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Macromolecular Synthesis in Cells Infected by Frog Virus 3. VII. Transcriptional and Post-Transcriptional Regulation of Virus Gene Expression

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Copyright©1977 AmericanSocietyfor Microbiology Printed in U.S.A.

Macromolecular

Synthesis in Cells Infected by Frog Virus 3

VII.

Transcriptional and Post-Transcriptional Regulation of Virus Gene

Expression

D. B. WILLIS,* R. GOORHA, M. MILES, AND A. GRANOFF

Division of Virology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101

Receivedforpublication28April 1977

We have used improved techniquesforseparatingindividual species of RNA

and proteintostudy the mechanisms that controlgene expressionbyfrogvirus

3, a eucaryotic DNA virus. Forty-seven species of viral RNA and 35 viral polypeptide species were resolved bypolyacrylamide gel electrophoresis. The

relativemolarratiosof virus-specificpolypeptides synthesized at various times

after infection were determined by computer planimetry and were compared

with the molar ratios of appropriate-sized viral RNAs to code for each

poly-peptide. Viral polypeptides were classified according to the time during the

growth cycle atwhichtheirmaximalrate ofsynthesisoccurred -early, 2 to 2.5

h; intermediate, 4 to 4.5 h; and late, 6 to 6.5 h. The viral RNAs, which were assumed to be mRNA's, couldnot beclassified according to time of maximum

synthesis; once their synthesis had begun, mostof the RNAs continued to be synthesizedatthesame orhigherrates. However, only10ofthe 47viralRNA

bands were plainly visible after electrophoresis ofextracts from cells labeled from1to 1.5hafterinfection; these10RNAsweredesignated "early" RNA. The earlypatternof both RNA and polypeptide synthesiswasmaintained foratleast

6 h in the presence of the amino acid

analog

fluorophenylalanine,

which indicates thatafunctionalviralpolypeptide was required for "late" transcription

and translation. The

presumptive

mRNA's for late

polypeptides

didnotappear

until2hafter infection, buttwoofthese"late" RNAs became themajorproducts of transcriptionby4h intotheinfectious cycle.In contrast tothedecliningrate ofsynthesis of the earlyproteins, corresponding earlyRNAspeciescontinuedto

be synthesized at the same orhigher rates throughout the replicative cycle. Although the synthesisoflatevirus-specificproteinsappearedtoberegulatedat

the level oftranscription, ourresultssuggestthat thesynthesis of bothearly and intermediateproteinswasregulatedatthepost-transcriptional level.

With a requirement for a nuclear function

(11)butwithmaturation inthecytoplasm(12),

frog virus3(FV3)differsinmode ofreplication fromall other knownDNAviruses. This virus

has several unusual features that assist in

analysis of virus-directed macromolecular syntheses. Treatment ofhost cells with heat-inactivated FV 3

(AFV

3)suppresses host RNA, DNA, andprotein synthesis without affecting the subsequent replication of active virus (8, 33). Therefore, very early viral events are

un-maskedinthe absenceofhostcell

macromolec-ularsynthesis.

Virus-specific polypeptide synthesis proceeds inasequentiallycontrolled fashionduringthe

growthcycleofwild-typeFV 3 (8). Goorhaand

Granoff (8)demonstratedthatoneclassof FV 3-specific proteins was synthesized at maximal rates at the beginning of infection (1 to 2 h),

whereas other proteins attained their highest

rates ofsynthesistoward the end of the growth cycle (6 to 8 h). The decrease in the rate of

synthesis

of atleastoneearlyprotein

appeared

to be controlled at the post-transcriptional

level. The increase in late protein synthesis was not dependent on viral DNA replication (8); therefore, we usethe terms "early" (<2 h) and"late" (>6h)inrelationtotimeafter infec-tion rather than onset of DNA replication. In contrast to theorderly replicationofwild-type

virus, certain temperature-sensitive mutants of FV3 exhibitderangedpatterns of protein syn-thesis (10), providing evidence for the role of virus-specificregulatory factorsinprotein syn-thesis.

Todefine the role oftranscriptionalcontrol in theregulationof FV3protein

synthesis,

Willis andGranoff(33)

analyzed

viralRNAon sucrose density gradients and by

hybridization

inhibi-tion.The resultssuggestedthat thesynthesisof

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viral messages varied both qualitatively and

quantitatively withtime. However, because of

the poor resolution of RNA obtainable on su-crose gradients, they could not determine the role of transcription in the regulation of any

individual polypeptides. We have recently

achievedsharpresolution ofbothpolypeptides and RNAs with polyacrylamide gels, and we

haveusedthese techniques to compare directly

the rates of synthesis ofindividual species of virus-specific RNA and the polypeptides for

which eachmay code. Most of the FV 3 RNAs

lacksegmentsof polyadenylic acid [poly(A)] at

their 3'-termini (34), and, as we will

demon-strate, the absence of variable-lengthtracts of poly(A) permits superior resolution of viral RNA onformamide-polyacrylamide gels.

Our results clearlydemonstrate that: (i) FV 3

RNA synthesis is controlled

quantitatively-not all RNA species were synthesized in the

samemolar ratios; (ii) FV 3 RNAsynthesis is controlled qualitatively-a virus-induced

pro-tein was required to turn onsynthesis of late RNA; and (iii) post-transcriptional regulation

is a majorstrategyfor control ofprotein

synthe-sis in the FV 3-infected cell-transcription of early RNA species continued unabated

throughout infection.

MATERIALS AND METHODS

Cells and virus. Fathead minnow (FHM) cells

were grown as monolayers at 330C inEagle

mini-mum essential medium containing 10% fetal calf

serum(MEM-10). A clonal isolate ofFV 3 wasgrown

and titratedinFHMcellsat250C;concentrated

vi-rus stocks were prepared as described previously

(22).

Reagents. [355]methionine (specific activity, 600

Ci/mmol) was produced by New England Nuclear

Corp.; [3H]uridine (specific activity, 20 Ci/mmol)

was purchased from Schwarz/Mann. Formamide,

obtained from BDH Laboratories, was deionized

with5%Amberlite MB-1 until theconductivity

mea-sured less than 3 mmho. All formamide solutions

werestoredat-200C.

Isotopic labeling ofvirus-specific RNA and

poly-peptidesininfected cells. To inhibit host RNA(33)

and proteinsyntheses(8),weexposedcellstoAFV3

(560C, 15min)in amountsequivalentto20PFU/cell

for 60 min at room temperature (230C). The cells

werethenwashed,overlaid with MEM-10, and

incu-batedat30°C for2h.The mediumwasremoved,and

active virus wasadsorbedat 50 PFU/cell (230C, 60

min). The cells were washed, overlaid with MEM

without serum, and incubated at 30°C. This time

wastakenas 0 hforthe infectiouscycle.It was not

necessaryto useAFV3 on thecells takenfor

analy-sisat6 or 8hbecause active virusalone inhibited hostcell RNAand proteinsyntheses by thattime(8, 33).

