Measles Virus Ribonucleic Acid and Protein Synthesis: Effects of 6-Azauridine and Cycloheximide on Viral Replication

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Measles

Virus Ribonucleic Acid and Protein

Synthesis: Effects of 6-Azauridine and

Cycloheximide

on

Viral

Replication

ALLEN PORTNERl AND ROBERT H. BUSSELL

DepartmentofMicrobiology, The University of Kansas, Lawrence, Kansas66044

Received forpublication30August1972

Cycloheximide and 6-azauridine were employed tostudy the time course of

measles virus protein and nucleic acid syntheses in AV3 cells. Synthesis of ribonucleic acid (RNA) essential for infectivitywasfirstdetected at6hr and

in-creased concurrently with the formation of essential protein. Maximum levels

ofvirus-specific RNA and protein were presentby 18hr, a time whenonly 5%

ofprogeny virus was detected. Essential RNA and protein syntheses preceded

the formation of infectious virusbyatleast 10 to 12 hr. The timecourseof RNA

and protein syntheses essential for the formation of complement-fixing (CF) antigen and salt-dependent agglutinin (SDA) was also determined. RNA

syn-thesis essentialforthe formation ofSDAwasfirst detectedat2 hr andwas

pres-ent maximally by 6 hr, whereas SDA-protein increased concurrently with the protein essential for infectivity. This suggested that the last protein essential for infectivity may be SDA. RNA synthesis essential for the formation of CF antigen was first detectedat 4hr, while CF-protein increased at 5 hrand

pre-ceded SDA-protein and protein essential for infectivity by approximately3 hr.

Reversal of inhibitionofprotein synthesis by cycloheximideindicated thatearly protein synthesis (1 to 3hr) was required for the formation of infectious virus.

The data suggest that the relatively long eclipse period observed with measles virus is relatedtoalong maturation period rather thantolate formation ofearly proteins, viral RNA, orstructural proteins.

There is much information on the physical and biological properties ofmeasles virus (16, 18, 28), but knowledge of the mechanism of replication is limited. Prior studies have re-vealed the general characteristics of the growth cycle (6, 16, 17) and the sequence of formation of infectious virus, hemagglutinin, complement-fixing antigen, and

hemolysin

(19). The present study was undertaken to determine the time course of ribonucleic acid (RNA) and protein synthesis necessary for the formation of infectious virus, salt-depend-ent agglutinin (24), and complement-fixing activity in measles virus-infected cells. An inhibitor of RNA synthesis, 6-azauridine (10), was used to determine the time course of RNA synthesis. Cycloheximide, a protein synthesis inhibitor (8), was used to determine the time course ofviral protein synthesis.

IPresent address: LaboratoryofVirology, St. Jude

Chil-dren's ResearchHospital andthe University of Tennessee, Memphis,Tenn. 38101.

46

MATERIALS AND METHODS Cells and media. The AV3 continuous human amnioncell line (21) was used. The cells were grown

in monolayers in 32-ounce (.95-liter) prescription bottles in medium 199 containing 0.25% lactalbumin hydrolysate and 15% heat-inactivated (60 C, 30min) newborn calf serum, and sodium bicarbonate was usedtoadjust the pH to 7.2 to 7.4 (GM). Eagle

mini-mum essential medium (MEM) with 5% newborn agammacalf serum wasusedfor maintenance (MM). Dialyzed newborn agamma calf serum was used when theeffect of6-azauridine was determined.

Virus. The Edmonston strain of measles virus

(7) wasused throughout this study. Stock virus was prepared in 48-hr cultures of AV3 cells. The virus inoculum (3 ml, 2.0 x 105 PFU/ml) was adsorbed at

36 C for 2 hr. After adsorption, 45 ml of MM was added, and the cultures were incubated at 36 C for

48 hr. The MM was exchanged, and the cultures

were incubated at 31 C for an additional 24 hr

be-fore they were harvested by freezing and thawing threetimes.

