Fine-structure mapping of herpes simplex virus type 1 temperature-sensitive mutations within the short repeat region of the genome.

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JOURNAL VIROLOGY, July 1981,

0022-538X/81/070150-12$02.00/0

Fine-Structure

Mapping

of

Herpes

Simplex

Virus

Type 1

Temperature-Sensitive

Mutations

Within the Short

Repeat

Region of the Genome

V. G. PRESTON

Medical ResearchCouncilVirology Unit, University ofGlasgow,GlasgowGll5JR, Scotland

Received 12December 1980/Accepted31 March 1981

Cloned herpes simplex virustype 1(HSV-1) DNA fragmentswereusedto fine-structure mapthetemperature-sensitive (ts) lesions from fourmutants, tsT,D, c75,andK, by markerrescue.Thesemutantsalloverproduced immediate-early viral polypeptides at the nonpermissive temperature.

Although

one of these viruses, tsK,gave a morerestrictedinfected-cellpolypeptide

profile

under these conditions than the other three, no complementation was detected between pairwisecrossesofthese mutants intheyieldtest.Recombination, however,was obtained between all mutant pairs except ts T and D. In

physical mapping

experiments, ts+viruswasrecovered fromcells coinfected with DNA oftsT,D, or c75 andBamHI fragmentk fromwild-type strain 17 HSV-1 DNA cloned in pAT153, whereas ts K wasrescued by cloned HSV-1 BamHI-y. Both of these clonedDNAfragmentscontained sequencesfrom the shortrepeat

region

of the HSV-1genome. The tsmutationswere moreprecisely mapped by markerrescue, using restriction enzyme fragments within BamHI-k and-y from cloned DNA. Thesmallestfragment ableto rescue amutant was 320basepairslong.Theorder of thefour mutations derived fromthesestudieswasconsistent with the

assign-ment by genetic recombination. All four lesions mapped within the coding sequences of the

immediate-early polypeptide

Vmw IE 175 (ICP4) which lie outside the "a"sequence.Theresults showedthatmutations in different regions of thegeneencodingVmw IE 175could produce similarphenotypic effectsatthe nonpermissivetemperature.

Infection ofsusceptiblecells with herpes sim-plex virus type 1 (HSV-1) in the continuous presenceof theprotein synthesis inhibitor cyclo-heximide results in restricted transcription of the viral genome (5, 9). The

virus-specific

mRNA's transcribed underthese conditionsby thecellularRNApolymeraseII codefor imme-diate-early (IE) polypeptideswhich are synthe-sizedvery earlyin the normal growthcycle of the virus (8, 18). Cells infected with tempera-ture-sensitive(ts) mutant tsT,D, K, or c75 and maintained at the nonpermissive temperature (NPT) produce

abnormally

large amounts of these polypeptides and reduced amounts of othervirus-specific species (11, 14, 17). Of these four mutants, ts K has been themost extensively studied. This virus has a ts defect in an IE polypeptiderequired for the"switch on" of tran-scriptionof HSV earlygenes,including the virus-coded thymidine (pyrimidine deoxynucleoside) kinasegene, and the subsequent "switch off" of transcriptionof viral IE genes (16, 28, 29). The IE polypeptide may also have a role in the activation of late viral genes, since itsfunction

is required continuously throughout the viral growthcycle, both beforeandafter theonsetof viral DNA synthesis (29). At the NPT, ts K inducesa Vmw IE 175polypeptide with abnor-malproperties (16). On cellfractionation, large amounts of this

polypeptide

were recovered in thecytoplasmic fractionin contrast totheVmw IE 175induced incells infected withtsKatthe

permissive

temperature (PT) orwithwild-type

virusateither the PT orNPT, which wasfound predominantly inthenuclear fraction. Further-more, Vmw IE 175 induced by ts K at the NPT failed to be processed normally to species of lower electrophoretic mobility. Both of these aberrantpropertieswerereversedby transfer of tsK-infected cells from the NPT tothe PT in the absence of further protein synthesis, sug-gestingthat the ts Klesionwas in the Vmw IE 175polypeptideand not in aprocessing enzyme (16).Similardefects in Vmw IE 175 induced by tsTorD at the NPT have also beenobserved (D. MacDonald and C. M. Preston, personal communication).

Marker rescue of ts mutations isa

commonly

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151

used technique for locating ts lesions on the physical map of the viral genome (22). In this method, isolated restriction endonuclease frag-ments from wild-type HSV DNA are screened for their ability to rescue selected genetic markers. A number of problems associated with this approach have limited its use. In particular, difficulty in purifying specific DNA fragments, free of contaminating sequences from other re-gions of the viral genome, canlead to spurious rescue of selected markers. Furthermore, the efficiency of rescue may be low, especially when small DNA fragmentsareused to rescue virus, and therefore it can be difficult to map muta-tions which lie close together. In addition, ob-taining large amounts ofsmall fragments may be a problem. All of these difficulties can be overcomebyusing cloned HSV DNAfragments in physical mapping experiments. The greater precision in mapping genetic markers should facilitate theassignment of mutations to specific genes.