At variousintervals afterinfection, the overlaid

medium was replaced with methionine-free MEM

containing 25 UCi of[35S]methionine (to label

pro-teins) or50 uCi of [3H]uridine (to label RNA) per ml.

After a 30-minlabelingperiod,cytoplasmic extracts

wereprepared forelectrophoresis.

Polyacrylamide slab gel electrophoresis ofRNA.

RNA was extracted from the cytoplasm as

previ-ouslydescribed (21, 33). The finalLiCl-precipitated

pellet, free ofdouble-stranded RNA or DNA, was

dissolved at a concentration of200 to 1000ug/mlin 2

mMphosphate-buffered formamide containing 30%

glycerol (formamide sample buffer). The phosphate

saltswereadded directlytothedeionized formamide

inamounts that gave a pH of7.0 inaqueoussolvents

(5). Slab gels consisting of3.5% acrylamide in 20

mM phosphate-buffered formamide were cast in a

Hoefer slab gel apparatus (30 cm by 15 cm by 1.5

mm). Samples containing approximately 6 ug of

RNA in 10 ,u of samplebuffer with 0.01%

bromo-phenol blue were carefully layered under a well

filler of20mMphosphate-bufferedformamide.

Elec-trophoresis was carried out for20h at200Cat180V

with a buffer of0.04 Msodium phosphate, pH7.0.

Thegel wascut at the position of the tracking dye

and fixed for 1 h in a solution of 1% lanthanum

acetate in 1%aceticacid (1) and thenimpregnated

with 20% 2,5-diphenyloxazole (scintillation grade)

indimethyl sulfoxide according to Bonner and

Las-key (3). The gel was dried under vacuum with a

Hoefer gel-drying apparatus, covered with Saran

wrap,and exposed to presensitized Kodak RP X-ray

film at -70°C for various periods of time (18). Polyacrylamide gel electrophoresis of proteins.

Proteinsamples wereprepared from the cytoplasmic

extracts (8), andapproximately20 to 30 ,ug of

pro-tein (80,000cpmof[35S]methionine) was layered in

each well ofa 10-cm 5 to 15% acrylamide-0.1%

so-diumdodecyl sulfate (SDS) gradient slab gel with a

3%stacking gel. The gelwaselectrophoresedin0.05

MTris-glycine buffer, pH 8.3, for 6 h at 60 V and

fixed in a solution of 10% trichloroacetic acid-1%

glycerol for1h. Aftertheyweredried, the gels were

exposedtoX-rayfilmatroomtemperature for2to 5

days.

DNA:RNA hybridization. About 100

jzg

of RNA

(specific activity, 10,000 to 20,000 cpm/,ug),

ex-tracted from the cytoplasm of infected cells, was

hybridizedtotwo24-mmnitrocellulose filters

bear-ing100,g ofdenaturedFV3 orFHMDNA each (27)

for24h at370C inasolution containing 30%

form-amide, 1.2MNaCl,0.12 Msodium citrate, and0.1%

SDS. The filterswererinsed several timesin 100ml

ofthehybridization buffer. The hybridized RNA was

eluted with 1 ml of99% formamide-0.1% SDS at

500Cfor 10minand diluted 1:1 with 0.2 M sodium

acetate buffer, pH 5.4. The RNA wasprecipitated

overnightat -20°Cwith3volumes of absolute

etha-noland100,ugof carrieryeast RNA. The RNA pellet

wascollected by centrifugation (30 min, 20,000 xg)

and wasdissolved in formamide sample buffer for

electrophoresis.

Molecular weight determinations of RNAs and

proteins. Molecular weights of each of the viral

RNAandprotein bandswerecalculated by

compar-ing the distance migrated with the Rf of the

follow-ingstandards: 28SFHMrRNA,molecular weight, 2

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x 106; 18S FHM rRNA, 7.08 x 105; 23S Escherichia

colirRNA, 1.07x 106;16SE. coli rRNA, 5.5x 105;5S

FHM rRNA, 3.5x 104;4S FHM tRNA, 2.5 x104;and

reovirus genomic RNAdenatured in99% formamide

for 15 min at370C-L1,2,3,average molecular weight,

1.2 x 106; Ml, 7.6 x 105; M2,3, average molecular

weight, 7.08 x 105;

SI,

4.7 x 104;S2, 3.8 x 104; S3,3.3

X 104;andS4, 3.09 x 104. FHM cellular RNAs were

assumed to be comparable to similar species from mammalian cells (26). Labeled reovirus RNA was

prepared by growing reovirus inLcellsfor 3 days in

the presence of 5

jACi

of[3H]uridine per ml. Virions

were purified (30), and single-stranded reovirus

RNAs were prepared by denaturation in 99%

form-amide (15 min, 37°C). The molecular weights were

taken to be one-half of the values determined for

double-stranded RNA segments (30). The distance migrated by the standards was proportional to the logarithm of the molecular weight (5) and was used

toderive a fit for the curve y=aebx by the method of

least squares. The resultant curve, y=6.06e-0.00167x,

where y = log molecular weight andx = migration

distance, had a determination coefficient (r2) of

0.962.Themolecular weights of viral proteins were

determined inasimilar fashion with the following

standardproteins; 8-galactosidase, 130x 103;

lacto-peroxidase, 90 x 103; bovineserum albumin, 67 x

103;ovalbumin, 44 x 103;carbonicanhydrase, 29 x

103; andcytochrome c, 12.4x 103.Theresulting

best-fit curve, of the type y =ax + b, was y= -0.16x +

5.54, where y = log molecularweight andx =

dis-tancemigrated. The coefficient of determination (r2)

equaled0.99.

Computer analysis of data. The computer

analy-sis was similar to that described by Honess and

Roizman (13) for herpesvirus proteins. The

devel-oped filmswerescanned with an Ortec4310

densi-tometer interfaced with a PDP 8/1 minicomputer

(Digital Electronics Corp.). With the densitometer

operating in the percent transmission mode, the

analog voltage output generated by the darkened

filmswaschangedtodigitalinformationbyan

ana-log-digital converter (sampling rate of250pointsper

s) and storedonmagnetic tape. The computer

pro-gram converted the digital data into absorbance

values, corrected base-line levels, displayed the

scans on an oscilloscope (Tektronic, Inc. type 611

storageunit),andintegrated the selectedpeaks(see

Fig. 3, bottom rightpanel).

The operator determined the points of inflection

on either side of the peaks by inspection of the

oscilloscope screen and instructed the computerto

mark them with an arrow. The program was

de-signedto integrate theentire areaunder the peak

from the corrected base line. The area of minor

bands was determinedby scanning longer exposures

(96 h) and relating the areas to a reference peak

withinthelimits of resolution of both 36- and 96-h

exposures. The same rationale was followed with

24-h exposures to ascertain the areas of theheaviest

bands, which exceeded the linear capability of the filmduringthe36-hexposure.