Infection of cells. Approximately 1.8 x 106 cells

were inoculated into 2-ounce (5.8-ml) prescription

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MEASLES VIRUS RNA AND PROTEIN SYNTHESIS

bottles and incubated for 24 to 48 hr at 36 C. The GM wasremoved, and the cells were incubated with 0.5ml of virus for 2 hr at 36C. The input multiplicity of infection(MOI)wasapproximately 5 to 10 plaque-forming units (PFU) per cell unless otherwise indi-cated. The inoculum was removed, and the cells were washed with 5 ml of phosphate-buffered saline (PBS) containing 1% agamma calf serum and rein-cubated with 5 ml of MM at 36 C. The time after the 2-hr adsorption period was regarded as zero time for these experiments. For some experiments, tube cultures were seeded with 5 x 105 cells and inoculated with 0.2 ml of virus. All other conditions were as described above.

Plaque assay. Monolayers of AV3 cells, 70 to

80% complete, in 2-ounce prescription bottles were washed, and two cultures were inoculated with 0.5 mlofeach virusdilution.Adsorptionwas at 36C for 2 hr. Unadsorbed virus was decanted, and the monolayers were overlayed with 5 ml of sodium carboxymethylcellulose (NaCMC) medium. Cultures wereincubatedat36C for3to4days, atwhich time primary plaques developed. The overlay medium was decanted, and the monolayers were fixed with 8 ml ofFormalin(10%inPBS, pH7.2). Afteratleast

2hrat roomtemperature, the fixativewasdecanted,

and the monolayers were covered with 4 ml of Giemsa stain. Thirty minutes later the stain was

decanted, and the monolayers were washed three times with tap water and air dried. Plaques were

counted withadissecting microscope (12x), and the titerwas reportedasplaque-formingunitsper milli-liter. The NaCMC overlay (final concentration, 1%) was prepared by dissolving 10 g of powdered high viscosity NaCMC (General Biochemicals) in 840 ml of distilledwaterandmixingat 4C for24hr. The solution was autoclaved at 116 C for 10min. After

cooling, Eagle MEM and a final concentration of 5% newbom calfserum was added.

Salt-dependent agglutinin. Samples were as-sayed for salt-dependent agglutinin (SDA) (24) by

employing a microtechnique (27). Undiluted virus

samples were adjusted to 0.8 M citrate with 3 M sodium citrate.The diluentwas 0.8M sodiumcitrate in PBS. A 1% suspension of rhesus monkey erythro-cytes was employed. The SDA titer, expressed as the reciprocal ofthe final dilution of virus causing agglutination, wasreadafter 2hrat 37C.

Complement fixation. A microtechnique was

used for the titration of complement-fixation (CF)

antigen (4). Approximately two optimal units of monkey anti-measles antiserum and five 50% end-point units of commercial guinea pig complement were employed. The end points were considered to

be the last dilution ofantigen producing30% or less

hemolysis.

Chemicals. 6-Azauridine (AUR) and

cyclohexi-mide were purchased from Sigma Chemical Com-pany, St. Louis,Missouri.

Cell viability determination. Cells were diluted

1:2 in 0.5% trypan blue and counted in a hemo-cytometer. Cells which excluded trypan blue were considered viable.

RESULTS

Measles virus single cycle growth curve and the appearance of viral antigens. The growth characteristics ofmeasles virus and the appearance ofviral antigens in AV3 cells were

determined to serve as base lines for the

in-hibition studies. AV3 cells were inoculated with measles virus. Cell cultures were har-vested at intervals up to 28 hr and then

ti-trated for infectious virus, CF antigen, and SDA (Fig. 1). A significant increase in infec-tious virus was observed between 14 and 18 hr, and virus titers continuedto increase until 28 hr. An increase in CF activity was detected

at 8 hr, and titers continued to increase

GROWTH

CURVE

-s*INFECTIVITY

0-0 CF

L.~-~ SDA

100

t

J -J

z LL

L-0

B0

10-1.0

0 4 8 12 16

20

24

28

HOURS

FIG. 1. Growthcurveof measles virus in AV3 cells.

Monolayerswere infected withaninput multiplicity

of5 to 10PFUpercell;viruswasadsorbedat36Cfor

2 hr, washed three times, and incubated with MM.

