Entry of poliovirus into cells does not require a low-pH step.

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JOURNAL OFVIROLOGY,Aug. 1993,p. 4543-4548 0022-538X/93/084543-06$02.00/0

Entry of Poliovirus into Cells

Does

Not

Require

a

Low-pH Step

LUIS PEREZANDLUIS CARRASCO*

Centro deBiologia Molecular "Severo Ochoa"(Consejo Superior de Investigaciones

Cientificas-UniversidadAut6noma deMadrid), Universidad Aut6noma de Madrid,

CantoBlanco, 28049 Madrid, Spain Received 11 January 1993/Accepted 11 May 1993

The requirementofalow-pHstepduringpoliovirusentrywasinvestigated by using the macrolide antibiotic bafilomycin Al,which isapowerfulandselectiveinhibitor of thevacuolarproton-ATPases. Thus, virusessuch as Semliki Forest virus and vesicular stomatitis virus that enter cells through endosomes and need their acidification, are potently inhibited by bafilomycin Al, whereas poliovirus infection is not affected by the antibiotic. The presence oflysosomotropic agents such as chloroquine, amantadine, dansylcadaverine, and

monensinduringpoliovirusentrydidnotinhibitinfection,furthersupportingtheideathatpoliovirus doesnot depend on a low-pH step to enter the cytoplasm. The effect ofbafilomycin Al on other members of the Picornaviridaefamilywasalsoassayed.Encephalomyocarditisvirus entryinto HeLa cellswasnotaffected by the macrolide antibiotic, whereas rhinoviruswas sensitive. Coentry of toxins, such as ax-sarcin, with viral particles was potently inhibited by bafilomycin Al, indicating that an active vacuolar proton-ATPase is

necessaryfor theearlymembrane permeabilization (coentryofa-sarcin) induced bypoliovirustotakeplace. Poliovirus has a virion particle 30 nm in diameter with

icosahedralsymmetrythatiscomposedof 60copieseachof four structural proteins, VP1, VP2, VP3, and VP4, that surround the 7.5-kb genome covalently linked to

genome-bound protein VPg (15, 25). Thepoliovirus infective cycle

commences by interaction ofpoliovirus particles with

spe-cific cell surface receptors (20). Then,virus internalization proceeds by receptor-mediated endocytosis (31). Entry of poliovirus particlesdirectly through theplasma membrane, after binding to its receptor, has also been suggested as a portal of virus entry (8). Once the viral particles are in endosomes, theymustexitthisorganelleto starttranslation of thegenomicRNA in thecytoplasm. Itis stillamatterof debate to what extent poliovirus requires acidification of endosomes to allow passage of the genome through the endosomal membrane (16, 18, 31) or whether poliovirus accomplishes this task in a pH-independent fashion (12). Indirect experiments in which poliovirus particles were

complexedwithneutral red and incubatedwithweak amines

or monensin for several minutes and then incubated for a verylongtime(18to40h) suggestedthatpoliovirus requires a low-pH step for infectivity (16-18). Virus entry was

evaluated by measuring the cytopathic effect caused by poliovirus complexedwith neutralred after several rounds ofviralreplication (16-18). High concentrationsofamines, i.e.,0.3 mM chloroquineor70 mMNH4Cl (31), have been

used in some of these studies, in which poliovirus poly-merase production was strongly inhibited even when the drugswereadded 1 or2 h after virus entry(31). Certainly, thelysosomotropicagentsemployedhaveanumber of side effects that influence a wealth of enzymes and functions apartfromraisingthe endosomalpH (13, 27). Thus,itis not surprising that during the long incubation times, during which several rounds of viral replication take place, there

was inhibition ofpoliovirus, particularly if the drugs used

were irreversible (18). Shorter incubationtimes, testingthe

*Correspondingauthor.

inhibition ofpoliovirusby chloroquine ormonensin during

theuncoating period, ledto the idea thatpoliovirusentryis pH independent (12).

