Molecular Genetics of Herpes Simplex Virus VI. Characterization of a Temperature-Sensitive Mutant Defective in the Expression of All Early Viral Gene Products

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0022-538X/81/050539-09$02.00/0 Vol.38, No.2

Molecular

Genetics of Herpes

Simplex

Virus

VI. Characterization

of a

Temperature-Sensitive Mutant Defective in the

Expression

of

All

Early Viral Gene

Products

DAVID M.KNIPE,' WILLIAM

BATTERSON,'

CATHYNOSAL, BERNARDROIZMAN,2* AND ALEXANDERBUCHAN3

Department ofMicrobiologyandMolecularGenetics,HarvardMedical School,Boston, Massachusetts

021151; The Marjorie B.KovlerViral OncologyLaboratories, University of Chicago, Chicago, Illinois

606372;andDepartmentofMicrobiology, University of

Birmingham,

England3

Received14April1980/Accepted19January 1981

The herpes simplex virus 1 (HFEM) mutant tsB7 failed to express any detect-able viral polypeptides and did not significantly inhibit hostcellprotein synthesis in infectedcellsmaintained at the nonpermissive temperature. The mutant could complement the growth of a coinfecting temperature-sensitive mutant virus differing in plaque phenotype and thus appeared capable of penetrating doubly infectedcells.The yield of tsB7 wasenhanced by the coinfecting virus but not to

theextentthat the coinfecting virus was enhanced. Coinfection studies suggested

that the tsB7 defect was complemented in trans, butpoorly, by the wild-type parent and other

viruses.

Marker rescue of tsB7 by transfection with herpes simplex virus 2XbaI DNA fragments mapped the mutation between 0.45 and 0.70mapunits.Analysis of the DNA structure of the ts+ intertypic recombinants

generated bythis rescue showed that the herpes simplex virus 2 DNA substitu-tions all contained the region between 0.46 and 0.52 map units, thus further

definingthe mappositionofthe mutation. Analyses of the polypeptides expressed by these intertypic recombinants defined the genome location of the genes

specifying polypeptides2, 6, 10, 32, 43, and44 and indicated that the mutation

mapsinorneargenescoding for virion structuralpolypeptides.Thisregion of the

genome is represented as stable transcripts and cytoplasmic mRNAonly after viral DNAreplication (P. C. Jones and B.

Roizrnan,

J. Virol.31:299-314, 1979), and thus this gene appearstobealatefunction.These resultsareconsistent with the ts mutation in tsB7 being in a gene coding for a virion component which functions before expression of the alpha genes early in infection. Themostlikely explanationis that themutantisblocked at astageofuncoatingandthe defect iscomplemented, althoughpoorly, byacoinfecting virus gene product.

Inthis paper,wereportonthe characteristics of theherpessimplexvirus1 (HSV-1) tempera-ture-sensitivemutanttsB7. Thismutantfailsto

produce any detectable viralpolypeptides and hasnoappreciablelong-termeffectonthe

syn-thesis of hostproteinsatthenonpermissive

tem-perature.Pertinenttothis reportarethe follow-ing.

(i) HSV-1 andherpessimplexvirus2(HSV-2)

aregenetically closelyrelated(14). Both HSV-1

and HSV-2 specify approximately 50

polypep-tides (12, 23) which formatleast three groups whose synthesis is coordinately regulated and

sequentially ordered in a cascade fashion (12,

25). The apolypeptidesmadeimmediatelyafter

infection ofpermissive cells with HSV-1 attain

peakratesofsynthesisbetween2and4h

post-infection andrequirenoviralproteinsynthesis

for thesynthesisandprocessingof their mRNA. Thesepolypeptidesarerequiredfor the

synthe-sisof, polypeptides,and these in turn shut off the synthesis ofapolypeptides;the ,B

polypep-tidesareinvolved in thesynthesisof viral DNA and induce thesynthesisof structuraly

polypep-tides.

