Isolation and characterization of herpes simplex virus mutants containing engineered mutations at the DNA polymerase locus.

Copyright ©1990,American SocietyforMicrobiology

Isolation and

Characterization

of Herpes Simplex

Virus Mutants

Containing Engineered Mutations

at the

DNA

Polymerase Locus

ALICE I. MARCY, DORNE R. YAGER, ANDDONALD M. COEN*

DepartmentofBiological Chemistry and Molecular Pharmacology, HarvardMedicalSchool,

Boston,Massachusetts02115

Received 1 November 1989/Accepted 25 January 1990

We have derived Vero cell lines containing the herpes simplex virus DNA polymerase (pol) gene that complementtemperature-sensitivepol mutants. These cell lines were used to recover virusescontaining new mutations at the pol locus. Twospontaneouslyarising host-range mutants, 6C4 and 7E4, wereisolated. These mutants did not growefficientlyon Verocells or synthesize late polypeptides but formed plaques onacell line containing the polgene (DP6 cells). Whereas mutant 6C4specifieda

wild-type-size

Polprotein, wedetectedno

full-length Polprotein in 7E4-infected cell extracts.Complementationstudiesdemonstratedthat 6C4 and 7E4 contain different mutations and indicated that6C4 is in a complementationgroupdifferent from that ofpol

temperature-sensitivemutant tsC7 or tsD9.A mutant inwhich 2.2kilobases ofpolsequences were replaced

with theEscherichiacolilacZgene under the control of the herpes simplex virus thymidine kinase promoter was constructed. This mutant formed blue plaques on DP6 cells in the presence of

5-bromo-4-chloro-3-indolyl-o-D-galactoside. Using this virus in marker rescue experiments, weengineeredthree mutantscontainingdeletions

in thepolcoding region which grew efficiently on DP6 cells but not on Vero cells and whichdiffered in their

synthesis of Pol polypeptides. The lacZ insertion virus was also used to introduce a deletion in the region

upstreamof thepol longopen reading frame, which removes a short open reading frame that could encode a

10-amino-acid peptide. This mutant grew to similar titers on Vero and DP6 cells, indicating that these

sequencesarenotessentialforgrowthof the virus intissue culture.

Theherpessimplexvirus(HSV)DNApolymerase (Pol) is

essential for viral DNAreplication (1, 35, 40), serves as a

targetfor antiviral drugs(9),andprovidesanexcellentmodel

for studies ofeucaryoticreplicativeDNApolymerase inpart

because it is amenable to both biochemical and genetic

analysis. Understanding Pol functionswill begreatly

facili-tated bymutants defining protein domainsthatcontributeto

catalysis, interactions with other proteins, stability, and

intracellularlocalization.

Such a combined approach has already contributed to

effortsto correlatePol structure with itsfunctions. Genetic

mapping and sequencing of pol mutants exhibiting altered

sensitivities to nucleoside and

PPi

analogs and aphidicolin

indicate that amino acids lyingindiscrete regions between

residues 597 and 961 contributetosubstratebinding (17, 18,

27, 31, 32, 45). Furtherinsights intoPolfunctionhave been

provided by temperature-sensitive (ts)

pol

mutants (6, 10).

Four pol ts mutants map within a 4-kilobase region that

specifiesasingle majormRNA and longopenreading frame

(18,23, 48). Genetic analysisofthese mutants hasidentified

three complementation groups (groups 1-3, 1-4, and 1-14),

with complex overlapping patterns of complementation,

consistentwith the enzymehaving multiple functions (6, 23,

39, 46). The prototypic mutant in complementation group

1-4, tsD9, exhibits altered drug sensitivity and contains a mutation in a domain thought to participate in substrate

recognition(17, 18). Mutantsincomplementation group 1-3

specifythermolabile Pol activity in vivo (1) and in vitro (37)

and map to sequences that include the left end of the gene

(10, 23), suggestingthat theN-terminalportion of the protein

includes regions contributing to stability. The limits of

sequencestowhichcertain of these tsmutations havebeen

mappedalsoinclude a short openreadingframe which could

*Correspondingauthor.

encodea10-amino-acidpeptide (18). Thisshortopenreading

frame lies 75 basepairs (bp) downstream ofthepol

transcrip-tional start site and ends 93 bp upstream ofthe long open

reading frame (18,48). Itsrole isunknown.