Studies onthe requirementofa low-pHstep forentryof other members of the Picomaviridae family indicate that foot-and-mouth disease virus replication is inhibited by lysosomotropic compounds (2, 6, 7). However, a direct

actionof theseagentsinthe initialstepsof virus infection is still uncertain, because chloroquinewas still active in the inhibition offoot-and-mouth disease virus replication even

when added 2.5 h after infection (2), whereas monensin blockedtheinternalizationoflabeled rhinovirus particlesin

HeLa cells (22). It has been claimed that this ionophore might even increasethe infectionof cells by

encephalomy-ocarditis (EMC)virus whenlow-pH medium is used during viruspenetration (18).

Endosomes,likeother organelles of thevesicularsystem,

areacidified bythe actionof the vacuolarproton-ATPases (21, 26). Theseenzymespumpprotonsinto the organellesat

the expense of ATP hydrolysis in an electrogenic fashion (21, 26). The effect of specific inhibition of the vacuolar proton-ATPase on animal virus infectivity has not been examined.Onlythe action of theunspecific ATPase inhibitor N,N'-dicyclohexylcarbodiimide on poliovirus has been as-sayed (17, 18). This compound was effective only when several rounds of viral replication occurred, but it had no

effect on poliovirus entry when the amount of virus was

increased(17, 18). Thetwomajor classes of inhibitors used raise the endosomal pH by accumulation ofweak amines (NH4Cl, chloroquine, andamantadine, etc.) orby dissipat-ingthe proton gradient of endosomesby ionophores (mon-ensin and nigericin) (27). These two classes ofcompounds leave the endosomal proton-ATPase untouched, and in principle, this enzyme continues functioning even in their

presence. BafilomycinAl (BFLA1)isapowerfuland

selec-tive inhibitor of the vacuolar proton-ATPases that has re-centlybeendiscovered(3, 21, 32).Toelucidate the require-mentsofalow-pHstepforpoliovirus infectivity,wedecided to test the effect this new antibiotic, and also those ofthe

4543

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other two classes ofinhibitorsdescribed above, on poliovi-rus entry. Our presentresults suggest that although poliovi-rus enters cells via an endosomal pathway, it does not require a low-pH step to infect cells.

MATERLILS ANDMETHODS

Cell lines,viruses, and media.Poliovirustype1 (Mahoney

strain)and EMC virus werepropagated,grown,andtitrated

by plaque assay in humanepithelioid carcinoma(HeLa)cells and mousefibroblasts (Lcells), respectively. Baby hamster kidney(BHK-21) cells were used for growth and titration of

Semliki Forest virus (SFV) and vesicular stomatitis virus

(VSV; Indianastrain). Humanrhinovirus(serotype 14)was

kindly provided by M. G. Rossmann (Purdue University).

BHK-21and African greenmonkey kidney(CV2)cells were grown in Dulbecco modified Eagle medium supplemented

with 8% fetal calf serum. HeLa andAfrican green monkey

kidney(Vero) cellswere grownin DulbeccomodifiedEagle

mediumsupplemented with 10%newborn calfserum. Inhibitors. BFLA1 wasprovided by K. Altendorf

(Univer-sity of Osnabruck, Osnabruck, Germany). a-Sarcin was a

generousgiftfrom D. M. Shuurmans(Department of Public

Health; Lansing, Mich.). Monensin, chloroquine, and dan-sylcadaverine were purchased from SigmaChemical Co., St.

Louis, Mo.

Analysis

of proteins by sodium dodecyl sulfate

(SDS)-poly-acrylamide gel electrophoresis (PAGE). Cells grown in 24-well plates were infected at a multiplicity of infection (MOI) of 50 PFU per cell. After virus adsorption (min 30

postin-fection), the cells were incubated in Dulbecco modified

Eagle medium plus 2% calf serum. Protein labeling was

performed with 20 ,uCi of

[35S]methionine

per ml (1.45

Ci/mmol;

Amersham International, Amersham, United

Kingdom)inmethionine-free medium. Theradiolabeledcell

monolayers were dissolvedin samplebuffer (62.5mM Tris

[pH 6.8], 2% SDS, 0.1 M dithiothreitol, 17% glycerol,

0.024% bromophenol blue [as anindicator]). Samples were

heated at 90°C for 5 min and electrophoresed on a 15%

polyacrylamide gel overnight at 80 V. Fluorography was

carried outin 1 M sodium salicylate. Thegelswerefinally

dried andexposedtoAgfa X-ray films.