(ii) Inthe course of their

replication,

herpes

simplex viruses shut off the synthesis of host proteins andDNA and reduce the

synthesis

of

host RNA (22, 29, 30). The shutoffof the

syn-thesis of hostproteinsappearsto occur intwo

stages. Thefirstoccurs

immediately

after

infec-tion and is mediated by a structural protein

inasmuchasitdoesnotrequirethe

transcription

of the viral genome (6). The second stage

re-quires the expression of viral protein and ap-pears to coincidewith the synthesis ofJ8

poly-539

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peptides (12, 21). In general, HSV-2 isolates inhibithost proteinsynthesismorerapidlyand more completely than HSV-1 during the first

stage ofinhibitionofhostmacromolecular

syn-thesis(2,22).AnalysisofHSV-1 x HSV-2

inter-typic recombinants showed that the HSV-2

gene(s)responsiblefor the accelerated shutoffof

host protein synthesis maps between 0.52 and

0.59mapunits(5, 20).

MATERIALS AND METHODS

Viruses. The isolation andpropertiesof HSV-2(G) and HSV-1 (HFEM) ts+ syn are described in refer-ences4and 9,respectively.HSV-1(HFEM)tsB7 syn+ was isolated by bromodeoxyuridine mutagenesis of HSV-1 (HFEM)-infectedcells.HFEMoriginally con-tainedamixture of syn and syn+viruses,andthetsB7 mutantapparentlyarosefromasyn+virus. HSV-1(F)

is a primaryisolate usedextensively intheChicago

laboratoryand has beenpassagedno morethanfour

times in cell cultureat340C (4, 11). Wehave found that this strainreplicatespoorlyat39°Candthat the

efficiency ofplatingat 390Crelative to 34°C is less

than l0'. It doesreplicate well at 370C, asits effi-ciency ofplatingat370Crelativeto34°Cis0.5.This phenotype is not unusual for fresh isolates ofHSV-1, since many other isolates testedshowed similar prop-erties (D. M. Knipe and B. Roizman, unpublished

observations). The temperature-sensitive marker in

HSV-1 (F) maps in the repeated sequences of the S component, 0.83 to 0.865 and 0.965 to 1.0map units (P.J.Godowski andD.M.Knipe,unpublisheddata). The virus expresseslargely a polypeptides at 39°C, andtherefore thewild-typeHSV-1(F)strainproduces atemperature-sensitiveaproduct similartothose of ts mutantsincomplementationgroup1-2(28).

HSV-1 (F) ts502 synl,2 is the designation of a recombinant produced by marker transfer of the syn loci from HSV-1 (1061) to HSV-1 (F) (27). At the permissive temperature it fuses bothHEp-2and Vero cells.

Virus stockswereprepared andtitrated in Vero cell cultures. The infected cells were maintained in

me-dium 199supplemented with 1% inactivated calf

se-rum.

Complementation tests.Complementation tests

wereperformed by a modification of theprocedure of Schaffer et al. (28). After addition of virus to the cultures, theflasksweresubmerged ina390Cwater bath or a 390C convection incubator. After the 1-h

absorption period, the virus inoculum wasremoved

and 5 ml ofmedium 199-1% inactivated calf serum was added. After 18 h ofincubation, the virus was harvested and titrated. Equivalent results were ob-tainedinexperiments in which thecellswere or were notwashedattheend of theabsorption period.

Markerrescuemapping.Mappingofthets lesion in tsB7wasperformedby thecotransfectionofmutant DNA andindividual fragments of HSV-2 (G) DNA generatedbycleavageofthe DNAwithXbaI restric-tionendonuclease (6, 15). The flasks were incubated for 3 to 4 days at 340C, and the progeny virus was harvested and titrated at 34 and390C. The rescued

ts+ cloneswereisolatedbyfourcyclesofplaque puri-fication underagaroseoverlayat390C(19).

Purification ofviral DNA andanalysis of

re-combinant genomes. Viral DNA for the marker

rescue studies and for analysis ofrecombinant

ge-nomes waspurified byNaldensitygradient

centrifu-gationofinfected cell extracts(31).HsuI,BgllI, XbaI,

andHpaIrestrictionendonucleaseswerepreparedas describedpreviously(19).BamHI andKpnIrestriction endonucleases were purchased from New England

Biolabs.