Virus was harvestedat the indicated times and

as-sayedforinfectivity, CFantigen,andSDA. Absolute maximum values (28hr) were: infectivity, 1.9 x 106

PFU/ml;CFantigen,1to4;SDA,1 to 6.

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throu throu

tivel)

meas (6, 1 SDA Efl tion of cy infec after centr fectiN were The centr Addi 5 to per r

expel

if cy

infec to pr

igh 22 hr. SDA activity increased from 12 AV3 cells were incubated in MM containing Lgh 28 hr. These results confirm the rela- 0.5 ,g/ml for either 6 or 24 hr. There was no

y

long

eclipse

periods

observed with alteration in cell

morphology.

The cells were

;les

virus in various cell culture systems washed six times to remove the inhibitor and 6, 19) and show that CF antigen and then were infected. Cultures were harvested appear before infectious virus. at 24 hr and assayed. Virus yields in cultures fect of cycloheximide on virus replica- pretreated for 24 hr were 5.5 x 101 PFU/ml andcell viability. Various concentrations (control yields were 6.0 x 105PFU/ml). Virus cloheximide were added to measles virus- yields in cultures pretreated for 6 hr were ted AV3 cells (MOI 0.2) immediately 2.1 x 101 PFU/ml (control yields were 2.2 x virus adsorption to determine the con- 105 PFU/ml). This indicated that the effect

rations

of cycloheximide required to ef- of cycloheximide (0.5 ,ug/ml) on the capacity vely block virus replication. Virus yields of the cells to produce virus was readily

re-determined by plaque assay at 24 hr. versible.

drug inhibited virus replication at con- Effect of AUR on virus replication. Ex--ations that were not cytotoxic (Fig. 2). periments were performed to determine the tional experiments employing an MOI of concentration of AUR to be used in subsequent 10 showed that 0.5

qg

of cycloheximide inhibitor experiments. After virus adsorption nl inhibited virus replication by 98%. An (MOI 1), medium containing different con-riment was also performed to determine centrations of AUR was added. Virus yields cloheximide added to the cells prior to were determined by plaque assay at 24 and tion would alter their subsequent ability 48 hr. Measles virus replication was sensitive roduce virus in the absence of the drug. to AUR; 98% inhibition occurred in the

pres-ence of 5

gg/ml

at 24 hr (Fig. 3). However, at 48 hr, there was a decline in the amount of inhibition indicating some loss of inhibition

on continued incubation. No alterations in cell morphology or viability were observed with 5,ugofAUR per ml.

Time course of viral RNA and protein synthesis necessary for the formation of infectious virus. AUR or cycloheximide was added at various times after infection of AV3 cells to inhibit either RNA or protein syn-thesis. Virus yields at 28 hr were plotted against the time of addition of the inhibitors (Fig. 4). Comparison with the viral growth curve reveals that an increase in the synthe-£v-25 VIABLE CELLS sis of an essential RNA was detected at 5 to 6

/ML

hr and preceded the appearance of mature

O----O PFU/ML virus by

approximately

10 hr. An increase in

protein synthesis necessary for viral infec-tivity was detected between 4 and 7 hr and preceded the appearance of mature virus by approximately 9 hr. These results also show that nearly maximal levels of RNA and

pro-CYCOE tein essential for viral

infectivity

were

present

o 001 0.1 IO 1O several hours before the appearance of

infec-CONCENTRATION OF CYCLOHEXIMIDE (pg/ml) tious virus.

Time course of RNA and protein syn-2. Effect ofcycloheximide on virus yield and thesis essential for the production of virus

uiability. Immediately after virus adsorption * ml

heximidewas added at the concentrationsindi-

antign.

the

timetcou

of RN

snthesis

At 24 hr, virus was harvested and

assayed

essental for the formaton of CF

antigen

(CF-sond group of cultures was not infected, and RNA) and SDA (SDA-RNA) iS shown in

heximide was added as indicated above. At Fig. 5. The

infectivity

curve is the same as the

viable cell counts wereperformed by staining azauridine curve in Fig. 4. An increase in

trypanblue. SDA activity was detected at 2 hr and was

l0

4

-J ILJ

0.