In this study, we have used cloned HSV-1 DNA tofine-structuremaptslesions from four mutants, tsT, D,c75,and K.Previousworkhas assignedthemutations in tsT, D,and K tothe shortrepeat sequences ofHSV-1 DNA (22, 23) and the defect in ts c75 totheterminally redun-dant "a" sequences present in both the short and long repeat regions ofthe genome (10). It wastherefore of interesttoknowwhether these mutations were in the genecodingfor Vmw IE 175, known to mapentirelywithin theinternal (IRs) and terminal (TRs) short repeat (30) or within some other gene, forexample, the gene coding forVmw IE 12or68,whose 5'endmaps, in each case, within

IRs/TRs,

and whose 3' terminus maps with the short unique (Us)

re-gion.

MATERIALS AND METHODS

Tissue culture cells. BHK-21 clone 13 cells (12)

weregrown inEaglemediumsupplementedwith 10%

tryptosephosphatebroth and 10% calfserum.

Virus. The isolation of HSV-1 strain17andtsT,

D, and K has been described previously (2; I. K.

Crombie, Ph.D. thesis, UniversityofGlasgow, Glas-gow, Scotland, 1975). Nonsyncytial isolates of these

viruseswereusedinallexperiments.tsc75wasderived

from strain 13vB4(7),andbothwerekindly provided

by R. W. Honess. These ts mutants revert to ts+

phenotype at amoderate frequency, suggesting that

thelesionsarepointmutations.

Recombination and complementation tests.

Recombination andcomplementationtestswere

per-fonnedessentiallyasdescribedbyBrownetal.(2).In

therecombinationandcomplementationyieldassays,

cell monolayerswereused insteadofcellsin

suspen-sion.

Preparation ofviral DNA. DNA was obtained

from cell-associated and cell-released virions as

de-scribed by Wilkie (31).

Construction of recombinant plasmid DNA

molecules. BamHI fragments k and y fromwild-type

HSV-1 strain 17 DNA were cloned into the plasmid pAT153 (26) by direct ligation of BamHI cohesive ends (25).

Isolation of plasmid DNA. Cultures of Esche-richia coli strain K-12 HB 101, harboring pAT153 carrying HSV-1 BamHI-y (pGX1) or BamHI-k (pGX2), were grown in Luria broth containing

ampi-cillin (100 tg/ml). Plasmid DNA was amplified by

treating logarithmically growing cells with 25 ytg of

chloramphenicol per ml for 12 h. After harvesting, the cells were suspended in a 25% solution of sucrose in 50

mM Tris-hydrochloride (pH 7.9) and incubated at0°C

inthe presence of lysozyme (1.7mg/ml). After 30min,

EDTAwasadded to a final concentration of 0.05 M

andincubation was continued for a further 30 min at

0°C. NaCl and sodium dodecyl sulfate (SDS) were added to final concentrations of 1.0 M and 1% (wt/

vol), respectively. The lysate was then stored at0°C

overnight before centrifugation for 1 h at 20,000 rpm inaSorvallSS34 rotor. Thesupernatantwasdiluted

with an equal volume of water and extracted twice

with phenol and once withchloroform. The DNA was

concentrated by ethanol precipitation, suspended in

0.01MTris-hydrochloride-0.001 M EDTA (pH 7.9) in

the presence of RNase A (100 ug/ml), and dialyzed

extensively against the same buffer. The DNA solution was extracted twice with phenol and once with

chlo-roform beforebeing precipitatedby isopropanol

treat-ment (13). The pellet was resuspended in a small

volume of0.01 MTris-hydrochloride-0.001 M EDTA

(pH 7.5).

Enzymes. RNase A was obtained from Sigma

Chemical Co. Restriction endonucleases were

pur-chased from New England Biolabs, and the enzyme

reactions werecarried outunder the conditions

rec-ommendedbythesuppliers. ThedigestedDNA used

inmarkerrescueexperimentswasextractedoncewith

phenol and once withchloroform to remove the

re-striction endonuclease.

Purification ofrestrictionendonuclease DNA

fragments. Restriction endonucleasefragmentswere

separated byagarose gel electrophoresis (34) inthe

presenceof ethidium bromide(0.5

,g/ml).

DNAbands

werevisualized underlong-waveUVlightand excised

from thegels.The DNAwaseluted from thegelslice

by high-voltage electrophoresis and purified further byhydroxylapatitechromatographyasdescribed

pre-viously (32). The DNA solution was dialyzed

exten-sively against 0.01 M Tris-hydrochloride-0.001 M EDTA (pH 7.5)and concentratedbyethanol

precipi-tation in the presence of carrier E. coli rRNA(30ytg/

ml). Thepelletwassuspendedin0.01M

Tris-hydro-chloride(pH 7.5).Verysmall restrictionendonuclease

fragmentswereseparated bypolyacrylamide gel

elec-trophoresis (33).Thegelswerestainedwithethidium

bromide (10yg/ml) for 10min, andthe DNAbands

werevisualizedunderlong-waveUVlight.Thebands

were excised from the gel, and the gel slices were

crushedbyDouncehomogenizationin10ml of0.01M

Tris-hydrochloride-0.001M EDTA(pH 7.5).The

sus-pension ofpolyacrylamide was rocked gently

over-nightat4°Cbeforelow-speedcentrifugationtoremove

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152 PRESTON

thepolyacrylamide.The DNAwaspurifiedfurtherby

hydroxylapatite chromatographyasdescribed above.