Analysisofpolypeptidesininfected and mock-infected cells. Cells were labeled with

L-U-'4C-la-beled amino acids(leucine, isoleucine,andvaline)at

timesspecified in the text andunder conditions

de-scribedbyMorseetal.(20).At the end of thelabeling interval,the cellswereharvested, disruptedwith

so-diumdodecylsulfate,andsubjectedtoelectrophoresis

inpolyacrylamideslabgelsasdescribed(20).

RESULTS

Replication

ofHSV-1 (HFEM) tsB7. The

efficiency

of

plaque

formation

by

tsB7was 1 x

105-

to3x

105-fold

lowerat39thanat340C. The

yield

ofinfectious virus from cultures infectedat

a

multiplicity

of 5

PFU/cell

andmaintained at

390C

was 102_ to 103-fold lower than that

ob-tained from

replicate

cultures maintained at

340C.

The viral progenyobtained from the

in-fected culturesincubatedat390Cwerestill

tem-peraturesensitiveanddidnotrepresent revert-ants.

Therefore,

at

high

multiplicities of

infec-tion themutantshowedsomeleakiness.At

340C

the virus formed plaques which were much

smaller than those of its parent, HFEM, and whichincreasedin size

slowly.

Transient temperature fluctuations below

390C allowed the formation of very small

plaques and expression of viral polypeptides;

thus,

thephenotype of tsB7 wasvery sensitive

to temperature fluctuation. Forthis reason all

experimentsweredone inflaskssubmerged ina waterbath maintained at390Corin a

convec-tion-heated incubator with strict temperature

control.

Polypeptide expression by tsB7. To

ex-amine theproteins specified bythemutantvirus

at the permissive and nonpermissive

tempera-tures, weinfected cultures ofcells withHSV-1

(HFEM) andtsB7, incubatedthe culturesat 39 or330C,andlabeledthem with

[35S]methionine.

Thelabeledcell extracts were then subjected to

gel electrophoresis (Fig. 1). At 330C, tsB7

ex-pressedapatternof polypeptidessimilar to that

ofHFEMexceptthat certain early proteins

(in-fectedcellpolypeptides[ICP]6,-8, and -38) were

overrepresented and certain late proteins

(ICP10,-15, -19,and-39) wereunderrepresented

in the mutant profile, presumably because the

mutant infection had not progressed as far as J. VIROL.

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MOCK HFEM tsB7

39 39 39 33 33 33

FIG. 1. Autoradiographic images ofpolypeptides extracted from HEp-2 cells infected with HSV-1

(HFEM) tsB7orHSV-1 (HFEM) or mock infected

andelectrophoretically separatedinsodiumdodecyl

sulfate-polyacrylamidegels. Theinfectedcells were

incubatedattemperatures shown and were labeled

with "4C-amino acidsfrom 18 to 20 hpostinfection.

the wild-type infection. In

all

infections

per-formedat

330C,

theinfections initiatedbytsB7

progressed moreslowlythan thoseinitiated by

HFEM.This isconsistentwith theslowplaque

formation

bythemutant virus.

At

390C,

HFEMexpressed apattemof

poly-peptides equivalenttothat at

330C,

exceptthat some late proteins (ICP10, -19, and -39) were

madeatlower levelsat

390C.

However,at

390C

tsB7didnotexpress anydetectable viral

poly-peptides.The

pattern

ofproteins labeledincells

infectedwith tsB7at

390C

was very

similar

to

the

pattern

ofpolypeptides from mock-infected cellsat

390C,

withatmost a veryslightdecrease inintensity ofeach

cellular

polypeptide band.

Thus,tsB7 did notsignificantly inhibithost

cell

protein synthesisat

390C.

Complementation of other mutants by

tsB7.

Schaffer et al. (28) previously reported thattsB7couldcomplementother temperature-sensitive mutants, but in those studies the

vi-ruses were absorbed for 1 h at

370C.

