Proteins Specified by Herpes Simplex Virus XI. Identification and Relative Molar Rates of Synthesis of Structural and Nonstructural Herpes Virus Polypeptides in the Infected Cell

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Copyright 0 1973 AmericanSociety for Microbiology Printed in U.S.A.

Proteins

Specified by Herpes Simplex Virus

XI. Identification and Relative Molar

Rates of

Synthesis of Structural and

Nonstructural Herpes Virus Polypeptides in the Infected Cell

ROBERT W. HONESS AND BERNARD ROIZMAN

Department ofMicrobiology, The University of Chicago, Chicago, Illinois60637

Received for publication 14August 1973

Analyses of polypeptides made in HEp-2 cells infected with herpes simplex

virus type 1 by high-resolution polyacrylamide gel electrophoresisrevealed the

synthesis ofat least 49 infected cell polypeptides (ICP) ranging in molecular

weight from 15,000 to280,000. Evidence for virusspecificity basedon increased

rates of synthesis postinfection, immunological specificity, and viral

control ofmobility andrateofsynthesiswasavailable for 47 of the ICP. These 47

polypeptides can account for 75% ofthe virus genetic information assuming a

DNAmolecular weight of10"andasymmetric transcription. On the basis of their

mobility relative tovirion proteins, the ICP were classified asstructural (S, 23

polypeptides), nonstructural (NS, 16 polypeptides), and unassigned (U, 10 polypeptides). Analysis of the synthesis of the ICP revealed the following. (i)

Rapidposttranslational cleavages of HSV proteinswerenotdetected; inparallel

experiments rapid posttranslational cleavages were readily demonstrated in

poliovirus-infected cells and thesewere blockedbyprotease inhibitors. (ii) Slow

posttranslational changes in the mobility of at least two polypeptides were

observed.(iii) Analysis of theratesofsynthesis of ICP examinedatfour intervals

postinfection revealed regulation of the patternandamountof ICPsynthesized.

ICP formed six classes (AtoF) differing in their kinetics ofsynthesis. S and NS

ICP were distributed nonrandomly amongthese classes. Thus, of thesum ofS

protein amino acid sequences apportioned among these kinetic classes, 47%,

constituting class A and comprising "late" structural proteins, were

character-izedby progressively increasing ratesofsynthesis until at least 12 h

postinfec-tion; whereas "early" structural proteins constituting class C, amountingto31%

of the totalamino acidsequences, weresynthesized with initially increasingrates

until 4 hpostinfection and with decliningratesthereafter. NS polypeptides and

remaining S polypeptides were distributedamongthe other kinetic classes-B,

D,E, and F. Control of protein abundance wasevident in that the polypeptides

werenotmade inequimolaramounts.However, Sand NS polypeptides couldnot

be differentiated onthe basis of their molar ratesofsynthesis. The bulk of the

detectedpolypeptides didnotdifferbymorethaneightfold in their molarratesof

synthesis.

Herpesvirus replication requires the

im-plementationof two programs,the transcription of viral DNAand the translation ofviral

mes-sages. Recentpapers (6, 7, 21) from our

labora-tory have documented the existence oftemporal

controlsofviral DNAtranscriptionand controls

of molar abundance of the RNA transcripts

accumulatingin theinfectedcell. In this paper

wereport the results of experiments designed to

outline the time ofsynthesisand abundance of

thetranslational productsofthe virus.

As aprefacetothis report, it seems desirable

tosummarize the backgroundand some of the

problems encountered inthis work.

Previous attempts to analyze the

polypep-tides madeininfected

cells,

definedasinfected cell

polypeptides

(ICP),

both inthis

laboratory

(23) and in others (1, 10) arecurrentlyof limited

value for two main reasons. First, the

tech-niques of polyacrylamide gel electrophoresis employing the continuous buffer system (19), combined with the estimation ofradioactivity

within slices of fractionated gels, were

inade-quate to resolve the large number ofproteins

made in the

herpesvirus-infected

cell.

Second,

the procedure then used for the purification of

1347

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herpesvirus particles was inadequate (14), and

hence no meaningful classification of proteins as either structural or nonstructural could be

made. The availability of techniques for the

purification of enveloped nucleocapsids

(vi-rions) (24), forhigh resolution electrophoresis of

proteins on polyacrylamidegels (3, 12), and for

quantitative assay of separated proteins (5) has

enabledus toundertake the present studies.

The major difficultyencountered in the

cur-rent studies of ICP is the differentiation

be-tween polypeptides specified by the virus and

the host, although the bulk of host proteins

ceases tobe made after infection (23).

Compel-lingevidence that a polypeptide is virus specific

is its synthesis in a cell-free amino acid

incor-porating system programmed by mRNA

com-plementary to viral DNA. Sincethis is not yet

feasible,wehavedefined ICPasvirusspecific if

they meet one or more ofthefollowingcriteria,

i.e., (i) stimulation in the rate of synthesis

postinfection in the face of general decline in

host proteinsynthesis; (ii) variations in

proper-ties of ICP as a function of the virus strain

infecting the cell, and (iii) immune

precipita-tion ofICP

by

antisera reactivesolely withvirus

antigens.

In a similar manner, the differentiation

be-tween structural and nonstructural

polypep-tides

ideally

requires the rigorous demonstra-tion ofchemicalidentity between the structural

proteins ofthevirus, previouslydesignated(24)

as virion proteins (VP), and their putative

analogues amongtheICP.In practice we have temporarily defined ICP as structural or

non-structural solely onthe basis ofelectrophoretic mobility relative to VP, pending a more

com-plete characterization oftheseproteins.

MATERIALS AND METHODS Solutions and chemicals. Maintenance medium forinfected cellswasmixture199supplemented with

1%calfserum.Labelingmedium consisted of mixture

199 containing one-tenth the usual amountsof leu-cine, isoleucine, and valine and supplemented with

1% dialyzed calf serum. Labeled amino acids were

purchased fromSchwartz/Mann, Orangeburg, N. Y., andwere as follows: L-isoleucine-U-_4C,

L-leucine-U-14C, andL-valine-U-14C, all withspecific activities of

approximately 300mCi/mmol; L-isoleucine-3H,

L-leu-cine-3H, andL-valine-8Hwithspecific activities from

1to 30Ci/mmol;andamixture ofthirteen"4C-labeled

aminoacids(reconstituted proteinhydrolyzate,

com-posed of alanine, arginine, aspartic acid, glutamic acid, isoleucine, leucine, lysine, phenylalanine, pro-line, serine, threonine, tyrosine, and valine with

specific activities of about 150 to 450 mCi/mmol).

Tolylsulfonyl-lysyl chloromethyl ketone (TLCK), a

specific inhibitor of trypsin, and

tolylsulfonyl-phenylalanylchloromethylketone(TPCK),aspecific

inhibitor of chymotrypsin activities (20) were ob-tained fromNutritional Biochemical Co., Cleveland, Ohio.

Cells. Humanepidermoid carcinomano.2(HEp-2) cells were grown in Eagle minimal essential medium (EMEM) supplemented with 10%calf serum, 0.001% ferricnitrate, and 1%sodium pyruvate.

Viruses. Most of the experiments described in this paper were done with the F prototype of HSV-1 [HSV-1(F)]. Other strains analyzed insome ofthe studies were HSV-l(mP) and HSV-2(G) correspond-ing to a laboratory strain of HSV-1 and the G-prototype of HSV-2, respectively. The origin and some properties of these strains have been reported elsewhere (4). Procedures for production of HSV strains were asdescribedby Roizman and Spear (17) with the exception that HSV-1(F) and HSV-2(G) have beenpassaged a maximum of four times at low multiplicity priortotheir use inthehigh-multiplicity infectionsdescribed in this study. Titrations of infec-tious viruswere performedas describedby Roizman and Roane (15).

