Specific Effect of Guanidine in the Programming of Poliovirus Inhibition of Deoxyribonucleic Acid Synthesis

Specific Effect

of

Guanidine

in

the

Programming

of

Poliovirus Inhibition

of

Deoxyribonucleic

Acid

Synthesis

C. D. POWERS, B. A. MILLER, H. KURTZ, AND W. W. ACKERMANN

DepartmentofEpidemiologyandVirusLaboratory, School of PublicHealth,

University ofMichigan, AnnArbor, Michigan 48104

Received for publication31 January 1969

Inhibition ofHeLacelldeoxyribonucleic acid (DNA) synthesis, which occurred

bythe 4thto5th hr afterinfection withpoliovirus, could be blockedcompletely by guanidineonlywhenitwas presentbeforethe2ndhr. Atthe2nd hr, therewasno

significant ribonucleic acid (RNA)-replicase activity, and addition ofguanidine inhibited allproduction ofvirusbut allowed 57%of maximal DNAinhibition to

develop. Maximum DNA inhibition developed in cells infected for 4 hr in the

presenceofguanidine when theguanidinewasremovedfora10-mininterval.

RNA-replicase activity wasnotenzymatically detectableand viralmultiplicationdidnot

develop in these cells unless the interval without guanidine was extended to 60 min. Theinterpretationof thedatawasthat theeffectofguanidineonviral-induced

inhibition ofDNA synthesis wasdistinct andnot a consequenceoftheinhibition

ofRNA-replicase.

Poliovirus can replicate with or without

con-comitant inhibition of cellular

deoxyribonucleic

acid (DNA)

synthesis.

The two processes are

differentially dependent

upon the amino acid

composition

ofthe culturemedium

(1). However,

when inhibition of DNA

synthesis

does occur, it is first observed between the 3rd and 4th hr post-infection when the firstmaturevirus appears

(1,

18, 19); both it and viral

replication

areinhibited

by

guanidine

(1, 17,

25),

and theinhibition of each

is prevented

by dimethylaminoethanol (2, 22).

Some

guanidine-resistant

viral mutants replicate

and block DNA

synthesis

in the presence of guanidine

(unpublished

data).

Guanidine is pur-portedto be a

specific

virus effector

(17)

which inhibits virus

replication

by

virtue of its action against the formation of ribonucleic acid

(RNA)-replicase

(5,

6,12).

The presentstudies were

designed

todetermine whether the action of

guanidine

upon

viral-induced inhibition ofDNA

synthesis

isa

distinc-tiveoneor aconsequenceoftheknown actionon

RNA-replicase.

The

findings

support the view

that aseparate

guanidine-sensitive

eventis respon-siblefor the DNA inhibition and thatthe forma-tion of

RNA-replicase

is

obligately subsequent

to it.

MATERIAIS AND METHODS

Cells.HeLa cellsweregrowninmonolayersat37 C

with Eagle's basal medium (11), twice concentrated

withrespect toaminoacids and vitamins, and

supple-mentedwith10% calfserum.Cultureswerepassaged

every 7 days and, periodically, cells and fluid were

inoculatedintospecial mediatoensurethatthey were

freeofmycoplasmaand bacteria (9). The cells were

growninBlake bottlesforexperimentswhichinvolved

assay ofenzyme activity, and in 2-oz prescription

bottlesfor studiesofviralreplication and isolation of

DNAforchemicalanalysis. During theexperimental

period, the medium was replaced with one

supple-mented with 1% calfserum.

Virus. The Mahoney strain of type 1 poliovirus

was passaged routinely in HeLa cells, washed and

concentrated by centrifugation, and used as a

sus-pensionin phosphate-buffered saline (PBS, pH 7.0).

Virus titerwasdetermined, usingaplaque assay, on

monolayersof HeLacellsin 2-ozprescriptionbottles,

and concentration was expressed as plaque-forming

units (PFU).

DNAwasextractedfrom HeLa cells byamethod

similartothatof Schneider (24).Following

precipita-tion with cold 40% trichloroacetic acid and two

washes with cold 5% trichloroacetic acid, the DNA

was dissolved in another portion of the same acid

(5%) with heating and was used to determine the

incorporation of 3H-thymidylate. DNA so isolated

was quantitatively determined by the method of Burton (7).

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When the DNA contentof a series of samples was

to bedetermined spectrophotometrically at 260 nm,

the Schmidt-Thannhauser method (23) for isolation

of DNA, as modified by Fleck and Munro (14), was utilized.

