanked by T7 and T3

promoters. Using T7 (New England Biolabs) or T3 RNA polymerase (Stratagene), weobtained RNAprobes specific

tothe viral minus-orplus-stranded poliovirus RNA,

respec-tively.Theprobeswerelabeled with[32P]CTP (400 Ci/mmol; Amersham).

62~~~~~~~~~2

A a B

0

so 480

.20-ol . . ...__. L.

0 1 2 3 4 5 6 7 8 910 0 0.5 1 1.5

TIME (h.p.l.) GilotoxinILN

FIG. 2. Inhibition ofpoliovirus and cellular RNAsynthesis by

gliotoxin. (A)TimecourseofpoliovirusRNAsynthesis.Conditions

of infection and analysis of RNA synthesiswere as described in

Materials and Methods. Poliovirus-infected cells in the absence of

inhibitor(O)orin thepresenceof 1.5p,M gliotoxinfromzerotime

(0) or at 1.5 (Cl), 3.5 (-), or 5.5 (A) h p.i. Pulse-labelingwith [3H]uridinewascarried outevery hourp.i. (B)Effect of different

concentrations ofgliotoxinonRNAsynthesisin mock-infected cells

in the absence ofdactinomycin (0)orinpoliovirus-infectedcells in

thepresenceofdactinomycin (-). RNAsynthesiswasdetermined

between 4to5 hp.i.Gliotoxinwasaddedat1.5 hp.i.

A

GTX 1.5 pM

TIME (h.p.i.)

Actin

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GLIOTOXIN BLOCKS POLIOVIRUS 3DPo' 1973 + Polhovirus

hp.i.GTX 0 2 4

addition

- Poiiovirus

0

GTX added

-5

hp.i. GTX 0 2 4 o

addition __

a,

v

0

-a:

GTX added _- +

RNAexrcted 0 7 2 7 4 7 7 at hp.i.

,_ AI

40-3

30- L-W

20--1

ce

, 10 cU ID

-ssRNA

RNA extracted 0 7 2 7

ath pi.

GTX addition RNA extracied

0+ 2+ 4+ at7hpi

L IA

501

40

-

30-20

0- 2- 4-

7-L 7-LJ

f

Synthesis RNA 10

S-I

-RFRNA

4 7 7

RNA

-O

O-O- 0

0- o0 2- 2- 4- 4+ 7- 0- 0- 2- 2- 4- 4+

7-FIG. 3. Effectofgliotoxin(GTX)onthesynthesisofpoliovirusplus-orminus-stranded RNA. HeLacellsgrownin 60-mm-diameter dishes wereinfectedwith poliovirus at50PFUpercell. Total RNAwasextractedfromuntreated(-) cellsat0, 2, 4,or7 hp.i.Total RNAfrom cells treated with 1.5p.M gliotoxin (+)wasextractedat7 hp.i. The inhibitorwasaddedatthe indicatedtimes,andincubation continued until the 7thh. TheRNAwasextracted andanalyzedbyNorthernblottingasindicated inMaterials andMethods. Thesameblotwashybridized

topositive-stranded(RNA-)and negative-stranded(RNA+) poliovirus riboprobes.

Northern (RNA) blot

analysis

of poliovirus RNA. HeLa cellsweregrownin60-mm-diameterdishes and infected with

poliovirus at an MOI of 50 PFU per cell. At the indicated timesafterinfection, the cellswerelysedinabuffer

contain-ing10 mMTris-HCl (pH7.8), 1 mMEDTA, 150 mMNaCl,

and 0.65% Nonidet P-40. After removing nuclei by low-speedcentrifugation,supernatantsweremixed withanequal

volume ofabuffercontaining20 mM Tris-HCl (pH7.8),20 mMEDTA, 350 mM NaCl, and 1% SDS. Total RNAwas

extractedwithphenolandprecipitatedwithethanol.

North-ern blot analysis with a radiolabeled probe was performed (7). The quantitation was done with a 3,000-A computing

densitometer (Molecular Dynamics).