Measurement ofradioactivity incorporated into trichloro-aceticacid-precipitable material. The cellswereincubated for 1 h with the virus and a-sarcin (50 ,g/ml) in Dulbecco modified Eagle medium plus 2% calf serum, washed, and

pulse-labeledfor 1 hwith 2 ,uCiof

[35S]methionine

perml in

methionine- and serum-free medium. Radioactive medium

was removed, and the cellswere washed with

phosphate-buffered saline, treated with 5% trichloroacetic acid, and washed twicewith ethanol. The cellmonolayerwasallowed todrybeforeaddition of 0.1 MNaOH-1%SDS.Thesamples

weredissolved inliquidscintillation counting cocktail (For-mula 989; Du Pont, Boston, Mass.), and radioactivity was

quantitated inaliquid scintillation counter (1219 Rackbeta;

LKB, Bromma, Sweden).

RESULTS

Effects ofBFILA1 onentry of poliovirus. Comparison with enveloped RNA-containing viruses. To test the effect of BFLA1 on poliovirus entry, HeLa cells were treated with the compound in three different ways: (i) BFLA1 was presentcontinuously, from virus addition until the cells were labeledwith[35S]methionine; (ii) the compound was present

only duringthefirst 20 min of infection, and then the excess

POLIO

°1

BFL

w o

=:

u a b c

SFV

°- BFL

C°

u a b c

C

ED,.bA P

vsv

L& BFL

C:

_° _a _b _c

G _

-NS(P)Iw

-N

...*I 5 2

-2C

_ __-.

C-rn 40 M-m 1.aI4

-mm-1C

FIG. 1. Effects ofBFLA1addedatdifferent timesonpoliovirus (POLIO), SFV, and VSV. The MOI was 50 PFU per cellforthe three viruses.Labeling of infectedHeLacellswascarriedoutfrom 3.5 to4.5 h postinfection, asdescribedin Materials andMethods. Except in the controllanes,1,uMBFLA1 waspresent from addition of the virusto thecells(0hpostinfection) until the labelingperiod (lanesa), during only the first 20minofinfection(lanes b),orfrom 20 min postinfection (lanes c) until the labeling period. Lanes: control, infected cellsnottreatedwith thecompound; HeLa,protein synthesis inuninfected cells not treated with BFLA1.

virus and the inhibitor were removed; or (iii) BFLA1 was added 20 min after the virus and left throughout infection.

Figure 1 shows that BFLA1 did not prevent infection of

HeLacellsby poliovirusunder any of the threeconditions

tested. In contrast, SFV and VSVwerepotentlyinhibitedby

BFLA1, but onlywhen the compound was present

during

virusentry(i.e., conditionsi andii). Additionof BFLA1 20

minafter the virus

(condition

iii)didnotblock SFVorVSV infection, indicatingthat this agent doesnot inhibit subse-quent stepsof viralinfectionthat takeplaceafter the first 20

minof thereplication cycle.WetestedtheinhibitionofSFV

attachment and internalization with radioactively labeled virions and found no inhibition by BFLA1 (results not

shown).

Thereare two additional points inFig. 1 that should be

emphasized. One is that BFLA1 does not inhibit cellular

translation, evenafterlongincubationtimes (seelaneafor

both SFV and VSV). After 6 h of incubation at 1 ,uM

BFLA1, cellularproteinsynthesiswas 100%of thecontrol value (datanotshown). The second aspect is the very low concentrations of BFLA1used, i.e., 1 ,uM, whichis about 100-fold lower than the concentrations ofchloroquine

nor-mally used to block SFV replication. The concentration

dependence of the effect of BFLA1on SFV andpoliovirus

replicationis shown inFig.2. Concentrationsof the

antibi-oticaslowas0.5,uM inhibitedSFVreplication,whereasan

almost 10-foldhigherconcentration of BFLA1(4 ,uM)hadno effectonpoliovirus.

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POLIOVIRUS ENTRY 4545

SFV

;

BFL

[iM]

0 0.5 1

p107-

-p62- a.

E1,2-

&

C-_.-.

POLIO

0 0.5

1

2

4 BFL[tM]

-____- - 3CD

2C

a

(mM)

, BFL

i (pM) AMAN CLO D-CAD MON

.!

A2 .^C, A-. 1^

mM)

BFL

:> (uM AMAN CLO DOAD MON

U,- - .. -.