Marker rescue assay. Marker rescue was

per-formed essentially as described by Stow etal. (22).

Briefly, BHK cellmonolayersin 50-mm dishes were

transfectedwithamixture of mutantviral DNA (0.5

to1

jig)

andaclonedwild-typeHSV-1DNAfragment

and treatedwith 25%(vol/vol) dimethylsulfoxide for

5minat4.25 hpostinfection.Afterincubation for 3or

4daysat31°C,the infectedcells wereharvested,and

the viral yieldwasassayedat31and38.5°C.

SDS-PAGE. Virus-infected cell protein samples

wereanalyzed by SDS-polyacrylamide gel

electropho-resus (PAGE) onagradient gel (6to 15%

polyacryl-amide) witha stacking gelof 5% polyacrylamideas

described by Marsdenetal.(14).

RESULTS

Comparison of polypeptides induced by

tsT, D,K and c75 in BHK cellsunder per-missive and nonperper-missive conditions.

Al-though previous work (6, 7, 11, 14) has shown

thattsT, D, K, andc75 exhibitsimilar,restricted

polypeptide phenotypes at the NPT, no one

study has compared the polypeptides induced

by thesefour mutants. For thisreason,the poly-peptide profilesof BHKcellsinfected withtsT, D, K, and c75 under nonpermissiveand permis-sive conditionswere examined bySDS-PAGE.

Figure1showsanautoradiogramof electropho-retically separated virus-infected cell polypep-tides labeled with[35S]methioninefrom 6 to 8 h

postinfection.Cells infected with mutantorts+

virusat310Cgavealatepatternofvirus-infected

cellpolypeptide synthesis, asdid cellsinfected

with ts+ virusattheNPT.By comparison, cells

infectedwith tsT, D, K,orc75 underrestrictive

conditionssynthesized increasedamountsof IE

polypeptides (e.g.,Vmw IE175, 110, 68,and63) and reducedamountsofearlyviralpolypeptides (e.g., the major viral capsid polypeptide Vmw

155) and failed tomake detectable amountsof

latepolypeptides (e.g., Vmw 51).The pattem of

virus-specific protein synthesisin tsK-infected

cells at the NPT was more restricted than in

cells infectedwithtsT, D,orc75.Forexample, the Vmw 43polypeptide bandwasnot

detecta-blein ts K-infected cellprofiles, whereastrace

amounts of this species could be seen inother

mutantpolypeptide profiles.

Genetic studies with tsT, D, K, and c75. (i) Complementation. InHSV,themost

com-monly used complementationassayistheyield

test, in which the total amount of infectious

virus produced by cells mixedly infected with

twodifferentmutants atthe NPTiscompared with the yields of virus produced from cells infected with each mutant alone at the NPT

after one growth cycle. Another assay is the

infectiouscenter test,in which the abilityofcells

mixedly infected with two ts mutants to form plaques on uninfected cell monolayers at the NPT is compared with that of cells

singly

in-fected with eachtsmutant.

Althoughearly work,onthebasis of the infec-tious center test,

assigned

ts K to a different complementationgroupfrom thatoftsTandD (Crombie,Ph.D. thesis, 1975), subsequent stud-ies with these mutantsraised doubtsaboutthis findingsincenegative resultswereobtained from theyieldtest.For this reason,theability oftsT, D,K, and c75 to

complement

oneanotherwas checked byusing bothcomplementationassays. Inthe yield test, no complementationwas ob-served between

pairwise

crosses of these mu-tants

(Table

1). This result confirms previous observations from our laboratory (N. D. Stow and D. Dargan,

personal

communication) and is consistent withreportsfrom otherlaboratories using mutants of the same phenotype (6, 11). Positivevalues, however,wereobtained fromall but two crosses in the infectious center test. There are twomajor problems associated with this test. First, it involves more manipulations underpermissive conditions than the yieldtest; second,aconsiderableamountof recombination may occur atthesametimeas

complementation

(15). To test the

possibility

that leakiness of virusmayhaveresulted in false-positive values ofcomplementation, viruswasabsorbedtocells in suspension at two different temperatures in theinfectiouscentertest.By

increasing

the virus

absorption

temperature, the number ofpositive

values ofcomplementationwas reduced (Table 2).