3

le

10

FIG

cell X

cyclo)

cated A sec

cyclol

24 hr,

withi

48

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o6

10c5

-j

U-

a-2

0 0.01 0.1 1.0 10

CONCENTRATION OFAZAURIDNE (pg/mi)

100

FIG. 3. Effect of 6-azauridine on measles virus

yield. Immediately after virus adsorption,

6-azauri-dine was addedat the concentrations indicated. At

24and 48 hrafter infection, viruswasharvested and

assayed.

complete by 6 hr. An increase in CF activity

was detected at4 hr. Comparison of SDA, CF antigen, and infectivity curves reveals that an

increase in RNA synthesis essential for the formation of SDA and CF antigen was

de-tected prior to an increase in the RNA

syn-thesis essential for virus maturation.

The time course of protein synthesis neces-sary for the formation of viral antigens is

shown in Fig. 6. The infectivity curve is the same as the cycloheximide curve in Fig. 4.

An increase in protein synthesis essential for SDA was detected at 8 hr and was

com-plete by 12 hr. Protein synthesis essential for CF activity showed an initial rise at 5 hr and was complete by 8 hr. Comparison of SDA, CF antigen, and infectivity curves reveals

that protein synthesis essential forthe

forma-tion ofCFantigenwasinitiated priortoprotein synthesis necessary for SDA and infectious

virus. Proteinsynthesisessential for the

forma-tion ofSDA activity appears to coincide with

that essential for infectious virus formation. Time course of "early protein" synthesis necessary for viral replication. Cyclohexi-mide was employed to demonstrate a require-ment for early protein synthesis. Cyclohexi-mide (0.5 ,g/ml) was added at zero time to block protein synthesis, and at various times the inhibitor was washed out. Virus yields at 24 hr were determined, and the percentage of virus inhibition was plotted against the time ofreversalofcycloheximide inhibition (Fig. 7). After 1 hr there was a rapid decline in virus yield until about 4 hr. We interpret this to indicate a requirement for early protein syn-thesis, which begins at 1 to 2 hr and continues until 4hr. Between 4 and6hr therewas a pla-teau followed by a further decline in virus

100

I -j

0 z 0 0

1.0

O 4 8 12 16 20 24 28

HOURS

FIG. 4. Measles virus yield as a function of the

timeof addition of6-azauridine andcycloheximide.

AV3 cells were infected as described in Fig. 1. At

the indicatedtimes, 6-azauridine(5ug/ml) or

cyclo-heximide (0.5 gg/ml) was added. Virus was

har-vestedat28hr,and virusyieldswereplotted against the timeofadditionoftheinhibitor.Alsoincludedis

the control growth curveof virus in untreated

cul-tures. Absolute maximum value (28 hr) was:

infec-tivity, 1.9 x 106 PFU/ml. -A

,I- 48 HR. YIELD

O ---O 24 HR.YIELD

1 - *- AZAURIDINE

o-(o

CYCLOHEXIMIDE

L~-A GROWTH CURVE

49

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DISCUSSION

RNA and protein synthesis essential for

AZAURIDINE measles virus infectivity were detected at 5 to

6 hr and 6to 8hr, respectively, and bothwere

*@

* INFECTIVITY at near maximum levels before significant

O-O CF amounts of new infectious virus were

pro-duced. The synthesis of this essential RNA

S DA and protein preceded virus maturation by

approximately 10 to 12 hr throughout the

1(

A z growth~

cycle.

We should

emphasize

that the

100 methods employed detect only the last

essen-Oi / ; /tial RNA and protein needed to confer

infec-0

tivity

on thevirus.

F-

W t

lCycloheximide

appeared

to affect SDA

8 andviral infectivity withasimilar timecourse.