The studies described herewereinitiatedtofacilitateour

understanding of Pol functions by increasing thenumberand

type ofpol mutants available for biochemical and

genetic

analysis andtoexamine theimportance ofthe10-amino-acid

upstream openreading frame for viral growth. Wewishedto be able to introduce site-specific mutations in thepol gene

into the HSV genome to allow mutant Pol

proteins

to be

analyzed in the context of a viral infection. Since we

expectedto recovervirusescontaining lethal pol mutations,

we isolated a cell line able to support the growth ofthese

viruses. As these mutants would not necessarily have a

selectable phenotype, we also needed todevelop a system

for screening pol mutants. We demonstrate the use of a

complementing

cell lineand apolmutantthatproducesblue

plaquestoprovideasystemfortheefficientrecoveryof pol

mutants anddescribe the isolation and

preliminary

charac-terization of six newpol mutants, one of which lacks the

10-amino-acid openreadingframe.

MATERIALSANDMETHODS

Cellsand viruses. Verocells weregrown andmaintainedas

described previously (46). Nero

cells

are a cloned line of

G418-resistant

cells

derivedbytransformingVerocells with

pSV2neo (42). HSV type 1 mutants tsC4, tsC7, and tsD9

werederived fromwild-typestrain KOS and havemutations

conferring temperature sensitivity at the pol locus (10).

MutanthrR3, kindlyprovided bySandraWeller,has alacZ insert in the ICP6gene (20).

Plasmids.The

plasmids

usedinthisstudyare

depicted

in

Fig. 1. Plasmid pDP1 contains the 153 bpupstream ofthe

2208

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UL

MIap Units Restriction Sites

Transcripts

Open Reading Fra

ts Mutants

Plasmids

EJ

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

0.407 0.413 0.422 0.439

-I

III

Ss Sm Sm Bt Sf B Sm Bt G SmSK E X Ss K X SP B B K

Bg Sp Sm

NL2Kbp

ICPB DNA POLYMERASE AAA

ATG

imes UF

SORF

pKEF-P4

TGA

tsC7

tsC4 tsD9

i I

pKOS 29

BamH Q

pSGI7 EcoRl M BamHI V

pDP1

pDP2A

pDPaX

pDP&S

pDPtkcBG

pDP4

tk PROMOTER lac Z

Bc

pDPtB

-FIG. 1. Maplocation ofHSV pol. The locations ofpoltranscript, open reading frames, and relevant restrictionsites and themapped limits oftsmutationsareshown. The bottomportion of the figure representsstructuresofplasmidsused in thisstudy. Restrictionenzymesare

abbreviatedasfollows:B, BamHI; Bc,BcII; Bg, Bg!I; Bt, BstII; E, EcoRI; G, BgIII; K, KpnI; P, PstI; S,SaII; Sf, SfiI; Ss, SstI; Sm, SmaI; andX, XhoI. UL, Unique long sequence;Us,uniqueshortsequence;SORF,shortupstream openreadingframe.

firstATGof the HSV polopenreading frame,theentire pol

openreadingframe, and156bp downstream ofthe

termina-tion TGA codon (HSVmapcoordinates 0.413to0.439). The

plasmid was constructed by treating pKOS29 (10) with

BamHI, filling in the overhanging ends by using Klenow

fragment, ligating on HindlIl linkers, and digesting with

HindIII and PstI. The

3.2-kbp

HindIII-PstI fragment was

then ligated to HindIII-PstI-digested pUC19 DNA (50) to

give risetoplasmid p5'DP. Plasmid pSG17 (19)wastreated

with

KpnI

and then with T4 DNA polymerase to remove

overhanging ends, ligatedtoXbaIlinkers,anddigested with

PstI and XbaI. The 0.8-kbp PstI-XbaI fragment from this

plasmid was recovered and ligated to PstI-XbaI-digested

p5'DPtocreatepDP1. pDP1stillhasthe

original

BamHIsite

at 0.413 map units adjacent to the unique HindIII site.