Enzyme assay. Infected cells were trypsinized,

centrifuged, and suspended in sucrose-Mg. Viral

replicase (RNA-primed RNA polymerase; 5, 6, 12),

was prepared and assayed as described by Baltimore

and Franklin (6).

Radioactivity. For measurement of radioactivity,

0.5mlof sample was added to 9.5 mlofscintillation

fluid [320 g ofnapthalene, 20 gof

2,5-diphenyloxa-zole (PPO), and 200 mg of

1,4-bis-2-(5-phenylox-azolyl)-benzene (POPOP) in 4 liters ofp-dioxane]

and then added to one 4000-ml container of

thixo-tropic gel powder (Cab-O-Sil) and counted for 10

min in an automatic Ansitron model 1300 liquid

scintillation counter (16).

Sources. 3H-thymidine monophosphate (1.8 c/

mmole), 3H-uridine triphosphate, and unlabeled

nucleosidetriphosphateswereobtained from Schwarz

BioResearch, Inc., Orangeburg, N.Y.; pyruvate

kinaseandphosphoenolpyruvatefrom Sigma

Chem-ical Co., St. Louis, Mo.; guanidine

hydrochlo-ride and actinomycin D from Merck & Co., Inc.,

Rahway, N.J.;napthalene, PPO, POPOP,and

Cab-O-Sil powder from Packard Instrument Co., Downers

Grove, Ill.; and p-dioxane and

2-dimethylamino-ethanolfrom Matheson Co., Inc., East Rutherford,

N.J.

Results

Developmentpostinfectionof resistanceto guani-dine

(i)

viralinhibition of DNAsynthesis

(ii)

syn-thesisofRNA-replicaseand

(iii)

viral replication. Since guanidine can block the production of RNA-replicase

(5)

and also viral-induced inhibi-tion ofDNA

synthesis

(1), these events may be distinct orone

might

merely

beaconsequence of

the other. To clarify this point, the

guanidine

sensitivity of eachprocess,aswellasviral

replica-tion, was determined

during

thecourse of

infec-tion.

Cultures of HeLa cells were infected with 40

PFU of

poliovirus

per celland incubatedat37 C.

During eachhourly intervalfrom 0 to 5,the rate

ofDNA

synthesis

wasdetermined

by

the method

of 3H-thymidylate incorporation into DNA. A second set ofreplicate infected cultures received guanidine (75

,g/ml)

at0, 1, 2, 2.5, 3, and 4hr,

and the rate of DNA

synthesis

wasdetermined in cells in the 4 to 5-hr interval

postinfection.

Athird set, composedof threepooled replicate

cultures foreach of the time intervalsmentioned above, was assayed for RNA-replicase activity

(6). At the 2nd hr, guanidine (75 ,ug/ml) was added to three cultureswhich, atthe 4thhr,were pooled and

assayed

forenzyme.

Other infected cultures were washed at 1 hr

postinfection withHank's balanced salt solution toremoveunadsorbedvirus. Theresidual virus in one culturewas determined at this time. Guani-dinewas added to the remainder at 2, 2.5, and 3 hr.Virus which developedin these by the 9th hr was comparedwith aculture receiving no guani-dine.

[image:2.487.261.451.247.438.2]

Theresulting comparative data are plotted in Fig. 1 as percentages of the maximum activities observed (viralyield at 9hr, DNA inhibition at 5hr,and replicase activityat 4 hr).The position of

barrepresentations indicateswhenguanidine was

added; the height of the bar denotes the percent-age ofactivity. At the2nd hr, the rate of DNA synthesis remained at, or was slightly greater

than, the initial uninfected level, and the

RNA-replicateactivity wasjust barely detectable.

Cul-

100-Co

w 3

80->

41 a 60-4t 0

40-z

:

(L

20-1 2 3 4 5

HOURS POST-INFECTION

FIG. 1.Effectofguanidine addition at various times

postinfection upon viral-induced inhibition of DNA

synthesis, production of RNA-replicase, and viral

replication. Symbols: solid line, DNA synthesis in

infectedcells,expressedasper centofmaximalspecific

activity ('H-TMP incorporation) in uninfected

con-trolcultures; dashedline, viralRNA-replicase activity,

expressed as per cent ofmaximal replicase activity

detectableat 4 hrpostinfection (80counts/minperml);

crossedline, RNA-replicase activityat4 hr

postinfec-tionwithguanidine (75

gg/ml)

addedat 2hr

postinfec-tion;dottedbar, DNAinhibition, expressedaspercent

of maximal inhibition observed at 5 hr postinfection

with 3H-TMPaddedat 4hrpostinfection; [Guanidine

wasaddedattimesspecifiedongraph (0, 1, 2, 2.5, 3,

and 4 hr postinfection.)]; open bar, virusproduction, expressedaspercent ofmaximal net yield obtained

incontrol culturesat9 hrpostinfection in the absence

ofguanidine. Guanidinewasaddedtoinfected cultures

at2,2.5, and3hrpostinfectionand allowedtoremain

onthe cells untilterminationofthe experimentat9hr

postinfection.

