Analysis of Additional Interference Occurring After the Removal of Interferon

JOURNAL OFVIROLOGY,Dec.1967,p.1158-1163

Copyright ( 1967 AmericanSociety forMicrobiology PrintedVol.in1, No.U.S.A.6

Analysis

of

Additional

Interference Occurring

After

the

Removal

of Interferon1

ROYCE Z. LOCKART, JR.

CentralResearchDepartment,ExperimentalStation,E. 1. du PontdeNemoursandCompaniy,

Wilmington,

Delaware 19898

Receivedforpublication19June 1967

Cultures of chick cells exposedtointerferoncontinuedtodecreasein virus-produc-ingabilityduring incubationaftertheinterferonwasremoved. Therateof develop-mentofthe additionalinterference and the degree of viral interference finally mani-festedweredependentontheconcentration ofinterferonto which the cultureswere exposedandthe time ofexposure. Additionalinterference occurred also ininfected cells. Additional interference was inhibitedby actinomycin Dand puromycin. The best explanation ofadditional interference is that it results from interferon that is fixedtothe cellsduringtheir initialperiod ofcontact.

Vilcek and Rada (9) demonstrated that cells

exposedtointerferon at 4 C were not resistant if

challenged with virus immediately after the

removalof interferon. The cells became

resistant,

however, if they were incubated at 37 C for

several hours before challenge. More recently,

Levine (4) confirmedtheobservations mentioned

above and showed

additionally

that chick cells

which had been briefly exposed tointerferon in

the presence of puromycin, and then infected

immediately with Western equine encephalo-myelitis (WEE) virus, produced less virus than

didcontrol cultures which hadnotbeenexposed tointerferon.If,afterthe interferonwasremoved,

the cultures were incubated

additionally

for

several hours with

medium,

they produced

still less virus after their infection with WEE virus

(4). Levine interpreted his data to indicate that the additional reduction of viral

yields

was due

toresidual interferon

activity,

probably

resulting

from cell-fixed interferon.

Theexperimental results reportedhere confirm and extend those

previously reported.

They

sup-port the contentionthat theadditional reductions of viralyields bycellsaftertheir briefexposureto

interferon result from residual bound interferon because both

deoxyribonucleic

acid (DNA)-dependent ribonucleic acid

(RNA)

and protein synthesis in the cells are

required

for the addi-tionalreductions inviral

yields.

It is knownthat the antiviral effect

resulting

from interferon

re-quires cellular RNA and protein synthesis (2, 4, 6, 8).Also,residual interferon

activity

wasshown

totakeplaceininfectedcells.

1Thisworkwasdonewhile the authorwas a mem-ber of thefacultyoftheDepartmentofMicrobiology,

TheUniversityofTexas, Austin.

MATERIALS AND METHODS

Cell cultures, media, and solutions. Chick embryo cells were prepared from embryos 10 days of age. The cells weresuspended inEagle'smedium (1) containing 3% calf serum (EC medium) and distributed into 60-mm petri dishes. Cultures were used the day after their preparation, at which time each culture con-tained approximately 3 million cells. Phosphate-buffered saline containing bovine serumalbumin at a concentration of0.1% (PBSA) was used to wash the cell cultures and served as a diluent for viruses. Ac-tinomycin D was provided by Merck, Sharp and Dohme, West Point, Pa. It was dissolved in95%ethyl alcohol and further diluted in EC medium for use. Puromycindihydrochloride was purchased from Nu-tritionalBiochemicals Corp., Cleveland, Ohio.

Virus. The preparation of WEE virus stocks, their storage, and the assay method for virus titrations were previously described (5).

Interferoln.Thepreparation of chick interferon was described previously (6). Fluids containing nonpuri-fiedinterferon were used. For potencymeasurements,

thefluids werediluted in twofold steps in EC medium, and 2 ml was added to chick embryo cultures. The greatestdilution capable of preventing the appearance

ofcytopathological effects in the cultures when they were infected with a large multiplicity [>10

plaque-formingunits (PFU) per cell] of WEE virus 17 to 24 hrlater wasconsidered tocontain one protective unit ofinterferon.