Wehave

found that, under these conditions of infection,

tsB7willexpress manyof the lateviralproteins

and is therefore leaky (W. Batterson and B.

Roizman,

unpublished

data). Wetherefore

reex-amined the question of the ability of tsB7 to

perform

complementation under conditions where it expresses no proteins, i.e., continual maintenanceat

390C.

Inthisseries ofexperiments, cultures ofVero

cells

were

individually

or doubly infected with

tsB7,

ts502,

orHFEM.CellscoinfectedwithtsB7

showeda 100-fold increase in the yield ofsyn

plaques (ts502 marker) andatwofold increase in the syn+plaques (tsB7 marker) relative tothe singlyinfected cultures (Table 1). Theincrease in yield was not due to recombination, since

ts+recombinantsconstitutednomorethan1% of

the virus

yield

from thedoublyinfectedcells.It

should be noted thatmostof the ts+

recombi-nants weresyn+, liketsB7. Ahigh frequency of

the ts+ recombinants wouldbe expected to be

syn+

in that the synl,2 mutations (0.70 to0.83 mapunits)arecloserto

ts502

(0.83to0.865and

0.965 to1.0 mapunits) thantothemaplocation

for tsB7, shown below to be 0.46 to 0.52 map

units.

Theobservation thatin the

complementation

test the increase in titer of ts502 was greater

than the increase in the tsB7 titer raised the

possibilitythattsB7 containedacis-acting

mu-tation thatcauses ittoreplicatepoorly. To

ex-amine thisquestion,wefirstcoinfectedcellswith

tsB7and ts502at

340C

andexamined the

prog-Theinfectedcellpolypeptideswerenumbered

accord-ingtothenomenclaturepublishedelsewhere(12,20). VOL. 38,1981

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TABLE 1. Testsfor complementation between tsB7 andts502a Complementationtest

390C 340C

Virus(PFU/cell) Yield(105) Virus (PFU/cell) Yield(106),340C

340C 390C

ts502 tsB7 ts502 tsB7 ts502 tsB7

ts502 tsB7 ts502 tsB7

5 0.64 10-5 0 1 49

10 1.33 10-5 0 5 150

5 11.7 1.4x 10-2 1 0 96

10 16.0 1.5x 10-2 5 0 153

5 1 278 20

5 5 207 32.0 0.19 1.85 5 5 172 62

1 5 56 117

aCells

wereinfected with eachmutantaloneorwithmixturesof bothat

multiplicities

shown and incubated

at39and340C.Theviralprogenyproducedin thesecellswastitratedattemperatures shown. The titers for

ts502and tsB7 are basedontheplaquemorphology typesof theprogeny.

eny at 340C. Again the syn or ts502 progeny

were favored, although by a lower ratio than from the cultures infected at 390C. Thus, the

poor growth of tsB7, even at the permissive temperature, couldexplain inpartthepooryield

oftsB7from thecomplementation experiments. We also examined the ability of HFEM ts+

syntocomplementtsB7syn+ at

390C.

Theyield

ofsyn+ viruseswassignificantlygreaterin

cul-tures coinfected with HFEM ts+ syn than in

singlyinfectedcultures (Table 2).Thus, HFEM could complement the replication of tsB7 at

390C.

However, the yield ofsyn+ viruswas

ap-proximately 10% of the yield of the syn virus,

eventhough allcellswerecoinfected with both. Therefore, even the wild-type parent of tsB7

couldonlypartiallycomplementtsB7. This is in

contrast with the situation when HFEM and

ts502 coinfected cells. The progeny virus from cellsinfected with thesetwomutantsshowedan

efficiencyofplating

(39/340C)

ofapproximately

one-half that of the progeny from a culture infected with HFEM alone (Table 2). Thus,

approximatelyone-half oftheprogeny from the

doublyinfected cellsweretemperaturesensitive.

Undertheseconditions ofinfection,theprogeny

contained approximately equal amountsof the

twoinfecting viruses.