RESULTS

Separation andcharacterizationofinfected

cell RNAs. Previous results with sucrose

gra-dients indicated that the synthesis of a 17S viral RNAincreased quantitatively with time after infection (33), but this method was not preciseenoughto measuretherateofsynthesis

of individual RNA molecules. Using the

tech-nique ofpolyacrylamide gel electrophoresis in formamide coupled with fluorography of the gels, we determined the number of

virus-spe-cific RNA species and the relative amounts of

each that were labeled during a 30-min pulse

with [3H]uridine atvariousintervals through-out the infectious cycle. Monolayers of FHM cells weretreated with AFV 3to suppresshost

RNA synthesis and were then superinfected

with active virus asdescribed above. RNAwas

extracted from the cytoplasm and analyzed by polyacrylamide gelelectrophoresisunder dena-turing conditions (98% formamide). Figure 1 shows that radioactivebands of RNA werenot

detected withas much as 10

jig

ofcytoplasmic

RNAextracted fromcells receiving only AFV 3 for 4 h (Fig. la); inhibition of host cell RNA synthesiswasmaximumby this time.

We assigned molecular weights to the viral RNAs based on theirmigration distance rela-tive to standards (seeabove). The infected cell RNA (ICR) number represents the molecular weight x 103. Only the molecules of present in-terest are labeled in Fig. 1, but a composite diagram of all the ICRs ultimately identified is included forreference in Fig. 2. The molecular weights of the infected cell RNAs ranged from 52,000 to 950,000. Many, but not all, of the viral RNAs were synthesized at 1 h after infection (Fig. lb). By 2 h (Fig. ic), 47 bands of RNA could be resolved, althoughnotall of them are apparent on the 36-hexposure in Fig. 1. Witha

FIG. 1. Fluorogram ofpolyacrylamidegel containing RNAsfromFV3-infectedcells.RNA,labeledfor30

minwith[3H]uridineatvarious timesafterinfection, wasextractedfromthecytoplasmof infectedcellsas

describedinthetext.From 3to10pgofRNAwasplacedineach wellforatotalof40,000cpm. Anexception

was the extractfrom cells receiving only AFV 3, in which 10pg ofRNA (4,000 cpm) was used. AFV3 treatment wasnotnecessaryfor6-or8-hsamples, because bythat time active virus alone hadsufficiently

reduced host synthesis. Marker RNAwaspreparedfromuninfectedFHMcellsthathad beengrownfor3days

inthepresenceof0.1 ,UiCiof[14C]uridineperml;toretainthe4S tRNAspecies, wedidnotprecipitatethis

RNAwithLiCl.Thefilmwasexposed for36hat-70°C.(a)4-h AFV3;(b)3-hAFV3,1-h active FV3;(c)

3-hAFV3,2-hactive FV3;(d)3-hAFV3,4-hactive FV3;(e)6-h active FV3;(f)8-h active FV3; (g)6-h FV 3

RNAhybridizedtoandelutedfromFV 3DNA; (h) uninfectedFHMRNA.

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__ORIGIN

VOL. 24, 1977

ICR

597

577

560O

534

463-3

425

390

358

345

322

240

235-169

a

b

c

d

-283

-18S

-43

e

f

FIG. 1.

g

h

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RNA

BAND NO.

PROTEI N

2 3

14

15 16

17

8

19=

20 21

23

3

314r

5

I2

29

2

33 24 S 235

ICP

,- ORIGIN

or133

43

119 115

I0I 108

105

93

79

/75 72 68 0

650

62A

59 55 A

60 S 47 0

345 31

3\39 M

\ 37 5 3\36 0 \\3

29 27

26

L19^

18.5 18 0

7

14

FIG. 2. Schematic representation ofICRs and ICPs. Thisdrawingisacompositeof all bandsseen onboth

short- andlong-termexposuresofpolyacrylamide gelsandateveryintervalexamined.The symbolsrepresent the time during the virus growth cycle at which various ICPsare maximally synthesized. 0, Early; *,

intermediate; A,late. The ICRspostulatedtocodeforthe ICPswereassignedidentical symbols.

96-hexposure(datanotshown), the radioactive disintegrationsinthemajor bands exceeded the linearcapability of the film. Nonewviral RNA

bands were seen at4, 6, or8 h after infection

(Fig. id, e, and f).

From Fig. 1 itappears that the viral RNAs were not all synthesized atthe same rate and

thatsomeofthem-ICR560, 534, 390, 240, and

235(bands 10, 11, 18, 27, 28,andAontheFig.2

schematic)-werenotsynthesizedin detectable

amounts at 1 h after infection. As the RNA

molecules synthesized ininfected cells had no

counterparts incells receiving only AFV3, we

presumed that these moleculeswereinduced by

infection and were coded for by the viral ge-nome. Direct supportfor this assumption was

provided when we subjected an infected cell

RNA to electrophoresis after hybridization to

and elution from FV 3 DNA. Less than 0.5% of

the RNA hybridized to FHM DNA, whereas

35%hybridizedtoviralDNA.AsFig. igshows,

most of the prominently labeled RNA

mole-cules thatwereseeninextracts of cells infected

for 6 hhybridizedto viral DNA.

Molar ratios of viral RNA. Having shown

that thesynthesis ofmanyviral RNAs varied

with time, we next quantitativelydetermined

the relative number of molecules present in

each band. Bonner and Laskey (3)

demon-strated that thedensityof the blackenedareas

ofan exposed X-ray film was directly

propor-tionalto theradioactivedisintegrations inthe

gel. We confirmed the linear response of the

filmand determined theupperandlower limits

ICR BANDNO.

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of resolution with graded amounts of known

standards (see above). The voltage output of our Ortec densitometer during a scan of the film was then converted to digitalinformation and displayed on an oscilloscope (Fig. 3). The

total scan was divided into four sections; each section wasexpanded, and the areas under the

peaks were integrated by the computer (13). ICR-577 (early) and ICR 169 (intermediate) are

pointed out in the 1-h scan. ICR 534, a late

message, is just to the right of ICR 577, but was notdetecteduntil 2 h. The pattern established

by4hdidnotchange thereafter.

The molarratios of the RNAbands at each time interval are given in Table 1. All values

were normalized to thatofICR 322 (band 22),

which wasdefinedas 1 unit at 1 h after

infec-tion. This bandwaschosen becauseit was

eas-ily seen on all exposures and did not show extreme variations in rate of synthesis with time.Somebands(e.g.,ICR235/240)that could be distinguished visuallywere notresolved by the densitometer, and their areas were com-bined. The minorbands smaller thanICR 127 were not included in Table 1 because the gels weretoo long to be scannedconvenientlyinthis region. The relative molar rates of synthesis, or

molar ratios, were calculated as described in

the explanationofTable 1 (13).

