Fine-structure mapping and functional analysis of temperature-sensitive mutants in the gene encoding the herpes simplex virus type 1 immediate early protein VP175.

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JOURNAL OFVIROLOGY, Oct.1980,p.189-203

0022-538X/80/10-0189/15$02.00/0 Vol. 36, No. 1

Fine-Structure

Mapping and Functional Analysis of

Temperature-Sensitive

Mutants in the

Gene Encoding the

Herpes

Simplex

Virus Type

1 Immediate Early Protein VP175

RICHARD A.F. DIXON'2AND PRISCILLAA.SCHAFFER'*

TheSidney Farber CancerInstitute,HarvardMedical School, Boston, Massachusetts02115,1and

Department

of

Virology,

Baylor

CoUege of

Medicine,

Houston,

Texas770302

Herpes simplex virus (HSV)-specific proteins fall into at least three kinetic

classes whose synthesis issequentially and coordinately regulated.

Temperature-sensitive (ts) mutants of one complementation group (1-2) are defective in the

transition from immediate early to early and late protein synthesis. To elucidate thefunctionof the 1-2 gene product in the HSV type 1 replicative cycle, nine ts

mutants in this group were mapped by fine-structureanalysis and characterized

biochemically. Physicalmapping by homotypic marker rescue has shown thatall

members of the group lie within thetenninally repeatedsequencesof the S region

of the genome. Fine-structure geneticandphysical mapping permitted the

mu-tations to be ordered within these sequences.Because it has been shown that the

messagefor VP175 and the DNAtemplate

specifying

thisprotein extend beyond

the limits of thephysical map of the mutations, it follows that the mutations

mustlie within thestructuralgenefor VP175.Sodiumdodecyl

sulfate-polyacryl-amide gel electrophoresis analysis showed that most members of the group

overproduced the immediate early proteins VP175, -136, -110, and -63 and

markedlyunderproduced earlyandlateproteinsatthenonpermissive

tempera-ture. Intemperatureshiftup experiments,itwasfound that thesynthesisofearly

andlateproteinsceased, whereas the synthesis of immediateearlyproteins began

again. Thus, it is postulated that VP175 is (i) involved in the transition from

immediateearlytoearly protein synthesis, (ii) requiredcontinuouslytomaintain

early proteinsynthesis,and (iii)autoregulated, actingtoinhibit immediateearly proteinsynthesis.

The genome ofherpes

simplex

virus type 1

(HSV-1) is large

(molecular weight,

96 x

106)

and

structurally complex (1,

10, 11, 18, 38,

44).

Despiterapidprogress in the

genetic analysis

of

HSV, little is known about theregulationofviral

geneexpression. The observation that the

syn-thesis of three classes of viral polypeptides is

sequentiallyand

coordinately regulated

was

fol-lowedbythedemonisrationthat coordinate

reg-ulation isatleast

partly

controlledatthelevel oftranscription (7, 8, 13, 14, 17, 22, 24,25,

32, 43,

47). More

recently,.a

viral gene has been

iden-tifiedwhichactsvery

early

in the HSV

replica-tivecycle andcontrols the transition from im-mediate

early

to

early

and late

protein synthesis

(8, 24, 25,32).Thisgeneislocated in theterminal

repeats of S (20, 29, 33,

42)

and is

thought

to

encodeaviral

polypeptide

of molecular

weight

175,000,

designated

ICP4

(13)

orVP175

(8).

A

number of

temperature-sensitive (ts)

mutantsin

thisgene havebeenisolated(5, 28,

36).

Studies

with these mutants have revealed that VP175

controls a

transcriptional event(s)

which is

es-sential forthetransition fromimmediate

early

functionstoearlyandlatefunctions(28, 32, 47).

Additionally, the gene is diploid (20, 27, 33),

presentinganunique situation for genetic

anal-ysis.

It is currently believed that VP175 is the

product of gene 1-2; however, most of the

evi-dencetosupport this contention is

circumstan-tial. Thus,mutantsdefective in this gene

over-produce VP175, the mutant protein exhibits

al-tered intracellular compartmentalization and

migrationinsodium dodecyl sulfate(SDS)-gels,

and thetemplate

specifying

VP175 maps in the

same region of the genome as do mutants in

group 1-2(27,29, 41,42).Todetermine whether

the lesions in mutants in group 1-2 are in the coding sequence for VP175, we have mapped nine ts mutants in group 1-2 by physical and

geneticmethodsandcorrelated their order and

map locations with the known location of DNA

sequences whichspecify VP175 (27, 33) and the

mRNA which encodes thisprotein (48).

Other investigators have demonstrated that

onefunctionoftheproductofgene 1-2 involves

transcriptional regulation (32, 47). To further 189

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190 DIXON AND SCHAFFER

define therole of thisproteininregulation,we

have examined viral polypeptide synthesis by one mutantin the group,tsB21u,intemperature

shiftup experiments. Our results demonstrate

thatVP175 is involved inmediatingthe

transi-tion fromimmediateearlytoearly protein

syn-thesis, that it is required continuously for the

synthesis of some early and late proteins, and

that itacts toinhibit itsownsynthesis.

MATERLALS AND MErHODS Cellsandcellculture.Serially propagatedhuman embryoniclungfibroblasts(HEL), primaryrabbit kid-neycells(RK),andAfrican greenmonkeykidneycells (Vero) wereused. Cellsweregrown inEagleminimal essential medium(Autopow;FlowLaboratories,

Rock-vile,Md.) containing10% fetal bovineserum, 0.03% glutamine, and 0.025% NaHCO3. Cells were main-tained in thesamemediumcontaining5%fetalbovine serum.Virusstocksweregrown,andcomplementation

andrecombinationtests wereperformedin HELcells;

RKcellswereused for the isolation of viral DNA and for markerrescuestudies.Virusinfectivityassayswere performedin Vero cells.

Viruses and virus assays. The KOS strain of HSV-1 was used as the wild-type virus (40). The isolation ofmutants tsB2b, tsB21u, tsB27h, tsB28h,

tsB32h,tsJ12g,ts90l,tsLB2, tsD,and tsc75 has been

reportedpreviously (4,5, 12,15,20,34).The last four

mutants wereobtained from M.Levine,I.Halliburton,

J. Subak-Sharpe, and R. Honess,respectively. Virus stocks were prepared and infectivities were deter-mined as previously described (36). In all tests, the

permissivetemperaturewas34°Cand the

nonpermis-sivetemperaturewas390C.

Complementation tests and mapping proce-dures. Mutants tsB2b, ts9Ol, tMD, and tsLB2 were showninapreviousstudytodefinecomplementation

group 1-2 (36). Complementation analysis with the othermutantsused inthisstudyis describedherein.