The mutant tsB7 appearedto have a partial

cis-acting phenotype, although it could

appar-ently be complemented bycoinfecting viruses. As discussed further below, a partial

comple-mentation ofanuncoatingmutant would give a

partial cis-actingmutantphenotype.

MarkerrescueoftsB7 with HSV-2 DNA

fragments.The genomelocationof the ts lesion

in tsB7 was determined by the cotransfection

method ofmarker rescue (16). In these

experi-TABLE 2. MarkerrescueofHSV-1 (HFEM) tsB7by cotransfection with XbaI restriction endonuclease

fragments ofHSV-2(G)DNA

Ratio oftiters,

Transfection mixture

39/340C

(103)

tsB7DNA alone 0.01

+XbaI-A-C 1

+XbaI-D 25

+XbaI-G 1.8

+XbaI-H 0.3

+XbaI-I 0.1

+XbaI-J 0.5

ments,cellcultureswerecotransfected withtsB7

DNA andindividual XbaIrestriction

endonucle-asefragmentsofHSV-2 (G) DNA (Fig. 2).After

3to 4 daysofincubationat340C, the progeny viruswasharvested and assayedat34 and390C (Table 3). The most efficient rescue was

ob-tained withthe XbaI-D fragment, mapping from

0.45 to 0.71 map units. To further characterize

the site of therecombinational events,six

recom-binantsproduced bymarkerrescue with HSV-2

(G) DNA were analyzed with respect to the

domainsofthe HSV-2DNAsubstituted in the

recombinants.Theresults (Fig. 3 and 4) were as follows.

(i) Therecombinant RB7G1containedno

de-tectableHSV-2 sequences(Fig. 3) andexpressed

nopolypeptides which comigratedwith HSV-2

polypeptides (Fig. 4). Thus, RB7G1 was either

a revertant or a recombinant containing too

small areplacement of HSV-1 sequences tobe detected.

(ii) The recombinant RB7G2 retained the

HSV-1EcoRI-M-O and theHpaI-F-Ecleavage

sites but lost all intervening HSV-1 cleavage

sites. Therefore, the maximalleft boundary of

542

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HSV-2(G)

4 L >o S >

ab

UL

e Us.a

C D G H J I

45 26 12 I44..

45 26 12

I8.4

4.455 XbaI

0 0.2 0.4 0.6 0.8 1.0

FIG. 2. Schematicdiagram ofthesequencearrangementandXbaI restrictionendonucleasemapsofDNA fragmentstested in markerrescuestudies. L and Srepresentthelongand shortcovalentlylinkedcomponents

ofHSV-DNA, and ULandUsrepresent theunique sequencesofeach component,respectively; ab and b'a'

represent the inverted repeatedsequences bounding the L component, whereas a'c' andca represent the inverted repeatedsequences boundingthe Scomponent. The lettersabove the line denote thenameofthe fragment; thenumber below the line is the molecularweight ofthe DNAfragmentin millions.

TABLE 3. Coinfection oftsB7 and HFEM ts+syna

34°Ctiter 390Ctiter

Infecting virus 39/34°C ratio

syn synf+ 8syn syn+

tsB7 <103 <1i3

HFEM syn 5x106 3.5x106 0.7

ts502 105 <103 10-2

tsB7+HFEM 6x 106 6 x105 3.5 x 10c 0.58

ts502+HFEM 6.3x10c 2.5 x 106 0.39

aCultureswereinfected with the indicated virusesat

390C.

After18hofincubation,the progeny

virus

was harvested and titrated at 34 and390C.

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FIG. 3. Summary oftherescueofthe HSV-1 (HFEM)tsB7 with HSV-2DNA.Diagramshows the location oftheHSV-2sequencesreplacingHSV-1sequencesin recombinantsproduced bymarkerrescueoftsB7 with HSV-2 DNA. The topandbottom lines next tothedesignation ofeach recombinantrepresentHSV-1 and HSV-2 DNA, respectively. The heavy line represents the sequences identified in each recombinant. The

numbers above theheavylineidentifythe HSV-2ICPs produced bythe recombinants andshown inFig.4.