Important features of the data in Table 1 are asfollows: (i) at1hafterinfection therewas, at most, afivefold differenceinthemolar ratesof

synthesis ofmost of the observable RNA spe-cies. ICR 169 (band 33)and, possibly, ICR 577

(band9)might be consideredinrelativeexcess. In addition, several RNA species that became prominentlaterininfection-notably ICR 560, 534, 390 (bands 10, 11, 18), andall ICR smaller

than 169-were not detected at 1 h, even in

long-term exposures.

(ii) By 2 h after infection, the rate of synthe-sisof the threelargest RNA molecules (ICR895 toICR947) declined slightlyascompared with

the smaller species. Bands undetected in 1-h extracts were nowobserved, with ICR560 (band 10)and particularlyICR 534 (band 11) showing

markedincreases. Although ICR 169 (band 33) wasstillbeing synthesized at arapid rate, the

predominant species was now ICR 463 (band 14).

(iii) After 4 h of infection, a characteristic pattern of RNAsynthesis emerged that did not

change thereafter (Fig. 3). More molecules of ICR 534 weresynthesized than any other RNA species. Whereas at 1 h there was a fivefold

difference in the molar rates of synthesis be-tween the rarest molecules (ICR 240/235) and the most abundant (ICR 169), at 4 h ICR 534 had become the most abundant RNA species

andwassynthesizedat50 times the rate of ICR 947,the scarcest4-hspecies. At 4 h after infec-tionthere was also a greater difference noted in the molar ratios of all other RNA molecules than was observed at 1 h.

(iv) Theoverall rate of RNAsynthesis began to decline 6h after infection, but several indi-vidual species, ICRs 453/439, 390, 358, 345,322,

240/235, 155/153, and notably ICR 169, reached their peak rate of synthesis at this time (Table 1).

The wide variation seen in the molar ratiosof viral RNAs synthesizedover the course of the reproductive cycle indicates thatcomplex quan-titative transcriptional controls operate in FV 3-infected cells, whereas the appearance of new RNA species 2 h after infection suggests that qualitative controls are important also.

Viral RNAs synthesized in the presence of fluorophenylalanine. If a viral protein induced the changes in the RNA profile that occurred between 1 and 4 h, then interference with the synthesis of functional proteins should lock viral RNA synthesis into a 1-h pattern. The amino acid analog fluorophenylalanine (FPA) isincorporated into proteins that become

non-functional if a phenylalanine residue is

re-quired for activity (28). Since FPA does not

actually inhibit protein synthesis, it can be

used to study the necessity ofearly virus-in-duced proteins forsubsequent protein, as well as forRNA, synthesis. Figure 4 shows that 1 h after infection only 10ICRbands were promi-nent inthe extracts from both.untreated (Fig.

4b) andFPA-treated (Fig. 4c) cells,althougha few other large-molecular-weight RNAs were

faintlyvisible. By6hafter infection thetypical "late" RNA pattern was seen in extracts of

untreated cells (Fig. 4d), but thecellsinfected inthepresenceofFPA(Fig. 4e)synthesizedthe same 10RNAsatthe same rate as at1 h (Fig. 4b). These resultsimplythat a viralprotein(s), normally synthesized within the first hour or twoofinfection,wasrequired forthe character-istic changes in the patterns of RNAsynthesis thatwere observedfrom 1 to 4h.

Separation

and characterization of viral polypeptides. Inearlierreports from this

labo-ratory, Goorha and Granoff (8) identified 20 virus-specific polypeptides with cylindrical polyacrylamide gels. Theyobserved both a class of"early" proteins that reachedahighrate of

synthesisat2h(and thereafterdeclined)and a class of "late" proteins that did not attain a maximal rate ofsynthesis until 6to 8 h.

Because of the improved resolution of pro-teinsobtained ongradientslab gels, we

reana-lyzed the infected cell polypeptides (ICP) and

determined the rates ofsynthesis ofindividual

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332

1.0 1IX 0.8

0.6

0.4

0.2

0 2.0

1.6

LLJ z co 0 Cf)

mn

1.2

0.8

04

0 03.0

02.4

01.8

01.2

0.6

0

J. VIROL.

1.75

1.40

105

0.70

0.35

20 16

1.2

0.8

0.4

0 2.0

1.6

1.2

0.8

0.4

0

0 3 6 9 12 I5 40 4.6 5.2 5.8 64 70

DISTANCE MIGRATED (arbitrary units)

FIG. 3. Absorbancetracingsof fluorogramsof viral RNA. ThefluorogramsofFig. 1 werescannedbyan

Ortec densitometer interfacedto an analog-digital converter. The imageprojected by the computer onthe

oscilloscope wasphotographed withaPolaroidcameraandreproducedartistically. Labeled peaksareICR

577,early; ICR 169, intermediate; and ICR 534, late. The sharplineatthebeginning ofthe scanrepresentsa

line drawn2 cmfrom the origin. The bottom right panel showsanexpanded portion of the4-hsamplefrom4

to7migrationunits; arrowsdemarcatethelimitsof integration, which thecomputerextendedtothe base line.

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TABLE 1. Molar ratiosof viral RNAssynthesizedatvarioustimesafterinfection

ICR molwt Molarratio (h)

Bandno. (x103) 1 2 4 6 8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 947 926 895 845 817 746 720 597 577 560 534 506 489 463 453 439 425 390 372 358 345 322 299 290 261 254 240 235 226 204 196 180 169 163 155 153 136 127 1.10 1.22 2.10 {1.88 1.73 1.50 1.28 2.44 ND ND 1.42 1.55 1.30 {0.63 1.36 ND ND 0.95 0.75 1.00 ND ND ND 0.57 ND ND ND ND 3.16 ND ND ND ND 0.22 0.49 0.82 0.65 0.66 0.96 0.48 1.34 3.00 2.02 2.17 0.96 1.30 4.65 0.97 0.92 0.92 0.57 0.77 0.97 0.96 0.62 0.65 0.68 0.72 0.33 ND ND 0.43 4.19 0.64 0.87 ND 0.98 0.44 0.62 0.56 2.07 0.62 1.63 0.63 3.50 4.64 9.84 23.65 1.46 1.54 7.05 0.79 0.91 1.46 0.74 0.73 1.33 1.57 1.18 2.96 0.98 2.03 0.37 ND ND 0.69 5.01 0.70 2.51 0.85 1.16 0.44 0.79 1.80 1.79 1.32 1.92 0.97 2.49 5.14 6.60 14.54 1.84 2.26 6.81 1.29 1.05 2.35 1.37 2.00 3.63 2.57 1.28 0.81 1.67 3.22 0.57 ND ND 1.10 10.10 1.16 3.68 1.25 1.23 0.63 1.14 1.39 1.77 1.26 1.93 1.16 2.15 4.80 5.26 9.76 1.69 1.54 5.43 0.65 0.75 1.33 1.13 1.44 1.74 1.46 1.09 0.73 1.05 2.09 0.81 ND ND 0.49 8.25 0.85 2.55 1.08 1.30

aMolecularweightswere calculatedfrom theirrelativemigration distancecompared withthestandards