An attenuated vaccine strain ofpoliovirus type 1 was employed as a positive control in experiments dealing with rapid posttranslationalcleavages of virus polypeptides. Media and procedures employed for infection and radiolabeling of poliovirus-infected HEp-2 cells were identical to those described for

HSV-infected cells.

Purification of isotopically labeled, enveloped virusparticles. Infected cellswerelabeledfrom 4 to 24 or 30 h postinfection with either 3H-leucine, isoleucine, andvaline (1 to1.5MCiofeachaminoacid per ml oflabelingmedium)or"4C-leucine, isoleucine, and valine (0.08gCiofeach aminoacid perml)in50

ml of labeling medium per 2 x 108 infected cells. Virionswerepurifiedfromlabeled cellsessentiallyas

describedbySpear and Roizman (24), except that the flotationthrough the discontinuoussucrosegradient, which followed thebanding inadextran-10gradient,

was replaced by a second dextran-10 gradient sedi-mentation. Particlespurifiedby thismodified proce-dure had a polypeptide profile similar to that ob-tainedusing theoriginalprocedure. Particlespurified

in this way consisted of approximately 95% of en-veloped particles (unpenetrated by neutral phos-photungstate negative stain) with particleto infectiv-ity ratios ofapproximately100.

Labelingofproteinssynthesized byinfected and

uninfected cells. ConfluentHEp-2cellmonolayersin

tissue culture flasks(approximately2 x 106 cellsper flask)wereexposedto20PFUof viruspercell(in20 ml ofmaintenance medium) or were mock infected with2.0mlofthismedium. Inoculated cultureswere

incubated withconstantagitation for1h at 37C,and thereaftervirus or mock inoculaweredecanted. The monolayerswerethenrinsed(5.0 mlof maintenance

medium perflask), replenished with5.0mlof mainte-nancemedium perflask,andreincubatedat 37C.For

labeling, the cultures were rinsed and then replen-ished with labeling medium containing "4C-leucine,

isoleucine, and valine (0.3to 1.7 gCi ofeachamino

acid perml).Atthe end ofthelabeling periodthecells were either: (i) rinsed with ice-cold phosphate-buf-fered saline(3 x 5.0ml/flask)toterminate

incorpora-J.

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PROTEINS SPECIFIED BY HERPES SIMPLEX VIRUS

tion ("pulse") and then stripped from the flask, denatured and solubilized by heating at 80 C with a small volume of 1% sodiumdodecyl sulfate (SDS) for subsequent trichloroacetic acid precipitation or 2% SDS, 5%

0-mercaptoethanol,

0.05M Tris-hydrochlo-ride (pH 7.0) for subsequent polyacrylamide gel electrophoresis, or (ii) rinsed (3x5.0 ml), replenished with maintenance medium (at 37 C), and reincubated in theabsence of exogenous labeled precursors prior to terminatingincorporation, asabove ("pulse chase"). The timesatwhichlabeled precursors were added and removed, etc., are stated as the times in hours after theaddition of the virus inoculum.

Radio-immune precipitation. The radio-immune precipitation reactions for analysis of herpesvirus-specific polypeptides were performed as described by Honessand Watson (J.Gen. Virol., in press). Briefly, the supernatant fluid obtained after sedimentation (35,000 rpm, 1 h, in a Beckman SW40 rotor) of a homogenate of infected HEp-2 cells, which had been labeled with "C-amino acids from 4.5 to 6.5 h postinfection, was mixed with an excess of a rabbit hyperimmune serum specific for herpes simplex virus antigens and incubated at +4 C overnight. The re-sulting immune precipitate was sedimented (3,500 rpm for 15 min), washed thoroughly by resuspension and sedimentation in ice-cold phosphate-buffered saline, and finally resuspended and solubilized by heating in 2%SDS, 5% ,B-mercaptoethanol, and 0.5 M Tris-hydrochloride (pH 7.0) at 80Cfor 10min.The solubilized immune precipitate was then subjected to electrophoresison apolyacrylamide gel. The immune sera,kindly donated by P. Wildy and D. Watson, were hyperimmune rabbit sera prepared by inoculation with rabbitkidney cells infected with HSV-1,ashas been described in detail (27). These sera gave up to 12 precipitin bands in agargel radial immunodiffusion

testsagainst infectedHEp-2, RK13,orBHK-21cells, but hadnodemonstrable reaction against uninfected cells or componentsoftissueculturemedia, and did notprecipitate any significant radioactivity froman artificial mixture of labeled uninfected cells and unlabeled infected cell antigensamples (R. W.

Hon-essand D. H.Watson, J. Gen. Virol., in press). Analytical procedures. Incorporation of radioac-tive amino acids into acid-insoluble material was determined by precipitation with cold 5% trichlora-cetic acid. The precipitates were collected on mem-brane filters (Millipore Corp. type GA, 2.5 cm) and washed, and the radioactivity was estimated.

Polyacrylamide gel electrophoresis. The electro-phoretic, staining, and autoradiographic techniques were as described previously (W. Gibson and B. Roizman, J. Virol., submitted for publication, 24),

employing the discontinuous buffer system (2, 13)

modifiedbytheinclusion ofSDS (3, 12).

Densitometry of stained gels and autoradio-grams andcomputer-ai'dedanalysis of absorbance tracings. Absorbance tracings of stained gels and autoradiograms wereobtainedwith aGilford record-ing spectrophotometer equipped with a gel scanner

attachment. The voltage output from the photocell

was also recorded by a General Automation 16/45 digital computer anddisplayed as a plot of

absorb-ance (voltage) as a function of distance migrated

(time of scan) on a Tektronix oscilloscope. The computer wasprogrammed to expand regions of the initial profile as determined by the operator and to compute the areas under the tracing for individual bands defined by vertical lines set parallel to the absorbance (voltage) axis. The results of a typical analysis of proteins made in HSV-1(F)-infected HEp-2 cells are shown in Fig. 1; the upper panel shows thecomplete profile and the regions (A,B, C) selectedfor expansion are shown in the three lower panels. The lines intersecting theexpanded profilesin

the lower panels demarcate the areas ofthe bands calculatedby the computer. The bandsarenumbered inaccordance with the designationsofHSV-1(F) ICP which are described in Results. Thepeakof radioac-tivity migrating furthest towards the anode(the right side of all profiles shown in this paper) in this and subsequent gels represents material too small to be sieved at aparticular gel strength and which therefore migrates with the free-SDS front (3) close to the bromophenol blue (BPB) dye marker. In the 8.5% gel showninFig.1allICP smaller than ICP45migrate in this position.

RESULTS

Enumeration and differentiation between structural and nonstructural polypeptides synthesized in the infected cell. In the first

series of experiments the mobilities of labeled

ICP and VP were compared by electrophoresis

either as an artificial mixture

(coelectrophore-sis) or separately, but on the same polyacrylam-ide gel slab (parallel electrophoresis). Both

techniques (Fig. 2 and 3) showed that there

were moreICP than there were VP and that the

relative mobilities of ICP and VP were the same, whether obtained by a combination of coelectrophoresis and autoradiography (Fig. 2a

andb) or by autoradiography of parallel

separa-tion on slab gels (Fig. 3). Mobilities of ICP

relative to VP were independent of

polyacryl-amide gel concentration (Fig. 3).

However, as has been noted previously (24), autoradiography of whole gels resolved more

bands than measurements of radioactivity in

gel slices. For example, in Fig. 2a a maximum of

eight bands was defined by variation in the "C-radioactivity of fractions 15 to 40, and a

number of these peaks were defined by

eleva-tionofradioactivityinasingle slice.Incontrast,

12 well resolved bands were apparent in the

autoradiogramofthecorrespondingregion (Fig. 2b). On the basis of these results,

autoradiogra-phy of parallel separations was the method

chosen forsubsequent experiments.