PlasmidpDP2A,which containsthepol openreadingframe

andpromoter sequences, wasmade asfollows. The 363-bp

BstII-BamHI fragment containing thepolpromoter and 51

bp of5'untranslated sequences wasremoved frompKEFP4

(47), treated with Klenowfragment, and ligated to

HindIIl

linkers. The resulting fragment was digested with HindIII

andligatedtoHindIII-treated

pDP1

to createpDP2A. Viral

sequences in thisplasmiddiffer from theviralgenomebythe presence ofthe HindIII site and a deletion starting 95 bp

upstream ofthe mRNA start site and extending upstream

intooriLfor62bp (present in pKEFP4; 47, 48).Thisdeletion

does not remove the binding sites for

transcription

factors

Spland TFIID upstreamofthepolmRNAcapsite

(48),

and

thepolpromoterinpDP2A retainsactivity, as

judged

by its

ability to expresspolin transfected cellsupon HSV

super-infection.

PlasmidpDPtkBGwas madeby first

inserting

the800-bp

BamHI-BglII fragment containingtheHSVthymidine kinase

(tk)promoterfrom

plasmid pKG3.6

(26) into

BglII-digested

pDPl.Theresulting

plasmid

wasdigested with

Sail,

treated

with Klenow fragment, ligated to

BglII

linkers, and then

digested with

BgIH.

ThisDNAwasthen

ligated

to a

3.0-kbp

BamHI fragment containingthecodingsequencesfor

Esch-erichia coli

3-galactosidase

from pDP500, kindly

provided

by Dennis Panicali(34). Plasmids

pDPAS

and

pDPAX

were

madebytreating pDP1with

Sail

or

XhoI, respectively,

and

ligase.

Site-specific mutagenesiswasused to introduce aunique

BclI site (TGATCA)11bpupstreamofthepolopen

reading

frame in

pDP1

by a procedure developed by

Taylor

et al.

(43). Mutagenesis was

performed by using

the

oligonucleo-tide

5'-TCGGGTGIGAICAACCGC-3'

and a

636-bp

HindIII-SphI

fragment

of pDP1 cloned into the

bacterio-phage vector

M13mpl9.

The

oligonucleotide

hybridized

to

thenoncoding pol strand, and the altered basesare

under-lined. Reagents for this

procedure

were

provided

in a com-mercial kit(Amersham

Corp.).

Clones

containing

the muta-tion were identified

directly by

DNA

sequencing,

using

previouslydescribed

procedures

(18). Double-stranded

rep--- -

JF--I.

L-I

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TABLE 1. Efficiency of plating of HSV ts mutants onVero and DP6 cells

Verocells DP6cells Fold

Strain or Mutation increasein

mutant site Titerat Titer at Titer at Titerat titer at

340C 390C EOPa 34 C 39 C EOP 39oCb

KOS 4.2 x 107 2.3 x 107 0.55 3.8 x 107 2.4 x 107 0.63 1.1

tsD9 pol 9.3 x 106 <5 x 101 <5.4 x 10-6 1.1 X 107 3.4 x 106 0.31 5.6 x 104

tsC4 pol 3.8 x 106 <5 x 101 <1.3 X 10-5 1.1 X 107 2.6 x 105 0.02 1.4 x 103

tsC7 po/ 9.5 X 105 <5 X 101 <5.3 x 10-5 2.2 x 106 6.5 x 104 0.03 5.7 x 102

tsJ12 gB 5.5 x 106 <5 x 101 <9.1 x 10-6 5.0 x 106 <5 x 101 <1 X 10-5 NDC

a EOP,Efficiency ofplating(determined as titerat39°C/titerat34°C).

bEfficiency of plating on DP6cells/efficiency of platingonVerocells.

cND, Cannot bedetermined.

licative-form phage DNA was prepared, and the mutated

HindIII-SphI fragment was removed and ligated with

HindIII-SphI-digested pDP1. The presence of the new

re-striction site in the resulting clone, pDP4, was verified by

digesting plasmid DNA derived fromDam- cells (GM119,

kindly provided by Jacqueline Miller) with BclI. Plasmid

pDPAB was made by ligating the 363-bp HindIII-BamHI

fragment frompDP2A toHindIII-BclI-digested pDP4.