(ii) Recombination. The fourmutants were tested for their ability to recombine with one another.Allmutantpairs,except ts T and ts D, produced significant levels ofts+ progeny virus in two-factor crosses. Recombination values ranged from <0.001% (for thecross ts T x ts D) to 17.1% (for the cross tsT x ts K) in a single experiment. By usingrecombination data from three independent experiments, a linkage map of mutationswasconstructed (Fig. 2). It should be notedthatthets c75mutation does not lie to oneend of the genetic map as would be expected if this lesion mapped in the "a" sequence of HSV-1 DNA asreported by Knipe et al. (10).

Fine-structure mappingoftsT, D, K, and c75 mutations. The mutations in ts D and K have been located within the short repeat se-quences (11, 21), whereas the ts c75 lesion has beenreported to lie within theterninally redun-dant "a" sequence ofthe HSV-1 genome (10). Cloned HSV-1BamHI restriction endonuclease fragments fromthis region were therefore tested for their ability to rescue ts+ virus from cells transfectedwith tsmutant DNA. Figure 3 shows

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310 38

50

.)_ N PN- V,

- c

~~~~~N

(UX Q Y a l_- X ~a- I a

4E () (4 (4 U, 4.0 4. (4 U) (4 4-0

U)0 4-0 +.' 40 40 U0 (A 4' 4.' 4' 4.' U,

Vmw

IE 175 155 IE 136

IE

110 IE

68

IE 63 51

[image:4.493.47.443.63.520.2]

43

FIG. 1. Autoradiogram ofelectrophoretically separated

polypeptides

from

mock-infected

cells (MI) and

virus-infectedcells incubated atthePTand NPT. Cellswere labeledwith

['SJmethionine

from6 to 8h

postinfection.

the result ofamarker rescue experiment using clonedBamHI

fragments

k and y from HSV-1 strain 17. Themaplocation of these

fragments

on the HSV-1 genome is also included in the figure. When cells were coinfected with ts K DNA and

pGX1

plasmid

DNA

(which

contains theHSV-1 BamHI

fragment y),

a

high

levelof ts+ progeny was obtained. Since

wild-type

se-quences from

BamHI-y

wereableto correctthe

defect in the mutant, the ts Klesion must lie withinthisregion.By similar reasoning, ts T, D, andc75lesionswere

mapped

within the BamHI-k sequenceofHSV-1 DNA.Onthe basis of this experiment,the ts Klesionwasseparatedfrom the three other defects. Theseresultswere ob-tained by

using

plasmid DNA digested with BamHI.Ifuncleavedplasmid DNA(about75% supercoiled) wasused, the efficiency ofmarker

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154 PRESTON

TABLE 1. Complementation betweentsT, D,c75,

andKG

Virus T D c75 K

T 0.6 1.2 0.7

0.2 390.2 792.2

D 1.1 0.4

0.3 120.0

c75 0.7

4.0

K

aComplementation indices from theyield (top

num-ber) and the infectiouscenter(bottomnumber)tests.

Values were calculated from the formulas given in

Brown et al.(2). Indices greater than2.0 were

consid-eredpositive andareunderlined. Anabsorption

tem-peratureof38.5°Cwasused in theyieldtest, whereas

a temperature of 37°C was used in the infectious

[image:5.493.255.448.54.212.2]

centertest.

TABLE 2. Complementation indices from infectious

centertestsa

Virus T D c75 K

T 1.3 183.2 408.5

<0.1 38.7 1.8

D 0.7 285.5

0.9 37.9

c75 3.4

1.9

K

aA temperature of 37°C (top number) or

390C

(bottom number) was used.

rescue of ts+ progeny virus was markedly re-duced (Table 3). If, however, cells were coin-fectedwithplasmidDNAcleavedwithHindIII andwholetsmutantDNA, the proportionof ts+ progeny wasonlytwo- tofivefold less than val-ues obtained by using

plasmid

DNA digested withBamHI. These resultssuggest that linear-ized plasmid DNA molecules

(generated

by

cleavage with BamHI or

HindIII)

rescue ts+ virus moreefficiently thancircular, supercoiled DNA.

Effect of varying the concentration of plasmidDNA on markerrescueofts+virus. Tomaximize the efficiency ofmarkerrescue,a

dose-response curve was obtained by varying the concentration ofplasmid DNA in transfec-tion experiments. Increasing the concentration ofplasmidDNAmixed with aconstant amount ofmutant viral DNA increased the proportion ofts+ progeny (Fig. 4). A plateau level of ts+ progeny was reached when the relative molar ratio ofBamHI-y or -k to viral genomic DNA wasfiveorgreater.

Therefore,

inallsubsequent experiments a 10-foldor greater relative molar ratio of HSV-1BamHI-yor-k(orsubfragments

TD c75

T ~D --c75 K

-C0001. -C0001, 0

00%

3.86

2-0

171 5, 11-2

0.9. 0.7. 1.0 3-1. 10.. 2'4

[image:5.493.51.246.74.181.2]

1-6, 0o 1-4

FIG. 2. Linkage mapoftsT, D, c75, and K. Values

represent recombination frequencies (percentage)

from three independentexperiments andwere

calcu-latedbyusingtheformulafromBrownetal.(2). The

toplineofthe map isascalerepresentationof

recom-binationfrequencies (from oneexperiment) between

adjacent markers. The bottom lineofthe map

repre-sents the order of mutations without regardto the

relativephysicalmap distance.

of these sequences) to HSV-1 ts mutant DNA wasused.

Fine-structure

physical

mapping

oftsT, D, c75, and K within BamHI-k and-y. The

ts mutations were more precisely mapped by

marker rescue with restriction endonuclease fragments within cloned BamHI-k and -y. The results of transfectionexperiments using tsT,D, or c75DNAmixed withspecificsubfragments of plasmid pGX2 DNA (which containsthe HSV-1 BamHI-k fragment) are shown in Fig. 5 and summarized in Fig.6. The

fragments

HincII-c, SStI-b,and Sau3AI-b allrescued thetsT and D lesions. Since the smallest region of BamHI-k common toall threefragmentswasthesequence sharedby Sau3AI-b and

HincII-c,