Complementationtestsandrecombination tests using two-factorcrosses werecarriedout asdescribed

pre-viously(34,37).Markerrescueexperimentswere per-formedasreportedbyChuetal.(5). The restriction maps of strainKOS used todetermine the physical locationsof mutations in markerrescuestudies were taken from Skare and Summers (EcoRI) (39) and Wagner andSummers (HincII) (45) or weregenerated

in thislaboratory(BamHI).

Isolation ofviralDNA, digestion with

restric-tionenzymes, and agarose gel electrophoresis.

DNAwasisolated frompartially purifiedcytoplasmic andextracellular virionsasdescribedby Chu et al. (5). Restrictionenzymes were purchased from New Eng-land Biolabs (Boston, Mass.), and DNA was digested with2U ofenzyme per

jig

ofDNA. Fragments were separated byelectrophoresis on horizontal slab gels andisolatedaspreviously described (5).

Analysisofinfected cellpolypeptides.HEL cell

monolayerswereinfectedat amultiplicity of 100PFU/

cell andincubated in water bathsat either 34 or

390C

under the conditions describedin the text. Polypep-tideswerelabeled with 5pCiof

'4C-amino

acids per

ml (Schwarz/Mann, Orangeburg, N.Y.; amino acid

mixture, 50 to 60 mCi/matom) in maintenance

me-dium containing 10% of the normal amounts of all amino acids except arginine and glutamine, which wereused in normal amounts. Immediateearly pro-teins (31) were induced in infected cultures treated with 50 ug ofcycloheximide (Calbiochem, LaJolla,

Calif.) per ml from 0 to 7 hpostinfection. Thedrug wasremoved after7h,monolayerswerewashed five times withTris-saline, and mediumcontaining5 uCi of'4C-amino acids and 15pgofactinomycinD (Cal-biochem)permlwasadded.Cellswereharvestedat8 h postinfection by scraping into the medium. They

werewashed threetimes in Tris-buffered slineand suspended in water, and the resulting suspensions

werefrozen.Sampleswerelaterthawed,sonicatedfor 2min,adjustedto0.05MTris-hydrochloride (pH

6.7)-1%SDS-1%2-mercaptoethanol-0.5M urea, andboiled

for 2 min.Electrophoresiswascarriedout on7to15%

lineargradientSDS-polyacrylamideslabgels (23).The

gelswere fixed in7% acetic acid and 50%methanol, impregnated with Enhance (New England Nuclear

Corp.,Boston,Mass.),and dried. The driedgelswere

thenfluorographedat-70°ConKodakSB-5film.

RESULTS

Complementation studies with ts

mu-tants. Intwoseparatetests, eightof theninets

mutantsusedin thisstudywereshowntobelong

tocomplementation group 1-2 (5, 36).To

con-firn the assignment of these mutants and of

tsc75 (20) togroup 1-2,aquantitative

yield-of-progeny complementation test was performed

(Table 1); tsJl2g, a memberofgroup 1-9, was

used as the positive control (36). None of the

ninemutantstested complementedeach other,

whereas all complemented tsJ12g (Table 1).

Thus, all ninemutantsbelongedtogroup 1-2.It

should be noted that theninemutantsincluded

in this study behave as if they possess point

mutations inasinglegene;i.e.,theyrevertwith

lowtomoderatefrequency(datanotshown) and

exhibitnonoverlappingpatterns of

complemen-tation intestswith members ofothergroups (5,

36).

Physical mapping experiments. Marker

rescuestudieswereundertakentodeterminethe

physical map locations of the nine ts mutants.

We have reported previously that tsB21u lies

withinthe "c"sequencesin theterminal repeats

of the Sregionof thegenome(29).Thislocation

is in agreement with thefindings of Stow and

Wilkie(42) andKnipeetal. (20,21), whoplaced

tsLB2,tsD, andtsc75withinEcoRIfragment K,

which contains S-terminal sequences "c" and

Sta."Ps

Table 2 shows the results of marker rescue

experiments using purified EcoRI fragments

shown inFig. 1.tsLB2,tsD,and tsc75hadbeen

shown previously to bein fragment K (20, 21,

42). Efficientrescuewith the remaining six

mu-tants was observed with fragment K (0.966 to

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HSV-1 ts MUTATIONS IN VP175 191

TABLE 1. Complementation between members ofgroup 1-2

Virus B2 B21 B27 B28 B32 901 LB2 tsD tsc75 tsJl2g

tsB2b 1.5a 0.70 0.81 0.37 0.40 0.55 0.48 0.70 170

tsB21u 0.31 0.27 0.86 0.76 0.88 0.33 0.32 4.9

tsB27h 0.28 0.18 1.9 1.5 0.61 0.68 19

tsB28h 0.20 0.42 0.68 0.13 0.50 72

tsB32h 1.4 0.84 0.28 0.38 580

ts901 0.45 0.10 0.20 570

tsLB2 0.44 0.91 22

tsD 0.35 444

tsc75 30

aComplementation index=[yield (A +

B)]/[yield

(A)+(B)]; infected cultures wereincubated at 39°C, and yields were assayed at 34°C; values greater than 2.0 (in italics) were consideredpositive(34).

TABLE 2. Marker rescuetests with mutants in group 1-2: rescue with EcoRI fragments

Fragment' Virus

A,B,C D,E,F,G H I,J K None tsB2b 4.0 0.0 0.0 0.0 0.7 0.0

tsB21u 10.0 0.0 0.0 0.0 9.6 0.0

tsB27h 1.5 0.0 0.0 0.0 1.0 0.0 tsB28h 0.7 0.0 0.0 0.0 0.6 0.0 tsB32h 7.0 0.0 0.0 0.0 41.0 0.0 ts901 22.0 0.0 0.0 0.2 1.0 0.0

aValues represent the ratio of the yield of progeny

virusfrom transfectedcells when assayed at 39°C as compared with the yieldat340C(xlOO). Virus titers

at34°C were always greater than 105 PFU/ml.

1.000) and with amixture offragments A, B, and

C; fragments B and C are joint-spanning

frag-ments, both of which contain sequences in K.

TheEcoRI Kfragmentlies

entirely

withinthe

terminallyrepeated sequences of the Sregion.

Althoughwehavenotdetermined whether each

mutation is present within both repeats, it is

likely that this is thecase, sincefragments

rep-resenting both ends of S have been shown to

rescuetsB21u and thepresenceof the lesion in

only one end would permit self-rescue of the

mutant(29). Moreover,like

tsB21u,

theterminal

fragments of the L region (J and E) failed to

rescue,indicatingthatnoneof the mutationslies

within"a"sequencespresentatthe

joint

andat

the terminiof themolecule.

