Inhibition of host cell protein synthesis by UV-inactivated poliovirus.

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CopyrightC 1977 American Society for Microbiology PrintedinU.SA.

Inhibition of Host Cell Protein Synthesis by UV-Inactivated

Poliovirus

TIM HELENTJARIS* AND ELLIE EHRENFELD

Departments of Biochemistry andMicrobiology,* University of Utah Medical Center, Salt Lake City, Utah 84132

Receivedfor publication 1 July 1976

The ability of poliovirus that was irradiated with UV light at energies up to

2,160

ergs/mm2

to subsequently inhibit host cell protein synthesis was

mea-sured.Theinactivation of the host cell shutofffunction followed one-hit kinetics.

Increasingirradiation did not affect the rate ofinhibition until the multiplicity

ofinfectionafter irradiation was reduced toapproximately 1 PFU/cell. At higher

functional multiplicities,the rate was unchanged,but an increasinglag before

the onset ofinhibition was observed with increasing irradiation. The energy

levels required to inactivate virus-induced inhibition of host cell protein

synthe-sis suggestthatdamage to virus RNA rather than to virus capsid proteins is

responsible for the loss of function. When the inactivation of host cell shutoff was

compared with the inactivation of other viral functions by UV irradiation, it

correlated exactly withtheloss of infectivity but not withotherviralfunctions

measured. Guanidine treatment, which prevents detectable viral RNA and

proteinsynthesis, completely inhibited host cell shutoff by low multiplicities of

unirradiated virusinfection but nothigher multiplicities. When a high

multi-plicity of virus wasfirst reduced to a low titer by irradiation, host cell shutoff

wasstillevident in the presence of guanidine. The results demonstrate that the

complete inhibition ofhost cell protein synthesis can be accomplished by one

infectious viral genome per cell.

The inhibition ofinfected cell RNA and

pro-teinsynthesis bypicornaviruseswasrecognized

almost15yearsago, yetthe mechanismof this

viral function is still unclear. This inhibition

might be due to either input virion protein or

RNA without the need for viral RNA

transla-tion or replication or due to a product of a

functional viral genome, or possibly due to

some combination of the two. Conflicting

evi-dence has been presentedfor and against each

possibility. Penman and Summers (16), using

either puromycin orcycloheximide to

synchro-nizepoliovirusinfection, found that a period of

protein synthesis was required after the

re-moval of the inhibitor before the inhibition of

cell protein synthesis was evident. Baltimore

andco-workers(2)also found that critical levels

ofp-fluorophenylalanine, aninhibitor ofprotein

synthesis,couldblock theabilityofmengovirus

toinhibitbothcell RNAandproteinsynthesis.

These datasuggested that the inhibition of cell

protein synthesis required the translation of

the viral genome. However, Holland (7) and

Collinsand Roberts (4) havesubsequently

pre-sentedevidence demonstrating that inhibitors

ofproteinsynthesis(puromycin,

fluorophenyla-lanine, and azetidine) could selectively

disso-ciate host cell "shutoff' from detectable viral RNA and protein synthesis. Also, infection in

the presence of 1 mM guanidine, which

pre-vents detectable viral replication and

transla-tion, doesnot seem toaffect hostcellshutoffat

highermultiplicities of infection (MOIs) (7, 16).

Usingtemperature-sensitivemutants to

exam-inethisproblem,Steiner-PryorandCooper (20)

showed that the abilityto inhibithost cell

pro-teinsynthesis seemstomap in theregionofthe

structural protein genes. However, Cole and

Baltimoredemonstrated that defective

interfer-ing polio virions are able to inhibit host cell

translationdespite the fact that their

defective-nessoriginatedbecause of deletionsinthe

cap-sidgenes and resulted inaninabilitytospecify

for structuralproteins (3).

Perhaps the most compelling evidence

indi-cating the need for a functional viral genome

has been the ability of UV irradiation (1, 16)

and treatment by proflavine (7) to inactivate

theabilityof thevirus to causehost cell shutoff.

Atthe time these experimentswerereported,it

was presumed that the majorsite ofaction of

eachagentwas todamagetheviral RNA.