Analyses such as those showninFig. 2and 3

have shown that cells infected withHSV-1(F)

synthesize at least 49 polypeptides, whereas

under the same conditions only 27 bandswere

resolved inseparations of proteins frompurified

virions. TheseICPwereassigned numbersfrom

1349 VOL.12,1973

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Distance Corbitrary

units]

FIG. 1. Illustration ofcomputerplanimetry of the proteins separated in polyacrylamide gels. The top panelshows the absorbancetracing ofan

autoradio-gram of HSV-1(F) ICPlabeled with'IC-amino acids from3.5to8.7hpostinfection.A,B, andCindicate

those regions of theprofile shown in the expanded forminthe lowerthreepanels.In eachpanel theareas

boundedbytheabscissa,absorbanceprofile, and the broken lines parallel to the ab )rbance axis were

1to 49 in order ofdescendingmolecularweight,

and independent of the established numbering

system for virionproteins (i.e., VP 1-24 [24]).

One objectiveofthisstudywas toclassifythe

ICP as structural and nonstructural and to

examinetheirsynthesis.For the purposes of this

paper the basis for the classification was as

follows. (i) All parallel-run comparisons on

whichassignments weremade included proteins

of purified virions in amounts sufficient to

permit the detection ofeventhose VP presentin

smallamounts (e.g., VP4, VP6,and VP

9).

(ii)

AnICP with thesame electrophoretic

mobility

as a VPwasconsidered ananalogueofthis VP andwas

designated

a"structural"

(S)

polypep-tide. (iii) ICP with electrophoretic mobilities differingfromthoseofVP,but migrating under

a broad VP band, were designated

"unas-signed" (U)

polypeptides.

(iv) ICP with electro-phoretic mobilities clearly different from those of VP were designated "nonstructural"

(NS)

polypeptides.

Table 1 (columns 1 to 4) summarizes the

enumeration and classification of ICP

by

the above criteria. Of the 49

ICP,

10 were consid-ered

unassigned,

16were

nonstructural,

and the remainderwerestructural.

Evidence for the virus

specificity

of ICP. Evidence that mostoftheICPwehave identi-fiedare

specified

by viral genetic information is

based on three types of experiments dealing with comparisons of

polypeptides

synthesized in infected and uninfected cells, the specific precipitation ofICP

by

antiserareactingsolely with

virus-specific

antigens,andcomparisonsof

HSV-1(F) ICP with ICP made in cells infected with other strains and variants ofherpes

sim-plex virus.

Comparison

of

polypeptides synthesized

in

infected and uninfected cells.

Figure

4showsa

typical

result ofa

comparison

ofthe

polypep-tides

synthesized

overashort

period

ininfected and uninfectedHEp-2 cells. In this experiment replicate cultures of HEp-2 cells were labeled with

"4C-amino

acids from 4.5 to 5.0 h after

infection with HSV-1(F) or after mock

infec-tion.

Equivalent

samples of labeled infected anduninfected cellswere thenharvested, solu-bilized, and subjected to electrophoresis on 8.5% acrylamide gels. Visual examination of

gels stained with Coomassie brilliant blue re-calculatedbythecomputer. Thenumbers above the profile refertothe numericaldesignation of the ICP contained in each band. The arbitrary unitson the

ordinatearerelatedtoabsorbancebytheequation1.0

absorbance unit equals 1,139 arbitrary units. One arbitrary unit of distance(abscissae)wasequalto2.0

cm. r-i

1--

30-

6-F, .in LI

4D

1--.Jo

0

%A

.M

-W

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c c

386;