15AiF

asdiscussedinthetext.Molarratios werecalculated according to the formula MR=K*105 wWhere

Kis theoverallrateofRNAsynthesisatthe time of thedeterminationexpressedas afunctionof thehighest

observed rate, A and Marethe absorbanceofbandi and its molecular weight,respectively,F is afactor

normalizingall valuessothe ICR322equals 1unitat1hafterinfection, and 105 is a constant. ND, None

detected via densitometer.

polypeptides from densitometric scans of the autoradiogram. Figure2isacomposite

diagra-matic representation ofthe 35 ICPs

detected;

molecular

weights

ranged

from

14,000

to

143,000. Figure 5is an

autoradiograph

ofa

gel

after

electrophoresis

of

[35S]methionine-labeled

proteinsextracted fromcellsatdifferent times after infection. Notallof the35 viral

polypep-tideswere detectedonthis exposure; the num-bered ICPs are among those whose rates of

synthesis varied over the course of infection

and were

presumed

to be

regulated.

Because

the band numbers differed from those

previ-ously published, we assigned each protein an

ICP numberaccordingtoitscalculated

molecu-larweight x 103. Severalpreviously undetected features of theregulationof FV3protein

syn-thesis became apparent after we examined these autoradiograms. (i) Better resolution of

large-molecular-weight proteins was obtained on slabs than on cylindrical gels; 16 species

largerthan ICP55 (Fig.2, band 17)were

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334 WILLIS ET AL.

ICR

597

577-489

46

3-

425-358

322

169

a

b

c

d

e

f

FIG. 4. Fluorogram ofgel containing RNAs from cells infectedinthe presenceofFPA.MonolayersofFHM

cellsweretreated withAFV3for4hbefore infection with50PFUofactiveFV 3percell. The medium didnot

containphenylalanine. FPA waspresentthroughout infectionintheindicated cultures.Cultureswerelabeled

with 50,uCi of[3H]uridineper ml at1to 1.5 or 6 to 6.5 hafterinfection with active virus. RNAs were extracted

andsubjectedtoelectrophoresisasdescribedinthetext,and thefilmwasexposedfor 48 h at

-700C.

(a) 5-h

AFV 3;(b)4-hAFV3, 1-h active FV 3; (c) same as(b) but with100MgofFPA permladdedat 0hofinfection

withactivevirus; (d)6-h FV3; (e) same as(d) butwith 100pgof FPA per mladdedat 0h;(f) uninfected

FHM RNA (3-day label).

rated from purified virus (Fig. 5a), whereas beforeweonlysaw 4proteinsinthisregion. (ii)

In addition to the

"early"

and "late" protein classes identified on

cylindrical

gels,

we ob-served a third or "intermediate" class of pro-teinswhose maximalrateof

synthesis

wasat 4

h (Fig. 5c). (iii) If the cellsweretreated for5h

with AFV 3(Fig. 5f),there wasasmallamount

ofresidual hostproteinsynthesis, primarily of molecules with molecularweights of50,000 and 17,500. The 17,500-molecular-weight poly-peptidejust below ICP 18 was considered a host protein because its rate ofsynthesisat2h after superinfection (Fig. 5b) was the same as in the

ORIGIN

28

S

18

S

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REGULATION OF VIRAL mRNA AND PROTEIN 335

ICP

68

-657

=

6 2

55

*

47/45

-39/

37-36

I

9

I

8

---a~

a

b

c

d

e

f

FIG. 5. Autoradiogram ofpolyacrylamide gel containing virus-specific polypeptides fromFV3-infected

cells. Proteinswerelabeledfor30 min with25 ttCi of[35S]methionineperml,andthecytoplasmicextracts

werepreparedandsubjectedtoelectrophoresis as describedinthetext.From6to30 pgofprotein (80,000 cpm)wereplacedineach well. Thefilmwasexposedfor 72h at230C.(a)PurifiedFV 3structuralproteins,30

jg;

(b) 3-h AFV 3, 2-hactiveFV3;(c) 3-h AFV3, 4-hactiveFV3;(d)6-hactiveFV3;(e) 8-hactiveFV3;(f)

5-h AFV3.Heat-inactivatedvirus was notused for6- or8-hsamples, becauseactive virusalone inhibited host

cell proteinsynthesisatthattime.

heat-inactivated control (Fig. 5f). This band

was notgiven an ICPnumber. ICP 50 (Fig. 2,

band 18) had a small hostcomponent, but the polypeptide was considered to be predomi-nantlyviral inoriginbecauseits rateof

synthe-sisincreasedwithtime after infection (Fig. 5c,

d, ande).

To obtainquantitative data, we scannedthe autoradiographs with an Ortec densitometer

(Fig. 6) and instructed the computer to inte-grate the areas under the peaks; the molar ratios arepresented in Table 2.Almost one-half

of the detectable viral proteins were synthe-sized atmaximalratesearly ininfection(1 or 2

h). TheseincludeICPs 115, 118, 105/101, 93, 68,

65, 59, 47/45, 36, 35/31, 17, 15, and 14. The late

proteins-ICPs 62, 55, and 19

(bands 4, 17,

and

30)-were

barely

detectableat1or2 h butrose tohighratesof

synthesis

late in infection

(6

h). The newly identified third class of "intermedi-ate" proteins (ICPs 79, 75, 50,

39/37,

29/27, 26,

18,and17)reached theirhighestrateof

synthe-sis at4h.ICP 18(band 32) wasalwaysproduced

in greater quantities than any other protein, but the rate ofsynthesisdropped50%between4

and8hafter infection. ICP 18,the most abun-dant productoftranslation, and ICP 55, a late protein that is themost prominent viral struc-turalprotein, are identified on the densitomet-rictracings in Fig. 6. The proteins that we

clas--ORIGIN

_r...____....

...__...

..._.