thetsT and D mutations must lie within this DNA. Al-though ts c75 wasrescued by HincII-c, the re-sults ofmarker rescue experiments with sepa-ratedSau3AIfragments from BamHI-ksuggest that this lesion lies in a different region of BamHI-k from ts T and D defects, within the terminal Sau3AI fragment c. No significant levels ofts+ progeny virus were obtained with separated orunseparatedSstIfragmentsin coin-fectionexperiments withts c75 DNA. The ob-servation suggests that the ts c75 lesionmaps closeto an SstIcleavagesite.UnseparatedSmaI fragments from Bam-HI-k also failed to rescue this mutant. Since there is anSmaIsiteadjacent totheSstI sitedelimiting SstI-band c (A. Dav-ison, personal communication), thisresult indi-cates that the ts c75 defect is located close to thisSstIsite.

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MAPPING OF HSV-1 ts MUTATIONS 155

[I.

L

IRL

IRS4Us*TRS

~4- S

BamH I

bi

k Y n i I'l x q

I R-O 4-IR US -. 4- TR -.

L S S

Viral DNA Cloned HSV-1 fragment Relative eop (38.5° titre/31°titre ) x

103

tsK <0.01

BamHI y 10.3

[| I k <0.01

ts T

_

0.09

BamHI y 0.01

BamHl k 203

ts D <0.01

BcmHI y c0.01

BamHIk 31.6

ts c75 <0.01

BamHI y <0.01

BamHI k 3.5

FIG. 3. MarkerrescueoftsT,D, c75, and K. The HSV-1 genome contains two unique regions, UL andUs,

eachflankedbyinverted repeat sequences(TRL, IRL;TRS, IRS)andjoinedatIRL-IRs (20). The S component

of the viral DNA is shown in detail together with theBamHIphysicalmapof this region. Plasmids pGX2 and

pGXI, containingHSV-1 BamHIfragments k and y, respectively, werecleaved with BamHIbefore being

testedforabilityto rescue tsT,D,c75, K.

TABLE 3. Efficiency ofmarkerrescueoftsTandK,

using cloned HSV-1 in linearorsupercoiledform

DNA Plasmid DNA Relative

tsK None <0.001

pGX1 (containsHSV-1 BamHI- 0.04 Y)

pGX1digestedwith BamHI 0.9

pGX1

digestedwithHindIII 0.2

tsT None 0.009

pGX2(containsHSV-1 BamHI- 0.8

k)

pGX2digestedwith BamHI 10.5

pGX2digestedwithHindIII 4.3

CValues

represent relative efficiency of plating

(EOP), (38.5°C/31°C) x 100.

By using separated restriction endonuclease fragmentsofBamHI-y,the tsKlesionwasalso mapped to a smallregion of the viral genome.

The results ofmarker rescue experiments are

shown in Fig. 7andsummarizedin Fig.8. The

dataareconsistentwith thetsKdefectmapping

in BamHI-y close to the BamHI site which

delimits BamHI-m' and-y.

Figure 9 shows an overall summary of the

fine-map locations oftsT, D, c75, andK

muta-tions.AlthoughthemRNA for Vmw IE 175has

been shown to lieentirelywithinIRs/TRs (30),

the exactmappositionofthe mRNA is not yet

known. The 3' end of themRNA, however,has

been shownbynucleotide sequence analysisof

BamHI-k and nuclease S1 mapping (1) to lie

within BamHI-k, 3.2 kilobase pairs from the

restriction endonuclease site delimitingBamHI

fragments k and m' (A. Davison and F.Rixon, personal communication). Since the size of the

mRNA specifying Vmw IE 175 has been

esti-matedto be 4.7 kilobases (30) and there isno

evidence of splicing of the transcript for this

TRL

:3

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156

PRESTON

Wo

i

a

c c

0136 076 025 O

ug plasmid DNA ug plasmid DNA

FIG. 4. Relationship betweenefficiency ofmarkerrescueofamutantand the wild-typeDNAfragment

concentration.

(V)

MarkerrescueoftsT withpGX2cleavedwithBamHI;(2)markerrescueoftsKwithpGX1

cleaved with BamHI.

Mutant Enzyme Fragment

HincII - a b c

ts T 0.11 0-09 0.29 34

tsD0 0-01 -0 01 '0 01 17 tsc75 0.04 0-07 001 45

SstI - a b c

tsT 0.02 035 89 0 52

tsD .001 '0.01 11 0.01

tsc75 0.04 0.03 4001 0.01

Sau3AI - a b c d

ts T 0.04 0.02 1.63 0.06 0.07

ts D <0.01 0.02 0.34 40.01 40.01

tsc75 .01 '0.01 '0.01 0.26 .001

Smal - unseparated fragments tsc75 0.08 0 11

Sst I .01 003

FIG. 5. Marker rescue of ts T, D, and c75 with

restriction endonuclease fragments from pGX2 DNA.

Values represent relative efficiency of plating, 38.5/

31°Cx 103 of progeny virus from transfected cells.

Thephysical maps for pGX2 fragments generated by

SstI,HincII, and HindIII are shown above. Cleavage

polypeptidewithin theregion wherethe lesions map, tsT, D, K, andc75mutationsmustalllie within thecodingsequences of the gene.

Marker rescue ofts c75 with separated HpaI fragments from strain 13vB4. It has previously been reported by Knipe et al. (10) that the ts c75lesionis locatedwithin the "a" sequenceof HSV-1 since the terminaland joint-spanning DNAfragments from ts+ virus DNA rescuethismutant. Becausethese workers had used restriction endonuclease DNA fragments from strain MP andan intratypicrecombinant in markerrescueexperimentswith this virus, we attempted to confirm the original observations by using the parental ts+ virus strain 13vB4 DNAofts c75.Althoughtheresults in Figure 10 show some fragment contamination (for

exam-ple,

HpaI-i

iscontaminated withHpaI-g + h),

the data suggest that sequencesfrom IRL/TRL areunabletoconvert tsvirustots+ phenotype. This findingsupports the conclusion that the ts c75 mutationmaps within

IRs/TRs

but not in the"a"sequence.

DISCUSSION

Although tsmutationsin atleast29 cistrons of HSV-1 have been identified (3, 19; P. A. Schaffer,

unpublished

data), fewof thesedefects havebeenassignedtospecificgenes. Thispaper describes the fine-structure mapping of four ts mutationswhichliewithin the short repeat

se-sites of Sau3AIare given for HSV-1 BamHI-k

se-quencesonly (24, 27; Davison, personal

communica-tion). The solid region of pGX2 refers to HSV-1 BamHI-k sequences, and the open region represents pAT153 DNA sequences.

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HSV-1

BamHI

k

IR7

sequence

-Rs

0 0.4 04 1.2 1.6 2.0 32 3.6 40 44 44 52 54 6.0

a I