0

2004~~~~~~92000

6~~~~~~~~~~3

b F

10 9 26-27

c 39

~~~~~~~~~~~~~35

36

0

31

41

21 4445

32

1629

4 ~~~23 37

4243 6

0 20 40 60 80 100

Fractions

FIG. 2. Distributionofradioactivity inasliced gel(a)andtheautoradiogram ofanidentical polyacrylamide

gel (b)containingelectrophcretically separated proteins from 3H-amino acids-labeledpurified virions andan

infected celllysatelabeled with14C-amino acids from4.25to6.5hpostinfection. In the radioactivity profileof

the slicedgel (upperpartofFig.2a),polypeptides ofthevirion (3H-amino acids,opencircles and broken line)

are indicated by thelarger numerals enclosed withinrectangular frames, andthe infected cell polypeptides

(ICP), labeledwith "4C-amino acids,areindicated by the closed circles, the solid line,and smaller numerals. The lower part of the figure (b) is an absorbance tracing of the autoradiographic image (i.e., "4C-amino

acids-ICP) ofanidenticalgel. Thesmallnumeralsonthis profilearethose of the ICP. Electrophoresison8.5%

cylindrical gels.

vealed a large number of bands, too closely vealed any significant difference between the

spaced to be well resolved by densitometry. stained profiles ofelectrophoretically separated

Neither visual inspection nordensitometry re- proteins of total infected or uninfected cells

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7l7lS%gej

IC P

1-2

"ii

3

4 5 62

278 930 1110 r

312 16

19_

2

22

4243 25

26_4

2728

°

2930

32_

potnecin Sou 34_ 36_

38i3e

40'41__

BPB

p eG. 3. Autoradio,

phoreticallyseparatt

polypeptides (purifie

20hpostinfection)a

labeled withefC-a

postinfection.Solubi

infected cells were parallelon7.75%an

processed forautorao

ductions of photogr

gram,andtherefore,,

darkerareaatthetol

illumination during printedwithdifferen

twosamples.

(Fig. 4, middletrao

ographic images o

differentiated(Fig

The distribution

uninfected cell pr(

total protein (mic

Fig. 4), with the i

grating with

polyl

molecular weight

lysates contained

and their distribu

the distribution of proportion of "C-f

12.0%ge

l

into components with molecular weights greater than 50,000.

VP ICP VI In view of thevery

large

number of

polypep--W - tide

species

synthesized

in uninfected cells for

1-2- m 1-3 nearly every ICP there is a component in the

1-2 58 uninfected cell characterized

by

the same or

19

7mm-8

similar

electrophoretic mobility.

Attempts

to

21 U M 13-14 differentiate between

polypeptides synthesized

4

3026-2

-1516 in infected and uninfected cells solely on the

5

31

0

19-1

basis of direct

comparisons

oftheir

electropho-6A 34-36 retic mobilities therefore seemed unprofitable.

7 38-41 2

However,

the fact that the

profiles

of

proteins

8 22 synthesized are different may be used as

evi-44 dence for selective inhibition or stimulation

10

(induction) to account for these differences.

12

Quantitative

comparisons

of amino acid

incor-1314

poration over similar

regions

of infected and

uninfected cell

profiles (top

and bottom

trac-15 ings of Fig. 4) indicated a higher rate of

synthe-16 sis in the infected cell over regions of ICP other

17 than those indicated

by "h"

in

Fig.

4

(and

the

18

19

components travelling

at the

dye

front in

Fig.

20 4). Thus, incorporation into ICP 34, 35, and 42,

43 was

quantitatively comparable

to that ob-served over the same region ofthe uninfected cell.

22 This

type

of

comparison

may be termed the

23 criterion of induction; it provides some evidence

for virus specificity of ICP other than those

BPB BPB

BPB

comigratingwith prominentcomponents ofthe

graphic comparisons of electro uninfected cells. These conclusions are

rein-ed14C-amino acids-labeledvirion

forced

by

the results of analyses of the

kinetics

'd

fromcells and labeledfrom 3to of synthesis of ICP considered later in the text.

ndpolypeptides of infected cells Precipitation of ICP by antiserum specific

mino acids from 3.5 to 8.7 h for virusantigens.In thisexperiment, infected

ilized labeled virions andlabeled cells were labeled with "4C-amino acids from

subjected to electrophoresis in 4.25 to 6.25 h afterinfection withHSV-1(F). A

yd12% slabgels. These were then sample of the infected cell homogenate was

diography. The figures are repro- sedimented (35,000 rpm, 1 h, in a Beckman

,aphs of the original autoradio-

SW40

rotor), and the supernatant fluid, which

suffer fromlossofresolution. The contained 35% of the acid-insoluble radioactivity

pofthe7.75%gelis dueto uneven of the total cell

homogenate,

was mixed witha

photography. The

12%o

gel was o h oa elhmgnt,wsmxdwt

t

exposure

periods for each of the

sufficient

volume

of a hyperimmune rabbit

serum

specific

for

herpes simplex

virus anti-gens to

give

maximal

precipitation

oflabeled

components (R. W. Honess and D. H. Watson,

cing). However,the autoradi- J. Gen.

Virol.,

in

press).

The

resulting

immune

fthese

gels

could bereadily

precipitate

contained 25 to27% ofthe

radioac-4, topand bottomtracings).

tivity

presentin thesupernatantfluid.

Samples

of

"4C-amino

acids-labeled of thetotal infected cell

homogenate,

the

super-oteins coincided with that of natant

fluid,

and the immune

precipitate

were

idle

and bottom tracings of then

subjected

to

electrophoresis

on 8.5%

poly-majority ofradioactivity mi-

acrylamide gel cylinders. Autoradiograms

of

peptides less than 50,000 in these

gels

are shown in

Fig.

5. The

samples

of

In contrast, infected cell the total infected cell

homogenate

and the

su-fewer labeled polypeptides pernatant fraction were derived from a

com-tion in thegel didnotreflect parable number ofcells; thesample ofthe

im-thetotal cellprotein.Alarge muneprecipitatewasobtained fromavolume of

amino acid

incorporation

was supernatant fluid sixto

eight

timesgreater than J.

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1353

TABLE 1. Enumeration, classification and evidenceforvirusspecificity ofHSV-1 (F) ICP

1 2a 3 4b 5C 6 7e- 8'

Poly- Classifica- Precipitation by

Infected peptide tionofICP

Specific

antiserum F-ICP F-ICP Criteria

cellpoly- molec- Relationship and stabil- stimula- different different for virus

peptide ular toVP ityto tionand Avail- from from specificity (ICP) weight prolonged kinetics of able for Precip- G-ICP mP-ICP

satisfied

(x10-8) chase synthesis precipi- itated (columns)

tation 275 260 184 155 146 140 135 130 126 120 117 112 103 98 93 87 78 76 74 72 71 65 63 61 59 57 53 50 48 44 43 42 40 37 36 33 32 31 30 27 25 23 VP 1-2 VP 3 VP4 VP 5 VP 6 VP 6A VP 7 VP8, 8A broad virion glycoprotein bands) VP9 VP 10 VP11 VP 12 VP 14 VP 15 VP 16 VP17 VP18 VP19 VP20 VP21 VP 22 VP23 VP 24 S S S S S NS S U S U U U U S NS NS(D) S S S S NS NS(D) NS(I) S S NS S U S S S NS S NS NS NS NS S S NS U U U S U NS NS NS +A + +A +C +C + +C +B +A + +C +E +E +E +E +A +F +F +F +A + +F +D +D +F +F -E -E 0 0 0 + + + + + + + + 0 0 0 + 0 0 + + + + + + + + + + + + 0rc 0 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + F> mP

mP> F

F> mP

mP> F mP> F mP> F mP>F mF> F F> mP F>mP + F>mP + + mP> F mP> F mP> F 5,7 5 5,7 5,6,7 5,6,7 5,6 5,6 5,6 5,6,7 5,6,7 5,6 5 5 5 5,6,7 5 5,7 5,6,7 5,7 5,6,7 5,7 5,7 5,7 5 5,6,7 5,6,7 5,7 5,6,7 5 5,7 5 0 0 0 5,6 5,6 5,6 5,6 5,6,7 5,7 7 7 5, 7 5,7 5 5 5,7 5 VOL. 12, 1973

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 39 40 41 42 43 44 45 46 47 48 49

aMolecularweightsofICP obtainedby interpolationinpreviouslydetermined values for virionpolypeptides

ofSpearand Roizman(24).

S, structural; NS, nonstructural; U, unassigned. See text for criteria

applied

in the classification. In

additiontothe 24 polypeptidesdescribedby Spearand Roizman(24), theseanalysestookcognizanceoftwo additional polypeptidesfoundin purifiedvirions (Heine, Honess, andRoizman, manuscriptinpreparation).

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TABLE 1.-Continued

These were VP 6A, a minor virion polypeptide which co-electrophoresed with ICP 8, and a glycoprotein designatedVP 8A.Duetopoor resolutionofVP7, 8,and 8A, themobility analogueofVP 8Aamong the ICP is uncertain and alloftheICP included within this mobility rangearethereforeclassified as S (ICP 10 which co-electrophoreses with VP 8) or as U. (D), Components declining or disappearingon prolonged chase. (I), Components increasingorappearingon prolonged chase.

c+, Aminoacid incorporation into components migratingin these regionswas higherin infectedthan in

uninfectedcells. -,Aminoacidincorporation into componentsmigratingintheseregionsininfectedcellswas

comparable to,orlowerthan, that observed in thesameregions of uninfected cells. See Fig. 3 and relatedtext.

LettersA toF refertothe patternsofsynthesisasshown inFig.11.Absence ofaletterdesignation indicates that the patternofsynthesis isuncertain.