How-ever, theproflavineexperimentswere not

con-clusivesince, asthe authorindicated, he could

259

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not exclude the possibility that inactivation

wasdueto the oxidation ofcoat proteins. The

conclusions drawnfrom the UV light

inactiva-tion experiments could also be quesinactiva-tioned in

view of data presented by Miller and

Plage-mann(14). They investigated the effects ofUV

irradiation on mengovirus and found that the

inactivation of viral infectivity seemedto

corre-late with damage to the viral genome, but

that alterations in viral capsid proteins did

oc-curwithgreater amountsofirradiation. Other

data demonstrating the effects ofUV

irradia-tion on proteins have also been reported (11,

17).

Because other viruses have been shown to

inhibit hostcell processes afterUVlight

inacti-vation (8, 15, 19) and because of the recent

observations of Racevskisetal. (18) suggesting

that poliovirions can inhibit translation in

re-ticulocyte lysates, wereexamined the effect of

UV irradiation on the ability of poliovirus to

inhibit cellular protein synthesis. The principal

goal of this investigation was to determine

whether a functional viral genome was

neces-sary for host cell shutoff or whether shutoff

could be ascribed solely to input virus capsid

proteins. Our results indicate that the ability of

poliovirus to inhibit cell protein synthesis is

sensitive to UV irradiation atlevels ofenergy

which are consistentwith damageto the viral

genome but nottovirionproteins. The

inactiva-tionof the shutoffabilityfollowsone-hit

kinet-ics and demonstrates that approximately 1

PFU/cell is sufficient to cause host cell shutoff.

Increasing the MOI above 1 decreases the lag

time before the virus replication cycle begins.

Moreover, when compared with the

inactiva-tion of other viral funcinactiva-tions, loss of the shutoff

ability correlates exactly with loss of virion

infectivity.

MATERIALS AND METHODS

Cells and virus. Suspension cultures of HeLa S3 cells were grown at a density of 3 x 105to 8 x 105 cells/mlinEagleminimal essential medium(MEM) supplementedwith 5%fetal calf serum.

TheMahoney strain of poliovirus type 1 was col-lected bycentrifugation from infected cell extracts treated with 1%sodiumdodecylsulfate (SDS), and thevirus was further purified on CsCldensity gra-dients. Optical absorption of 1.00 at 260 nm was assumedequal to 1.3 x 1013virusparticles, and the infectivity of recovered virus was assayed on HeLa cellmonolayers under 0.7% agarose.

Irradiation of virus. Virus was diluted to 1.5 x

1010PFU/ml in MEM without serum and irradiated inmicrotiterplates at a distance of 5 cm by a broad-spectrum UVlamp. The energyatthe distance of 5 cmwas measured to be 36ergs/mm2*s.

Evaluationofproteinsynthesis in infected cells.

Cells at 5 x 106/ml in MEM in Spinner culture were infected with virus at an MOI ofapproximately 10 PFU/cell. After 30 min, fetal calf serumw'asadded to 5%. Guanidine,whenadded,wasusedat afinal concentrationof 1 mM. Every 30 min from 0.5 to 5.0 hpostinfection, 0.5 ml of infectedcells wasremoved andexposedto0.25,ICiof[35S]methionine(specific activity, 503Ci/mmol;NewEngland Nuclear Corp.) for 10 min at37°CinSpinnerculture. The incorpora-tion wasstopped by the additionof cold Earle salt solution, and the cells werewashed by centrifuga-tion.Cellprotein wasprecipitated by 5% trichloroa-ceticacidontoWhatman GF/C filters, and the incor-porationof[35S]methionine wasevaluated ina liq-uidscintillationcounter.

Viral protein production was also examined by SDS-polyacrylamide gel electrophoresis (PAGE) of infected cell lysates. HeLa cells were infected as above with virus that had been irradiated for var-iousperiods oftime.The MOI of each samplebefore irradiationwas 10. At 30 minpostinfectioncalf se-rumwasaddedto5%. Atspecifiedtimes, 50 ,tCiof 14C-labeled amino acids (proteinhydrolysate, New England Nuclear Corp.) was added and the samples wereallowedtoincorporatethelabeledaminoacids for a period of 2 h. At this point the cells were

washed in Earle solution and resuspended inRSB (10 mMNaCl-10 mMTris [pH7.4]-1.5mMMgCl2). Nonidet P-40 wasadded to 1%andthe nuclei were discarded after centrifugation. The cytoplasm was made 1% in SDS and analyzed on polyacrylamide gels bythe method of Maizel (12).