11111,....

*g.,, _-- loop-a C1

FIG. 2. Effects of different concentrations of BFLA1 on SFV

andpoliovirus (POLIO) infection of HeLa cells. Cellmonolayers

werepretreatedwith the indicatedconcentrations of BFLA1 15min

before infection with the virus(0hpostinfection; MOI,50 PFUper

cell). After 1 h of incubation with the virus and BFLA1, the monolayerswerewashed and incubated with fresh medium for 5 h postinfection. Labeling with [35S]methionine was carried out as

indicated in Materials and Methods. Proteinswerelabeled from 5to

6 hpostinfection with 15 pCiof[35S]methionine perml in

methi-onine-free medium andanalyzed bySDS-PAGE.

Comparison of BFLA1 and otherlysosomotropically active compounds. Although BFLAl potently blocked replication of the enveloped RNA-containing viruses tested, it was possible apriori that, for unknownreasons, thiscompound

was active only on these viruses but noton poliovirus. It could bespeculated thatpoliovirus enters cells inan endo-somal subpopulationwhere thevacuolar proton-ATPase is not susceptible to BFLAL. To assay the effects of other agents known toraise thepHinendosomes, wetested the aminesamantadine,chloroquine, anddansylcadaverineand the ionophore monensin against poliovirus. Amines are thoughttoaccumulate inendosomes and otherorganelles of the vesicular system, wheretheybecomeprotonatedandare

thus lesspermeable, leadingtoanincreaseintheendosomal pH (27). On the otherhand,monensin isan ionophorethat exchanges Na+/H+ in favor ofan H+ gradient, thus dissi-pating the ApH createdby the proton-ATPase pump (27). Since there isnonetmovementofcharges by monensin, AtI is not affected by the ionophore. These compounds were assayedover awiderangeofconcentrations.Figure3shows the results obtainedwith the lowest andhighest concentra-tions tested on poliovirus. None of these compounds blockedpoliovirus replication.Thehighestconcentrations of amantadine andchloroquineanalyzed, andalso dansylcada-verine, decrease the synthesis ofpoliovirus proteins, most likely because of the toxic effects that these agents have under those conditions (27). In no case was there even a slightincrease of cellularprotein synthesisthat wouldbean

FIG. 3. Effects ofseveralinhibitorsoninfection of HeLacellsby poliovirus(POLIO) (a)orSFV (b). Each compound was added to cellsattheconcentrationsindicatedat15 minbefore virus infection andweretreated asdescribedin the legend to Fig. 2. Synthesis of late viral proteinswasanalyzedby SDS-PAGE as described in the legendtoFig. 2. Proteinsynthesiswas measuredin cells infected withpoliovirusorSFV andtreated with the following compounds: BFL, BFLA1; AMAN, amantadine; CLQ, chloroquine; D-CAD: dansylcadaverine; MON, monensin.

indication that the entry ofpoliovirus was compromised by thesecompounds. As a control, the effects of these agents on SFV infection were alsotested (Fig. 3). All of them hadan inhibitory effect on SFV infection, although amantadine and dansylcadaverine were less effective than the other inhibi-tors at the concentrations tested (compare levels of viral peptideC). Therefore, entry of poliovirus is not affected by agents whichincrease the endosomal pH by three different

mechanisms, i.e., (i) blockade of the vacuolar

proton-ATPase with BFLA1, (ii) buffering of H+ by weak amines, and(iii) dissipation of the ApH by monensin.

Inprevious studies, HeLa S3 cells were used to analyze

therequirement for a low-pHstep forpoliovirus entry into

cells (16-18). Therefore, we decided to test the effects of BFLA1 onpoliovirus entry in different cell lines. Figure 4 shows that BFLA1 did not reduce poliovirus infection of HeLa S3, CV2, or Vero cells, suggesting that the uptake mechanismsarelikelytobesimilar inall of these cell types.

Coentry of a-sarcin with poliovirus. Effect of BFLA1.