Transformationof Verocells. Verocellsweretransformed with pSV2neo (kindly provided by Neal DeLuca) and a

10-fold molar excess of pDP2A by a modification of the

procedure described byDeLuca et al. (14). After

transfor-mation,thecells were incubated for2daysandthenplated

to adensityof 5 x 103/cm2in thepresenceof750

jig

ofG418

per ml(GIBCO Laboratories). Individual colonies appeared

after2to 3 weeksand wereamplifiedand assayedfortheir

ability tosupport the growthofpol mutant tsC7 or tsD9 at

the nonpermissive temperature (39°C). One cell line, DP6,

exhibited high complementing activity and was used for

further study.

Complementationanalysis. Complementation assays were

performedbyconductingmixedinfections under

nonpermis-sive conditions (growth at39°C on Vero cells) and assayed

under permissive conditions (growth at 34WC on DP6cells)

by procedures described previously (40, 46). The

comple-mentation indexwascalculated as (plaques for mixed A+ B

infection)/(plaques for A infection + plaques for B

infec-tion),where A and B aretwo mutants. An index of

.10

was

taken to beindicativeofcomplementation(46).

Marker transfer andrescue. AnHSV

expressing

,-galac-tosidase was recovered by cotransfecting DP6 cells with

infectious KOS DNA and pDPkBG by methods described

previously (7). Progeny from the transfection were plated

onto DP6 cells and stained after 3 days with 2 ml of

phosphate-buffered saline containing 600 to 750 ,ug of

5-bromo-4-chloro-3-indolyl-,-D-galactoside(X-Gal) per ml. At

1 or 2 days after staining, blue plaques were picked and screenedfor the ability togrow on DP6 cells and not on Vero cells. One suchvirus, HP66, was selected for further study. Marker rescue was performed as described previously (7),

using infectious HP66 DNA and the plasmids indicated in

Results.

Southern blot analysis. Viral DNA wasrecovered by the procedure described by Coen et al. (11). DNA samples were

digested with restriction enzymes and electrophoresed

throughagarose gels. The DNA was transferred to a nylon

membrane asdescribedby

Southern

(41). Membranes were

UVcross-linked (8) for 5 minimmediately after being blotted and then probed with

32P-radiolabeled

DNA fragments.

DNA probes were prepared by random priming of

gel-isolated restrictionfragments with[32P]dCTP(16).

Western immunoblot analysis and immunoprecipitations.

[35S]methionine-radiolabeled

or unlabeled extracts of

in-fected cells for sodium dodecyl sulfate

(SDS)-polyacryl-amidegels,immunoblots, and

immunoprecipitation

analyses

were prepared as described previously (26). For blotting,

proteinswere electrophoresedthrough

SDS-polyacrylamide

gels and then transferred to nitrocellulose as described

by

Towbinetal. (44). Thenitrocellulosewasthen reactedwith

a 1:1,000dilution ofa mixture ofpolyclonal rabbit antisera

directedagainst

P-galactosidase-polymerase

fusion

proteins

derived from bacteria (PP5 and/or BGG4; 49). Bound

anti-bodiesweredetected withagoatanti-rabbit

immunoglobulin

G-alkaline phosphataseconjugate, andvisualized withnitro

blue tetrazolium and 5-chloro-4-chloro-3-indolyl phosphate

(PromegaBiotec). ICP8 wasdetected withpolyclonal rabbit

antiserum 3-83, kindlyprovided by David Knipe.Thymidine

kinase wasdetected withpolyclonal rabbit antiserum

gener-ouslyprovided by William Summers.

RESULTS

Vero cellsexpressing DNApolymerase.As afirststep in the

isolationofnewpolmutants,wefirstclonedthepolgene and

its upstream sequences into plasmid pDP2A (Fig. 1). This

plasmid contains no other known intact HSV genes and

exhibited biological activity in a transfection assay (5) in

whichaplasmid containinganHSVorigin of DNA

replica-tion was used (M. Challberg, personal communication).

Since HSVpol isessential for viral

replication,

the

propa-gation ofviruses containing lethal pol mutations

requires

a

cell line expressing wild-type Pol. To thisend, we

cotrans-formed Vero cells with pDP2A and pSV2neo, which

con-tains the neo geneunder the controlofthe simianvirus 40

promoter(42).G418-resistant colonies of cellswerepicked,

expanded,andassayed for

their

abilitytosupport thegrowth

of polmutanttsC7 or tsD9at 39°C.