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tures which received guanidine at the 2nd hr showednoincrease in

replicase

by the 4th hr and therewas noviralmultiplication by the 9th hr, but therate of DNAsynthesis in these cultures

indi-cated 57% ofmaximalinhibition by the 5th hr. Addition of guanidine before the 2nd hr

pre-vented viral inhibitionof DNA synthesis. There-fore, inhibition ofDNAsynthesis doesnotrequire viral replication or synthesis of replicase. The guanidine sensitivity of the inhibition is duetoan

actionupon an eventwhich islargely

completed

before replicase activity appears. The expression of the early event interms of therate ofDNA

synthesis requires several additional hours.

By 2.5 hr, replicasewasdetectable and

guani-dine additionatthis time allowed

development

by

the9th hr of3 % of the maximal viral

yield.

Cul-tureswith

guanidine

at3rd hr

reached

30%

of the maximal value. Thus, it is clear that whereas

guanidine acting

upon the

early sensitive

event

may prevent the

sequential

production

of

repli-case,thereis alsoadirect actiononthereplicase

systemwhichcanbe demonstrated after the

early

event islargely

complete.

Thisdirect effect is in

agreementwith the mode of viral

inhibitory

action proposed by others

(5, 13,

17).

Effectofa delayedreversalof

guanidine

inhibi-tion onsubsequentdevelopment of

(i)

DNA inhibi-tion,

(ii) replicase activity,

and

(iii)

resistance of

DNAinhibition andviral

replication

to guanidine. Certain events in the

programming

ofthe viral infection, such as

attachment,

penetration,

un-coating

and inhibition of cellular

protein

and

RNA

synthesis

(3, 10,

13,

15, 21), proceed

inthe

presence of

guanidine.

In

time,

all infected cells

should be arrestedatthe first

guanidine-sensitive

reaction. Reversal of the

guanidine

should allow

allto

proceed again

in

synchronous

fashion. The

blockade of

guanidine

against

viralDNA inhibi-tion and

replicase

can be removed

by

dilution

or

by

the addition of

dimethylaminoethanol

(DMAE)

(2,

22).

In such

cultures,

the

develop-ment of DNA inhibition and its resistance to guanidine,aswellastheappearance of

replicase,

can befollowed to determine whether

they

pro-ceed inan

obligately

sequential

order.

Virus and

guanidine

(75 ,g/ml)

were added simultaneously tocultures of cells. Fourhr after

infection,

at atime whenDNA inhibition inthe

absence ofguanidine would have been

attained,

DMAE (90

gg/ml)

was added to each infected

culture. Thesame level of

compound

was added

touninfectedcellsas acontrol. DNA

synthesis,

as

it occurred ineach of the next 5

hr,

was deter-mined by the

incorporation

of 3H-TMP

(thy-midine monophosphate; 0.125

,c/ml).

Inhibitionwasprevented while

guanidine

only

was present (4

hr),

and the addition ofDMAE

initiateda reversal of this effect during the 1sthr

after its addition (Fig. 2). However, nearly 5

additional hr were required before it reached a

level of effectiveness which nearly equaled that seen in the infected control cultures (guanidine-untreated) at 5 hr postinfection. Thus, it would appear that inhibition of DNA synthesis in a

guanidine-treated, infected culture can be

post-poned for several hours and yet be expressed

subsequently; i.e., the potential or message for

inhibition isnot lost during thearrestedperiod.

These results were duplicated merely by

washing guanidine out of the system at 4 hr

postinfection. This was accomplished by re-moving the guanidine-containing medium from

the cultures, washing them twice with warm

medium (containing no guanidine), and adding

back prewarmed (37 C), guanidine-free medium

for

theduration ofthe experiment. Neither

guani-dinenor DMAEhadanyeffect on DNA synthesis

inuninfectedcontrol cultures.

HOURS POST-INFECTION

FIG. 2. Effectof guanidine removal(orreversal) at 4hrpostinfectionuponviral-inducedinhibition ofDNA

synthesis and theproduction ofRNA-replicase.