dInthe left column("Available forprecipitation"), 0 and + indicate, respectively, undetectable amounts and the presenceof aparticular ICPinthesupernatant fluid. In theright column, + indicates precipitated and - indicatesnotprecipitated, by antiserum. See Fig.5and relatedtext.

eIn the comparisonofHSV-1(F) andHSV-2(G) (left column), + indicates any HSV-1(F) ICPforwhich there

was no exactcounterpart in mobilityoramount in HSV-2(G)-infected cells. Comparisons of HSV-1(F) and HSV-1(mP) (right column) indicate significantquantitativedifferences between ICP with identical mobilities producedincellsinfected with thetwoviruses, i.e., F > mP referstoapolypeptide producedinlargeramounts inFthaninmP-infected cells.

fNumbers refer to the columnssummarizing those criteria forvirusspecificity which were satisfiedby a

particular ICPofHSV-1(F). Forexample, ICP5satisfiedthe criterionofinduction(column 5), possessed viral antigenic specificity (column 6), and its mobility was affected by virus type-specific variation (column 7). Symbol 0 indicates thatnoneofthese criteria werefulfilled.

that subjected to electrophoresis on the

com-paniongel.

By inspection of Fig. 5 it may be seen that

certain polypeptides of the homogenate

parti-tionedselectivelywith thepelletafter

sedimen-tation and were in relativelylowconcentration

(ICP9 to 12, 20, 21,and31)or werenotdetected

(ICP44andICP 45)inthesupernatant fluid.Of

the polypeptides present in the supernatant

fluid,some,i.e., ICP 5, 6, 7, 8, 9 to 12, 19, 26, 32,

36, 38, and 39 were

precipitated

efficiently,

whereas others, i.e., ICP 16, 21, 28, 29, 37, and

40 were

precipitated

less efficiently, and some,

ICP22 to 24and41, were notprecipitatedatall.

Although precipitation

by

such antisera is

additive evidence for virus specificity, the

fail-uretoprecipitatea

particular

componentisnot

evidence that thiscomponent is host

specified.

Virus type and intratype

(strain)

specific

differences inICP. In theseexperiments

repli-catemonolayersofHEp-2cellswereexposedto 20 PFU of either

HSV-1(F), HSV-l(mP),

or

HSV-2(G) per cell and labeled with "C-amino

acids from 3.5 to 8.7 h postinfection. The cells

were then harvested and solubilized, and the

proteins were subjected to electrophoresis in

parallel. Theresults ofautoradiographic

analy-ses (not shown) may be summarizedbriefly as

follows. (i) ICP

synthesized

in cells infected

with HSV-1(F) and HSV-l(mP) were largely

congruent in both number and electrophoretic

mobilitybutdifferedinthe relative amounts of

a small number of polypeptides which were

made within the same time interval. Thus,

significantly moreofICP1-2, 10, 26, 27,and31

were present in

HSV-1(F)-infected

cells.

Con-versely,HSV-1 (mP) -infected cells accumulated

moreofICP4, 21, 22, 23, 24, 42, 43,and 48than

did HSV-1(F)-infected cells. (ii) Although the general appearance of profiles ofproteins

syn-thesized in HSV-1(F)- and HSV-2(G)-infected cellsweresimilar, thereweredifferences bothin

mobility and molarconcentrations of polypep-tides migratingin corresponding regions ofthe twoelectrophoretic separations.For ourpresent

purpose ofestablishing the virus specificity of

the ICP in HSV-1(F)-infected cells, it is

suffi-cient to indicate thoseHSV-1(F) ICPforwhich

an exact counterpart, either in mobility or

molarconcentration,was absent fromlysatesof

HSV-2(G)-infected

cells. Thus, HSV-2(G)-infected cells lacked exact counterparts to

HSV-1(F), ICP5, 6, 10, 11, 16, 18, 19, 21, 27, 28,

29, 40, 41, 44, and45.

The evidencefor virusspecificityofHSV-1(F) ICP which we have adduced in the preceding section is summarizedincolumns5, 6, 7, and8 ofTable 1.

Evidence that

HSV-l(F)

ICP are the

pri-mary products of translation: comparison of

HSV-1(F)

ICP labeled duringshortand long pulses and after a pulse chase. In these experiments replicate cultures of cells were

exposed to "C-amino acids at 4.5 and 6.5 h

postinfection forperiods of 10, 30, and 60 min.

Some of the additional cultures labeled for 10

min wereincubated for anadditional40min in

maintenance medium without labeled

precur-sors. The proteins oftheharvested, solubilized

cells were then subjected to electrophoresis in

parallel on an 8.5% acrylamide gel slab. Com-parisons of the autoradiograms (not shown)

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=

Cl -M -Ic

0 10 30 50 70 90

Distance

(m

m)

FIG. 4. Absorbance profiles of autoradiographic images of electrophoretically separated proteins from infected (toptracing)oruninfectedcells(bottomtracing) pulselabeled from 4.5to5.0hafter infectionormock infection. Theprofile ofthetotalelectrophoretically separated infectedcellproteins determinedby scanning the Coomassie brilliant blue stainedgel is alsoshown.Theproteinswereseparatedon 8.5%polyacrylamide gels. Numbersonupperprofileareof selected ICPtofacilitatecomparison withprofiles. The letter "h" identifies proteins in the infected cell which were synthesizedat rates comparable toproteins of similar mobilityin uninfected cells.

revealednosignificant differences inthe

mobil-ityorrelativequantity ofpolypeptides

accumu-lating in the cells during 10- to60-min pulses

administered at a given time postinfection.

Neither were there any significant differences

between the autoradiograms ofa 10-min

pulse

1355 VOL.12,1973

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Total

Sup.

cell

flui

'(P

4. 5ji.

16

0

21

16 ...

21

sm.;

22

234

25 ..

...

....

2526

2728

29

30

31

32 ...i

35

37

1

39 :

41

42

43 ,44

46 I

mmune anda

10-min

pulse

followed

by

a

40-min

chase. Asdiscussedbelow and shown in Fig. 6, similar d p pt. pulse and pulse-chase experimentsclearly

dem-onstrated rapid posttranslational processing of

4

|(I

P

polypeptides

in

poliovirus-infected

HEp-2

cells. §2' Moreover, a prolonged chase resulted in rela-tively minorchangesintheprofileofHSV-1(F)

ICP (compare the 5.0- to 5.25-h pulse with the

5.0- to5.5-h pulse chased to 9.0 h; Fig. 9).

Failure tomodify the pattern of synthesis

of HSV-1(F) ICP by exposure to specific

6 inhibitors of trypsin (TLCK) and

chymo-6 trypsin (TPCK) proteases. Chloromethyl

ke-8 tone derivatives inhibiting proteolysis by

tryp-sin and

chymotrypsin

have been shown to

1112 preventthe rapid posttranslationalcleavages of

16

poliovirus proteins inanumber ofcell lines (11,

26) and to cause the accumulation of

"poly-protein" precursors. The effect of these

inhibi-19 tors on the synthesis of HSV-1(F) ICP was

21

therefore examined in conjunction with

experi-ments on poliovirus-infected HEp-2 cells as a

positive control system.

Replicate HEp-2 cellcultures were infected at

high multiplicityeither withHSV-1(F) or

polio-26

virustype 1, labeledwith "C-aminoacids for a

15-mininterval beginning at 5.0 h postinfection,

29

either in the absence of inhibitors or in the

presence of 10' M TPCK or of TLCK, and

thereafter removed and processed for

electro-phoresis. An additional culture of cells infected

with poliovirus was pulse labeled for a similar

32

15-min

period

in the absence of inhibitors and

thereafter incubated for 40 min with medium

36 lacking labeled precursors. At the end of this chaseperiod these cellswere also processed for

37 electrophoresis. All samples were subjected to

8,

39

electrophoresis on the same (9.0% acrylamide)

40

_lJ^-7 gel slab. The autoradiograms of these samples

40v (Fig. 6)showed the following. (i) Control

experi-ments with poliovirus-infected cells readily

demonstratedrapid

posttranslational

cleavages which were effectively blockedby inhibitors of

protease activity. Thus, comparisonsoflysates

fromcellsgiven apulse and

pulse

chase(Fig. 6)

showthat, in the absence ofinhibitors,

radioac-tivityrapidly disappeared frombands NCVP0

and NCVP11/2 andappearedinbands VP 0, VP

1 (VP 2), and VP 3. In thepresence of 10' M

FIG.5. Autoradiograms of8.5%polyacrylamide gels containing electrophoretically separated (i) total

in-fected cell lysatelabeled with 14C-amino acids from 4.25to6.5hpostinfection (total cell), (ii)the

superna-tantfluidobtainedafterhigh-speed sedimentation of

thelysate (sup.fluid), and (iii) animmune

precipi-tateformed by the addition of antiserumspecific for

virus antigens (immune ppt), to the supernatant fluid.Numbers to theleftof the three gels refer to ICP in the total celllysate, those to the right indicate the

ICP precipitated bythevirus-specificantiserum.

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PROTEINSSPECIFIED BY HERPES SIMPLEX VIRUS

Polio-I

HSV-1

Pulse Pulse, Pulse Pulse Polio-.I Pus Ple Ple

proteins

TPCK

TLCK

Chase TLCK TPCK

IC P

NCVP 00

7

NCVP 0 L' 8

11149-12

-7~~~~~~~~51

-_NCVP

1I

:'

15-17

;;;

FI~CVP

1

72

" ww 19

21

_ _ _