RESULTS

Kineticsofinterferon action. The rate at which culturesof CEcellsexposed to interferon at 37 C losttheir ability toproduce virus varied directly with the concentration of interferon to which they wereexposed. This dependencyis illustrated in Fig. 1. The ratedifferences shown are readily reproducible and may serve as a sensitive way to 1158

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3

HOURS WITH INTERFERON

FIG. 1. Kineticsof interference resulting from

inter-feron. Cultures ofchickembryocellswereincubatedfor

1,3, and5hrat37Cwith2, 8,and 32 protective units

of interferon. At each interval of time, cultures were washed andinfectedby the addition ofmore than 10

PFUpercellofWEE virus. Virus yields ofthecultures

wereplottedrelative to thosefromcultures which had

receivednointerferon.

measure relative interferon concentrations. In the experiment illustrated, replicate cultures of

chickembryocells wereincubated with interferon

diluted in EC medium. Each culture received 2

ml. At the times indicated, each culture was

washed three times with3 ml ofPBSA,carebeing

taken to remove all fluid between washes. Each

culture was then infected by the addition of at least10PFUpercell ofWEEvirus. Thetitersof

virusin thefluidsremoved 16 hr postinoculation were determined and areplotted in Fig. 1

rela-tive to the titers found in control fluids. Cells

generally produced between 1,000and3,000PFU

per cell. Therefore, itcanbeseenthatcells were

rendered able to produce an average of only a

few PFU when incubated for 3 to 5 hrwith 32

units of interferon, but produced considerably morewhen incubatedwith less interferon.

Additional interference after removal of

inter-feron. Replicate cultures of chick embryo cells were exposed to2, 8, and 32 units of interferon

for1 hrat37 C. Theinterferonwasremoved,and

the cultures were washed four times with warm PBSA. Two cultures exposedto each concentra-tion of interferonwereimmediatelyinfectedbythe

additionof >10PFUpercell of WEEvirus. Two

ml of warm EC medium was added to the re-mainder of the cultures. After 2 and 4 hr of additional incubation at 37 C in medium only, two cultures which had been exposed to each

concentration of interferon for the initial hour were removed and infected. The concentrations of virus in the fluids of the cultures were

deter-mined 20 hr after theirinfection.The viral yields obtained from the cultures exposed to interferon

were compared with those from the control cultures (Fig. 2). Acomparison ofthe viralyields

relativeto controlsafter1 hrof incubationinthis

experiment with those shown inFig. 1 reveals a closecorrespondenceofthedegreeofinterference

in the two experiments, thus illustrating the

reproducibility ofthe technique. The data show

that cellcultures incubated withinterferon for 1 hrcontinuedtodecline in their abilitytoproduce

virus,evenafter the interferonwasremoved. This

continued loss ofability toproduce virus will be

referred to as "additional

interference"

through-out the remainder of this paper. The additional interference was most pronounced in the 2 hr

after removal ofthe interferon and was almost

completeby4hrafter its removal. Themaximal degree to which the cultures were inhibited in

theirabilitytoproduce viruswasdependentonthe

concentration of interferon to which they were exposed. Note the ability of cells exposed to 2 units of interferon to produce more virus when

they were incubated longer than 2 hr after the removal oftheinterferon. The rapid recovery of

virus-producing ability after a briefexposure to

o

wshed3 PU

0

hd

0 3 5

HOURS VIRUS ADDED

FIG. 2. Demonstration of additional interference.

Culturesofchickembryo cellswereincubated with2, 8!

and32protectiveunitsofinterferonforIhrat37C.All cultures were washed,andsome wereinfected

immedi-ately.Otherswereincubated2and 4hrlongerinmedium

at37 Cpriortobeinginfected. Yieldsofvirusobtained 20 hrafter infection areplottedrelative to the yields

from cultures which hadnotbeenexposedtointerferon.

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LOCKART

uI

-I

n,

a:)

>r

TIME(hrs.)VIRUS ADDED

FIG. 3. Additional interferenceafter10minof

incu-bation withinterferon. Theexperimentwasthesameas

that illustratedin Fig. 2,except thathigher

concentra-tionsof interferonwereused and theperiod ofexposure

ofthe cellstointerferonwas10 min.

small amounts of interferon and further

incuba-tion hasbeen noticedrepeatedly.