Approximately

30% of

the G418-resistant colonies expressed detectable

comple-mentingactivity.One cellline,DP6, which hadhighlevels of

complementing activitywas chosenforfurtherstudy.

Plaque formation by wild-type KOS and various ts

mu-tants onDP6 and Vero cellsat 34 and 39°C was

compared

(Table 1). Similartiters of

wild-type

strain KOS were

ob-tainedbyusingeither DP6 orVerocells at either 34 or39°C,

indicating thatexpression oftheresidentpolin DP6cellsis not detrimental for viral infection. DP6

cells

didnot comple-ment mutant tsJ12, whichcontains aglycoprotein B

muta-tion (13). Polymerase mutantstsC4, tsC7, and tsD9did not

formplaques onVerocells atthe elevatedtemperature but

formedplaquesonDP6cells under the sameconditions. For

tsD9, essentially wild-type levels ofplaque formationwere

obtained at 39°C. The extent of

complementation

differed

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TABLE 2. Plaque formation byHSVmutantsonVero andDP6cells

Titeron:

Strain or pimtto

mutant ~~polmutation

mutant Verocells DP6cells

KOS 4.2 x 107 3.8 x 107

6C4 Spontaneous host range <5 x 101 2.5 x 107

7E4 Spontaneous host range 5 x 101 1.6x 107

HP66 2.3-kbp deletion, lacZ 1.3 x 102 4.0 x 107

insertion

ASi 2.0-kbp deletion <5 x 101 9.8 x 106 AX14 1.2-kbp deletion 5.5 x 102 2.7 x 107

AX17 1.2-kbp deletion 1.2 x 102 1.1 x 107 AB10 144-bp deletion 6.0 x 106 2.7 x 106

among thepol mutants but was reproducibly the highest with tsD9 and lower with tsC4 and tsC7. DP6 cells contain

approximately30 copies of pol DNA percell,asdetermined

by Southern analysis of cellular DNA, and showed comple-menting abilitythrough 25 passages (data notshown).

Recovery of spontaneous pol host-range mutants. Having isolated a complementing cell line, our efforts were next directed toward obtaining apoldeletion mutant. For these experiments, DP6 cells were cotransfected with infectious wild-type viral DNA and pDPAS, which contains a 2-kbp deletion in thepolgene (Fig. 1). Two mutants isolated from the progeny of this transfection, 6C4 and 7E4, formed plaqueson DP6 cells but not on Vero cells (Table 2). These mutants were plaque purified andamplifiedforfurther study.

Todeterminewhether these host-range mutantviruses

con-tained the expected deletions, the BamHI restriction pat-terns of the mutant viral DNAs were compared with plasmid

DNA containing either the wild-type pol (pDP1) or the

deleted gene (pDPAS). Unexpectedly,both of the host-range mutants showed wild-type BamHIrestriction patterns when

probed with radiolabeledpolDNA (datanot shown).

Complementation analysis of spontaneous mutants. Since

the spontaneous mutants could becomplemented by a cell

line whose only viral sequences are the pol gene, we examined their ability to complement the growth ofpol ts mutants at the nonpermissive temperature (39°C) in Vero cells. Vero cells werecoinfectedwith thehost-rangemutants and either tsC7or tsD9 at 39°C. The progeny of this infection were assayed on DP6 cells at 34°C, and the results are shown in Table 3. As previously reported (39), tsD9 and tsC7

complementedone another for growth at thenonpermissive

temperature. 6C4 complemented both of thepolts mutants

and 7E4, while 7E4complemented neither pol ts mutant.