Themapping ofthe DNAsequencesin the recombinantsis basedontherestriction endonucleasemapsfor HSV-1 and HSV-2 shown on the bottom. The HSV-1 KpnI and BamHIofHSV-1 (HFEM) tsB7 were

determinedfromthemapsforHSV-1(F) produced byLocker and Frenkel(18).

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38,1981

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544 KNIPE AL.

FIG. 4. Autoradiographic images ofthe polypep-tidesextractedfromHEp-2cellsinfectedwith HSV-2 (G), HSV-1 (HFEM) tsB7, andrecombinants

pro-ducedby markerrescueoftsB7 with HSV-2(G)DNA andelectrophoreticallyseparatedin sodiumdodecyl sulfate-polyacrylamide gels. The cells were labeled with "C-amino acids from 10to 12 hpostinfection. Thepolypeptidesareidentified bynumberaccording tothe nomenclatureofHoness and Roizman(12)and Morseetal.(20).The HSV-2 ICPsareidentified bya

dash under the number. The insert on the bottom illustratesaportion ofagelshowingabetter sepa-rationofHSV-1 and HSV-2 ICP6.

the recombinational event was the HSV-1

EcoRI-M-O cleavage site,andthe minimal left

boundary was the HSV-1 HpaI-B-H cleavage site.Themaximal rightboundarywasthe

HSV-1HpaI-E-Fcleavage site, and theminimal right boundarywastheBamHI-O-Icleavagesite(Fig. 3). This recombinant expressed the ICP2, -6, -43, -44,-10,and -32 of HSV-2 (Fig.4).

(iii) The recombinant RB7G3 retained the HSV-1 EcoRI-O-L cleavage site and the BamHI-O-I cleavagesite but lost allintervening

HSV-1 cleavagesites. Themaximal left

bound-ary was the HSV-1 EcoRI-O-L cleavage site,

andthe minimal leftboundarywasthe

EcoRI-L-Acleavage site. The maximal right boundary

was the HSV-1 BamHI-O-I cleavage site, and

the minimal right boundary was the HSV-2

HpaI-D-Ecleavage site. This recombinant

spec-ified theHSV-2ICP10 (Fig. 4).

(iv) The recombinants RB7G4 and RB7G5

retained the HSV-1 EcoRI-M-O cleavage site

and the HSV-1 BamHI-D-H cleavage site but lost allinterveningHSV-1 cleavagesites.

There-fore, the maximal left boundary ofthe recombi-national events was the EcoRI-O-L cleavage site, and the minimal left boundary was the HSV-1 HpaI B-H cleavage site. The maximal right boundary was the BamHI cleavage site, and the minimalrightboundarywastheHSV-1 EcoRI-L-A cleavage site. These recombinants gained one new KpnI cleavage site consistent with theposition of the HSV-2KpnI-G-D

cleav-age site. These recombinants expressed the

HSV-2 ICP2,-43, and-44 (Fig. 4).

(v) The recombinant RB7G6 retained the HSV-1HpaI-B-HandHpaI-F-Ecleavage sites but lost all intervening HSV-1 cleavage sites.

The maximal left boundary was the HSV-1 HpaI-B-H cleavage site, and the miniimal left boundary was the HSV-1EcoRI-L-A cleavage site. The maximalrightboundarywasthe HpaI-F-Ecleavage site, and the minimal right

bound-ary was the HSV-1 BamHI-O-I cleavage site.

This recombinant expressed the HSV-2 ICP6,

-10,and-32 (Fig. 4).

In summary, the region of HSV-2 DNA

re-placementcommon toall recombinantswas the

region betweenthe HSV-1EcoRI-O-Lcleavage

site and theBamHI-D-H cleavage site,ormap

positions 0.46 to 0.52. This further defines the

map position of the ts mutation in tsB7. The

maximal boundaries for thetype-specific

deter-minants of thepolypeptideswereICP2, -43, and

-44(the EcoRI-M-O cleavagesiteto the EcoRI-L-A cleavage site); ICP10 (the EcoRI-L-A cleavage site totheBamHI-O-I cleavage site);

andICP6and -32(theHSV-2HpaI-D-E

cleav-age site totheHSV-1HpaI-F-E cleavage site).