Sym-bols: interrupted line (top left), DNA synthesis in

infectedcells with guanidine (75iug/ml) presentfrom

0 to4 hr; solid line,DNA synthesisin infectedcells,

expressed as per cent of maximal specific activity

(3H-TMP incorporation)inuninfected controlcultures;

dashed line, viral RNA-replicase activity, expressed

aspercentof maximal replicase activity detectableat

7 hrpostinfection (112 counts/minperml); diagonal

bars, DNAinhibition,expressedaspercentofmaximal

inhibition observedat9 hrpostinfection with 3H-TMP

added at 8 hrpostinfection. Guanidine was addedat

zero-hour, removedor reversed at4 hrpostinfection for designated time intervals (10, 20, 30, 40, or 60

min), and then added back for the duration of the

experiment.

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Other cultures treated similarly were assayed for RNA-replicase 10, 30, 60 and 180 min after guanidine was removed by washing. At 60 min,

replicase first appeared at the lowest detectable level. By 180 min,the activitywascomparableto uninhibitedcultures.

In a similarexperiment, guanidinewasremoved

by washingatthe 4th hrpostinfection; then,after afurtherinterval(10, 20,3040,or60

min),

guani-dine was returned to themedium. Such cultures were tested at the 9th hr for the rate of DNA

synthesis,andothersat22 hrfor virusproduction. An interval ofonly 10 minresulted inthe

de-velopment by the 9th hr of 80% of themaximal DNAinhibition achieved (Fig. 2), whereas much longerintervals without guanidine were required

for viral multiplication and replicase synthesis.

For example, a 30-min interval allowed no viral

multiplication, and a 60-min interval resultedin

only0.4%of the maximalnormalyield. Likewise,

synthesis ofreplicasecould bedetected only after guanidinehad been leftoutfor 60 min. If

guani-dinewere notadded back,theyieldofvirus

prod-uct by 22 hr was maximum, indicating the

re-versibilityof theguanidineeffect(Table1). Thus, whenguanidine wastemporarily removed from a viral-infected culture, resistance to the guanidine blockade of the viral-induced DNA

inhibitiondevelopedinjust10min.Virus replica-tion, ontheother hand, proceededinthefurther

presence of guanidine only when the interval

without guanidinewas extended to 60min. This

lag suggests that certain guanidine-sensitive

eventswhich werearrestedmustproceed priorto

synthesis or activation ofreplicase. The first ap-pearanceofreplicaseactivity, approximately 1 hr

after theremoval ofthe guanidine block instead ofat2.5hr (asinthe untreated cultures), is

con-sistent with the view that certain events in the

programming priortothatcontrollingDNA

syn-thesis are guanidine-resistant and proceeded during the first4 hrof guanidinetreatment.

TABLE 1.Effect

uponI

viralmultiplication of temporary

removalof guanidinefor variousintervals of time

at4hr

postinfection

Guanidine (40

pg/ml)

schedule

(hourspostinfection) |Virus titer ( X 104)

at22hr

In Out In

None 12,900

0-22 2.85

0-4 4-4:10 4:10-22 2.00

0-4 4-4:20 4:20-22 3.00

0-4 4-4:30 4:30-22 4.00

0-4 4-4:60 4:60-22 74.3

0-4 4-22 16,000

DISCUSSION

From this as well as earlier data (1, 18, 19),

maximalinhibition by poliovirus of cellular DNA synthesis occurs by 4 to 5 postinfection. This inhibition could be blocked completely by guani-dine but only when the latter was present before the 2nd hr.By2hr,when therewasnoobservable inhibition of DNA synthesis, the infected culture

wasalready

57%c/0

committedtoultimate inhibition of DNA synthesis (Fig. 1). The commitment represents thecompletion of an early guanidine-sensitiveevent.Thesuccessive stages of the

proc-essareinsensitivetoguanidine. The first

recogni-tion ofadistinctearly event in theprogramming ofDNA inhibitionwasbased upon the kinetics of theblocking effectat2 hrofcanavanine, an amino acidanalogue (1).

Synthesis ofaninhibitory protein could be the early event described above, since it would be sensitive to guanidine as well as canavanine and amino acid deletion (1). Further, results have demonstrated that the ability of virus to inhibit DNA synthesis is inactivated by ultraviolet ir-radiation (unpublished data). In light of this and the occurrence of thesensitive event priorto (Fig. 1)orintheabsence of (Fig. 2) detectablereplicase activity, the inhibitory protein is viewed as a

product of the functioning of the RNA of the infecting parental virionrather thanthat of newly replicated or progeny RNA.