Evaluation of RNA synthesis in infected cells. Cells wereinfected asbefore,but at 30 min postin-fection 5% serum, actinomycin D (gift of Merck, Sharp, and Dohme) at 10

jig/ml,

and ['4C]uridine

(specific activity, 50 mCi/mmol; NewEngland Nu-clearCorp.) at 1.3 ,Ci/mlwere also added. At 30-minintervals from 0.5 to 5.0 hpostinfection, 0.2 ml ofinfectedcells was removed and the incorporation wasstopped bythe addition of cold Earle solution. The cells were washed by centrifugation, and the RNA was precipitated by 5% trichloroacetic acid ontoWhatmanGF/C filters.The amount of incorpo-rated [14C]uridine was then determined in a liquid scintillation counter.

Virion production by cellsinfected with irradi-ated virions. Cells wereinfectedasbefore and,at 30 minpostinfection, 5% serum, actinomycin D at 10 ,ug/ml, and ["4C]uridinewere addedto 1.3 ,Ci/ml. At 6 hpostinfection, the cell cytoplasm was collected by treatment of the cells with 1% Nonidet P-40 and subsequent centrifugation to remove nuclei. SDS (1%) was added to the cytoplasmic extract along with purified poliovirus labeled with [3H]uridine, which served as the marker. The extract was lay-ered onto a 7 to 47% sucrose gradientinRSB and centrifuged at 40,000 rpm in an SW41 rotor for 2.5 h. The gradient was fractionated and each fraction was assayedfor 3H and 14Cacid-precipitable radioactiv-ity in a liquidscintillation counter. The 14C in the peak corresponding to irradiated virus was

com-paredwith thepatterndemonstrated by 3H-labeled virus that had notbeenirradiated. Since no obvious changeinthe sedimentationprofile was seenwith

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increasing irradiation,all of the 14C radioactivity in the virus peakwastotaled and used as a relative

indicator of virionproduction.

In other experiments unlabeled progeny virus

from irradiatedvirus-infectedextractswasprepared and titered on HeLa cell monolayers under 0.7% agarose.

RESULTS

Effect of irradiation on host cell shutoff.

Polioviruswas irradiatedasdetailed above for

periods of 0, 10, 20, 30, 40, and 50 s. These

irradiated preparationswereusedtoinfect cells

at an MOI that corresponded to 10 PFU/cell

before irradiation. Protein synthesiswas

evalu-ated inthese cells and the resultsareshownin

Fig. 1. The rate of [35S]methionine

incorpora-tion in the uninfected cells increases linearly

from 0.5 to about 3.5 h, where it decreases

slightly until 5.0 h. Incorporation by the cells

infected with unirradiated virus appears

nor-mal until 1.0hpostinfection. At this time, the

incorporationrate declines linearly until 2.0 h

postinfection, where it again beginstoincrease.

6-

5-

4-r;T)

I0

z

s

3-

C-"I

r

2-

1-\50SEC

\40 SEC

\30 SEC

20 SEC

MO SEC 'O SEC

2 3 4 5

TIME POST-INFECTION IHRS.)

FIG. 1. Host cellshutoff by irradiatedvirus. Vi-rus was irradiatedas indicated above and used to

infect HeLa cells. Protein synthesis in the infected

cells was evaluated by 10-min pulses with

[35S]methionineevery0.5 hfrom0.5to5.0 h postin-fection. Samples were trichloroacetic

acid-precipi-tated andevaluated inaliquid scintillationcounter. Control line represents protein synthesis in

unin-fectedcells.

This increase continues for a short time and

then it, too, decreases. From examination ofthe

proteinsmadeatthislater timeby SDS-PAGE,

ithas beendemonstrated that nearly all of the

proteinsmade by the cell after 2.0 h

postinfec-tion are virus specific (data notshown).