In vitro synthesis of poliovirus RNA. HeLa cells were

infected with poliovirus at an MOI of 200 PFU per cell, incubatedat37°C, and harvestedat 7 hpostinfection (p.i.). Preparation of the crude membrane fraction and its treat-ment with DEAE cellulose were performed as previously

described(20, 25).Invitro RNAsynthesiswascarriedoutin

a reaction mixture containing 50 mM N-2-hydroxyethylpi-perazine-N'-2-ethanesulfonic acid (HEPES)-KOH (pH 8),

3.5 mMmagnesiumacetate, 50mM

KCI,

10

pg

of dactino-mycin per ml, 2.5 mM phosphoenol pyruvate, 2 ,ug of pyruvate kinase, 10 p,g of DEAE-cellulose-treated mem-branes, and compounds at the concentrations indicated in each experiment. This mixturewasincubated for 30min at 4°C. Then, 50 p,Ci of

[3H]ATP

(56 Ci/mmol) per ml-1 mM UTP-1 mMCTP-1 mM GTP-100 pMATPwasadded, and incubation continuedat30°C for various periods of time. The reactionwas stoppedby the addition of 10% trichloroacetic acidat4°C.The RNAwascollectedonWhatmanGF/C fiber

filters, and theradioactivity incorporated in RNAwas deter-mined inascintillation counter.

Enzymatic assay of poliovirus RNA polymerase. Purified poliovirus RNApolymerase was kindly provided by S. J. Plotch (Lederle Laboratories, Pearl River, N.Y.). Poly(A)-dependentoligo(U)-primed poly(U) polymerase activitywas

measured at 30°C for 60 min as previously described (18),

with the modifications that dithiothreitol (DTT) was not added and[3H]UTPwas used asthe labeled nucleotide (45 Ci/mmol;Amersham).

RESULTS

Effect of gliotoxin on poliovirus protein synthesis. Glio-toxin, a fungal metabolite, inhibits the growth of several animal viruses, including picornaviruses. Earlier evidence indicated that it was a selective inhibitor of picomavirus

RNAsynthesisand had lesseffectonprotein synthesiswhen added laterduringinfection(15, 23). However,thosestudies

onlyrelied upon theincorporationofradioactiveprecursors into trichloroacetic acid-precipitable material. Thus, itwas ofinteresttoexamine the effects ofgliotoxinoncellular and viral protein synthesis and to analyze the proteins synthe-sizedbypolyacrylamide gel electrophoresis. Concentrations ofgliotoxin above 0.6 ,uM are effective in the inhibition of viralprotein synthesis (Fig. 1B)if presentfrom thebeginning

ofinfection, butno effectwasobservedonviral translation when gliotoxinwas added 4hp.i. Gliotoxin concentrations of 3 ,uM had almostno effecton cellularprotein synthesis. On theotherhand,although gliotoxin effectivelyreducedthe

synthesis of viral proteins, itwas ineffective inpreventing VOL. 66,1992

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1974 RODRIGUEZ AND CARRASCO

+Poliovirus -Pohiovirus

.Ej C)

h p.i. compound 0 2 4 0 a,

addition - C} I

Compound - +G±R - +G+R - +G +R

-added

* _M -ssRNA

RNAextracted 0 7 7 2 7 7 4 7 7 7

at hp.f 30-"

- RNA+

vw a

E!

20-CA 10 a

U,_

0.2

o o

0-OGOR2-2G22R4-4G4R

7-h p.i. compound 0 2 4 o

c-addition

Compound +G+R - GGRRG -R

added

,A&

_ _

RNA extracted at hp.i. 50'

40

30

20

10

0

..v...

0 7 7 2 7 7 4 7 7 7

RNA

-O-OGOR2-2G2R 4-4G4R

7-- RF RNA

FIG. 4. Effect ofguanidineand Ro 09-0179 onthesynthesis ofpoliovirus plus- or minus-stranded RNA. The experiment was carried out asdescribed in thelegendtoFig. 3, exceptthat3mMguanidine (+G)or3,uM Ro 09-0179(+R)wasadded instead ofgliotoxin.

the shutoff of host translation induced by the infection of

poliovirus athigh multiplicities (Fig. 1A). These results are

similartothosefound for other inhibitors ofpoliovirusRNA replication (5, 7).