Rather,

the

muta-tions lie within the "c"sequences

adjacent

to

Us

(20, 29). In the case of ts901, the low level of

rescueobservedwithcombined

fragments

Iand

J wasprobably duetocontamination of theI,J

mixturewithfragmentK,becausealthough

frag-mentsJ andKsharesome commonsequences, rescuewas notobserved withfragmentE.

To further refine the map positions of the

mutants, EcoRI fragment K was isolated and

redigested with BamHI and

HincII;

each of these enzymesmakesa

single

cutwithin theK

fragment,resultingin threefragments

(2.0,

1.85,

and 1.25 kilobases

[kb])

which are

easily

re-solved(Fig. 1).When these fragmentswereused

in marker rescue tests, the results shown in

Table 3 were obtained. With tsB2b, tsB21u,

tsB27h, tsB28h, and tsB32h, rescue occurred

maxrimally

with the tenninal HincII fragment

K2 (0.988 to 1.000), whereas with ts901,rescue

occurred only with the middle fragment, K3

(0.979 to 0.988). Fine-structure mapping of

tsLB2 and tsc75 demonstrated that with the

formermutant rescue occurredonlywith

frag-mentKl,whereas with thelattermutantrescue

occurred with both K2 and K3.

Asummaryof thephysicalmap locations of

all nine mutants is presentedin Fig. 2. Inthis

figure, the limits of fragments which exhibited

rescue areshownasbars. Thewhiteportion of

each bar indicatessequencesinwhichmutations

are thoughtnot to lie bythe process of

elimi-nation;e.g.,failure of the termini of Ltorescue

tsB32h, tsB28h, tsB27h, tsB2b, and tsB21u

ex-cludestheirlyingin the "a"sequences,whereas

rescue oftsc75 by all four terminal fragments

excludesits lying in the "c"sequences. Because

direct tests of the sequences excluded by the

processof elimination havenotbeenconducted,

theoutermostlimitsof each barshould be

con-sidered definitive. The location of tsD (0.966to

0.900) was taken from Stow et al. (41, 42). It

should be mentioned that Wilkie and his

col-leagues haverecently refined the limits of tsD

and of tsK, another member of group 1-2, to

coordinates, 0.979 to 0.990 and 0.966 to 0.978,

respectively (personal communication).The

se-quence of mutations on the

physical

map is

therefore tsK-tsLB2,tsD-ts901,

tsB32h-tsB28h-tsB27h-tsB2b-tsB21u,andtsc75.

Thelocation of thesequencespecifyingVP175 asdeternined byheterotypicrecombinationand marker rescue (27, 33) and the 4.7-kb mRNA

which codes for VP175 (48) are also shown in

Fig.2.Becausethemapcoordinatesof the mu-tantsliewithinthelimitssetforthe mRNAand

VP175 structural polypeptide and because no

evidence for

splicing

ofthegene forVP175 has

been reported, it is assumed that each ofthe

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FIG. 1. EcoRI restriction map of HSV-1, strain KOS. (1) L andS componentsofthe viral genome; (2) sequenceorganization; (3)fractionaldistance; (4)EcoRI restriction map in the P orientationofthe genome

(27)(fragments B andCarejoint-spanningfragments containingEplusK and JplusK, respectively); (5)

BamHI/HincIIrestriction mapofEcoRIfragmentK. TheterminiofK,aredeterminedbyEcoRI andBamHI

sites, theterminiofK3aredeterninedbyBamHI andHincII, and theterminiofK2aredeterminedbythe

HincIIsite and thephysicalendofthe Sregion. Thegel profileontheleftis apreparative gel of EcoRI-cleavedHSV-1 DNA fragments. The Kfragmentwasexcised and digestedwith BamHIandHincII. The

resultingfragmentsareshownin thegelprofileontheright.

mutants is defective inthe structural gene for

VP175.

Genetic mapping experiments. Because

markerrescue testsdidnotsucceed inseparating

all mutations physically within the repeat, a

linkage map was constructed by two-factor

crosses in an effortto order the mutantswith

respect to one another (Table 4, Fig. 2). The

following observations concerning the linkage

map are notable. First, the mutants clustered

into four groups, each separated by 1.4 to 1.9

recombination units. The firstgroup contained

mutantstsB2b, tsB21u,andtsB27h; the second

contained mutantstsB28h, tsB32h, and tsLB2;

the third contained tsD; and the fourth

con-tainedts9Ol.Second, although themutants

clus-tered into groups, no mutational hot-spotting

wasevident(i.e.,allmutationsinducedwithone

mutagen were not located in one place). Both

thymidine-specific (bromodeoxyuridine and

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VOL. 36, 1980

UV) andcytosine-specific (hydroxylamine) mu-tagens wereusedformutantinduction, and

mu-tationsderivedwitheithertypeofmutagen were

randomlyarrangedonthemap.Third, although

the physicalmap is notsufficiently detailed to

establish the order ofmutantswithin eachof the

groups, thegenetic andphysicalmaps are

coli-nearwith respect tothe order ofseven of the nine mutants tested: tsD-ts901 and

tsB32h-tsB28h-tsB27h-tsB2b-tsB21u.Only tsLB2 isout

of orderonthegeneticmap.Fourth, the

recom-TABLE 3. Marker rescue tests with mutants in group 1-2: rescue withBamHI,HincII-cleaved

EcoRI-K Fragmenta Virus

Ki K2 K3 None

tsB2b 0.0 2.1 0.0 0.0

tsB21u 0.0 4.5 0.3 0.0

tsB27h 0.0 0.3 0.0 0.0

tsB28h 0.0 0.1 0.0 0.0

tsB32h 0.0 1.4 0.0 0.0

ts901 0.0 0.0 5.4 0.0

tsLB2 7.3 0.0 0.0 0.0

tsc75 0.0 14.0 7.6 0.0

aSee Table 2.

c

0950 0.960 0.9,66 0.970

binationfrequencies observed were largerthan

one might expect given the total physical

dis-tanceinvolved (3.1kb). In previous studies, the greatestdistancebetween mutations inasingle

geneinuniquesequencesin L was 3.3

recombi-nation units (37) (unpublished data). The high

frequencyofrecombination between mutants of

group 1-2could reflect (i) auniquepropertyof the repeated "c" sequences in that they may constitute a region of very activerecombination,