Irradiation of poliovirus prior to infection has

two effects on the host cell shutoff pattern.

First, there is a significant delay before the

inhibition of cell protein synthesis is seen, and

this delay increases withincreased irradiation

up to 30 s. For instance, whereas unirradiated

virusbegins to inhibit cell protein synthesisat

about 1.15 hpostinfection, 10 s of irradiation

de-lays the onsetofinhibition to about 1.50h and

protein synthesis in cells infected byvirus

ir-radiatedfor 20 s appears normal until 1.85 h

postinfection. A total of 30 s of irradiation de-lays the shutoff until about 2.20 h postinfection,

butincreased irradiation of theinfecting virus

failed to further delaythe shutoff past 2.25 h

postinfection. It is evident from the effect on

viral proteinsynthesis as well asfromdata to

be described below that the viral replication

cycle as awholeisdelayed by irradiation in a

manneranalogous tothedelayonshutoff. The

second effect of irradiation on shutoffcan be

discerned fromtheslopeof the decrease in the

rateof cell proteinsynthesis. After10and20s

ofirradiation, although theonsetof inhibition

was delayed, the rate of inhibition of cell

pro-teinsynthesis was notsignificantly altered.

Be-ginning at 30 sofirradiation,however, therate

ofinhibitionbegins to decrease with increasing

irradiation. This change in slope continues

un-til 50 sof irradiation, which was the greatest

amount of irradiation used in these

experi-ments.

Figure 2 summarizestheeffect of increasing

UVirradiation ofinfecting virions on the rate

ofhost cell shutoff. The slopes ofthe shutoff

curvesfrom the onsetof the inhibition,

ignor-ingthelag,until the beginning of viral protein

synthesis havebeentaken as a measure ofthe

rateofshutoff. The slope of the line generated

by unirradiated virus was expressed as 1.00,

and theslopesoftheother lines were calculated

as fractions of 1.00. As shown in Fig. 1, low

levelsofirradiation(less than 25 s) resulted ir

nochangeintherateofinhibition. Thus,Fig.2

shows a noticeable shoulderonthe curve until

about 25 sofirradiation, wherethe inhibition

decreased logarithmically. This shoulder does

not appear to be due to multiple-hit

inactiva-tionkinetics,sincethe cells were infected at an

MOI greater than one before irradiation.

Extrapolation of the slope of the straight-line

segmentinFig.2tothe ordinateinterceptsat a

point that would suggestapproximately 10-hit

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262

cell. Thus, inhibition of host cellprotein

syn-thesis occurs at the same rate, albeit after a

variablelag, aslongasthere isapproximately

one infectious particle per cell; thereafter the

rateof inhibition isreduced.

Effect of irradiation on viral RNA and pro-tein synthesis. Another function of the viral

genome,virus-specific RNA synthesis, was

ex-amined after irradiation. Cells were infected

with irradiated virus at an MOI of 10PFU/cell

before irradiation and the viral RNA synthesis

in these cells was measured. The results are

shown inFig.4.Therewas nosignificant

incor-porationby uninfected cells over the period of1

to 5 h after the addition ofactinomycin D and

[14C]uridine (data not shown). In the cells in-fected with unirradiated virus, no viral RNA

synthesis is evident until 2.25 hpostinfection.

There is then a linearincorporation until about

4.00hpostinfection when synthesisceases. As

with host cellshutoff, the irradiation of virus

before infection demonstrates two effects on

viral RNA synthesis. First, there is a

signifi-cant delay before the onset of viral RNA

syn-thesis until about 3.50 h with 30 s ofirradiation,

10 20 30 40 50

TIME OF IRRADIATION (SECS.)

FIG. 2. Host cell shutoffby irradiated virus. The slopes of thestraight-line segments during host cell shutoff by irradiated virus inFig.1 wereplottedas fractionsoftheshutoffdemonstrated byunirradiated virus(solid line).Thedashed line represents extrap-olation ofthestraight-linesegmentfrom30to 50 s.

inactivation kinetics of the input. Since the

cellswereinfectedat anMOIofapproximately

10, this corresponds to one-hit kinetics in the

inactivation of the host cell shutoff ability.