Figure 3 illustrates anotherexperiment of the

samenature,but which differed from thatshown inFig. 2in that the concentrations ofinterferon

were higher and the period ofexposure was

re-ducedto10 min.Again,those culturesexposedto

to the two highest concentrations of interferon attained the maximal degree of inhibition when

they were incubated at 37 C withmedium for 3 hr and 50 min after the removal of interferon. The degree to which virus production was

in-hibitedat thattime again depended onthe

con-centration ofinterferon to which they had been

exposed. Also, as before, the cultures incubated

with the least concentration of interferon (10 protective units) showed an initial decrease in

their abilitytoproduce virus and thenagradual

increase in this ability. The time of initialcontact with interferon can be even shorter. Cells

in-cubated with 500 units of interferon for only 1 min were reduced in their ability to produce virusby 92.4 to 96.7% in threeseparate

experi-ments after an additional 4 hr of incubation in

warmmedium which contained nointerferon. Evidence that the additional interference was cell-fixed. The experiments just reported do not permitonetodecidebetween the alternatives that the additional interference was due to (i) cell-fixed interferonor(ii) the release into the medium

ofa small amount of reversibly adsorbed inter-feron. Therefore, the following experiment was performed. Six CE cultures were incubated with 16units of interferon for 1 hrat37C and subse-quently treated in the following three ways. (A) Twocultureswerewashed 12 times and immedi-ately infected withmore than 10 PFUpercell of

WEEvirus. (B) Twocultures were washed three

times after the removal of interferon and three times after each hour of incubation at 37 C in medium until they had received 12 washes. The cultures were then infected with virus. They had

been incubated with interferon for 1 hr and medium for 3 more hours after the removal of interferon. (C) Two cultures were washed 12

times after the removal ofinterferon, incubated additionally for 3 hr at 37 C, and infected with virus. Two cultures which hadreceived no

inter-feron were included in each of the scheduled

treatments (A to C) outlined above. Virus yields

ofthe cultures were determined 20 hr after they were infected. The results are shown in Table 1.

[image:3.471.63.209.73.275.2]

Additional interference occurred during the 3-hr period of incubation andto verynearly thesame

TABLE 1. Cell-fixednatureof"additional" interference

Virus titer(PFU/ml)

Viralyields(per Treatment No previousincubation 1-hrincubation with16 cent of control)

withinterferon units of interferon

Expt1 Expt2 Expt1 Expt 2 Expt1 Expt 2

(A) Washed12 times, then infected

immediately... 1.0 X 109 7.7 X 108 5.7 X 107 5.4 X 107 5.7 7.0 (B) Washed three times

immedi-ately; three times after each of threehourly periods of

in-cubation; infected... 1.3 X 109 1.6 X 109 1.5 X 107 5.9 X 106 1.2 0.4

(C) Washed 12times; incubated 3

hr; infected... 1.7 X 109 1.3 X 109 1.3 X 107 6.3 X 106 0.8 0.5

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OCCURRING INTERFERON

O . Adhed

intE cutue ploteas clse cices an desinate

C ~~~3~ 5

HOURS

FIG. 4. Inhibition ofadditional interference by

ac-tiinomycinD. Additionalinterference wasdemonstrated

in the culturesplottedasclosed circles anddesignated

VA. Those cultures had been incubated withinterferon

for 1 hr andwere infectedwith WEE viruscontaining actinomycinD inthe inoculum eitherimmediatelyafter

the removalofinterferonorafter2 and 4 hrofadditionlal incubation with medium. The closed circle with a V

indicates theyield ofvirusfrom cultures incubatedfor

1 hrwithinterferon andinfected immediately afterits removalwith viruswhoseinoculum didnotcontain

ac-tinomycinD. Theprevention ofadditionalinterference

byactinomycinisshownby thepoints designatedA V. Afteraninitial hourofincubation withinterferon,itwas

removed and these cultureswere incubatedadditionally

with medium containing actinomycinD (I pg/ml). Cell

cultureswhich hadnotbeenexposedtointerferonwere incubatedfor2 and 4 hr withactinomycinDtoserveas

controls. Theyare represented by the open circles fol-lowedbyV.

extent,

regardless

of whether the cultures were

washed

12 times

initially

or 3 times each hour

until they had been washed 12 times. It seems

unlikely thatthe additional interference was due

to

reversibly

adsorbed interferon.