Poliovirus is able to cointemalize and efficiently deliver protein toxins during entry into cells (1, 4). The exact mechanismbywhich this occurs is not fully understood, but virionuncoating is necessary for this process to occur (1). It has beenspeculatedthatdelivery of protein toxins by animal virusesinvolves disruptionofendosomes (5, 10), but other more selective mechanisms are possible. a-Sarcin had no effectonprotein synthesis when addedtouninfected HeLa cells, withor without BFLA1 (Fig. 5c), whereas in

agree-ment with previous findings, the toxin efficiently blocked

translation when added togetherwith virion particles (Fig. 5a).Thiscointernalization of theproteintoxinby poliovirus

waspotentlyblockedby BFLA1. These results suggest that

anactive vacuolar proton-ATPase is necessary for efficient

delivery of ax-sarcin to the cytoplasm. In addition, they

constitute indirect evidence that poliovirus entry occurs

through the endosomal pathway, because the coentry of

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CV2

BFL[pM] 5 0 2

4' _ _ -1

-_ -3CD

- 2C

-1C

Vero

_ze

M 0 2

i.

.-._-1 '-- -3CD

..-GM-2C

-lom-1C

FIG. 4. Poliovirus infection of different cell lii BFLA1.CV2, Vero, and HeLa S3 cellswereinfecte rusatanMOIof50 PFUpercell. The cellswerepr

2)ornot(lanes 0) at15min before infection with 2 indicated and incubated in thepresence ofthecom

virus for 1 h. At that time (1 hpostinfection), the r

washed and then incubated with fresh medium fo postinfection, protein synthesisinthe infectedcellsv

asdescribedin thelegendtoFig.2.

a-sarcin induced by poliovirus does notoccuu ence ofan inhibitorofan endosomal enzyme

coentry of alpha-sarcin induced by Sendai blockedatallby BFLA1,because this virusen fusion with the plasma membrane (14, 19) shown).Theseresults leadtotheconclusionthE all,thetoxindeliveredin thecytoplasm bypol

comesfromendosomes,becausepoisoningofI

a-sarcinwastotallyprevented byinhibition of proton-ATPase with BFLAL. For a model f entry illustrating the coentry of a-sarcin an

action of theinhibitors, seeFig. 7.

Effect ofBFIA1 on replication of other me

Picornaviridae family. The Picornaviridae fami into four genera: the enteroviruses, (poliovii

ruses,cardioviruses(EMC virus),andaftovirur mouth disease virus). The enteroviruses and

arecloselyrelatedbyanumber ofcharacteris the othertwogeneraare moredistant inevolui (24).Wetested theaction of BFLA1againstEl rhinovirus and compared its effect against ti RNA-containing virus VSV (Fig. 6). Infection4

by EMCvirus didnot seemtobe blocked byt antibiotic (e.g., compareviralpeptide alpha w outBFLA1), as occurswithpoliovirus, where or VSV infection was inhibited by BFLA1.

treatment ofEMC virus-infected cells with I

feredwith theinhibition of hosttranslation byI

did not investigate this effect. Figure 6 als( a-sarcinefficientlyentered thecytoplasminth the three virus particles. BFLA1 efficiently coentrymediatedbyanyof thethree viruses.A detailedanalyseswitheach of these virusesare

more firm conclusion regarding their mode (

HeLa S3

U

0

m 0 2

a-(1)0

I-u

0

z_

U) o

, 0

(L

LCi

2 Hela

i< -S

Iii

Ac

-POLIO

a-S + - + BFL

1 ..-

-3CL--_

-2C

-iin_ -2C

--1 C z t:°

X i

[E3FL.LIMs

nes. Effect of FIG. 5. a-Sarcin andpoliovirusentryinto HeLa cells. Effect of d withpoliovi- BFLA1.(a)Permeabilization of HeLa cells for a-sarcin at different -etreated(lanes MOIswith poliovirus. Symbols: El, poliovirus-infected cells incu-,uMBFLA1 as bated with 50,ugof a-sarcinpermlduringvirusentry(60min) bythe ipound and the protocol described in Materials andMethods; 0,cells treated with monolayerwas 1 FM BFLA1 during incubation with the virus and a-sarcin. Ir 4 h. At 5 h [35S]methionine incorporation into mock-infected cells (6.0 x 105 vasdetermined cpm)was 100% of the control value. Infection with the virus or treatmentwithBFLA1 ora-sarcininuninfected cellsyielded values of over85% ofthe control.(b) Effects of different concentrations of BFLA1 on thepermeabilization ofpoliovirus-infected HeLacells (MOI,100PFUpercell) fora-sarcin.BFLA1wasaddedtothe cells