Actionofgliotoxin on viralRNAsynthesis.Figure 2 shows the ability ofgliotoxin to block poliovirus RNA synthesis

when addedat different timesp.i. When gliotoxin (1.5 ,uM) wasadded at zero time or 1.5 hafter infection, viral RNA

synthesis was completelyblocked. Addition of thedrug at 3.5 h p.i. significantly reduced the peak of viral RNA

synthesis observed at 5 h (Fig. 2A). A concentration of

gliotoxin of 0.6 ,uMinhibited poliovirus RNA synthesis in infected cellsby80%,whereas cellular RNAsynthesiswas only slightly affected by 1.5 ,uM gliotoxin. These results agreewell with previous

findings

on

gliotoxin activity.

Ina similar assay, the flavone Ro 09-0179 was twofold less

efficacious,whereasconcentrations of 200 ,ug of HBB per ml or1.5 mMguanidinewererequiredtoblockpoliovirusRNA

synthesis by 80% (results notshown).

We foundrecentlythatflavones, suchas

3-methylquerce-tin or Ro 09-0179, were selective inhibitors of poliovirus

plus-stranded RNA synthesis, whereas the formation of minus-strandedRNA was evenstimulated in the presence of Ro 09-0179 (7). To test the action ofgliotoxin on plus- or minus-stranded RNA synthesis, specific riboprobes were made and the formation ofplus-orminus-stranded poliovi-rusRNA wasmeasuredthroughout infection.Inparallel,the inhibitorgliotoxinwas added at different timesp.i. and the RNAwas extracted at the end ofpoliovirus infection (7 h

p.i.). Figure3 shows thatgliotoxinefficientlyinterfereswith thesynthesisof bothplus-and minus-stranded RNAchains when present at any timep.i. It is ofparticular interestto focusonthe inhibition inducedbygliotoxinatthe 4th h

p.i.

Atthat time mostof the

plus-stranded

RNAs have

already

been synthesized, but the increase in plus-stranded RNA from 4 to7 h ishampered bygliotoxin. On the otherhand,

mostviral minus-stranded RNAismade from the 4th tothe

7th hp.i. This increase innegativeRNAis also blocked by the toxin. In a similar assay, guanidine had no effect on negative RNAsynthesis, whereas Ro 09-0179increased its formation by 200%(Fig. 4).

Inhibition of in vitro poliovirus RNAsynthesis. Noneof the

available inhibitors ofpoliovirusRNAsynthesisis effective

in cell-free systems. A system that faithfully synthesizes

both poliovirus plus- and minus-stranded RNA in vitro has been developed by purifying the replicative complexes

bound to membranes, the so-called crude replication com-plex (CRC) (25). Curiously enough, gliotoxinwas apotent inhibitor of this reaction (Fig.

5A).

Gliotoxin (750

p.M)

inhibited RNAsynthesisin CRCby60%,whereasguanidine

and Ro 09-0179 had littleor no effect

(Fig. SA).

The RNA made in these systems by crude replicative complexes mainlycomesfromelongationcarriedoutbythe

polymerase

3DP"°,

although initiation ofnewchains in such systems has beendescribedpreviously (19, 25). Thus, although

gliotoxin

is the first compound shown to inhibit

poliovirus

RNA synthesisin theCRC, itcannotbeconcluded that itactson polymerase

3DP"°.

In orderto testthis

possibility,

theeffect of gliotoxin was assayed in cell-free systems that used

purified 3D polymerase

(18). Figure

6A, shows that the

incorporation of [3H]UTPdirected bypoly(A) in the pres-enceofoligo(U)is alsoblockedbygliotoxin.Theinhibition of thisreactionbygliotoxinismorepotentthan theblockade ofthe CRC. Thus, 150 ,uMgliotoxinblocked the

incorpora-tion of

[3H]UTP

in this assayby 80%.On the other

hand,

this inhibitionwasspecificforgliotoxin,since otherinhibitors of

poliovirus RNAsynthesis, such asguanidineor R09-0179,

had no effect (Table 1). The presence of a reducing agent J. VIROL.