~~~~NCVP

2 r_-~2323

_

NCVP3Ow:

~~~~~25-27

30 31

32

NCVP

X VPO

VPI

-?P VP 2

VP3

4_IW

34-36

_jW

38-39

AMM 40-41

42-43

1*0v 44

45 46

FIG. 6. Autoradiogram ofa9.0%polyacrylamide slab gel containing electrophoreticallyseparated polypep-tidessynthesizedinHSV-1i(F)andpoliovirus type 1-infected HEp-2 cellslabeled with "4C-amino acids inthe presence and absence ofspecific inhibitors of trypsin (TLCK) and chymotrypsin (TPCK) proteases. Left, proteins synthesizedinpoliovirus-infected cells, pulsed for15min in the absence of inhibitors (Pulse), pulsed for15 minand chasedfor30 mininthe absenceofinhibitors (Pulse, Chase), andpulsed for15 min in the presenceof10-4MTLCK(Pulse TLCK) and TPCK (PulseTPCK). Samplesontherightareof HSV-1(F)ICP synthesizedinthe absence(Pulse)orpresenceofinhibitors(TPCK, TLCK).Thepoliovirustype1proteinsare designated accordingtoJacobson,Asso,and Baltimore(9).Thenumberstotheright of HSV-1i(F) samples refer to selectedHSV-1(F) ICP. Theoriginal autoradiogram was cut for thepreparation of this figure, and the

exposure interval employed for theprinting ofthe autoradiograms of HSV-1(F)-Pulse TPCK samplewas longerthan thatemployed for other samples.The molecularweightestimatesof poliovirus proteins agreed well with those of virion proteins subjected to electrophoresis inperipheral chambers of thesamegel slab but removedtosimplifythisfigure.

TLCK, thepoliovirus protein profilewas

modi-fied in a rather minor way (compare sample TLCK with the controlpulse), leadingto detec-tion ofa novel polypeptide slightly larger than NCVP 0 with a molecular weight of

approxi-mately 175,000andtoaslightreduction in label

in the regionof bandsNCVP 3a and NCVP 3b

in comparison to the untreated control.

How-ever, 10' M TPCK drastically reduced

incor-porationinto totalprotein,almostabolishedthe

appearance ofsecondary products, and led to

the accumulation ofpolypeptides of molecular

weight235,000(NCVP 00?) and160,000(NCVP

0). In general our results concur with those of

Summerset al. (26).

(ii) In contrast, at 5.0 h postinfection, no

rapid posttranslational cleavagesweredetected

in herpesvirus-infected HEp-2 cells. Thus, in

*::bqx

E._A%,

*bc ^

1357 VOL.12,1973

4110mo 11--Aww.'

low

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samples infected with HSV-1 no difference either in the overallrateof protein synthesisor

theelectrophoretic profilesofpolypeptides

syn-thesizedwasdetectedbetween thecontrol

sam-ple (Fig. 6, pulse) and that labeled in the

presence of 10-4 MTLCK. Although overall

in-corporation was reduced in the culture labeled

in the presenceof10-4 M TPCK, there was no

clear difference between the mobilities ofICP

madeinthe presenceandabsenceofthe

inhibi-tor. With the exception of glycosylated ICP,

which were made in disproportionately lower

amounts, the remaining polypeptides were

la-beled inthe same relative proportions asthose

in the untreated control culture. More signifi-cantly, inviewof the

objectives

ofthese

experi-ments, therewas noaccumulation ofany poly-peptide ofhigher molecular weight than those detected during the control pulse. We exclude the possibility that a polyprotein too large to

enter the separation gel was produced under

these conditions since the radioactivity at the

interface between 3.5% "stacker" gel and 9.0%

"separation" gelwas aslow, relativeto radioac-tivity within the9.0%gel, asthatof the control

gel. Moreover, the exclusion limitof 9.0%gels is

> 335,000, basedontheentry ofthethyroglobin

polypeptide into this gel strength.

Changes

inthesynthesis of ICP during the

virus growth cycle. Three series of

experi-ments were done with the object ofexamining

changes

in overall proteinsynthesis and inthe synthesis ofindividual groups ofICP

through-outthevirus growthcycle. Inthe firstseries we

measured the overall rate ofprotein synthesis

by labeling

cells for 2-h intervalsthroughout the growth cycle ofthevirus. The incorporationof

"C-amino acids into acid-insoluble material

per 2 x 108 cellsper 2hinonesuch experiment

is shown in Fig. 7, along with the titers ofthe

infectious virus accumulated intracellularly

throughout

the same experiment. The overall

rate of synthesis first declined rather rapidly, reachingaminimumat 2 to 3hpostinfection, it

then recoveredto a maximum ratecomparable

tothat ofmock-infected control cultures at 5 to

6 h, and thereafter declined in a nonuniform manner.

In the preceding experiments and in those

which follow, ICP were labeled with leucine,

isoleucine,andvaline.Quantitative estimations

ofthe relative rateoramountofsynthesisofICP

could therefore be in error iftheir content of

these three amino acids was not uniform. To

examine this

possibility,

in thesecond series of

experiments two sets of

replicate

cultures of

cells infected with

HSV-1(F)

werelabeledwith

mixtures of

'4C-leucine,

isoleucine,

and valine

=

1-

4-Z._

l4

12

In

10

o

x 8 .C

E

4

.0

.s,

12

'

Time

(hrs)

FIG. 7. Accumulation of infectious virus (A) and changes in therateof amino acidincorporation into acid-insoluble material in infected (0) and mock-infected cells (0). Amino acid incorporation was measuredasacid-insoluble radioactivity at the end of 2-h labeling intervalsduring infection or mock infec-tion and plotted at the midpoint of the interval. Pointsforinfectedcultures are the mean of duplicate determinations (2 x 106 cells perdetermination) and thosefor mock-infected cells are results of a single determination. Accumulation of infectious virus was measuredby plaque titrations of duplicate samplesof homogenatescontaining 2 x10 infected cells for each timeinterval.

and with a mixture of thirteen

"C-labeled

amino acids (reconstituted protein

hydroly-zate), respectively. Labeling medium in

experi-ments with

leucine,

isoleucine,

and valine was

as described in Materials and

Methods;

the medium used for labeling with the "C-protein hydrolysate was EMEM with one-tenth the

normal concentrations of amino acids supple-mented with 1% dialyzed calfserum. Cultures

were labeled for 15-min intervals

beginning

at

7.25 and at 22.25 h postinfection. The labeled

samples were subjected to

electrophoresis

in parallelon a

polyacrylamide gel

slab.

Compari-son ofabsorbance tracingsofthe derived

auto-radiograms of the 7.25-h (Fig. 8) and of the 22.25-h (notshown)

labeling

intervalsindicated that, although the population of

polypeptides

synthesizedat the twotime periodswas

mark-edly different, at neither time was there any

significant difference between estimates ofthe

relative amounts of ICP synthesized based on

incorporation ofleucine, isoleucine, and valine