Thatthis is the case was verified bydoing the

sameexperiment at anMOIbefore irradiation

of about 1,000. Under these conditions, the

shoulderwasextended from 25 sof irradiation

toapproximately threetimesthat value (70 s),

as expected ifthe shoulderwere MOI

depend-ent.

Effect of irradiation on viral infectivity.

The same virus used in the previous

experi-mentwas assayedforinfectivity by plaque

as-say before and after irradiation for 10 to 60 s

andthe resultsare showninFig. 3. No

shoul-der is evident and one-hit inactivationkinetics

areobserved. The37%survivaldose(D37)is306

ergs/mm2, and about3 logs of viral infectivity

arelost within 60 s of irradiation.Inagreement

withthe results showninFig. 2,25 sof

irradia-tionreduced theinfectivity of the virus inputin

these experiments to approximately 1 PFU/

11

U-Cl.

0D

0

z

z 0

C-0

CL

.1

-.01

-

.001-10 20 30 40 50

TIME OF IRRADIATION (SECS.)

FIG. 3. Infectivity loss after irradiation. Virus samples wereirradiatedfortheindicated times and

theinfectivitywasassayedonHeLacellmonolayers.

Theresults (0)wereplottedasfractionsof the

unir-radiated samples.

7-0 z

5

C,)

z

0

CL.

* \

60 J. VIROL.

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4-3- O SEC

_ 20SEC

z

O0SEC

30 SEC

c-I,

C-)~~ ~ ~~~~~0SEC

50 SEC 60 SEC

2 3 4 5 6

TIME POST-INFECTION (HRS)

FIG. 4. Viral RNA synthesis by irradiated virus. Virus was irradiated as indicated above and used to infect HeLa cells. At 0.5 h after infection actinomycin D (to 3.3

mg/ml)

and[14C]uridine (to 125 Xilml) wereadded. Incorporation was allowed to continue and0.2-ml samplesweretaken every0.5hfrom0.5 to 6.0 h postinfection. Incorporation was stopped with cold Earle solution, and the trichloroacetic acid-precipitable radioactivity was assayed by a

liquid scintillation counter.

and furtherincreases inirradiationareunable

to further delay RNA synthesis. Second, the

rateof RNAsynthesis, asdemonstratedby the

slope of theincrease inuridineincorporation, is

decreasedby increasingamountsofUV

irradia-tion.

As before we have ignored the delay factor

and havetakenthe

slope

of the line

generated

byunirradiated virustobe1.00 andexpressed

theothers as fractions of it. The loss of RNA

synthetic activity with increasing irradiation

was plotted in Fig. 5, as are the other results

presented inthis report. As can be seen, viral

RNAsynthesis isinactivatedby one-hit

kinet-ics, with noshoulder evident. The D37 for this

activity isapproximately 1,150

ergs/mm2.

The RNA synthesized in cells infected with

irradiatedvirus was examinedby sucrose

gra-dient velocity sedimentation and displayed a

normal sedimentationprofileand RNase

sensi-tivity(datanotshown). Noevidence for smaller

RNAfragmentswas detectablebythisanalysis.

Viralprotein synthesis was also evaluated by

SDS-PAGE andanautoradiographof thegel is

showninFig. 6.Cells infectedwith virus

inac-tivated for 0, 20, 40, and 60 s were labeled with 14C-amino acids for 2-h periods to coincide with

thepeak of viralprotein synthesisineach case

(Os, 2.25 to 4.25 hpostinfection;20s,2.50to 4.50

h postinfection; 40 s, 3.50 to 5.50 hpostinfection;

60 s, 4.00 to 6.00 hpostinfection). The gel

pat-tern of samples infected with virus after

in-creasing amounts of irradiation is not

signifi-cantly affected, except foraslightly increased

incorporation in several detectable host cell

proteins, possiblydue to less effective shutoff.

Somewhatunexpectedinview of thelarge loss

ofinfectivityinthe input virus duetothe

irra-diation, there is a significant amount of viral

protein being made even in the sampleinfected

with virusirradiated for 60 s.