IL

0

o

0 CYCLOHEXIMIDE

i0

0* * INFECTIVITY

O-O CF

6-- SDA

1.0~~~~~~~~~0

1.0L

I

IC2

0

4

8

12

16 20 24

28

8

HOURS

FIG. 5. Virus yield, SDA, and CF activity as a o

functionofthe timeofadditionof6-azauridine. Pro- e cedureasinFig.4.Infectivity curve same asazauri- o

dine curve inFig.4.Controlequalsyieldat28hr(no 10- 1 6-azauridine added). Absolute maximum values (28

hr) were: infectivity, 1.9 x 106 PFU/ml; CF anti- T

gen, 1to8;SDA, 1to6.

yield. In other

experiments,

higher

concentra-tions of

cycloheximide

were

employed

to more

rapidly and

effectively

block protein

synthesis,

allowing a better determination of the time course of early protein

synthesis.

Cyclohexi-mide (5

Ag/ml)

was added at various times and then removed from all cultures at 5 hr.

Virus yields were determined at 2-hr intervals 0 4 8 12 16 20 24 28

and plotted against the time of harvest.

Fig.

8 HOURS

shows that inhibitor added at zero time and

2 hr

..

displaced the early portion ofthe curves

FIG.

6. Virus yield, SDA, and CF activity as a

hrdisplaced the early portion ofthe

curvesfunction

of the time of addition of cycloheximide.

to the right.

However,

when inhibitor was Procedure as

in

Fig.

4.

Infectivity

curve same as

cy-added at 3 and 4

hr,

the

growth

curves were cloheximidecurve in Fig. 4. Control equals yield at similar to that of the control. This confirms 28 hr(nocycloheximide). Absolute maximum values that early protein synthesis occurs and is es- (28 hr) were: infectivity, 1.9x 106

PFU/ml;

CF anti-sentially complete

by

3 hr. gen, 1 to8;SDA, I to6.

50

PORTNER AND BUSSELL J. VIROL

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This suggests that the protein responsible for SDA activity may be the last protein

needed for the formation of infectious virus.

However, RNA synthesis essential for SDA

ac-tivity was detected very early in the growth

cycle (approximately 2hr) and wascompleted

by 6 hr. Thus, the inhibition of RNA synthesis does not affect the formation of SDA activity

with the same time course as the formation

of infectious virus and CF activity. This

sug-gests that more than one RNA species may

be involved in the formation of these viral constituents. We should point out that the times given for the appearance of various

activities (infectivity, CF, SDA) in the in-hibitor experiments are only approximations

because we do not know how long it takes the inhibitors to block virus-specific RNA and protein synthesis. What we wish to emphasize

in these experiments is the relative time

course of the various activities and not the

precise times given.

Multiple species ofvirus-specific RNA have been observed in cells infected with New-castle disease virus (NDV) and Sendai virus (1, 2, 13). In addition, quantitative and quali-tative differences have been recognized in

these RNAs that are synthesized early and

late during the growth cycle (14). If SDA repre-sents a precursor of a portion of the viral

envelope (24), then one of the first processes

detected in measles-infected cells was the

synthesis of RNA essential for the formation of a virus-specific envelope protein.

How-ever, this is difficult to reconcile with the

cycloheximide inhibition experiments that indicated that the actual formation of SDA coincided with the synthesis of the last

pro-tein required for virus infectivity. Although the time course of SDA-RNA appears to differ from that of CF-RNA and RNA essential for infectivity, their rates of appearance are

nearly thesame.

RNA synthesis essential forCF activity was

first detected at 3 hr and preceded the for-mation of protein essential for CF activity by

2 hr. We made no attempt to distinguish

between the various kinds of CF antigens. However, the S antigen (internal nucleopro-tein component) was the major CF

compo-nentfoundearlyinthegrowth cycleofmeasles

virus (20). Thus, the early increase in CF

ac-tivity we observed is probably due to the

production of S antigen.

Experiments were performed to determine

whether early protein synthesis was essential

for the replication of measles virus.

Experi-z

m

20

30

40

50

60

70

80

90

100

0 4 8 12

TIME OF REMO\AL OF CYCLOHEXIMIDE (HOURS)

FIG. 7. Virus yield as a function of the time of

reversal of cycloheximide inhibition. AV3 cells were

infectedas described in Fig. 1. The MM contained

0.5 usg of cycloheximide per ml. At the indicated

times, the cultures were washed sixtimestoreverse

cycloheximide inhibition. Virus was harvested at

24 hr. Zero time represents virus yield from

un-treated cultures (5.5 x 105 PFU/ml).

ments involving the reversal of the effects of cycloheximide indicated that early protein(s) synthesis was required and takes place

dur-ing the first 3 hr after virus adsorption (Fig.