(ii) the diploid nature ofthe gene, or (iii) the

large size of thestructural gene for VP175. In

TABLE 4. Recombination betweenmutantsingroup

1-2

Virus B2 B21 B27 B28 B32 901 LB2 D c75

tsB2b 0.36a 0.51 3.2 4.1 4.0 1.4 2.2 0.87

tsB21u 0.91 4.2 2.8 7.7 2.3 2.1 1.6

tsB27h 4.9 2.7 6.1 1.4 1.3 1.8

tsB28h 0.11 3.8 0.73 4.5 0.31

tsB32h 3.4 0.71 1.45 0.55

ts9Ol 2.0 1.9 0.89

tsLB2 3.5 0.40

tsD 0.0

tsc75

a D~omomaun irequency=! r /IA

-Recombmation

frequency

=

[yi4

39°C]/yield (A+B)at34°C]x2 x ]

a

0.9790.980 0.9880.990

B32B28,B27,B2,B

901

D

eld (A + B) at

100. SEQUENCE ORGANIZATION

VP175CODING SEQUENCE

4.7kb mRNA

1.000 FRACTIONAL

DISTANCE 121

c75 PHYSICAL MAP LB2

GENETIC MAP

FIG. 2. Physicalandgeneticmapsofmutantsincomplementationgroup 1-2. TheVP175codingsequence wastakenfromPrestonetal. (33) andMorseetal. (27). Thelimitsofthe4.7-kb mRNA anddirectionof transcriptionaretakenfrom Watsonetal.(48)andClementsetal.(6). Thefractionaldistance barindicates thepositionsof cleavagesitesofrestrictionenzymesusedin thisstudy:0.966=EcoRI,0.979=BamHI,and

0.988=HincII. The maximum limitsofthesequencesinwhich themutationsmaparepresentedassolid bars

(indicatingsequencesin whichthemutation isthoughttolie)andopenbars(indicatingsequences inwhich themutationprobablydoesnotlieasdeducedbytheprocessof elimination; seetextfor explanation). The geneticmapwasconstructedfromthe datapresentedin Table3.

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contrast to most mutants, low recombination group1-2. Todetermine whether all members of

frequencies wereconsistentlyobservedbetween the group examined in this

study

exhibit this

tsc75 and other mutants (20) (Table4). More- property,cellswereinfectedwith eachmutant,

over, tsc75 appearsto becloselylinkedto

tMD,

incubated at

39°C,

andlabeled from5 to 24 h

yet it is located in the "a"sequencesatthe end postinfection. All of the mutants exhibited a

of thephysical map. It may be thatmutanttsc75 similarbutnotidentical

polypeptide

phenotype

contains multiplemutations,thepossibleorigins at

390C

(Fig. 3).Thus,for allmutants,

immedi-ofwhich will be discussed later. ateearlypolypeptides

VP175,

-68,and-63 were

Polypeptide synthesis in ts mutant-in- overproduced. These

polypeptides

correspond

fected cells. We have shown

previously

that to

ICP4,

-22, and

-27,

respectively,

of Honess

tsB2b andtsB21uoverproduce immediate

early

andRoizman

(13).

Although

VP136

(ICP6)

was

polypeptides VP175, -136, -110, and -63 (8, 9). overproducedbyallmutantsexcept tsB28h and

Similarfindingshave beenreportedby Marsden tsB32h, VP110

(ICPO)

was overproduced only

et al. (26) and Knipe et al. (21) with

tsD, tsK, by

mutants not derived from strain

KOS,

i.e.,

tsT, andtsLB2, all members ofcomplementation

tsLB2,

tsc75, andtsD.

Additionally,

someearly

F M KOS82 B2 B27 LB2 B28 B32901 C75 D ;Std

A&MIL ~ | 168 175 175 184i4;

148 155 154

153,515

135 136 148 146(6

- '16 P0 *34 '135O.

968 76762F

62 64 65 g 26;

60 63 64 27

_U

00:

o6

, *$ , ;l

FIG. 3. SDS-PAGE analysis of viral proteins synthesized in cells infected with ts mutants in complemen-tationgroup 1-2. HEL cells wereinfected with100PFU/cellat39°C, labeled with14C-amino acids from 5 to

24hpostinfection, andprocessed for PAGE as described in Materials and Methods.Abbreviations: M, mock-infected cells; IE, immediateearlypolypeptides; KOS, wild-type virus; std, molecular weight standards in

decreasingorder(220,000,93,000, 69,000, 44,000,30,000,and12,000). Themolecular weights of viral proteins presented are thosereported here and by otherinvestigators: A, this study; B, Marsden etal.(26)andPreston (31); C, Courtneyetal. (9);D,Honess andRoizman (13)andPereira etal. (30). Valuesindicatemolecular weightsx103.Numbers inparentheses are ICP designations ofHonessandRoizman (13).

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andlateviralpolypeptides(e.g.,VP64 and-155,

respectively) were synthesized by all mutants.

That the variations may reflect strain-specific

differences in gene expression cannot be

ex-cluded. However,analtemativeexplanation for

the observed variation concerns the fact that

somemutantsexhibitgreaterleakat39°C than

othersderived from thesamestrain. Thus, for

example, tsB28h and tsB32h synthesizedgreater

amounts oflate proteins at 390C than did the

otherKOSmutants (datanotshown). Figure3

alsoincludes the various molecular weights and

nomenclaturesused in the literaturetodescribe

thesepolypeptides. Inthisreport, wehave used

thenomenclature of Preston (31)(Fig.3,column

B).

Temperature shiftexperiments.To

deter-mine whetherfunctional VP175 isrequiredonly

transientlyor throughout the replicative cycle,

temperature shiftup experiments were

per-formed with tsB21u. In the first experiment,

tsB21u-infectedcellswereincubatedfor various

lengths of time at34°C before shiftupto

390C;

allcultureswerelabeledatthetimeof shift and

harvestedat24h (Fig.4).

When cultures were incubated continuously

at 390C (Fig. 4, lane b), overproduction of

im-mediate early polypeptides VP175, -136, -110,

and -63 wasevident. Late polypeptides VP154

and VP100 and early polypeptide VP64 were

minor species in these cells. In contrast, early

andlatepolypeptides predominatedincultures

incubated at340Cfrom 1,2,4, or 8through24

h(Fig.4,lanesc, e, g,andi).