The ts mutation in tsB7 was alsoclosely linked

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1981

totheICP10 type-specific determinant(three of

sevenrecombinants) and the ICP2,-43, and -44

type-specific determinants (three of seven

re-combinants).

DISCUSSION

Wehavepresentedevidencethat the mutant

HSV-1 (HFEM) tsB7 expresses no detectable

viral polypeptides and doesnotsignificantly

in-hibit host cell protein synthesis in infectedcells maintained at the nonpermissive temperature.

Furthermore, the mutant cancomplement the

growth ofacoinfectingvirus. Thus, the mutant

viralgenome canpenetratedoublyinfected cells.

The block at the nonpermissive temperature

appears to occurafterentryof thevirus into the

host cell and before the expression ofthealpha

geneproducts.

Complementationstudieswith tsB7. The

mutanttsB7wasclearly showntobe capable of

complementing the replication ofacoinfecting temperature-sensitive mutant which maps

within the region of thegenomeencoding ICP4

(Godowski and Knipe, unpublished data) and which expresses largely alpha proteins at the

nonpermissive temperature (L. Pereira and B.

Roizman, unpublished data). Therefore, in order for tsB7tocomplement ts502, tsB7must express

ICP4. Because tsB7 does not express ICP4 in singly infected cells at any detectable level, it

appears that ts502 must supply the defective

genefunction in order for tsB7toexpressICP4. The mutated tsB7 gene function must be (at least in part)atrans-actinggeneproduct.

How-ever, we observed that the yield of tsB7 was

alwayslower than the

yield

ofthe

coinfecting

virus when cellswere infectedat

390C.

There-fore, it appears that the mutation in tsB7 is

partially cis-actinginnature; i.e.,the mutation

cannotbefully complemented byacoinfecting

virus. It should be noted that a defect in

un-coating which is partially complemented by a

coinfecting virus would give reduced yields of

themutantvirus defective inuncoatingbecause

theeffective number ofgenomesparticipatingin

replicationwould be less for theuncoating

mu-tant.Because of the time of theblock in

repli-cation intsB7-infectedcells,adefect in

uncoat-ing of the tsB7 genomeinside infected cells isa

possible explanation for the mutantdefect. An

alternative explanationthatwe cannotruleout

completely at the present time is that a very

small, undetectableamountofearlyproteinsis

made from the tsB7 genome whichcan

comple-mentthe growth ofts502. If theblockin

repli-cation of tsB7were inuncoating, this

explana-tion would also lead to higher yields of ts502

than tsB7.

ThetsB7mutation differsfrom thetwoother

MOLECULAR GENETICS OF HSV. VI. 545

classes ofviral mutantsthat show a complete

lack of viral gene expression under nonpernis-siveconditions, i.e., the ts3 mutantof polyoma

virus (3), the tsD mutants of simian virus 40

(24), and the group I host range mutants of

adenovirus (1, 10). The simian virus 40 and polyomamutantsmapinthe lateregion of the

genome (8, 17) and appear to be defective in

uncoating of the viral DNA (3, 24). Therefore, the DNAof thesemutants cannotbe transcribed

toyield early mRNA. Thesemutantsareunable

to complement other coinfectingmutants, and thusthisuncoatingfunction isacis-acting func-tion (3).

ThegroupIhostrange mutantsofadenovirus

map in the early regionI of the viral genome,

andonly the mRNA's for thatspecificregionare

expressed from themutant genome in

nonper-missive cells (1, 7). Thisappearsto be a "pre-early"genefunction whose synthesisis needed forexpression of theearlygenes.The mutation of tsB7 differs from these because it can be complemented by other viruses,and the existing

evidencestronglyarguesthatthe a gene prod-uctsrequirenoprior viral proteinsynthesis (12,

13).Several different inhibitors ofalpha protein

synthesis have been usedtoallowaccumulation

ofamRNA. There isnoevidenceto suggest that a"pre-early"protein gene product must be

syn-thesized beforeexpression of the agenes.