Ability of irradiated virions to produce

progeny virions. The synthesis of relatively

larger amounts of virus-specific proteins and

RNA by cells infected with virus, which was

demonstrated by plaque assay to be markedly reduced in infectivity, prompted us to measure

the production ofassembled viral particles in

these cells. Cells infectedwith irradiated virus

in the presence of actinomycin D and

[14C]uridinewereharvestedasdescribedabove,

andcytoplasmic extracts were analyzedon

su-z

c-cn

z

0

cr-LL.

10 20 30 40 50 60 TIMEOF IRRADIATION (SECS)

FIG. 5. Loss ofviralfunctions after irradiation. Lossofinfectivityandshutoffareplottedas before. Lossofviral RNAsynthesiswasdeterminedby plot-tingtheslopesofthe increase in viral RNAsynthesis (fromFig. 4) for irradiatedvirus asfractionsof the slope demonstrated by unirradiated virus. Virion productionwas determinedasdescribed inthe text, and the total counts from irradiated preparations

wereplotted asfractions ofthecountsfrom unirra-diated viruspreparations. PFUproductionwas de-terminedasdescribedinthe text, and the virusyield forirradiatedsampleswasplottedasfractionsof the virusyield fromanunirradiatedvirussample.

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264 HELENTJARIS AND EHRENFELD

VPI

VP2

VP3

FIG. 6. Viralproteinsynthesisafter irradiation. Virus wasirradiatedfor 20, 40,and 60sand used to

infect cells, as detailed in the text. Atspecified times 50 XCi of 14C-labeled aminoacids was added, and incorporation wasallowedto continue for 2 h. Thecytoplasms were made 1%SDS andanalyzed on10%

polyacrylamidegels.ArrowsindicateVP1, 2,and 3asmarkers. Thefourcolumnsrepresent cellcytoplasms

infectedwith virusirradiatedfor 0, 20, 40, and 60 s,respectively.

crose velocity gradients for the production of

virion particles. There were no differences in

the sedimentation of unirradiated virus and

progenyviruswhoseparentalvirionshad been

irradiatedforupto60s.Theamountof

radioac-tivity in thepeakof theprogenyfrom

nonirra-diated virus was expressed as 1.00, and all otherpeakswerecalculatedasfractions of 1.00.

The loss of the ability of irradiated virus to

specifyforprogenyviruswithequivalent

sedi-mentation properties is plotted in Fig. 5. The

curveislinear withintercept 1, indicating

sin-gle-hit inactivationkinetics. Interestingly, the

slope oftheinactivationcurvedoesnotcoincide with lossofinfectivity but with the slope of the

inactivationofviralRNA synthesis.

Unlabeled progeny virus wasprepared in a

likemannertoassayfortheloss of the ability of

irradiated virus to specifyfor infectious

prog-eny. The progenyvirus was

assayed

for

infec-tivityonHeLacellmonolayers (Fig. 5). There

isanoticeableshoulderinthecurveuntil30sof

irradiation, where a decrease in infectious

progenyproductionisseen.There appearstobe

noclearexplanationofwhy infectious progeny

production should have a higher D37 than

150s particle production or why the former

inactivationcurvehasashoulderonitwhereas

thelatter appears linearinitsinactivation.

Effect of guanidine on inhibition of cell

protein synthesisby irradiated virions.

Infec-tionofcellsinthe presence of1mMguanidine

resultsinnodetectablepoliovirus-directed

pro-teinand RNA synthesis (7, 16). Athigh MOIs

in the presence ofguanidine, host cell shutoff

occurs at near-normal rates, whereas at low

MOIs

the inhibition is

significantly

decreased.

This suggests that infection at alow MOI

re---so

um

.-.