Inculturesshiftedup at 1, 2,and4h (Fig.4,

lanesd,f, andh),immediateearly polypeptides

VP175, -136, and-63 werepredominant,

consid-erablymorebeingsynthesizedthan in the

340C

controls.Thus,immediateearly protein

synthe-siswascontinuous (i.e.,notshutoff) after

shif-tup. VP110 was also present, but to a lesser

extent than the other three polypeptides. In

cultures shiftedupat4h(lane h), VP175,-136,

-110, and -63 were present in greater amounts

than inthe340Ccontrols(lane g),but increased

amountsof VP154 and-64 were also observed. The synthesis ofVP110 is difficultto evaluate

intheseexperiments. Althoughlittlewasseenin

theshiftat2h andmore was seeninthe shifts

at 4and8h,thisincrease may be duetothefact

that anearlypolypeptide comigrateswithVP110

(30). Thus, theproteinseen atlater timesmay

bethe immediateearly polypeptide,VP110,the

earlypolypeptide,orboth.Clearly,however,the

synthesis of VP175, -136, and -VP63 was not

shut off after shiftup even though many early

andlateproteinswerebeingsynthesized

simul-taneously.Thus,despitethecurrenthypothesis

thatearly viralproteinsarerequiredtoturnoff

immediate early protein synthesis, these

pro-teins continuedtobe synthesized inthe presence

of early proteins. Because the only essential

lesion in tsB21u is in the gene for VP175, these

observationssuggestthe possibility that VP175

is involved in the inhibitionof immediate early

protein synthesisor,alternatively, thatVP175 is

required continuously to produce a factor

re-quiredto shut off the synthesis of this class of

polypeptides. Even whenthe shiftoccurred as

late as 8 h (Fig. 4, lane j), the expression of

VP175wasstillgreaterthan in the

340C

control

(lane i).Additional differenceswerenotedwhen

proteins were synthesized after shift to

390C

(lane j) compared with unshifted cultures (lane

i;seedots between lanes i and j). Thus,it appears

thatfunctional VP175 is requiredevenafter8h

for theexpression of these proteins, butnotfor

theexpression of all early and late proteins.

Anadditional observation made fromthisgel

concernsvariationintheelectrophoretic

mobil-ity of VP175.Inallcases,the VP175polypeptide

made at

340C

migrated moreslowly than that

madeat

390C

(mostclearlyseeninlanese,f,g,

and hofFig. 4). This observation is inagreement

with the observations ofPreston, who showed

that incells infected with tsK, different forms of

VP175weresynthesizedatpermissiveand

non-permissive temperature (31), and of Pereira et

al. (30) and Wilcox etal. (49), who foundthree

forms ofwild-typeVP175 inpulse-chase

experi-ments.

Functional VP175 has been shown to be

re-quiredonlytransientlyfor viral DNAsynthesis,

therequirementbeing bypassed bya2-h

prein-cubationat

340C

(35). The results of thepresent

study demonstrate that continued synthesis of

VP175 isrequiredfor the expressionof certain

late viralgeneproducts (see above).The

require-ment for functional VP175 in the synthesis of

infectious virus, however, has not been

exam-ined.Therefore, the yield of infectious virus in

cultures infectedasforshiftuptests(Fig. 4)was

measured(Table 5).

Inunshiftedcultures,tsB21uwastemperature

sensitive for growth when compared with the

wild-type virus, as expected. When

mutant-in-fected cultureswere shiftedup at1, 2, 4, and8

handyieldswereassayedafter24h, theywere

only10-foldhigherthan theyieldfrom the

cul-tureincubatedat

390C.

Thus,unlike viral DNA

synthesis, functional VP175 is apparently

re-quiredafter8hfor theproductionofinfectious

virus. Moreover,VP175appearedtoberequired

throughoutthe18-hgrowth cycle,since theyield

of infectious viruswasreduced 10-foldcompared

with unshifted cultures when cultures were

shifted up at 18 h (data not shown).

Alterna-tively, given the greatly diminished amount of

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i< b d e f g h

D--p!

Am " ..