Map position of the tsmutation oftsB7.

The markerrescuemapping of thetsmutation of tsB7 by analysis of intertypic recombinants

determined themapposition of the lesionto be

within0.46 to 0.52 mapunits.This location has

been confirmedby fme-structure mappingusing

cloned HSV-1 (F) DNA fragments (Batterson

andRoizman, workinprogress).Therefore, the

tsmutationseemslikelytomap inthis region of the viral DNA. This region of the genome is represented in stable cytoplasmic mRNA only after viral DNA replication (13). It therefore

seemslikelythat the tsB7mutation is withina

geneexpressed afterDNAreplication.

Further-more,becausemostof thegenesexpressed after

DNAreplicationcodefor virion structural

poly-peptides, it alsoseemslikelythatthe tsB7lesion

iswithinastructuralpolypeptide gene.

We have used the intertypic recombinants generated by rescue of tsB7 with HSV-2DNA

to determine the viral polypeptides encoded

nearthetsB7lesion. None of the viral

polypep-tides which show type-specific mobilities on

polyacrylamide gelscorrelatedexactlywith the

tsB7lesion;thus,none canbe saidtocontain the

lesion.However,thetype-specific determinants

forICP2,-10, -43, and-44 weretransferredalong

with therescuingsequences in three of theseven

recombinants analyzed. Thus, the genes for

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these proteins are closely linked to the tsB7 locus.

As indicated in the beginning of this paper, theshutoff of hostprotein and DNA synthesis

occurs intwo stages.The mutanttsB7 did not

significantly inhibit host cell protein synthesis

as assayed by thepattern of host cell proteins displayedonpolyacrylamidegels.Inadditionto

theexperiments cited in the text, other

experi-mentsinvolving infection ofsparselyseededcell

cultures incubatedatthenonpermissive

temper-atureindicated that the infected cellswereable

to multiply foratleast3 days, but that

subse-quent shiftdowntothepermissivetemperature

resulted in the destruction of the cells(Batterson

andRoizman, work inprogress).Thus,the effect

onthe cells ofinfection with tsB7atthe

nonper-missive temperatureseems to be

minimal.

Pre-vious studies have mapped the accelerated shutoffby HSV-2 virionstotheregionbetween

0.52and0.79mapunits(5,20) ornearthe tsB7 lesion. Therefore,it isconceivable that thetwo

functionsare encoded within thesamegene. It isstill unclear whether the lack of host shutoff

bytsB7isduetothe lack ofexpressionof viral

geneproductsin cellsinfectedatthe nonpermis-sive temperature.Thus,we cannotdetermineat

this time whether thetwofunctions arewithin the same gene, whether they are in different

genesandbotharelostby tsB7,orwhetherthe

twofunctionsareindifferentgenesandonlyone

islostby tsB7. Further studies are in progress

todiscriminate among thesepossibilities.

Nature ofthe tsB7 defective gene

prod-uct. The current evidence suggests that the

defectivegene product in tsB7is avirion

com-ponent that can be complemented by a gene

product contributed by anothercoinfecting vi-rus. Thisgeneproduct couldactuponthetsB7

capsidtouncoat theviral DNAor toallowit to

expresstheagenes.Alternatively,it could alter

the host RNA polymerase so that it can tran-scribe the viralgenomeefficiently. These func-tionswould be required only forinfections

ini-tiatedby virions because it is knownthat rigor-ouslydeproteinized HSV isinfectious, albeitat

lower efficiencies than virions. Therefore the

tsB7 gene function appears to be a late viral gene product that functions early in the viral

replicationcycle.

ACKNOWLEDGMENTS

These studies were aided by grants from the National

CancerInstitute,Public HealthService(CA98494, CA19264, and CA26345) and the American CancerSociety (MV-02).

D.M.K. was a special fellow of the Leukemia Society of

America,Inc.W.B. isapredoctoral trainee of the National Institute ofAllergyandInfectious Diseases (AI 07182).

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