I IAPMM _WSW

-- VP ..%

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quires some amplification of a viral function

necessary for shutoff. To examine what effect

guanidine treatmentmight have onshutoff by

irradiated virus, the inhibition of cell protein

synthesis was evaluated in the presence and

absence of guanidine in cells infected by

un-treated virus at MOIsof 10 and 1 and virus at

anMOI of10 that had been irradiated for 30 s

(Fig. 7).The inhibition by unirradiated virus at

anMOI of10 isreduced by the guanidine

treat-ment, but shutoff is still evident. Inhibition

begins at the same time in both

guanidine-treated and untreated, infected cells, but both

the slope and the degree of inhibition are de-creased by treatment. In the absence of guani-dine, cells infected with untreated virus at an

MOI of 1 show a significant delay in both

shu-toff and viral protein synthesis, as compared

with infection at an MOI of 10. This suggests

that at lower MOIs complete host cell shutoff

andviralreplicationmight requirethe

produc-tionofsome component,with the kineticsofits

I-| I+ GUANIDINE

____ _-10+ UV+ GUANIDINE

*\_..-..\- \_;.

*AK-

\.

* \ \. +u

(

~~\I10+

UV

v \

-- 10+GUANIDINE

\ 10

1 2 3 4 5

TIME POST-INFECTION (HRS)

FIG. 7. Effect of guaindine treatment on shutoff by irradiated virus. Untreated virus was used to

infect cells at an MOI of10 (- - ) and 1 (....) both with and without1 mMguanidine. Virus was

also irradiatedfor30sand usedtoinfectcellsatan

MOIof10 before irradiation (---) both with and without 1mMguanidine.Proteinsynthesiswas

eval-uatedandplottedasdescribed in thelegendtoFig.1.

production being dependent upon the MOI.

Guanidinetreatment atthis MOI not only

re-ducesshutoff but also completely aborts it. The

curvegenerated bythe combinationof infection

at an MOI of 1 withguanidine treatment does

not significantly differ from uninfected,

un-treated cells.

When thecells areinfected inthe absence of

guanidine with virus at an MOI of 10 that has

been irradiated for 30 s, the shutoff and viral

protein synthesis curve coincide exactly with

untreated virus infection at an MOI of 1. After

30 s of irradiation, there remains

approxi-mately 0.4 PFU/cell, as determinedby plaque

titration. That this sample generates a curve

similar to the one by untreated virus at a

higher MOI suggests that thedamaged genomes

aresomehowabletoparticipate inshutoff.This

possibility is also suggested by examiningthe

effect ofguanidineon the inhibitionby

irradi-atedvirus.Although guanidinetreatment

com-pletelyaborts theinhibitionbyuntreatedvirus

at anMOIof1, thereis stillsignificant

inhibi-tionbythe irradiated virus at aneffectiveMOI

of 0.4 PFU/cell. Again, the sample manifests

moreofaneffectthan wouldbeexpectedonthe

basis of the remaining plaque-forming ability

alone.

DISCUSSION

The principal goalof this investigation was

toexaminethe effect ofUV irradiationon the

abilityofpoliovirus to causeinhibition of host

cellproteinsynthesis.Effective shutoffrequires

one infectious virus particle per cell, and the

kinetics of inhibition are independent of the

MOI, except for an increasing delay before the

onsetof inhibition at decreasing MOIs. If the

delayfactor is ignored, theUV inactivation of

theability of the virus to induceshutoffcanbe

shown to follow one-hit inactivation kinetics,

with aD37of 306ergs/mm2, avalueidenticalto

that forthe inactivation ofinfectivity.

The energy required to inactivate the host

cell shutofffunction ofpoliovirushasbeen

com-paredwith valuesreportedinthe literature for

theinactivation of a variety of biological

func-tions and activities (Table 1). It is apparent

thattheD37for host cellshutoffis quitesimilar

to the D37 for the destruction ofmengovirus,

poliovirus, vesicular stomatitisvirus, and

mu-rineleukemia virusinfectivity, butitis

signifi-cantly smaller than theD37for the inactivation

ofcarboxypeptidase A, reverse transcriptase,or

the hemagglutination activity of mengovirus. The former is thought to be due to damage to the viral genomes, whereas the latter involves

damagetotheproteins themselves. The

dispar-ityin energies required forthe inactivation of

5-

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AND EHRENFELD

TABLE 1. UV irradiation survivalof RNA and protein functions

Target (reference) D37(ergs/mm2)

Mengovirus (14) 700

Poliovirus (this investigation) 306 Vesicular stomatitis virus (15) 52.3 Rauscherleukemia virus (11) 2,000