_ iF_g~~~~~~~~~~~~~~~~~~~~~~~~~-41

4_ 4m4_

4000 404M 4mm~~~~~~~~~~~~~~~~~~~~~~~~-t-~~~~~ VTl/:X; VP]5sa VP13C3p

VPIL

Vtp r

~~~~s 4

FIG. 4. SDS-PAGEanalysisofviralproteins synthe8izedin cellsinfectedwithtsB2lu aftertemtperature shiftup. (a) Mock-infectedHEL cells labeledfr-om4to24hat3900;(b) tsB2lu-infectedcellsincubatedat 3900 andlabeledfrom4to24h; (c-i)cellsinfected at3400 and held continuouslyatthistemperature or

shiftedupto3900 atdesignated times; (c,e,g,andi)cells incubatedat3400 andlabeledat1, 2, 4,and 8h,

respectively; (d,f, h, andj) cellsshiftedupandlabeledat1, 2, 4,and 8h,respectively; (k and1)wild-type

virus-infectedcells labeledfrom4to24hat34and3900,respectively.Allsamipleswereharvestedat24h and

prepared forPAGE and virusassay(TableS5)asdescribed in Materials and Methods.

VP175 synthesized after shiftup at 8 h (Fig. 4, lanej), it is possible that the same sequences whichspecifyVP175 alsospecify a later function

requiredforinfectivity,and thatamutation in

the sequences specifying VP175 could also in-activate thisputativelater function.

The experiment illustratedin Fig. 4

demon-strated that VP175 synthesis was notshut off J. VIROL.

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HSV-1 ts MUTATIONS IN VP175 197 TABLE 5. Yieldof tsB21u and wild-type virus after

temperature shiftup Initial

temp of Time of

Virus tiona shiftup (h) Virusyield

(OC)

tsB21u 34 -a 6 X107

39 - 8x 103

34 1 3x 104

34 2 3x 104

34 4 4x 104

34 8 8x 104

Wildtype 34 - 1 x l08

39 - 1 x 108

a

-,

Not shifted.

whencultureswereshiftedup at 1 to 4h(lanes

fandb,respectively; notclear from thisgel,but

seeninotherexperiments),indicatingthat

max-imumsynthesis of VP136occurslater than other

immediate early polypeptides, an observation

thatled Pereiraetal.toclassifythispolypeptide

in the group of early or ,B polypeptides (30).

Whencells wereshifted up at4h andlabeled

from4 to8h(laned), immediate early (VP175,

-136, -110, and-63), early(e.g., VP64), and late

(e.g.,VP154)polypeptidesweresynthesized.

Be-causelittlesynthesis of VP175wasobserved at

340C

during this period (lane f), this finding

suggests that continued (or possibly renewed)

expression of immediateearlypolypeptides

oc-curredinconjunctionwith theexpressionoflate

polypeptides.Pulse-labelingforperiodsasshort

as 30 minproduced thesame results (datanot

shown). Shiftingup atlatertimes (i.e.,8 to 12

and 12 to 24h; lanesgandj) didnotresult in

theaccumulation of VP175orVP136; however,

the synthesis of VP63 (and, to alesser extent,

VP110)wasenhancedrelativetoitssynthesisin

34and

390C

controlcultures (laneshand i and

lanes k and 1). Asin the case of infected cells

shiftedupfrom4 to8h,thesynthesis of

imme-diate early polypeptides (VP63 and -110)

oc-curredsimultaneouslywith thesynthesisoflate

(VP100 and-154) polypeptides.

Totestthepossibility that theaccumulation

of immediate early polypeptides in cultures

shifted to

390C

was duetotheirincreased

sta-bilityatthis temperature, mutant-infectedcells

werepulse-labeledat

390C

from4 to4.5h (Fig.

5, lane m) and chased from4.5 to 8 hat

390C

(lane n)orafter shiftdownto

340C

(lane o).The

labeled,chasedpolypeptideswereequallystable

atbothtemperatures(lanesnando),supporting

theconcept that theaccumulation ofimmediate

early proteins at

390C

aftershiftup wasdueto

continuedorincreasedsynthesisrather thanto

decreased turnover. Although it appears that

somesynthesis continued duringthe chase

pe-riod(Fig. 5), suchsynthesis was not observed in

other experiments. Therelative stability of the

protein at the two temperatures was identical,

however.

Additional evidence for the reexpression of

immediate early polypeptides after shiftup

comesfrom the observationthat when

tsB21u-infected cellswereshiftedat 4 or 8 h and either

pulse-labeled immediately for 1 h or

pulse-la-beled 2 h after the shiftup, the same level of

synthesis of VP175 was observed (data not

shown). Because the half-life of the early

mRNA's isonly1 h (46), it would benecessary

to synthesizenew mRNA in order to maintain

thesame rateofprotein synthesis.In this

exper-iment, expression of VP175 in cultures shifted

up at 8hwasobserved, whereasno expression

of VP175 was observed in cultures labeled at

340C during the same period, again indicating

that VP175 is reexpressed and therefore

in-volved ininhibiting itsownsynthesis.

Anadditional observation made from the gels

shown in Fig. 5 concerns the active form of

VP175.ItisapparentfromFig.4and5thatthe

form of VP175 whichaccumulatedat340Cwas

ofhigher apparent molecular weight than the

form which accumulated at 390C. After

pulse-labeling, afast-migratingform wassynthesized,

and this form could be chased to a

slower-mi-grating form which accumulatedat390C (Fig.5,

lanesmandn). After shift from39to340Cand

chase (lane o), aslight changeinapparent

mo-lecularweightwasevident. As mentionedabove,

the finding of three electrophoretic variants of

VP175is consistent with thereportsof Pereira

etal. (30), Preston (31), andWilcox etal. (49),

who have described three forms of theprotein

(VP175a, -b,and -corICP4a, -b,and-c) which

shareaprecursor-productrelationship. Preston

noted thatuponshift from34to

390C,

VP175c

wasconvertedto VP175b, with loss ofactivity

(31).Consistent with thisobservation,wefound

that VP175b was converted to VP175c upon

shiftdown. Of additional interest is Preston's

findingthatupon shiftdown in the presence of

cycloheximide, no new protein synthesis was

required forearly transcription(32). From these

observations,itappearsthat the "c"forn is the

active form of VP175. Although the nature of

the modification in VP175 isnotknown,Wilcox

etal. (49)havepresentedevidence which

impli-catesphosphorylationinthemodification proc-ess.Whatever themechanismofmodification,it

isessentialforfunctionalactivity.

DISCUSSION

Theresultspresentedheresupport and

com-plement previousfindings concerningthe

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a b c d e f g h i

I

k I m n o

.VP1.4

VP136

.i~~~~~~~~~~~p-.0

* ~ _

.,A

_._Jss

1.

0

*. 44

- -

VP4

-4

VPP53

-*m.

FIG. 5. SDS-PAGEanalysis ofviralproteinssynthesizedin cellsinfectedwith tsB21uaftertemperature

shiftup andpulse-labeling. HEL cells wereinfectedwith 100PFU/celland labeledasfollows: (a)

mock-infectedcells labeledfrom4to8hat34°C; (bandc)tsB21u-infectedcellslabeledfrom1 to4hat34and 39°C,respectively; (d)tsB21u-infectedcellsshifted from34to39°Cat4hand labeledfrom4 to8h;(eandf) tsB21u-infectedcellslabeledfrom4to8hat39and34°C,respectively; (g)tsB21u-infectedcellsshiftedup from34 to39°Cat8h and labeledfrom8to12h;(handi)tsB21u-infectedcellslabeledfrom8to12hat39

and34°C,respectively; (j) tsB21u-infectedcellsshiftedfrom34 to39°Cat12h andlabeledfrom 12to 24h;

(kand1)tsB21u-infectedcellslabeledfrom12 to24 hat39and34°C,respectively;(m-o)tsB21u-infectedcells labeledat39°C from4to4.5h andharvestedat4.5h(m)orchased with coldamino acid-containingmedia

from2.4 to 8hat39°C (n)or34°C (o).

tion and function of the gene encoding VP175.

Theoriginaldescriptionofcascaderegulation in

theHSV-1replicativecycle(13) ledtothe

iden-tification of a group of proteins (immediate

early,ora, proteins)whicharesynthesized

im-mediatelyafter infection and whosesynthesisis

required fortheinitiationofsynthesis of a

sec-ond group ofproteins, theearly, or,B,proteins.