~~~~~b

[ c a

a b d c

a _

ts

D*ts

T

a

a b d c

tsc75

summary:

FIG. 6. Summary ofmarkerrescuedataontsT, D, andc75.Thesolid linesrepresenttheregions of

HSV-1BamHI-kwhichrescuetsT, D,orc75.

quences ofthe HSV-1 genome and show that

these lesions mapwithin the coding sequences

of thegenespecifying theimmediate-early

poly-peptide Vmw IE 175 (ICP4).

By using cloned HSV-1 DNA fragments in

marker rescue experiments, I have overcome

manyof theproblemsencounteredwhen using

DNA fragments isolated from viral DNA. The

improved efficiency of rescue of ts+ progeny

from cells coinfected with mutant viral DNA

and ts+ HSV-1 fragment DNA allowed

muta-tionstobe located withinverysmallregionsof the viralgenome.Inthecaseof tsK,rescuewas

obtainedwitha320-base-pairSau3AIfraginent

fromcloned HSV-1 strain 17BamHI-y.Todate,

this is the smallest DNAfragment reported to

rescueatslesion,andclearlymarkerrescuewith this sizeoffragmentisapproachingthe limits of

thetechnique.Theabilitytomapamutation to

averysmallregionof the viralgenomebyusing

clonedfragmentsfromwild-typeHSVDNAnow

makesitpossibletodeterminebyDNA

sequenc-ingmethods theprecisebasepairchangecausing

the defect.

AsimilaranalysisoftsmutationswithinIRs/

TRs

hadbeen made byDixon and Schaffer(6).

Theymapped the lesions ofviruses

belonging

to

complementation group 1-2tothree regions of the short repeat, probably corresponding to

BamHI-yandHSV-1 sequenceswithin

HincII-a and -c in pGX2. Although they assigned ts LB2 mutation to sequences within BamHI-y,

thephysicalmaplocation of thisdefect didnot

correlate withthepositiononthegeneticmap.