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GLIOTOXIN BLOCKS POLIOVIRUS 3DP°' 1975 E

0)

i

E 120

0

1004

0

4-

20-!

n-0 20 40 60 0 150300400600750900

inme nln Glktoxln pi

FIG. 5. Effect of gliotoxin, guanidine,and Ro 09-0179oninvitro

synthesis of poliovirus RNA. (A) Preparation ofthe crude

mem-brane fraction and in vitro RNA synthesis were carried out as

described in Materials and Methods. The DEAE-cellulose-treated membraneswereincubatedat30°Cfor variousperiodsof time inthe absence of inhibitor (0)orinthepresenceof750 p.Mgliotoxin(0), 30 mM guanidine (E), or 750 puM Ro 09-0179 (-). (B) Effect of different concentrations of gliotoxinoninvitro RNAsynthesis. such as DTT prevents the inhibition of poliovirus RNA synthesisinintact cells(23), suggestingthat the blockadeby gliotoxin involvestheformation of disulfidebridgesbetween gliotoxin and essential sulfhydryl groupson theviral poly-merase. The data of Table 1 indicate that the presence of DTT in the cell-free system partially reduced itsinhibitory potential, in agreement with the idea that reducing the sulfhydrylgroups abolishes theactivityofgliotoxin (23).

DISCUSSION

Elucidating the exact steps involved in replication of poliovirus RNA is an important goal. Although the major

biochemical events are known, there are still a number of

stepsthat remain obscure(3, 19,25). Thus, theexactmode of initiation of viral plus-andminus-stranded RNA synthe-sis, the coupling between membranes and replication com-plexes, and the identification of host proteins, if any, in-volved in this process need to be clarified (8, 19, 22). Elucidation ofsomeof theseprocessesatthemolecularlevel will be facilitatedbythe use ofselective inhibitors of viral RNAsynthesis. Inordertousethese inhibitorsproperlywe

need first to carry out detailed studies of their mode of action.

Few inhibitors ofpicornavirus RNA synthesis are

avail-I

0 20 40

Tlm min.

60 0 150 300 450 600

Glktoxinpm

FIG. 6. Effect ofgliotoxinoninvitro poly(U) polymerase

activ-ity.(A) Enzymaticassayofpoliovirus RNApolymerasewascarried

out as described in Materials and Methods. Poly(U) polymerase

activitywasmeasured forvariousperiods of time inthe absence (0)

orpresence(-)of600,uM gliotoxin. (B) Effect of different

[image:5.612.64.298.78.203.2]

concen-trationsofgliotoxinonpoly(U) polymerase activity.

TABLE 1. Effect ofinhibitors of poliovirus RNAsynthesis oninvitro

3DPo0

activity'

[3H]UTP incorporated

Incubationconditions

cpm %Control

Time, 0 min 373 0

Time,60 min + 5 mM DTT 20,063 95

Time, 60 min

Minus DTT 21,075 100

Minus

3DPO1

524 0.7

Minuspoly(A) 423 0.3

Minus oligo(U) 2,134 9

Plus 2% dimethyl sulfoxide 20,468 97

Plus 30 mM guanidine 16,973 80

Plus 750 ,uM Ro 09-0179 22,676 107

Plus 600 ,uM gliotoxin 1,697 6

Plus 600 ,uM gliotoxin + 5 mM 7,291 33 DTT

a Poly(U) polymerase activitywasmeasured at30'Cfor60minasdescribed

in thelegendtoFig. 6. Inhibitorswereaddedat zerotime. Dataarethe means

ofduplicate samples.

able at present(2). Only the modes of action of guanidine and some flavones arepartially known (7, 21). These com-pounds inhibit polioviruspositive-stranded RNA synthesis (7). Flavones stimulate the synthesis of minus-stranded RNAininfected cells byastill unknownmechanism(7). The poliovirus protein target of flavone's action still remains to beelucidated. In fact, theonlyknown targetforaninhibitor ofpoliovirus RNA synthesis is protein 2C, which is the site of action of guanidine (17). This constitutes the major evidence implicating this protein in viral RNA synthesis.