Inhibition of the additional interference by

actinomycin D. The effect of

actinomycin

D on the additional interference

just

described was

determined. Two cultures were infected with WEE virus, and the

yield

of virus 20 hr later served as thecontrol value. Other cultureswere

infected after incubation with

actinomycin

D

(1,ug/ml)for2and4 hr.The viral

yields

fromthe cultures incubated with

actinomycin

served as a measure of the loss of

virus-producing

capacity

bythecells

resulting

fromthe

drug

(open

circles,

Fig. 4). Other cultures were incubated with 16

units ofinterferon for 1 hr. Four cultures were washed and infected

immediately.

Two of the cultures contained

actinomycin

in the virus

inoculum

(VA

in

Fig. 4);

two did not

(V

in

Fig. 4). Thosecultures which had been incubated

withinterferon for 1 hr and which were infected

immediately with a virus inoculum containing

actinomycin produced only about 50% as much

virusasthe controls (whichalso were challenged with actinomycin in the virus inoculum). Those cultures incubated with interferon for 1 hr and

infected immediately with a virus inoculum

con-taining no actinomycin produced only 11% as much virus.Therefore, cultures of cells incubated

with interferon for 1 hr produced one-ninth as much virus when infected with virus alone, but virus production was reduced by only

one-half when actinomycin D was present in the

inoculum. The discrepancy in virus yields indi-cated that additional interference probably occurred after infection. Thispoint will be dealt with later. Four cultures which had been incu-bated with interferon for 1 hr were washed and incubated further at 37 Cwith medium. After 2 hr, andagain4 hr later,two of thecultures were

removed, washed, and infected with a virus inoculumcontainingactinomycinD. The yields of virus 20 hr after infection were determined.

Whereas the cultures which were infected with

virus in the presence of actinomycinimmediately after the removal of interferon produced 50% asmuch virus ascontrols, those culturesincubated

an additional 2 and 4 hrprior to infection

pro-duced 11 and 7.3%, respectively, as much virus asthe controls. Thisdemonstrationofadditional

0

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z 0

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C-0 0~

C,)

5;

0 3 5

HOURS

FIG. 5. Inhibition ofadditionalinterference by puro-mycin. AfterI hrofincubation with 32unitsof

inter-feron, cultures wereincubatedadditionally foreither2 or4 hrwith mediumlacking (0) andcontaining (0)

puromycinat

50,Sg/ml.

Atthetimeperiods designated,

they were infected with virus. The virus inocula

con-tained actinomycinD (5ug/ml).Theclosed circlewith

a Vindicates theyield ofvirusfrom cultures incubated

forIhr with interferonandinfectedimmediately after itsremoval with virus whose inoculum didnotcontain

actinomycinD.

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LOCKART

interference is shown in Fig. 4 (closed circles labeledVA).Fourothercultures which had been

incubated with interferon for 1 hr were washed and incubatedfurtherat37 Cwithmedium con-tainingactinomycin D (1 ,ug/ml) and 32 units of interferon.Theinterferonwasadded to show that the actinomycin D was working. Two cultures

were removed after 2 hr of

incubation,

and the finaltwocultures wereincubated for4hr. At the

time oftheir

removal,

they were washed and in-fected. The amounts of virus produced by the cultures at 20 hr after infection were deter-mined and are represented in Fig. 4bythe open

triangles. The action of the 32 units of added interferon and any additional interference were

preventedbythepresence ofthe actinomycin D. The slight decline of virus production found in

those cultures was mostprobablydue tothe loss

of

virus-producing

ability

resulting

from the presence of the actinomycin, as the yields of virus from control cultures

(open circles, Fig.

4) decreased at the same rate when incubated

withactinomycin.

Inhibition of the additional interference by

puromycin.Theproceduresfollowedtodetermine

the effect ofpuromycinonadditionalinterference

were the same as described for the

experiments

in which

actinomycin

was used.