If the extent ofDNA inhibition observed at 5 hr postinfection is interpreted as an indicator of the extent or rate of theearlyevent (synthesis of in-hibitory protein) prior to addition of guanidine, the reaction is perceived tobegin (following cer-tain prior events) rapidly sometime between the 1st and 2nd hr andthen toproceed more slowly until the 3rd hr. Itprecedes thesynthesis of

repli-case, butfrom 2.5 to 3.5 hr this reaction and the appearance of RNA-replicase seem to occur concurrently. This may result merely from an asynchrony of the infection. The two processes maynotproceedconcurrentlyinthe same cell.

These sequential activities appeared at more distinctly spaced intervals following a delayed reversal ofa guanidine-inhibited infection, which would allow greatersynchrony oftheinfectionto be established. Removal ofguanidine after 4 hr fromaninfected culturefor onlya10-min interval subsequently (after 5 hr) allowed development of maximal DNA inhibition inthe continued

pres-ence ofguanidine; however, neither replicase ac-tivity nor subsequent viral replication (Table 1) could be detected until guanidine had been

re-moved for 60 min(Fig.2).

Onecannoteliminate thepossibility that

repli-case activity appeared during the first 60 min

following removal of guanidine in amounts

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neither enzymatically detectable nor sufficient for any virusproduction,butcompletely adequatefor production ofinhibitoryprotein.However,

extrap-olationof thecurves describing the rate of pro-duction ofreplicase, inthedetectable range (Fig. 2) tozero-production, does not suggest this

possi-bility. The detection of infectious virus is a

sensitive procedure. In these experiments, some virus production was alwaysdetectedwhen repli-case activity had been present, but nearly complete DNA inhibition was observed without evidenceof virusproduction.

The action of guanidine on viral inhibition of DNAsynthesis is not convincinglyexplained as a consequence of its previously described effect on the viral RNA replicase system. Rather, it appears as a distinctprior effect.

Recently, ithas been suggested that synthesisof

severalviralproteinsmaybe initiatedatoncewith formation of long polypeptide sequences, which

subsequently are segmented and processed into

several different proteins (20). Ifthis becorrect, the sequential appearance of specific biologic activities with differential sensitivity in time to

inhibitors, such as guanidine, indicates that the programming of the infection occurs at a sub-sequentlevel whenthepolypeptidesare

processed

into functional protein. Comparative studies of

the kinetics of inhibition by cycloheximide and

guanidine indicatethat the action ofthe latter is not at the stage of polypeptide synthesis (4).

Othershaveproposed that guanidinepreventsthe conformation ofone ormorepolypeptides of the replicase complex, preventing functional

activity

(17).

The lag between the

guanidine-sensitive

reac-tion related to DNA inhibition and the appear-anceofreplicase

following guanidine

removal

(or

reversal) suggests thatthe processes occur inan

obligately sequential

order.

Thus,

the

guanidine

blockade ofreplicase

activity

mayoccur not

only

by direct action on the conformation ofthe

en-zyme but alsoby actiononthe

inhibiting protein.

That asingle type of

guanidine

effectisinvolved

is supported

by

the similar concentrations of guanidine which areeffective inthe blockade of

both viral replication and DNA

inhibition,

as

wellas the

reversibility

of both

by

DMAE

(1, 2;

Fig.2). Guanidinemaybea

specific

virus

effector,

butonlyinthe sensethat it hasa

specific

type of

effect on

synthesis

of a number of

early

viral

proteins.

The lag between

guanidine

removal and the appearance of

replicase

activity

and the

prolonged

synthesis ofreplicaseareincontrast towhat one

would predict from a

previous

study

(17)

of a

guanidine-requiring

mutant of

poliovirus.

Inthe

lattersituation, replicasesynthesis wasinterpreted

asoccurring preciselyin a10-mininterval around the 3rd hrof infection. However, other reports are in agreement withthe present findings (5, 7, 13).

Thelag is not related to the rate of diffusion of guanidine from the cell since the response of the DNA inhibitory process is prompt (Fig. 2).

With the wild-type poliovirus, the early event

(formationofinhibitory protein) and the synthe-sis or activation of replicase may occur in an

obligatelysequentialorder, and both are sensitive to guanidine, whereas the mutant of Lwoff (17) may be guanidine-requiring only with regard to the replicase. Hence, in the absence of guanidine, the programming of the mutant may proceed

through theformationof inhibitory protein (and

otherearly events) tothe specific point of replicase

formation. Addition of guanidine would be re-quired only for a short interval to initiate

con-siderable replication of the mutant.

LITERATURECITED

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14. Fleck, A., andH. N. Munro. 1962. Theprecisionofultraviolet absorption measurements in the Schmidt-Thannhauser procedure for nucleic acid estimation. Biochim. Biophys. Acta 55:571-583.

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