VOL. 24, 1977

0

I---MMM-MW qmmup

qmmw qmmwaft*&

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0

0.80 064

0.48

0.32

016

0 0 80

064

048

0.32

0.16

0 080

064

048

0.32

0.16

6 9 12 15 0 3 6 9 12 15

DISTANCE MIGRATED (arbitrary units)

FIG. 6. Absorbance tracings ofautoradiograms ofviralproteins.DetailsasinlegendtoFig.3. Thelabeled peaksareICP18, intermediate; and ICP 55, late. VSP, Viral structuralprotein.

sified as early and intermediate displayed

de-creased molarratesof synthesis after2and4h of infection, respectively (Table 2). Table 1shows

no similarreduction in the rate ofsynthesisof

most of the early RNAs. The continued high

rate of synthesis of early and intermediate

RNAsextendsthe earlierworkofGoorha and

Granoff(9), who suggested that theinhibition

PURIFIED FV 3 _ VSP 55

2.0 1.6

1|2

08

0A4

FV 3

1

._.-

-.

..

0 0.80

0.64

048

0.32

LLJ z co 0

(f) co

0.16

0 080

0.64

048

0.32

016

8 hr.

ICP55

ICP 18

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TABLE 2. Molar ratiosof viralpolypeptides synthesized at various times after infectiona

ICPmolwt Molar ratio (h)

Bandno.

((x

103) 1 2 4 6 8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 143 133 119 115 111 108 105 101 93 79 75 72 68 65 62 59 55 50 47 45 41 39 37 36 35 31 29 27 26 19 18.5 18 17 15 14 ND 0.015 0.011 0.030 0.115 0.088 {0.104 0.12 0.12 0.19 ND 0.47 0.37 ND 0.29 0.39 1.01 {2.10 ND {0.61 1.64 0.76 0.75 ND 0.63 ND 4.32 0.65 0.49 1.27 ND 0.032 0.025 0.09 0.20 0.24 0.88 0.25 0.24 0.48 ND 1.18 0.65 0.40 1.0 1.08 2.46 7.2 ND 1.82 6.12 2.72 2.58 1.20 5.88 ND 20.65 5.2 10.59 6.16 ND ND ND ND ND 0.24 0.61 ND 0.30 1.10 ND 0.47 0.50 2.36 0.68 4.8 7.3 3.78 ND 6.75 4.38 1.20 3.18 2.86 12.99 ND 26.48 5.41 5.93 2.52 ND ND ND ND ND 0.11 0.54 ND 0.21 0.96 ND 0.34 0.40 2.89 0.79 5.71 5.69 1.73 ND 5.53 3.57 1.32 1.98 1.79 16.96 ND 16.3 2.76 4.93 ND ND ND ND ND ND 0.19 0.59 ND 0.13 0.71 ND ND 0.24 2.4 0.75 5.7 4.47 0.76 ND 4.38 2.83 ND 1.4 ND 8.4 ND 12.0 ND 2.23 ND

a Molecular weights werecalculated from relative migrationdistancescomparedtostandard proteinsas

describedinthe text. The molar ratios were determined according to theformula given in the footnote to

Table I; the termFnormalizedall data to ICP 59 at 2 h postinfection. ND, Nondetectable by densitometer.

ofat least oneearly protein took place at the

post-transcriptionallevel.

Viral

polypeptides synthesized

in the pres-ence of FPA. Ifthe RNA species

synthesized

after infectioninthepresenceof FPAwerethe

true early viral mRNA's, andFPA didnot

in-hibit theirtranslation, then the viral polypep-tidessynthesizedinthepresenceofFPAshould represent the translation products of these RNAs. Figure 7 shows that viralpolypeptides synthesized after6hofinfection in the presence

ofFPA(Fig. 7e) were the same ones thatwere synthesized earlyininfection in theabsenceof

drug (Fig. 7b). Some of these polypeptides

(ICPs 68, 65, 50, 47/45, 39/37, 36, 18) were the

rightsize tobetranslation products ofthe

spe-cificearly RNAs

synthesized

inthepresenceof

FPA (Fig. 4e; ICRs 597, 577, 463, 425, 358, 345, 169). The polypeptides that were notobserved

after6 hof infectioninthepresenceofFPAbut

werereadily detected after infectioninthe

ab-senceofdrug (Fig. 7d; ICPs 62, 55, 19)werethe

rightsize tohavebeen coded forby RNAs that

only

appeared

iffunctional protein

synthesis

was

permitted

(Fig.

4d;

ICRs560, 534, 240/235). This result strengthens the proposed coding correlation between those RNAs andproteins.

DISCUSSION

Correlation ofspecificviral RNAs and pro-teins. We have shownthatvirus-specific RNAs

isolated from cells infected with a large DNA virus, FV 3, can be resolved into at least 47 component bands by polyacrylamide gel

elec-trophoresis in formamide. The molecular

weights ofthe viral RNAsranged from 52,000

to 947,000, whereas the molecular weights of the viral polypeptides ranged from 14,000 to

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ICP

68

65

-

ww-62

55

50

47/45

39/37

36

19

-ORIGIN

Avow --K

18

a

b

C

d

e

FIG. 7. Autoradiogram ofgel containing polypeptides from cells infected in the presence ofFPA.

Monolay-ersofFHMcellswereinfectedasdescribedinthelegendtoFig.5.Polypeptides were labeled for 30 min with

25

ILCi

of[35Slmethionineperml, and the cytoplasmic extractswereprepared and subjected to electrophoresis

asdescribedinthe text. (a)PurifiedFV 3structural proteins;(b) 3-h AFV 3, 1-h active FV 3; (c) same as (b)

but with100pg ofFPA per ml addedat0hofinfection;(d)6-hactiveFV3; (e) 6-hactive FV3with 100

Mg

of

FPAperml added at0hof infection.

143,000. Only 40% of the single-stranded ge-nome equivalent was accounted for from the totalmolecularweight of the RNAs determined

directly from

theRNAelectrophoresisor calcu-lated from the molecular weights of the pro-teins. Therefore, some of the bands of both RNA and protein probably contained two or moremolecular species. Slight errors inmolar

ratio determinations were also introduced by the integration process, which did not

distin-guishoverlap intoneighboring peaks.

One of thegoals of this investigationwas to identify selected

RNA

bands as the messengers for specific viral polypeptides by size and by coordinate temporal behavior in rates of

syn-thesis, and thereby to assist inexplaining the controlmechanisms usedbythis virus.The size of the mRNAnecessary tocode for each of the

polypeptides was calculated assuming the aminoacid residueto haveamolecularweight

of 100 andthe nucleotide that of 300. This as-sumption seems reasonable because FV 3

mRNA hasnopoly(A)tracts(34),andwehave not found any evidence ofeither glycosylation orpost-translational cleavage ofviral polypep-tides (unpublished data). Vesicular stomatitis virus mRNA (32)andreovirusmRNA (4) have both been translated in vitro and appear to

possessnolargeuntranslatedregions. The seg-ments of the influenza virus genome are also

therightsize tocode for the influenzaproteins (29).

Table3lists 10 RNA:protein pairs for which

wepresently havereasonablygood circumstan-tial basesfor assigning arelationship, such as size, rate of synthesis, and synthesis in the

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presenceof FPA. Fourpairsareearly, threeare

intermediate, and three are late. All of the

calculated and observed molecular weights are

withinarange of 10%.