Early proteinswereshowntoplay arolein the

shutoff of immediateearly protein synthesis(13,

14).

Amongthe immediateearly proteins first

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scribed were VP175, -110, and -63 (13). The

existence ofa mutant, tsB2b, which

overpro-duced VP175 andwasdefectivein thetransition

from immediate early tolate protein synthesis

was reported simultaneously (8). The latter

study also demonstratedthat in tsB2b-infected

cells, VP175wasnottransportedto thenucleus

asefficientlyasin wild-typevirus-infectedcells

at the nonpermissive temperature. Both the

transition fromimmediate early to late protein

synthesisandtransport tothe nucleuscould be

reverseduponshiftdown and bypassed by

prein-cubationatthepermissivetemperature,

indicat-ing that the defective function in this mutant

was bothearly and reversible. Subsequent

ex-periments demonstrated that functional VP175

was essential for viralDNA synthesis and that

therequirementcould be bypassedby

preincu-bation for 2 h at the permissive temperature

(35). More recently, it was shown that upon

shiftdown, new transcription was required for

viral DNAsynthesis in cells infected with tsB2b

(28).Otherinvestigators have isolated additional

tsmutantsofcomplementationgroup 1-2(D, T,

K, and LB2) (36) whichpossesssimilar

pheno-typicproperties. Preston (32) has reported that

at least six immediate early polypeptides

(VP175,-136, -110,-68, -63,and -12) are

synthe-sized in vivo after release of a cycloheximide

block and that in vitro translation of immediate

early mRNA yields thesesameproducts.

Pres-tonhasalsocompared the synthesis of

immedi-ate early polypeptidesin tsK andwild-type

vi-rus-infected cells and has obtained evidence

which demonstrates that (i) immediate early

proteins are directlyinvolved in the transition

from immediateearlytoearly protein synthesis,

(ii) three forms of VP175 exist, and (iii) upon

temperature shiftdown, the multiple effects of

themutationsarereversible. Incontrast,Pereira

etal. (30) placed VP136 in the group ofearly

proteinsdespitethe fact that it issynthesizedin

the presence of cycloheximide or canavanine.

Thisclassificationwasbaseduponthe

observa-tion that VP136 wassynthesized slightly later

than the other immediateearly proteins.

To confirm that the product ofgene 1-2 is

indeed VP175 andtofurther define the function

of thisproteinin theregulationof viral

transcrip-tion, studieswereundertakentomapphysically

andgeneticallyaseries ofmutantsingroup 1-2

andtocharacterizetheresidualactivityof

mu-tantgene productsin infected cellsatthe

non-permissivetemperature.

Mapping studies. Previously, studies have shown thattsB21u (29), tsB2b (3), tsD(41, 42),

tsLB2 (21), and tsc75

(22)

lie in the

repeated

sequencesbounding Us.Inthisstudy,we have

attemptedtorefine the mappositionsofseveral

mutantsin complementation group 1-2 and to separate them physically within these se-quences.

It is our contention that the data presented

heretogether with thefindingsof other

investi-gators place themutants within the structural

gene for VP175. The pertinent arguments for

thiscontentionare asfollows: the ninemutants

(i) fail to complement each other, (ii) exhibit

similarviralDNAandprotein phenotypes, (iii)

produceanaltered form of VP175, (iv)are

sep-arable by physicaland genetic mapping

proce-dures, and (v) map within the DNAsequences

which encode VP175. Moreover, VP175 is the

only protein which has been shown to be

en-codedwithin thesesequences, and (vi) a4.7-kb

immediateearly mRNA coding foraprotein of

molecular weight 175,000 also maps in these

sequences(6).Therefore,unless extensive splic-ing occurs in production of the message for

VP175, andnoevidencefor such splicing hasyet

beenreported,the ninemutants mustlie in the

structural gene for VP175. Mapping studies

therefore exclude the possibility that the

phe-notypic properties ofmutants ofgroup 1-2 are

due to mutations in a gene which regulates

VP175synthesis.

As described above (Results), mostmutants

map nearertheterminal "a"sequencesthan the

unique region of S. Two possible explanations

for this observation exist: (i)thissegmentof the

VP175 gene, ifmutated, results in significant,

observable phenotypic changes (e.g., this

seg-mentencodesanactivesite),or(ii) some

prop-erty of the DNA makes ithighlymutable. We

cannot testthe forner possibilityat this time;

however, the latter lends itself to speculation.

Knipe et al. (21) have demonstrated regionsof

identity between the terminal "ca"sequencesin

S and have proposed a copying mechanism

whereby,duringDNAreplication,oneterminus

serves asthe templatefor the other. They fur-therpostulatedthat "c"sequencesfurther from

the terminal "a" sequences can tolerate

se-quenceheterogeneitymorereadilythan the "c"

sequences nearer "a." Thus, the former

se-quences have been designated variable "c"

se-quences, and those nearer terminal "a"

se-quences have been designated invariable "c"

sequences. These investigators also

demon-strated that ts lesions in VP175 are recessive. Thus, sequences atonly one terminusneedbe

wildtype togenerate the wild-type phenotype,

whereassequencesatbothterminimustbe

mu-tant to generate the ts phenotype (21, 31, 33).

Our dataareconsistent with thishypothesisin

that a mutation induced in invariable "c"

se-quenceswouldbemorelikelytobecopiedinto the opposite terminus, yielding a mutant phe-VOL. 36, 1980

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notype, than would a mutation in variable "c"

sequences, which could produce a"silent"

mu-tant phenotype. Thus, perhaps, the copying

mechanism of DNA synthesis constitutes the

underlyingreasonfor the isolation ofa

predom-inance of ts mutations ininvariable,terminal"c" sequences. Further analysis of the remaining

members ofgroup 1-2 and efforts tointroduce

mutations into the variable and invariable

se-quences in oneterminus butnotthe other will

benecessary to prove thishypothesis.

Thegeneticmap of the nine mutantsexhibits

strikinglinearity and additivity, much more so

than linkage maps of markers in unique

se-quences in L (37; Dixon and Schaffer,

manu-script in preparation). Moreover, the

recombi-nation frequencies observedamong mutants in group 1-2 were muchhigherthanthose observed

between markers within a single gene in the

uniqueregion (Dixon andSchaffer, manuscript

inpreparation). Thediploid natureof thegene

may accountfor the high frequency of recom-bination sinceonlyoneterminus need recombine

toyield ats+ recombinant: crossovers ateither

end wouldproducetwice theeffective recombi-nationfrequency.

Another interesting observation which arose

from recombinationstudies concerns the

behav-ior of tsc75. This mutant recombines

ineffi-ciently with other members of group 1-2. In

addition, it behaves as if itwere closelylinked

totsD,yet it hasbeenmapped physicallyin the

terminal "a" sequences (20). A possible

expla-nation for thesefindingsis thatthe"a"sequence

may, infact, berepeated within "c"sequences.

Thissuggestionwasfirst madeby Wadsworthet

al. (44), who demonstrated small lariat

struc-tures at the ends of molecules which had been

digestedwith exonucleaseIII,andproposedthat

the sequencearrangement of the Sregion may be ...a'x'ac'---Us---ca'xa.We will assumefor the

sake ofargument that this arrangement is cor-rect. Ifthe "a"sequence isdiploidin eachrepeat