Carboxypeptidase A (17) 300,000

Reversetranscriptase (11) 23,000-31,000

Mengovirus hemagglutina- 80,000

tionactivity (15)

the two groupsof functions is wide enough to

determine withsomecertainty that the

inacti-vation of thepoliovirus host cell shutoff activity

is due to damage to the viral RNA. Also, as

poliovirions are thought to contain 60 copies

each of four different capsid proteins, itseems

unlikely thatonehitpervirionwouldreflectan

effect of irradiationon aparticular capsid

pro-tein. In addition, we can detect no gross

changes in poliovirus capsid proteins by

SDS-PAGE afteras muchas 10minof irradiation.

The requirement, therefore, ofa functional

viralgenomefor the inhibition of host cell

pro-teinsynthesisseemsimperative, but the

partic-ipation of capsid proteins by a combinational

mechanism cannotbedisproved. To exclude a

role forcapsid proteins inhost cell shutoff, we

have attemptedtoexamineprotein synthesisin

cells infected withpurified viralRNA, but we

have been unabletoachieve asynchronous

in-fection of a large enough fraction of the cell

populationtoreliablyevaluate the rates of

pro-teinsynthesis.

By comparing the D37 of the poliovirus host

cellshutoffactivitywiththeD37 forother

polio-virusfunctions, it should be possible to

deter-mine the target size of the responsible RNA segment.Figure5showsthat theD37 forshutoff

is identical to that for infectivity, indicating

thatahit anywhereinthegenomeis sufficient

to inactivate that genome's capacity to effect

shutoff. However,ifone assumesthat the

syn-thesis of poliovirus proteinsoccursby the

cleav-age ofa single precursor polypeptide, then it

might be possible thatdamage anywhereinthe

RNAwouldpreventthepropercleavageofany

translation product as well as prevent the

translation ofsequences distal tothe UV hit.

In this case, all viral functions would display

identical inactivation curves. The slope

ob-served for the inactivation of viral RNA

syn-thesis, however, is approximately four times

that observed for the inactivation of host cell

shutofforinfectivity (Fig. 5),whichapparently

dissociates host cell shutoff from viral RNA

synthesis. One interpretation of this data is

that viral RNA synthesis is inactivated only

after a hit within the gene for the

virus-specified component(s) of the RNA

polymerase

and that this information occupies

approxi-matelyone-fourth of the genome. However,we

have not been able to rule out the possibility

of "product complementation" between

dam-aged genomes. Possibly the D37 for RNA

synthesis is actually much smaller, but,

be-cause the experiments were done at an MOI

of10, complementation between damaged

ge-nomes may slow the observed inactivation.

This possibility is suggested by two other

re-sults.Althoughinfectivity decreases withaD37

of 306 ergs/mm2, the ability to produce

infec-tious virions after infection at an MOI of 10

decreasesat amuchslowerratethanexpected

(Fig. 5). That no complementation is seen in

theinactivation ofinfectivityisnotsurprising

sinceaplaque titration by design is doneat an

MOI of much lessthan 1. The slower rate for

infectious virus production may actually

repre-sent complementation between damaged

ge-nomes or it may represent a shoulder due to

infection at an MOI higher than 1 and still be

consistent with one-hit inactivation kinetics

(analogousto host cell shutoff). Finally, when

cells wereinfected with irradiated virus andin

the presence ofguanidine under conditions in

which the effective MOI was reduced to less

than 1by irradiation, shutoff was still

demon-strable, whereas unirradiated virus at an MOI

of 1 in the presence ofguanidine resulted inno

detectable shutoff. Again, the irradiated sam-ple manifests more of an effect than would be

expectedonthebasis ofinfectivity alone. Both

oftheseare consistent withsome sortof

comple-mentation between or participation by

dam-aged genomes.

ACKNOWLEDGMENTS

This work was supported by grant GB18026 from the National Science Foundation and Public HealthService grant AI12387 from theNational Institute ofAllergy and Infectious Diseases.E.E. isthe recipient of Public Health Service careerdevelopmentaward AI 00096from the Na-tionalInstitute ofAllergy and Infectious Diseases.

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