rin the pres- 15 min before at-sarcin and virus infection. a-Sarcin entry was . In fact, the determined as inhibition of protein synthesis measured as described virus is not for panel a. (c) Electrophoretic analysis of[35S]methionine-labeled

terus

by

diret

proteins in cellsinfected with

poliovirus (POLIO)

in thepresenceof iters

by

direct ot-sarcin and BFLA1. Mock-infected or poliovirus-infected HeLa

(results not cells were incubated with orwithout 50

p,g

ofa-sarcin(a-s) per ml

at most,ifnot with (+) or without(-) and 1 ,uM BFLA1 for 1 h. The cells were

liovirus entry washedand incubated with fresh medium until labeling of proteins

HeLa cellsby from 5to6hpostinfectionasdescribedinthelegendtoFig. 2. Ac, the vacuolar cellularproteinactin.

.or poliovirus

d the site of

results certainly suggest that only rhinovirus requires a Xmbers of the low-pH step forefficiententryintocells.

iily is divided

rus), rhinovi- DISCUSSION

ses

(foot-and-rhinoviruses Themechanisms usedbyanimalvirusesto crosscellular

,tics,whereas membranes and delivertheirgenomesinto thecytoplasmto

tionaryterms start infection is a matter of intensive research (14, 19).

MC virus and Animal viruses containing lipid membranes

accomplish

this

he enveloped task by fusion of the external envelope, either with the of HeLa cells plasma membrane, as occurswithSendaivirus,herpesvirus

the macrolide orvacciniavirus,orwith the endosomalmembrane,aswith

rith

and with- SFV, VSV, or influenza virus (14, 19). In the latter case, asrhinovirus fusion is promoted by conformational modifications of the

Remarkably, viralglycoproteins after receptorbindingandacidification of

BFLA1 inter- endosomes (28, 29). Prevention of this acidification by

the virus. We lysosomotropic agents leads to inhibition of infection by D shows that SFV,VSV,orinfluenza virus(14). Modelstoaccountforthe

epresenceof entryofnonenveloped viruses,suchaspoliovirusor adeno-blocked this virus, are less well developed. We support the idea that

Ithoughmore attachment of poliovirus and internalization in endosomes

-needed fora leads toconformational changes in the virion particle,

par-of entry, our ticularlyinVP1 (11),whichcaninteract with the membrane

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POLIOVIRUS ENTRY 4547 EMCV

sarcin

BFL - + - +

E--I

EY- -_ _

HRV sarcn

p92-

p75-VPO .

vpI-

VP3-vsv

sarcin

mock

'a c C

u_

0 %1

X-FIG. 6. Action ofBFLA1 onthe entry ofseveralpicornaviruses into HeLa cells. Effectona-sarcin coentry. Monolayers of HeLa cells (Ohio strain) were mock infected (mock) or infected with human rhinovirus serotype14(HRV)at anMOI of 100 PFU per cell orwith EMC virusorVSVat anMOIof 25 PFU per cell. Lanes: +, treatmentof cells with 2 ILM BFLA1 (BFL) as described in the legend to Fig. 2; -, cellsnot treated with BFLA1. As indicated (sarcin), 50 p1g of a-sarcin per mlwasaddedtothe mediumatthe same time asthe virus. After incubation for 1 h, the cells were washed and incubatedwith fresh medium. [35SJmethionine-labeled

viralproteins wereanalyzed by PAGE. Samples containing equal amountsof totalproteinwereloaded in all of the lanes. Thecells werelabeledat5.5 hpostinfection for VSV and EMC virus andat 7.5hpostinfection for rhinovirus. Ac,cellularprotein actin.

and aid the insertion of

myristoylated

proteinVP4 into the

membrane (11). This insertion not only destabilizes the

particle butmayalso allow interaction ofsomemoieties of

other viral

proteins

with themembrane

(11).

Such an

inser-tion couldopenaporein the membrane that allowspassage

of thegenome tothe

cytoplasm.