andif tsc75 doesliewithin the "a" sequence, at

least two copies of the mutant "a" sequence

wouldbepresent in order to preventself-rescue.

Although Knipe et al. (20) have proposed that

thecodingsequences for VP175includethe

ter-minal "a" sequence, this need not be the case. Infact, it may be a mutation in the internal "a" sequence which produces the mutant pheno-type, or perhaps mutations in both "a" se-quences. Ourgeneticandphysicalmapping data support this contention. If the mutation in the

internal "a" sequence were essential for the

expression of the ts phenotype, and ifthis

se-quence were adjacentto tsD, then the position oftsc75 foundbyrecombinationanalysis would

be predicted. Moreover, if "a" sequences were

also present in "c" sequences, one wouldpredict

that tsc75 would be rescued by fragment K2

(containing the terminal "a" sequence) and by

fragment K3 (containing the putative "a"

se-quencein"c"). Indeed,this is precisely the result weobtained.

Alternatively, the low frequency of

recombi-nation observed between tsc75 and other mem-bers of group 1-2 may reflect the fact that recom-bination occurs with decreasedfrequencyatthe ends of molecules.Thus, low recombination fre-quencies with markers in the terminal "a" se-quencecould result inmismapping.This expla-nation is inconsistent, however, with rescue of tsc75by both K2 and K3.

Function of VP175. Having demonstrated that all nine mutants in complementation group 1-2arelocated in thestructuralgeneforVP175,

further functional analysis of this polypeptide

was undertaken.

We first demonstrated thatall nine mutants exhibit similarphenotypes withregardto poly-peptide synthesis at the nonpermissive temper-ature. Minorvariationsintheamounts of certain polypeptides were observed, owing perhaps to strainvariation orleak,but in most casesVP175, -136, -110,-68, and -63 were seen to be overpro-duced. Intsc75-infectedcells,however, increased

amounts ofVP110 accumulated relative tothe

other mutants.Althoughthismaysimply reflect

a strain-specific difference, it may also reflect

someunusualproperties ofVP110.Clementset al. (6) and Watson etal. (48) have shown that VP110 is encodedby the terminalrepeats ofthe

Lregion. Because the "a"sequence is common totherepeatsof bothLandS and because the

mRNAcodingforVP110may contain aterminal

"a"sequence, it ispossible that tsc75 is mutant for both VP175 and VP110 and is,therefore, a

nonresolvable double mutant. If tsc75 contains

a mutation in the gene for VP110, then the

accumulationof VP110 inmutant-infected cells

mayreflectarole forVP110initsownregulation

or,alternatively,itmayreflectafailureto

proc-ess the mutant form of VP110 analogous to mutants in VP175. Because nosingle mutant in VP110 is available, we cannot distinguish be-tween the twopossibilities.

Severalgroups have reported thatfunctional

VP175 is required to switch from immediate

early to early protein synthesis and that the requirement is probably at the level of

transcrip-tion (7, 8, 13, 14,22, 24, 25, 32, 43, 47). What is not clear is the roleofearly proteinsinregulating

immediate early protein synthesis and the role ofVP175 inregulating its ownsynthesis. Tem-perature shiftup experiments have shown that VP175 aswell as otherimmediateearly proteins canbesynthesized in the presence of both early J. VIROL.

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HSV-1 ts MUTATIONS IN VP175 201 and late functional proteins, indicating that

VP175 isinvolved either directlyorindirectlyin

turningoff itsownsynthesis andperhaps that of

other immediateearly proteins. This conclusion was particularly apparent in the case of shifts

lateininfectionwhich resulted in the

reexpres-sion or increased synthesis of immediate early

proteins (Fig.4).If this isindeed the case, then

themodel ofcascaderegulation mustbe

modi-fiedtoinclude autoregulation of immediateearly

proteins in addition to feedback inhibition by

early proteins.

Anotherpoint tobe madeconcernsthe

exis-tence ofmultiple control groups in each

poly-peptide class. Pereiraet al. (30) firstsuggested

that subgroups of immediate early and early

proteinsexisted,aconceptsupported by Preston

(32). Our experiments have defined a group of

proteins whichareactivelateininfection (after

8h) andyetrequirethecontinuedexpressionof

VP175. This is apparent from the differences

seenin polypeptide synthesis aftershiftuplate

in infection and from the observation that no

viruswas producedeven thoughmostproteins

were synthesized (35). Inaddition, VP136, first

classifiedas animmediateearly protein by

Mars-denetal. (26)andanearly protein byPereiraet

al. (30), does not appear to be under VP175

control, yet it accumulates after VP175, -110,

and -63 reach maximum synthesis. Thus, as

pointedoutbyothers, VP136synthesisexhibits

thekinetics ofanearly proteinand the control

requirements ofanimmediateearly protein.

A model consistent with all available data

would require that all promoters on the HSV

genomebeactiveatalltimes but that promoters

differinthefrequencywith whichtranscription

isinitiated. PromotersforVP175, -110,and -63

would have the highest initiation frequencies,

whereas those for VP136 and other early and

lateproteins would havealowerfrequency,but

still berecognized by the

transcriptional

appa-ratus. One function of VP175 would be to

in-creasethefrequencyoftranscriptionfromsome

earlyor even late promotersand decrease the

frequency from its own promoters. Consistent

withthis model arethe

following

observations:

(i) certain late proteins (e.g.,

VP154)

can be

expressed in the absence of functional VP175

(this study), (ii) VP175 is

required

for certain

late viralfunctions (thisstudy), (iii)theresident

HSV TK gene in biochemically transformed

cells is expressed in the absence of VP175 (19,

24,25), (iv) functionalVP175isabletoenhance

HSV TK expression in HSV TK-transformed

cells (19, 24, 25), (v) VP175 is

reexpressed

in

group 1-2mutant-infected cellsafter

shiftup (i.e.,

after inactivation of theautoregulatory function

ofVP175) (thisstudy),and(vi) theactive form

of VP175 has DNA binding properties (49). If

thismodelwerecorrect, onemightexpect VP175

tobind directlyto promoter sequences or

per-haps toform complexes with the RNA polym-erase toalteritsspecificity. Thistranscriptional

model is similarin many ways to theregulatory

mechanism operative during adenovirus

repli-cation inwhich pre-early proteinsare required

toinitiate the synthesis of early proteins,which

in turn initiate later protein synthesis (2, 16).

However, duringtranscription in vitro,the ma-jorlatepromoter(s) is recognized in preference

to the early promoter(s) (J. Manley, personal

communication). This indicates that although

early functionsareneededtoenhance late

pro-moter activity, they can be bypassed by the cellular transcriptional complex.

ACKNOWLEDGMENTS

We thank L. B. Sandner and P. A.Temple forexcellent

technical assistance, J. G. Driscoll forpreparation of the manuscript, and D. M. Coen forhelpful discussions and sug-gestions. We areespecially indebted to R. J. Courtney for constructive comments and continued interest and

encourage-ment.

This investigation wassupported byPublic Health Service research grant CA20260 and programproject grant CA21082 from the National Cancer Institute.

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