However,

the

cultureswereincubated with 32 units ofinterferon rather than 16, and noadditionalinterferon was

added during the additional incubation in the presence of

puromycin.

Puromycin

was usedata

final concentration of 50

,g/ml.

Also,

all those

cultures incubated for additional

periods

after

the removal of interferon were infected with virusinocula

containing

actinomycin

D

(1 ,ug/ml).

As shownin

Fig.

5,

puromycin,

like

actinomycin,

prevented the additional interference which

oc-curred in the presence of medium alone when cultures were incubated for 2 and 4 hr after the

removal ofinterferon.

Occurrence

of

additional

interference

in

infected

cells.After brief

periods

of exposureto

interferon,

virusyieldswere

nearly

10-foldgreaterwhen

acti-nomycin D was present in the virus inoculum

(Fig.4and6)oraddedjust

prior

toinfection (6).

Inlight ofthe

foregoing

results,themostprobable explanation is that the reduced

yields

of virus

produced when the cultures are infected with virus alone area result, atleastin part, of addi-tional interference

taking

place in the infected

cells.

Actinomycin

Dwas usedtoshowthat this didoccur. Culturesofcellswere incubated for 1 hrwith 16 units ofinterferon.Theinterferonwas

removed and the cultureswerewashedwithwarm

PBSA. Two cultures (closed circle, Fig. 6) were

infected

immediately

with virus and two (open

circle,

VA, Fig.

6)

with virus inaninoculum

con-0

D VA

0L

0 6ashe, Infect

Act.

v ~~~~~~~~Act.Act

-1 0 3 6 9 12

Time of addition of ACTINOMYCIN(Hrs. Post-infection)

FIG. 6.Additional interference in infected cells.

Cul-turesof CE cellswereincubated with16 unitsof

inter-feron forI hr, washed, and infected. To the medium, actinomycinD wasaddedtoaconcentration ofI,ug/ml

atthe timesindicated. The closed circle witha V

indi-catesthe yield ofvirusfrom cultures incubated forIhr

with interferon and infected immediately after its re-movalwithviruswhoseinoculum did not contain actino-mycin D. Viral titers were determined 20 hr after in-fection.

taining

actinomycin

D (5 ,ug/ml). An additional eight cultures which hadbeenincubated with in-terferon for 1 hr were washedand

infected,

and

mediumwasadded to each.At2, 5, 8, and 11 hr afterinfection, sufficient medium with

actinomy-cinwasaddedsothatitwaspresentat a concen-trationof 1 ,ug/ml. The concentration ofvirus in thefluidsremoved 20 hrafterinfectionwas deter-minedforallthecultures. The results are plotted in Fig. 6 (open circles, Act.). Additional

inter-ference ininfected cells accounted for the

differ-ence in virusyields found between those cultures

whichwereincubatedwithinterferonfor 1 hr and

infected, withor withoutactinomycin. The addi-tional interference was reflected only gradually andwasinhibitedby the addition of actinomycin

totheinfectedcultures at time intervals prior to 9hrafter infection.

DISCUSSION

Thedata inFig.2and 3 show that cellcultures which have beenincubated with interferon only

briefly, sothat only a small amount of virus

in-hibition had occurred, continued to decrease in their abilitytoproduce virus whenincubated for

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additional periods of time. Additional

interfer-ence was described previously by Levine (4).

Several otheraspects of the additionalinterference

became apparent in the present work. The rates at which additional interference occurred were de-pendent on the concentrations of interferon to which the cultures were exposed, but were less than those observed if theinterferon was left on the cultures (compare Fig. 2 with Fig. 1). The final amounts by which virus yields were decreased were alsodependent on the concentra-tion of interferon to which the cultures were

initially exposed. Those cultures exposedto low

concentrations (Fig. 2), or morebriefly (Fig. 3),

were decreased in their virus-producing ability

upon additionalincubation, butregainedsomeof thatabilityon further incubation. Itwas demon-strated that the continued interference was

cell-fixedbyshowingthat12washes,whether applied

immediately upon the removal of interferon or intermittently fora 3-hrperiodoftime, failedto