In this

model, proteins

of

the virus

particle

act

similarly

to the B chains of some

protein toxins (30). In fact, we have reported that animal

virions, including poliovirusparticles,areabletoreplacethe

Bchains ofprotein toxins and promote the entry of the A

chain or effector

moiety

of the molecule

(5,

9,

23).

We

recently showed that for thisco-entryoftoxinswith

polio-virus, uncoatingof theparticleisrequired,in agreement with

themodel describedabove

(1).

Ourpresent

findings

onthe

involvement of the vacuolar proton ATPase inthis

phenom-enonare of interest in

understanding

not

only

the entryof

nonenvelopedvirusesbutalso thecoentry ofproteintoxins

inside cells.Thus,wecannotdissociatepoliovirusuncoating

from the coentry mechanisms,

arguing against

during

virus

entry

(1,

4, 5,

10). Therefore, poliovirus

enters from

endo-somes into the

cytoplasm

and maintains the endosomal

membraneintact,because this rupture would lead toleakage

of the endosomalcontent,

including

oa-sarcin, into the

cyto-plasm. Thus, after orduring poliovirus entry, the

modifica-tion introduced in the cellular membrane is usedbya-sarcin totraversethe membrane.Our present results favor the view that the force that pushes a-sarcin through the membrane may be the proton gradient generated by the vacuolar

proton-ATPase. Therefore, instead ofasimple mechanistic

model ofcreation ofaporethroughwhich molecules pass, other forces

(proton

motive,

electrical)

may be involved in

cX-sACINH

IBAFILOHYCINA

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N a-SARCIN H

a-SUrCIN H

FIG. 7. Schematic model of two potential mechanisms of polio-virus entry into cells. Poliopolio-virusinternalizationthrough endosomes ordirectcrossing of the plasma membrane would lead to insertion of structuralproteins intothe membrane. These proteins, together with the protongradient between endosomes and the cytoplasm gener-atedby theH'-ATPase,wouldtranslocate the toxin a-sarcin to the cell interior.

protein translocation.Inconclusion, the virus capsid protein

would be able tocouplethese forces to thetranslocation of

theproteinspresentinendosomes.

Two major questions must be answered to explain the

mechanism ofpoliovirus entry into cells.One iswhetherthe virus is internalizedthrough endosomes, and the second is

whetherendosomes need to beacidified for the virus to enter

cells. Webelieve that thesecondquestionhas beenclarified

by the present work. Since none of the agents known to

increase the endosomal pH prevent poliovirus infection

when they are present during entry, we conclude that a

low-pHstepisnotrequiredforpoliovirustoinfect cells. We

emphasize theunique mode of action ofoneoftheinhibitors

used in ourwork,i.e., BFLAL. Since this agentselectively

blocks thevacuolar proton-ATPases (3, 21, 32) and shows

activity at micromolar concentrations, we suggest that it

should replace other compoundswidely employed to

ana-lyze the low-pH requirements for animal virusentry(14, 19).

However,ourresults donotprovideadefinitiveanswer to

thefirstquestionraisedabove,i.e., whetherpoliovirus gains

access tothecytoplasmvia endosomes. We believethatthe

two possible mechanisms of poliovirus entry, i.e., direct

entry throughthe plasma membrane (8) and internalization

in endosomes (31), are still open. It is even possible that

poliovirususesboth of them.Althoughpoliovirus

permeabi-lizescells fora-sarcinandthisevent occurs inendosomes, it

is possible a priori that the virus has already released its

genomeintothe cytoplasm afterbindingtothe cellsurface

receptor and the virion proteins present in the membrane

andinternalized in endosomesarethe ones thatpermeabilize

the membrane for a-sarcin once the ApH has been created

(model in Fig. 7). Alternatively, it is possible that the

poliovirus particle can traverse the cell membrane only

during the internalization process. Recent electron

micro-scopic evidence illustrating entry of poliovirus into cells

favors the second mechanism(31). Furtherexperiments are neededtoclarifythis issue andgive us abetteridea of how the genome of anonenveloped animal virus is ableto cross

alipidmembrane to start infection.

VOL.67, 1993

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ACKNOWLEDGMENTS

LuisPerez is the holder of a Comunidad Aut6noma de Madrid fellowship. Plan Nacional project BIO 92-0715, DGICYT project PB90-0177, and theFundaci6n Ram6nAreces areacknowledged for financialsupport.

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