In Fig.8,wehaveplotted the relativeratesof

synthesisatvarious timesafterinfectionof the

RNAsandproteins selected for Table 3. A fur-ther discussion of these data will be foundin

the sections on transcriptional and

post-tran-scriptional regulation. We recognizethe

limita-tionsof the data in the estimation of molecular weights and assignment of particular RNAsas messagesforparticular proteins. Nevertheless,

until isolated FV 3 mRNA's are translated in

vitro and the products are identified as FV 3

polypeptides, the postulated protein relation-ships provide a foundation for useful

discus-sion. Although some of the RNA to protein

correlations may be incorrect, our

interpreta-tionof the data regarding post-transcriptional

control is unambiguous.

Regulation at the level of transcription. The data presented in Table 1 suggest that transcription isregulated in both aqualitative

and quantitative manner. With the exception of ICR 169 (band 33) and ICR 577 (band 9),

mostof the RNAsseenat1hpostinfection (Fig.

4b)orafter6 h in thepresenceof FPA(Fig. 4e)

were produced in approximately equimolar

amounts-at most there was only athree- to

fourfold difference in themolarratesof synthe-sis of any ofthe other species. By 4 h after infectionnotonlywereadditional species being

synthesized, but therewasalsoasmuchas a 50-fold differential between the relative molar

rates ofsynthesis of the most scarce and the

most abundant RNAs. More molecules of ICR 534(band 11)weremadethan ofanyother viral

RNA. We consider this RNAtobethe messen-gerfor themajor viral structural protein,

ICP-55 (band 17), because of its size and rate of synthesis.

These results support our earlier

hybridiza-tionstudies (33), which demonstrated the

exis-tenceof both temporal and abundance controls

of viral RNA synthesis. The new techniques,

however, allow us to giveattention to the

be-haviorof specificmolecular species.

We identified three viral RNAs (ICRs 560, 534, and240/235) that could potentially codefor

three late proteins, ICPs 62, 55, and 19 (Table 3). TheseICRs werenotdetected at 1 h

postin-fection and wereinhibitedby FPA. The small

amount of ICR 240/235 synthesis that was

ob-served under these conditions could be

ex-plained ifoneofthesebandswasthe mRNA for aprotein other than ICP 19.

In Fig. 8 (bottom panel) we show that the

rates of synthesis of both late RNAs and

pro-teinsacceleratedtoabout thesamedegree

dur-ing infection. This concomitant increase

indi-catesthat synthesisof late proteinswas

regu-lated atthelevel oftranscription.

Regulation atthe level oftranslation. The results clearly demonstrate the importance of

post-transcriptional control in theswitch-off of early and intermediate proteinsynthesis in FV 3-infected cells. The only RNAs whose molar

rate of synthesis declined with time were the

large-molecular-weight species, ICR 947

through ICR 817 (Table 1). There was also a

decrease inmolar ratios of the largest infected

TABLE 3. Correlation of FV 3-specificmRNA's andproteins

RNA-calculatedb Mol RNA synthe-ICPmol wta (x103) Classification

RNAWcalciat

dX

mol ICRbmol wt (x103) sized inpresence

wt(x10~~~~~~) ofFPA

68 Early 612 597 +

65 Early 585 577 +

47/45 Early 405, 423 425 +

36 Early 324 345 +

50 Intermediate 450 463 +

39/37 Intermediate 333, 351 358 +

18 Intermediate 162 169 +

62 Late 558 560 0

55 Late 495 534 0

19 Late 270 240,235 0

aMolecularweights of proteins were determined by relative mobility compared with standards in

SDS-polyacrylamidegels.

bMolecularweights of RNAs required to code for such proteins were calculated from an average amino

acidmolecular weight of100andthat of 300 for nucleotides, with three nucleotides per amino acid. Observed

molecular weightof RNAs (ICR) weredetermined by electrophoretic mobility informamide-polyacrylamide

gelsasdescribed in the text.

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PROTEIN EARLY RNA definite increase (Fig. 8). Therefore, if these EAL; ;\q>O RNAs are the actual messages for these

pro-teins,

a

negative

control must

operate

at the

O t -\ i\ \ /

post-transcriptional

>\\

level.

The RNAs assigned tothethreeintermediate

#

proteins

also continued to be

synthesized

atan

-*\0i~i1/ increasedrate at6

h,

whereas from 4 to 6hthe

synthesis

of the

corresponding proteins

went down, again implicating post-transcriptional

\_______

INTERNEDIATE

control (Fig. 8).

Goorhaand Granoff

(9)

have

presented

evi-INTERMEDIATE

dencefor the post-transcriptional regulation of

MA1

\\

\wv-

/ R \&. ICP 18 (then termed VSP 13). The present re-/ port supports//

\

their

findings

by

demonstrating

l.

that therate ofsynthesis ofICR 169, the

pre-.sumptive

mRNA for

ICP

18, remains high

throughout

infection

(Fig.

8).

/0

7/ General

significance

ofthis

study.

Most of

the

experimental

evidence favors

transcrip-l________________________

lltional

_ control as the primary mode of

regula-LATE tion

};and

by

DNA

bacteriophage

and

by

both RNA DNAanimal viruses

(2,

7, 17,19, 23, 24, 31,

scrip

\ 32).Theclassical

hypothesis

oncontrol of

tran-scription

by

DNA viruses was established

by

N\X

.o

investigators

who used

hybridization

data to

- divide RNA

qualitatively

into

early

and late

o categories, depending on time of initiation of

-/1l] viral DNA synthesis. Anelementofqualitative

transcriptional

controlwasalso

apparent

inFV

HOURS

3-infected

cells,

but in this

system

a

virus-in-0 2 4 6 8 0 2 4 6 8 duced

protein,

rather than DNA

synthesis

per

HOURS POST INFECTION se (8), was required to turn on late

transcrip-. 8. Molar rate of synthesis of early, interme-

tion.

and late FV 3 RNAs and proteins. The values After analyzing the kinetics of DNA:RNA

pressed as percentage of the maximum molar

hybridization

in

liquid,

Frenkel and Roizman

fsynthesis. Identical symbols are used for the

hybridizato iquaid Frenkel

'.andcriz

?ptide

and its presumptive mRNA. Early pro- (6)

postulated

quantitative control of transcrip ICP 68, 0; ICP 65, 0; ICP

47/45,

U;ICP 36, tion in herpesvirus-infected cells; Wold et al.

rlyRNAs: ICR 597, 0;ICR 577, 0;ICR 425,

(35)

didthesamewithadenovirus. Such

experi-FR

332, El. Intermediate proteins: ICP 50,

*;

ments inform us only ofthe number of

abun-9/37,

0;ICP 18, U. Intermediate RNAs: ICR dance classes of RNA that differ by10-fold. We I; ICR 358, 0;ICR 169,

E.