or of the mixture of thirteen labeled amino

acids.

It should be emphasized that these

findings

do not indicate that the amino acid

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(13)

PROTEINSSPECIFIEDBY HERPES SIMPLEX VIRUS

tions ofICP are uniform. In the same

experi-mentthereweresignificant differencesbetween the profiles of ICP labeled with the thirteen

amino acids and those labeled with single

amino acids (35S-methionine or "C-tyrosine).

However, in the context of this paper, the

pertinent observation is that

HSV-1(F)

ICP do

not differ significantly in the sum of their

contents of the three amino acids which we

routinely employedaslabeled precursors.

In the third series of experiments, replicate culturesofinfected cells were labeled from 2.5

to2.9, 3.75 to 4.0, 5.0 to 5.25,and11.0 to 11.5h

postinfection, respectively. These times were

chosenonthebasis of Fig.7andothertemporal features ofthevirus growth

cycle.

The interval

from 2.5 to 2.9h coincided with the end ofthe

initial decline in protein synthesis and before viral DNA synthesis is normally detected (14, 16). The next two intervals were

just

before (3.75to4.0h) andat (5.0to5.25

h) the

timeof maximal overall protein

synthesis

and

high

rates ofviral DNA synthesis. The last interval

(11.0to 11.5 h) was at atime atwhich rates of

protein and viralDNA

syntheses

were

declining

Distance [arbitrary units]

FIG. 8. Absorbance tracings of the autoradio-graphic images of polyacrylamide gelslabscontaining electrophoretically separated polypeptides from HSV-1(F)-infectedcells labeled with "4C-leucine,

iso-leucine,and valine(top panel)andwithamixtureof 13 "4C-amino acids (bottom panel) for a 15-min

interval from 7.25 h post-infection.

and the intracellular rate of accumulation of

infectious virus was near maximal.

Figure9showsanautoradiogram of a polyac-rylamide gel slab on which these samples were

subjected to electrophoresis. To facilitate the

presentation and interpretation of the data

available from this figure it is convenient to

express the amount of each ICP synthesized

during the various intervals as a "relative molar

amount synthesized," or a "relative molar rate

ofsynthesis" (RM). This datum was obtained

as follows: the autoradiograms of the gel slab

(Fig. 9) were scanned withthe aid of a Gilford spectrophotometer connected to a GA 16/45

computer. The latterdisplayed the absorbance

tracing, the absorbance ofeach band

(A,.

An)

and of the sum of the bands

(A,

... An)*

Since electrophoresis on more than one gel

strength wasnecessary toresolve certain of the 49ICP, the absorbanceofthosebands known to

containmorethan onepolypeptidewas divided

by

the number of known components, on the

formalassumption that theband was composed ofequal masses of its knownconstituents. The

relativemolar amount,RM, of protein i

synthe-sized was then obtainedfrom the relation:

RMi=

107

Kt.

At

Mi

(ZAi

.

.An)

where

Kt

istheoverallrateofprotein synthesis

atthetime ofthe determinationexpressed as a

fraction of thehighestobservedrate,

Ai

and

Mt

arethe absorbanceofband i andits molecular weight, respectively, and107 is a constant.

The RM

profiles

of the

polypeptides

detected

intheautoradiogramsofFig.9 areshowninFig.

10.

Figure11shows themannerinwhich theRM values of synthesis of polypeptides vary

throughout infection.

The results

presented

in Fig. 9 to 11 may be summarized as follows. (i)

During

any one

pulse-labeling

interval,the RM formost S,

NS,

and U polypeptides were within an eightfold

range; S and NS polypeptidesweredistributed

throughout this range. The most significant exceptions were the RM values ofthe S

poly-peptides, ICP1-2andICP 31, which differed

by

130-fold during the 11.0- to 11.5-h labeling

interval (Fig. 10) and formed the extremes of theobservedrange of values.

(ii) The variation in the relative proportions

ofS, NS, and U classes of

polypeptides

synthe-sized throughout the interval of the growth

cycle analyzed in this

study

was less than the

variation between polypeptides within the S

grouping. Thus, of thetotal amount ofprotein

VOL.12,1973 1359

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(14)

HONESSAND ROIZMAN

Lysates

of infected cells

7 ouised

over

intervals

as

indicated

-I

i5.0

Vi

r

i

o n

s

V

i

r

i

o n s

Vp r

p

C P I V P

1-2

1l2

3

Q

.... 1

14 l

1

^Q

...

'a!g_- 21

j22

. 23

2

3

23~~~~~~3

15 256

_

2625

16

l17 1

19 ^ * 31 l 31

-32 20

3

3534

st

3635

*

356

6423 j

42343

BPB BPB

FIG. 9. Autoradiogram of a part of a 7.75%polyacrylamide gel slab containing electrophoretically separated proteins pulse labeled in HSV-1(F)-infected cells during intervals shown on top of each sample. The legend on top of the third gel from the right indicates that cells were labeledfrom 5.0 to 5.5 h and then incubated in medium lacking labeled precursors until 9 hpostinfection. Peripheral samples are virion proteins, identified by largenumbers to the extreme right and left of the figure. The autoradiogram was cut in the preparation of this figure to permit convenient location of numbering for the ICP (smaller numbers between sample positions 2 and 3 and 5 and 6 of thisfigure).

synthesized, the S polypeptide fraction

(ex-pressed in moles percent) increasedonly

two-fold fromearly(2.5to2.9h)tolate(11.0to11.5 h) times in infection, whereas duringthesame

intervalthe molar ratios of SpolypeptideICP 5

to that of S polypeptide ICP 4 increased more than 30-fold.

(iii) Analysis of the kinetics ofsynthesis of

ICP (Fig. 11) showed sixpatterns ofsynthesis,

designated by the letters A toF. The

segrega-tion of ICP among these six patterns was

independentof the maximum rate ofsynthesis

of the polypeptides. However, the distribution

of S, NS, and U polypeptides among the six

groups was nonrandom. All but one of the

polypeptides presently included in group Aare

S. The exception, ICP 17, may bea long-lived

precursor since it disappeared on prolonged

chase (compare 5.0-to 5.25-hpulse and5.0- to

5.5-h pulse chased to 9.0 h shown in Fig. 9). Group C contained S polypeptides only. The S

ICP included in the analysis presented in Fig. 11 had an aggregate molecularweight of 2.3 x 106, and this S protein molecular weight was

4

5

6

7

8 9

11 ,

z

14

_mw _"w 8

_9

_w10

4 12 16

r::: .V

18 17

-19

:,~ _4 19 .;iw_ 20

21 M

4

5 6

6A 7 8 9

1°11

1211

1413

1360 J. VIROL.

fE% n

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(15)

PROTEINS SPECIFIED BY HERPES SIMPLEXVIRUS

2.5-2.9hr 2.7 32 7

23

4 i I

0~~~~~2

3.75 -4.0Ohr389

6 ~~~~~~~~2627 6

8 32

19 23

20 129 137~

25hr