Suchanomalousbehaviorwasnotobserved with

the tsKmutation withinBamHI-y. Both tsK

andLB2havemorerestrictedpolypeptide

phe-notypesatthe NPT than othermutantswithin this complementation group, and therefore it would beinterestingto determine whether the

ts LB2 lesion maps in the same region of

BamHI-y as the tsK mutation. In contrast to

resultspresentedin thispaper,Dixon and

Schaf-fer(6)obtainedrescueoftsc75 with two

differ-entfragments from the shortrepeat.This

find-ingled them tosuggestthattsc75mightcontain

two mutations within each short repeat, one

within the"a"sequenceattheIRL/IRs junction

or end of TRs and one within a second "a"

sequencepresentinthe HSV-1 HincII-cregion ofpGX2.Iwasunabletoobtainrescueof tsc75

withthe HincII-afragment (Fig. 5) and

there-kilobase pairs

SstI

Sau3AI

HincII

summary:

Hinc1I

Sau3AI

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Enzyme fragment

Pvul1/BamHI - a b c

0.01 0 01 1536 0*001

Hinfl - a b c.d e f g h.i j.k

<00<004324-001 <0*01 cOOl <001 001 <001 010

SmaI/BamHI - a b. c d e f g

<001 0.02 0.57 <0.01 0.01 <001 003

Sau3AI - a b c d e f

i 006 0o09 172 <0*01 <0.01 <0.01 <0.01

FIG. 7. MarkerrescueoftsKwith restriction endonucleasefragmentsfrom pGX1DNA. Values represent

relativeefficiencyofplating, 38.5/31°Cx103, ofprogenyvirusfrom transfectedcells. Thephysicalmapsfor

pGX1 generated by PvuIIplusBamHI,Hinfl, HindIII, and BamHIplus SmaIare shownabove. Cleavage

sitesof Sau3AIaregivenforHSV-1 BamHI-ysequencesonly(24; M.-J.Murchie,personalcommunication).

Thesolidregion represents HSV-1BamHI-ysequences, andthe openregion referstopA T143DNAsequences.

Note thatpAT153is present in thisplasmidas adimer in head-to-tail arrangement.

HSV-1 BamH I y

0 0.2 0.4 06

[ . a a 0B

1.0 1.2 14 1 6 175

I . *

b

c

]~~~Q-

c

I

e

_

I

f e b

I

d

b

if

I

a

I

djle

[image:9.493.100.445.29.327.2]

tsK summary:

FIG. 8. Summary ofmarkerrescuedataontsK. The solid linesrepresenttheregionsof HSV-1BamHI-y

whichrescuetsK.

158

k

ilobase

pairs

Pvu II

Hinf

I

Sma I

Sau3A

I

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[image:9.493.89.417.393.653.2]

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1 3 I 4

I

-m a

III

Ie.n 11 v n

I

_i

5I

3

5

:

3

rS

I

n

'I,

m'

1 7 11

Y'

I

v

11

n

,DT

K

T,D

'c75

c75

08 09

'~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.______os___ 0

fractional

genome length

FIG. 9. Physical map locations of ts T, D, c75, and K mutations. The orientation and map positions of IE

mRNA's in the S componentof HSV-1 DNA are based on data from Watson etal. (30), and Clements etal.

(4)andanunpublished data of Davison and Rixon. IE mRNA's 1, 3, 4, and 5 specify the polypeptides Vmw

110, 175, 68,and 12,respectively.

k u

m o l p ? n i t

HpaI

I I

i

,

b h f e qsvrm c g

I

- a b cde f gh i j I m n k? u?opqr s t v

0.01 7.06 0.50 19.74 0.25 8.60 1.34 0.56

0.15

0.20 0.50 0.20 0D2 0.05 0.04 0.02

FIG. 10. Markerrescueofts c75with separated HpaIfragmentsfrom 13vB4 DNA. The HpaIphysical map

ofstrain 13vB4 isalmost identical to that of strain 17 except for the apparent absence of the cleavage site

delimiting HpaI fragments k anduand the presence of a new site within this DNA region. To avoid confusion,

the nomenclatureforthe strain17HpaI digest has been used. The joint-spanning DNA fragments HpaI-a

and darecomposedoffragmentsm+candm +g,respectively. Values represent relative efficiency ofplating,

38.5/310Cx 103, ofprogenyvirusfromtransfected cells. The underlined figures refer to values obtained by

usingfragmentscontaining sequences from the repeated regions of HSV-1 DNA.

forecannotsupporttheirproposal. In addition, Ifailedtoshowconvincingrescueof ts c75 with sequencesfrom thelongrepeatof strain 13VB4 DNA and suggest that this mutant does not haveadefect in the "a"sequence.This conclu-sion is

supported

byDNA sequence

analysis

of a cloned HSV-1 DNA fragment spanning the

IRs/TRs

region of strain 17viralgenome.This

work showed that inthe cloned DNA fragment, the 3' endofthe gene for Vmw IE 175lieswithin the short repeat but at least 800 base pairs outside the "a" sequence (Davison and Rixon, personal communication). Hence, iftsc75had a mutation within the "a" sequence, it would not beexpectedtohaveaphenotypelike thegroup 1-2mutants.Themap location of thismutation can therefore nolongerbe used asevidence in

support of the circular

replication

model pro-posed byKnipeetal. (10)in whichthe terminal "appsequencesare

regenerated by repair

synthe-sis, using the internal invertedrepeatsas a

tem-plate.