Late proteins: ICP have provided direct evidence foramorevaried

;ICP 55, 0;ICP 19,

*.

Late RNAs: ICR 560, quantitative control of FV 3 RNA synthesis.

R 534, 0; ICR

240/235,

S. Separation of

F-V

3 RNA onpolyacrylamidegels clearly demonstrates that themolecularspecies proteins (ICP 133 through ICP 111, Table were not all synthesized at thesame rate at any

Loseratesof

synthesis might, therefore,

be

specified

time and that the same size classes

lively

controlled at the transcriptional were synthesized at differentrates atdifferent

All RNAs smaller than ICR 746 contin- times.

ibe synthesized at 4 and 6 h after infection A paramount finding of this study was the

-es comparable to or greater than those key role of post-transcriptional regulation in

wishedat 2 h. the viral growth cycle; over one-half of the seven RNAs assigned the coding poten- detectable infected cell polypeptides appeared

r the early and intermediate proteins (Ta- to be regulated at this level. The evidence for

) were of the correct size and met the translational control ineucaryotic systems has

Lonal

criterion of being synthesized in the accumulated slowly over the years since

Mc-ace

of FPA. The rate of RNA synthesis for Auslan (20) first demonstrated that vaccinia ur early proteins did not decrease between virus-induced thymidine kinase synthesis was 6h and, in all but one instance, showed a not switched off inthepresence ofactinomycin

0

100 7 5

50

25

0

FIG diate,

are ex

rate o) polype teins:

El.Ea

*; IC

ICP31

463, 4 62, 0,

0;ICE

cell pi

2),wh negati

level.

uedto

at rat establ The

tial fo:

ble 3)

additi

preset thefor

2and

100

75

50

25

0

100

iL

F-x

'I

75

50

25

340

WILLIS ET AL. J. VIROL.

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D. Honess and Roizman (14, 15) suggested a "cascade" regulation of herpesvirus-specific

proteins. As they found early herpesvirus

RNAs present late in infection, Frenkel and Roizman (6) postulated post-transcriptional regulation for the switch-offofearly herpesvi-rus proteins. Oppermann and Koch (25) have

also strengthened the hypothesis for transla-tional control in eucaryotic virus infection by

demonstrating that messages for the prerepli-cative polypeptides ofvaccinia virus persisted

lateininfection. However,asimultaneous

com-parison ofthe rates of

synthesis

of individual viralRNA species with those of viral polypep-tidessynthesizedthroughouttheinfectious

cy-cle was only achieved with FV 3. Our data

demonstrate thatearlyRNAsarenot

only

pres-entlate in infection but are stillactivelybeing

synthesized, despiteanapparentinabilitytobe

translated. Therefore, there isnonegative con-trolof mostof theearlyFV 3 RNAs. Theproof thataparticular proteinis

specified by

a

partic-ular RNA willbe provided when isolatedviral

RNAsaretranslatedinvitro, andwe nowhave

the means to accomplish this. We will then assayforthe factors thatregulate translation,

eg., ofICP 18.

ACKNOWLEDGMENTS

We wish to thank Frank Morris of the Department of ElectricalEngineering at Memphis State University for his invaluable aid in the computer analysis. Ramona Tirey and Susan Carrprovidedskilled technical assistance.

This research was supported by a research project grant CA07055and a Childhood Cancer Research Center Grant CA 08480 from theNational Cancer Institute and by AL-SAC.

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2. Aloni, Y., E. Winocour, and L. Sachs. 1968. Character-ization ofthe simian virus 40-specific RNA in virus-yielding and transformed cells. J. Mol. Biol. 31:415-429.

3. Bonner, W. M., and R. A. Laskey. 1974. A film detec-tionmethodfortritium-labeled proteins and nucleic acidsinpolyacrylamide gels.Eur. J.Biochem. 46:83-88.

4. Both, G.W., S. Lavi, and A. J. Shatkin.1975.Synthesis ofall the geneproductsof the reovirus genomeinvivo andinvitro.Cell 4:173-180.

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8. Goorha,R., and A. Granoff. 1974. Macromolecular syn-thesis in cells infected by frog virus 3. I. Virus-spe-cific protein synthesis and its regulation. Virology 60:237-250.

9. Goorha, R., and A. Granoff. 1974. Macromolecular syn-thesis in cellsinfected by frog virus 3. II. Evidence for post-transcriptional control of a viral structural pro-tein.Virology 60:251-259.

10. Goorha,R., R. F. Naegele, D. Purifoy, and A. Gran-off. 1975. Macromolecular synthesis in cells infected withfrogvirus 3.III.Virus-specificproteinsynthesis bytemperature-sensitive mutants. Virology 66:428-439.

11. Goorha, R., D. Willis, andA.Granoff.1977. Macromo-lecular synthesis in cells infected by frog virus 3. VI. FV3replication is dependentonthecell nucleus.J. Virol. 21:802-805.

12. Granoff, A., P. E. Came, and D. C. Breeze. 1966. Viruses and renal carcinoma ofRana pipiens.I.The isolation and properties of virus from normaland tumortissue.Virology 29:133-148.

13. Honess, R. W.,and B.Roizman. 1973.Proteins speci-fiedby herpes simplex virus. XI. Identification and relative molar rates of synthesis of structural and nonstructural herpesvirus polypeptides in the in-fected cell. J.Virol. 12:1347-1365.

14. Honess, R. W., and B. Roizman. 1974. Regulation of herpesvirus macromolecular synthesis. I. Cascade regulation of the synthesis of three groups of viral proteins. J. Virol. 14:8-19.

15. Honess, R. W., and B. Roizman.1975. Regulation of herpesvirus macromolecular synthesis: sequential transition of polypeptide synthesis requires func-tional viral polypeptides. Proc. Natl. Acad. Sci. U.S.A.72:1276-1280.

16. Kelly, D. C., and R. J. Avery.1974.Frog virus3 deoxy-ribonucleic acid. J. Gen. Virol. 24:1-10.

17. Kourilsky, P., M. Bourguignon, M. Bouquet, and F. Gros. 1970. Early transcription controls after induc-tionof prophage. ColdSpring Harbor Symp. Quant. Biol.25:305-314.

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Figure

FIG. 2.short-theintermediate; Schematic representation ofICRs and ICPs. This drawing is a composite ofall bands seen on both and long-term exposures ofpolyacrylamide gels and at every interval examined
FIG. 3.577,Ortectooscilloscopeline 7 Absorbance tracings offluorograms of viral RNA. The fluorograms ofFig
TABLE 1. Molar ratios of viral RNAs synthesized at various times after infection
FIG. 4.containcells Fluorogram ofgel containing RNAs from cells infected in the presence ofFPA
+7

References

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