affectsignificantlythe amount ofadditional ference that occurred after the removal of inter-feron. Having established that the additional interference was cell-fixed, several

possibilities

were considered. Additional interference

might

have resulted from (i) cell-fixed

interferon,

(ii)

messenger RNAwhich had accumulated during

theinitialperiodofexposuretointerferon, or

(iii)

some other process set in motion

by

the initial exposure to interferon. As the additional

inter-ference was stopped by both actinomycin and

puromycin, it almostcertainlywasnotaresultof anearlyaccumulationof interferon-induced

mes-senger RNA. And, by being sensitive to both

drugs, the additional interference reacts like the interference which occurs in the continuing pres-ence ofinterferon.

Finally, itwas

interesting

to seethatadditional interference occurred eventhough the cells were

infected. Such a

finding

is consistent with the demonstration that interferon can inhibit arbo-viruses when added after infection

(7, 10),

and it suggests thatWEE virus has very little effect on

cellularRNA and

protein synthesis.

Other

experi-ments

(Sreevalsan, personal

communication)

have

indicatedthat infection of chick cells with WEE

virus

only

slowly

inhibits cellular RNAand pro-teinsynthesis,with

50%

inhibition

being

apparent at about 8 hr

postinoculation.

That additional

interference occurs in infectedcells also

explains

why

approximately

10 times more virus is

pro-ducedwhen cultures which have been incubated withinterferonareincubated with

actinomycin

D

at the time of or before their inoculation with WEEvirus (6).Thislarge difference in viral yields due to the presence ofactinomycinoccurred, how-ever, only duringrelatively short periods of incu-bation with interferon when the cultures were

infected priorto thetime that theinterferon pres-ent had exerted its maximalinhibitoryeffect.

Although the data presented still fall short of proving that interferon is taken up by the cell, they are most consistent with that explanation. Further, they indicatethat, once taken up, inter-feron can continue its induceractivity for several hours,eventhough the cellsareinfected.

ACKNOWLEDGMENT

Iwish toacknowledge the excellent technical assist-ance of Barbara Horn.

Thisinvestigation was supported byPublic Health Service grantsAI-03538 and5-K3-AI-19,385 from the National Institute of Allergy and Infectious Diseases.

LITERATURE CITED

1. EAGLE, H. 1955. Nutrition needs of mammalian

cells intissueculture. Science 122:501-504.

2. FRIEDMAN, R. M., AND J. A. SONNABEND. 1964. Inhibition of interferon action by

p-fluoro-phenylalanine.Nature 203:366-367.

3. LEVINE, S. 1964. Effect of actinomycin D and

puromycindihydrochloride on action of inter-feron. Virology 24:586-588.

4. LEVINE, S. 1966.Persistence of active interferon in cells washed after treatment with interferon. Proc.Soc.Exptl.Biol. Med. 121:1041-1045. 5. LOCKART,R. Z. 1963. Production of an interferon

byLcells infected with Western equine

enceph-alomyelitisvirus. J. Bacteriol. 85:556-566. 6. LOCKART, R. Z. 1964. Thenecessity for cellular

RNA andprotein synthesis for viral inhibition

resulting from interferon. Biochem. Biophys. Res. Commun.15:513-518.

7. LOCKART, R. Z., AND T. SREEVALSAN. 1963. The effect of interferon on the synthesis of viral nucleic acid. Viruses,Nucleic Acids, and Can-cer, 17th AnnualSymposium on Fundamental Cancer Research, University of Texas M. D. Anderson Hospital and Tumor Institute, Houston, p.447.The Williams and Wilkins Co., Baltimore.

8. TAYLOR, J. 1964. Inhibition of interferon action byactinomycin. Biochem. Biophys. Res.

Com-mun. 14:447-451.

9. VILCEK, J.,ANDB. RADA. 1962. Studies onan in-terferon from tick-borne encephalitis virus-infected cells (IF). III. Antiviral action of IF. Acta Virol. 6:9-16.

10. WAGNER, R. R. 1961. Biologicalstudies of

inter-feron. I. Suppression of cellularinfection with Eastern equine encephalomyelitis virus. Virol-ogy 13:323-337.

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