Itshouldbenoted thattsc75wasrescuedless

efficiently by

HSV-1 strain 17 BamHI-k

se-quencesthan were tsT and D. Thereason for therelatively poorrescueoftsc75 isnotclear. It ispossiblethat ts c75containstwo tslesions that map close together in BamHI-k. Alterna-tively, viral strain differences may accountfor the inefficient rescue ofts c75. Another

expla-nation is that thereareDNAsequences between the ts T andc75mutations that enhance

recom-bination.

Precise physical mapping of mutations

en-IEmRNA

BamHI

mutation

.4

-IR

L

--

*--I

Rs

-0

Lis

it,-t

TRs

90

I

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160 PRESTON

ables recombination

frequencies

to be inter-preted in terms ofphysical distance. Fromthe genetic linkage map of the HSV-1 thymidine kinase locus whichlies within

UL,

Smiley

etal. (21)calculatedaminimum value of 3.2% recom-bination perkilobase

pair

of DNA. The maxi-mumdistance betweentsT and K is about 2.14 kilobasepairs.

Taking

ageometricmean recom-bination frequencyof11.8% between thesetwo lesions(from threeindependent

experiments),

a value of 5.5% perkilobase pair ofDNA is ob-tained.

Although

this

figure

is

considerably

higher thantheestimate of

Smiley

etal.

(21)

for recombination in the

unique

region

of HSV-1 DNA, there is enormous variation in the fre-quency of ts+ recombinantsfroma

given

genetic crossperformedatdifferent

times,

whichmakes itdifficulttocompare results fromdifferent lab-oratories. For

example,

the cell type used in geneticexperimentsappearstoinfluence recom-bination values. In this

study

and the one

by

Honessetal.(7),significant values ofaround 1% were obtained for thecross ts D x tsc75 per-formed inBHK cells. By contrast,

Knipe

etal. (10) and Dixon and Schaffer (6),

using

rabbit skincells and humanembryonic

lung

fibroblasts,

respectively, failedtodetect recombination be-tween these two ts mutants. In

calculating

re-combination

frequencies,

a number of assump-tions are

made,

including

the

following:

(i) a

single crossover event is sufficient for the for-mationofvirus withats+phenotype (provided intermolecular recombination occurs between linear andnotcircular DNA

molecules);

(ii) ts+ virushad arelative

efficiency

ofplating (NPT/ PT)of 1; and(iii)thedoubletsmutantreciprocal class arises atthe same frequency as does the ts+class. Although the tsKandTlesionsmap withinadiploid gene,twosourcesofinformation suggest thatonly onefunctional copy is neces-sary forthe ts+phenotype. First, only oneend ofthe S component of HSV-1 strain1061rescues

tsD

(11),

and

second,

HSV-1/HSV-2

recombi-nants, containing one HSV-1 and one HSV-2 short repeat,expressboth VmwIE175 of HSV-1 and the HSV-2 counterpart polypeptide. Therefore, asinglecrossoverevent intheshort repeatsequence shouldbesufficientfor the gen-eration of a ts+ recombinant. On this assump-tion, Dixon andSchaffer (6) suggested thatthe recombination frequency between two muta-tions in adiploid gene shouldbetwicethe fre-quency expected for a haploid gene. Detailed analysisof thegenomestructuresofHSV inter-typicrecombinants withheterologous short re-peats has shownthatthesevirusesareunstable. Recombination between the heterologous re-peats cangenerateDNAmolecules with differ-ent arrangements of HSV-1 and HSV-2

se-quences fromtheparental recombinant genome

(18, 35; Davison, personal

communication).Itis

therefore

likely

thatan

intratypic recombinant,

heterozygous

for a ts lesion within the gene

encoding

Vmw

175,

would also be unstable and

upon

subsequent

intermolecular recombination

segregate to

give

virus

homozygous

for ts or ts+ markers inadditionto

heterozygous

ts/ts+ virus. For this reason, the recombination

frequency

between markers in a

diploid

gene would be

predicted

tobelessthan twice thefrequency of

the samemarkers ina

haploid

gene. Inspiteof the difficulties ininterpreting thegenetic

data,

it is clear that there is a high frequency of recombinationwithin the shortrepeatsof

HSV-1,

but it is still uncertain whether this

region

undergoes

enhancedrecombinationin

compari-sonwith the

unique regions

ofHSV-1 DNA.

ACKNOWLEDGMENTS

Iamgrateful to J. H.Subak-Sharpe for his encouragement andvaluablecriticism of the manuscript. I also thank A. J. Davison and M.-J. Murchie forpermission to cite the

unpub-lishedfine-structure restriction endonuclease cleavage maps of HSV-1 strain 17BamHI k andy fragments and M.Buckley

for excellenttechnical assistance. Inaddition, Iamgratefulto B.Honess forproviding stocks of the HSV-1 strain 13vB4 and tsc75 andtoP.A.Schaffer for a copy of themanuscriptby

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