Otherwise, the exactrole ofprotein 2C in poliovirus RNA replication is still puzzling (13, 25). This protein may be involved in thephysicalconnection between membranes and the viralreplicationcomplexes(1). Of interest in this respect is the recent discovery of helicase activity in a potyvirus

protein similartothatofpoliovirus2C(12).

Gliotoxin iscertainlythemostpowerfulinhibitor of polio-virus RNA synthesis in cultured cells(15, 23). The action of gliotoxin differs from the effect of guanidine and flavones, since thesetwoagents preferentiallyinhibit thesynthesisof

poliovirus positive-stranded RNA (7). On the contrary,

gliotoxin does notshow this selectivity and interferes with thesynthesisofbothminus-andplus-stranded RNA synthe-sis.Our studies of the effect of gliotoxin in cell-free synthesis

clearly show that it is a potent and unique inhibitor of

poliovirus RNA synthesis in these systems. Moreover,

gliotoxin inhibited the activity of purified

3DP°o.

Therefore, two agents that block two different poliovirus proteins involved inRNAreplicationare nowavailable: acompound that interferes with the activity of poliovirus protein 2C,

guanidine, and another one that blocks protein 3DPoI, gliotoxin. Futurestudies of the action and the characteriza-tionofthe target for theother inhibitorsofpoliovirus RNA synthesis will complete ourunderstanding of the effectsof these agents.

ACKNOWLEDGMENTS

The expert technical assistance of M. Chorro and M. A. Sanz is acknowledged.We thankS. J. Plotch(Lederle Laboratories, Pearl River, N.Y.)forkindly providingpurified3D°".

P.L.R. isthe holder of an F.P.I. and of a Residencia de Estudi-antes,Ayuntamientode Madridfellowship. Plan Nacional, project numbersB1088-0233andPB90-0177,andFundaci6n Ram6nAreces areacknowledgedforfinancialsupport.

B

E 302

20 B

1625

a 20

15

I

5

0

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1976 RODRIGUEZ AND CARRASCO REFERENCES

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2. Came, P. E., and L. A. A. Caliguiri. 1982. Chemotherapy of viral infections.Springer-Verlag, Berlin.

3. Carrasco, L., M. J. Otero,andJ.L. Castrillo. 1989. Modifica-tions ofmembranepermeabilitybyvirus infections. Pharmacol. Ther.40:171-212.

4. Castrillo, J. L., and L.Carrasco. 1987.Action of 3-methylquer-cetinonpoliovirus RNA replication. J. Virol. 61:3319-3321. 5. Castrillo, J. L., D. Vanden Berghe, and L. Carrasco. 1986.

3-methyl-quercetin isapotent and selective inhibitor of polio-virusRNA synthesis.Virology152:219-227.

6. Eggers, H. J.1982.Benzimidazoles,p.377-418.In P. E.Came, and L. A. Caliguiri (ed.), Chemotherapy of viral infections. Springer-Verlag, Berlin.

7. Gonzfilez, M. E., F. Martinez-Abarca, and L. Carrasco. 1990. Flavonoids: potent inhibitors of poliovirus RNA synthesis. Antibiot. Chem. Chemother. 1:203-209.

8. Guinea, R., and L. Carrasco. 1990. Phospholipid biosynthesis and poliovirus genome replication: two coupled phenomena. EMBO J.9:2011-2016.

9. Ishitsuka, H., C. Ohsawa, T. Ohiwa,I.Umeda, andY. Suhara. 1982.Antipicornavirus flavoneRo 09-0179.Antimicrob.Agents Chemother. 22:611-616.

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