~~~~~38-31

26

6~~~~~~~~~~3

I 1371

1~~~~~30i1

10~~~I~33

II i2I9

I

110-1155hr

26

21 32

4 S~~~~~~~~~~~83

36

1719 3

2 1L..,:1i

~~~~~J

250 ISO

10-

SO - 60 40

Molecular weight X

10-3

FIG. 10. Relative molar amounts of in} polypeptides synthesized duringshort labe

distributed in groups A, C, B, F, E, and D in

descending

order in amounts of 47, 31,8.1, 8.0,

4.2, and1.6%, respectively.

(iv) The rates of synthesis of polypeptides

0-43

comprising groupsA, B, C, and D showeithera

gradual increase (A), or an initial increase followed bya levelingoff(group B) or adecline

(groups C and D). As we noted earlier in the

text,

the initial stimulation in their rate of

synthesis

isadditionalevidence thatthese

poly-peptides

are virus

specific.

However,

polypep-tides of group E could be "very early"

virus-specific products or host polypeptides with

gradually diminishing rates of synthesis. It is

noteworthythat several polypeptides which do notfulfill the generalcriterion ofinduction, and

which are not precipitated by the antiserum

(e.g.,ICP34, 35 andICP42, 43;see Fig. 4 and

Table 1, columns 5and6), fall into groupE.

Patterns

F of Fig. 11 were probably conse-quences of

superimpositions

ofthe patterns of

synthesis

oftwoormore

polypeptides,

ofwhich

L.

one or morebelongedtoclass Eand the others

toclass

A, B, C,

orD. This artifactual

pattern

wasobserved for

polypeptides

migrating

in

gels

of a

particular

strength in

multiple,

incom-pletelyresolved bands. In the electropherogram illustrating this analysis (Fig. 9, 7.75% gel) the

groupsofICP34 to 36and40 to 43maybe cited

asexamplesof

poorly

resolved species

which,

in

40-43 the absenceofother

data,

would beincludedin

Analyses

of the same and similar

samples

on other

gel

strengths

(7 and 9%)

..

permitted

classification

ofICP34,35,42,and43

asgroup Epolypeptides (Table 1, column 5).

L

. DISCUSSION

Autoradiographic analyses

of

electrophoreti-cally

separated

polypeptides

in

polyacrylamide

gels

revealed that HEp-2 cells infected with HSV-1(F) makeat least49

polypeptides,

num-bered in order of decreasing size from 1 to49.

Some evidence forvirus

specificity

isavailable for47ofthese

polypeptides

and consists ofone

or more of the following: (i)

synthesis

of the

polypeptide

is

stimulated

after infection,

(ii)

the amount and in some instances the

electro-ffi phoreticmobilityare

determined

by thevirusin

40-43 that these parameters vary

depending

on the

virus type and intratype variant infecting the

vals computed from data shown in Fig. 9 by the

lli

procedure

described in the text. The relative molar il amountsof polypeptides (RM, see text for methodof 1.ra

computation) classified

as

S,

U, and NS are

repre-36

D sented by "solid"lines, "dotted" lines, and "dashed" lines,respectively.Thepolypeptidesareidentified by

fected cell ICP number (above bars) and themolecular weight

?ling inter- estimate

(abscissa).

VOL. 12, 1973

1361

-W 4D "d

4A

4D

z

21-%A

4D

7m Z. i

-LI',

10

.a..

a

0-

1--4D

E

6..cli

-2

40

E

IU

Z

4D 09

I

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[image:15.497.71.231.54.640.2]

(16)

0 2 4 6 8 10 0 2 4 6 8 10 0 2 4 6 8 10 Time (hrs)

FIG. 11. Patternsof synthesis of ICPinHSV-1(F)-infected cells. The ICPweregrouped bythepattern of synthesis designatedAtoF. SincetheRMfor the ICPcomprisingeachgroupare notthesame, theplotswere

normalized. However, themaximum RMobservedduringthereproductive cycle is shownunder column Min the boxedlegendnext to thenumber andsymbolicrepresentation of the ICP.

cell, and (iii) the polypeptides are precipitable by anti-HSV-1 antisera of known specificity.

The remaining two polypeptides (ICP 34 and

35) are probably host polypeptides, a

con-clusion reinforcedbytheobservation that these polypeptides comigrate with major proteins of

the uninfected cell.

These 47 virus-specific polypeptides havean

aggregate molecular weight corresponding to

that of about 41,000 amino acids. Assuming

that the distribution of "sense" information in

the HSV-1 DNA is asymmetricassuggestedby

analyses of transcription (6), HSV DNA can

specify the sequence ofa maximum of 55,000

amino acids. On the basisofthesecalculations

the47polypeptidesaccountforabout75% ofthe

maximum information contentofHSV-1 DNA.

Of the 27 known virion polypeptides, 24

"ana-logues," i.e., polypeptides with identical

elec-trophoretic mobilities, were provisionally

iden-tified among the47 ICP. Of the remainder, 15

did not correspond to any known virion poly-peptide and 9 remained unclassified, although

it islikely that most are nonstructural.

Polypep-tides specified by the remaining 25% of HSV

genetic information areeithermadeinamounts

too small to be detected or are obscured by

major polypeptides. Although the resolution

andidentification ofpolypeptides is incomplete

and their classification is provisional pending

furtherstudies, severalaspectsof the synthesis

of ICP are apparent from results of this paper

and meritdiscussion.

Posttranslational modification of viral polypeptides. At least three kinds of

posttrans-lational modifications may alter the mobility

ofviruspolypeptides, i.e., (i) rapid

posttransla-tional cleavages such as those seen in

picor-navirus-infectedcells (9, 25, 26), (ii) slow

post-translational cleavages determined by intracel-lular translocation or occurring during assembly

ofthevirion, and (iii) conjugation or addition of

prosthetic groups, e.g., glycosylation,

phospho-rylation, amidation, acetylation, methylation, etc. Studies presented in this paper have

ex-cluded rapid posttranslational cleavages of the

type seen in picornavirus-infected cells.

How-ever, a prolonged chase revealed modifications

inthemobilityofsomepolypeptides, notablyof

ICP 17and of a component ofICP 23 (Fig. 9).

The magnitude ofthese

changes

suggest that

ICP 17 is cleaved to asyet unknown products.

Thenatureofthe conversion ofacomponent of

ICP23 (to ICP24) is uncertain. Ingeneral, the

operation of slow modificationsrequiresfurther

study in view of the considerable number of HSV polypeptides which are substrates for

glycosylation (18,22) and

phosphorylation,

and

particularlysince wehavenotidentifiedprotein

22A ofB-capsids,theputative precursor toVP

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PROTEINS SPECIFIEDBYHERPES SIMPLEXVIRUS

22 (8; W. Gibson and B.

Roizman,

J.

Virol.,

submitted

for

publication)

inourpresent

analy-sis.

Kinetics of synthesis of structural and nonstructural polypeptides.

Analysis

of the kinetics ofsynthesis ofICP revealed six basic patternsdesignatedAthroughF.Of these six, F

isprobably artifactual, aconsequence of multi-ple comigrating polypeptides synthesized

ac-cordingtopattern E and

B,

C orD. Pattern E could describe equally well host proteins with progressivelydecliningratesof

synthesis

aswell

as virus proteins synthesized very

early

in

infection. Most of virus-specific polypeptides follow pattems A

through

D,

but not in a

random fashion. Thus, structural proteins and theirlikelyprecursorsalmost all followpatterns A and C, whereas NS polypeptides

clearly

identified by two or more criteria as virus-specific follow patterns B, D, and E. Several

commentsshould be made

regarding

patternsof

synthesis ofthese

polypeptides

and their

func-tion. Briefly, (i) it is convenient to regard S

polypeptides comprisinggroupC and the virus-specific NS polypeptides comprising groups E

as early.

Conversely,

the S

polypeptides

com-prising group A arelate, whereas the NS poly-peptides ofgroups Band D

might

be

designated

stable and

unstable, reflecting

their rates of synthesis late in infection. (ii)

Comparison

of

the rates of synthesis of the S polypeptides at

varioustimeintervalswiththeamountsofthese

polypeptides

in the virion show that

they

are notsynthesizedinproportiontotheir incorpora-tion intovirions. As seen fromdatapresentedin

Fig. 9, the probability of incorporation is not

uniform and appears to be much

higher

for

membersofgroup A

(late)

polypeptides

thanfor

group C (early)

polypeptides.

The

interpreta-tion of this observation is

presently

uncertain;

wemayspeculate that

early S polypeptides

may

have functions unrelated to their role in the

virion in addition to

having

aggregation rate

constants

markedly

different from those of

group Apolypeptides.

Temporal and abundance control of viral protein synthesis. In parallel with studies on

the control oftranscription (6, 7, 21), we are

concerned with two aspects of viral protein synthesis, i.e., temporal control ofprotein

syn-thesis and control of viral protein abundance.

Theexistence ofatemporalcontrol ofprotein

synthesis is apparent from analyses of the

kinetics ofsynthesis of viral proteins

summa-rized in Fig. 11. As discussedearlierinthetext,

the distribution of S, NS, and U

polypeptides

among the different kinetic classes is

nonran-dom, with NSproteinsfollowingpatternsB, D,

and

E,

and Sproteins largely following patterns

A and C. Because S polypeptides are

synthe-sized both early (pattern C) and late (pattern

A), there is no clear temporal differentiation

between S and NS proteins.The incorporation

of amino acids into S polypeptides early is therefore appreciable; S polypeptides incorpo-rated 53, 67, and 72% of total amino acids

during the labeling intervals beginning at 2.5,

3.75, and 11.0 h postinfection,respectively. We

conclude from the foregoing analyses that (i)

temporal controls ofHSV-1 gene expression do

exist, (ii) these controls definethesynthesis of atleast fivegroupsofproteins, and (iii)thereis no simple differentiation of proteins intoearly nonstructural and late structural. Structural

proteins are members of both early and late

groups and form a substantial fraction of total

proteins madethroughout infection.

The search for abundance control ofprotein

synthesis arises fromtheobservation that viral

transcripts form two classes differing in their accumulated mean molar abundance. The

abundant class is transcribed from 14 to 16%

and 19 to 22% of virus DNA at 2 and 8 h

postinfection, respectively, and is from 140- to atleast 15-foldmore