7 and 8). The early protein(s) was formed

prior to the proteins essential for CF antigen, SDA, and infectious virus. The synthesis of early and late proteins in cells infected with

myxo- and paramyxoviruses has been

demon-strated by others (25, 26, 29, 30). These stud-ies indicated that the late proteins represent

virus structural protein, whereas the early protein was essential for viral nucleic acid

synthesis as well as structural protein

synthe-sis. An RNA-dependent RNA polymerase has been observed in cells infected with Sendai virus (15) and has also been found in purified NDV and Sendai virus virions (11, 22). Cycloheximide blocked the synthesis of Sendai virus 57S RNA but not the 18S and 35S RNAs (23). This suggested that the 18S and 35S RNAs were formed by the

poly-merase carried by the virion, whereas the 57S

RNA could be synthesized by a different

en-zyme. Since similar species of RNA ('18,

35, and 57S) have been found in measles-in-fected cells (Bussell and Robinson,

manu-script in preparation), it is possible that the early protein we have reported in this paper is also involved in the synthesis of measles 57S RNA.

One of the most striking differences

be-tween the growth cycles of measles and the

40'IR

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not due to lengthy delays in the formation of early protein(s) or RNA and protein. While measles RNA andprotein syntheses appear to be somewhat slower, this does not entirely account for the longer eclipse period. The results indicate that the rate-limiting step in the formation of infectious measles virus is not the rate of synthesis of essential RNA or protein, but is some subsequent stage such as the maturation process.

The experiments employing AUR to

measure the time course of RNA synthesis essential for infectivity do not give any clue as to the species of RNA that is primarily in-volved. This RNA could be the parental RNA or some other species of RNA needed for the synthesis of other viral components. The latter could be virus-specific comple-mentary RNA similar to that described in NDV- andSendai virus-infected cells (1, 2, 13).

ACKNOWLEDGMENTS

Wewishto expressourappreciationtoLyndel Kingand

Mary Portner for excellent technical assistance. This in-vestigation was supported by Public Health Service re-searchgrantAI 07143 from the National Institute ofAllergy and Infectious Diseases, Public Health Service Training Grant 5T GM-703 from the National Institute of General 12 14 16 18 20 22 24 Medical Sciences, and NASA Training Grant NSG(T)-55.

HOURS

FIG. 8. The inhibitionof protein synthesis and de-lay in appearance ofinfectious virus after reversal.

AV3 cells were infected as described in Fig. 1. At

various times, cycloheximide was added (5 osg/ml)

andthen removed fromallcultures 5hrafter

adsorp-tion. Virus yieldswere determinedat 2-hr intervals

and plotted against the time ofharvest.

structurally related myxoviruses and

para-myxoviruses is the length of the eclipse period. Considerable variation in the eclipse period of measles virus has been observed (16);

how-ever, in all cases it was considerably longer

than that observed with myxoviruses and paramyxoviruses such as fowl plague (3, 25),

NDV (9, 12, 26, 30), influenza A, strain

WSN (9), and SV5 (5). It is difficult to

com-pare our data with those cited above because

of differences in either the inhibitors

em-ployed or the methods employed to

deter-mine sequential synthesis of viral

compo-nents. However, the results obtained with measles virus differ in one major respect

and that is the lag observed between the synthesis of the various virus components

and virus maturation. Thus, the relatively long eclipse period appears to be mainly due

to a relatively long maturation process and

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decar-i0d

05+---J

IL a.

CYCLOHEXIMIDE ADDED (TIME IN HOURS)

O-O 0

*--4 2

A-A 3

L] ] 4

A- -A CONTROL / /

/7

~~ /~//'/

/'

/

/ .

104-

-id'

j

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[image:7.495.61.252.59.374.2]

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MEASLES VIRUS RNA AND-PROTEIN SYNTHESIS

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