Polypeptides synthesized in spontaneous mutant-infected

cells. To determine whether the mutations in 6C4 and 7E4

resulted in the failure of the mutants to synthesize late

polypeptides,Nero and DP6 cells infected with either of the

mutants or the wild-type virus were labeled with

[35S]methionine

from 2 to 6 h postinfection. In Nero cells,

[image:4.612.58.299.105.223.2]

these twomutantsfailed tosynthesizedetectable amounts of

TABLE 3. Complementation ofpol host-range and ts mutants

Complementation indexwith mutant: Mutant

tsC7 6C4 7E4

tsD9 11 11 0.5

tsC7 66 1.0

6C4 110

true latepolypeptides such as ICP1,2 (Fig. 2A, top

arrow-head) and synthesized reduced amounts of other late

poly-peptides, relative to KOS. This pattern resembled that of

Nero cells infected by KOS in the presence of 200 ,ug of phosphonoacetic acid per ml, an inhibitor of viral DNA

synthesis.In contrast,in DP6cells,the two mutants

synthe-sized polypeptides that wereindistinguishable from those of the wild-type virus (results not shown). Thus thepol

muta-tions inthese two mutants led toadecreasein thesynthesis

oflate polypeptides, presumably due to the failure to syn-thesize viral DNA. Indeed, dot blot analysis (results not

shown) indicated that the two mutants were profoundly

impaired in the synthesis of viral DNAin Nero cells. Toexamine the Pol proteins specified by these host-range mutants, we probed blots containing proteins from KOS-,

6C4-, and 7E4-infected Nero cells with antiserum PP5, which is directed against a fusion protein consisting of ,B-galactosidase and theC-terminal 261 amino acids of Pol (49).

6C4 synthesizedanimmunoreactiveprotein the same size as

wild-type Pol, while no detectable equivalent was specified

by 7E4 (Fig. 2B). Similar results were observed when PP5

and BGG4 antisera were used to immunoprecipitate [355] methionine-labeled proteins from KOS-, 6C4-, and 7E4-infected Nero cell extracts(data not shown). 6C4- and 7E4-infected Nero cell extracts contained levels of ICP8 similar to those found in KOS-infected cells (Fig. 2B); thus the absence offull-length Pol polypeptide in 7E4-infected cell extracts was notdue to inefficient infection.

Recovery of apol insertion mutant expressing

j8-galactosi-dase.The resultsof the effortstoobtain apol deletion mutant suggested that animproved screeningprocedurewas needed to enhance the efficiency of detecting recombinant viruses. Wechose to use the E. colilacZ gene in a manner similarto that used byPanicali et al. (34) and Chakrabarti et al. (4) for vaccinia virusand by Weller andcolleaguesfor HSV (3, 20, 21). With this approach, a virus that forms blue plaques in the presence ofX-Galis used as ascreening marker in order

torecover recombinants that form white plaques due to the

replacement oflacZwithsequences froma plasmid contain-ing knownpol mutations.

Plasmid pDPtkBG (Fig. 1) was used in a marker transfer

experiment with wild-type infectious DNA to obtain viral recombinants that grewonlyonDP6 cells because of thepol

deletion-lacZ insertion and formed blue plaques in the presence of X-Gal due to the expression oflacZ. One such mutant, HP66, which grew efficiently on DP6 cells and poorly on Vero cells (Table 2) waschosen forfurther study.

The position of the lacZ gene was confirmed by Southern

analysis of wild-type KOS and HP66 viral DNAs. HP66 contained a5.4-kilobaseBamHIfragmentthathybridized to

both thelacZ gene andpol(BamHI Q-fragment) probes, as expected (Fig. 3). Mutant hrR3, which contains lacZ in a

3.0-kilobase BamHI fragment in the HSV ICP6 gene (20),

was included in this analysis to confirm thespecificity of the

lacZprobe.

The ability of HP66 to grow in DP6cells and not onNero cells demonstrated that the lethal mutation inHP66 is in the

pol gene. This was verified by marker rescue experiments

using wild-type pol sequences (pDP1). Transfections of

HP66infectious viral DNA with pDP1 gave rise to

approx-imately 100-fold more recombinants that grew on Vero cells than transfections with no plasmid DNA (data not shown).

The presence of

P-galactosidase

in HP66-infected cells was

confirmed by Western blot analysis of cells infected with KOS or HP66, using a serum specific fora

3-galactosidase-polymerase fusionprotein (49).HP66specifiednodetectable

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-Y

0

v2

(0~

COz m

Y cr I

CLr (fl 14 41-0 0 C) W6

o oe to N

Y, Y

-.4

_ .& .

n_

_ )! _NCD

5.4kb- u

3 3kb- *4. *

*.

Probe: BamO

cn CO Q

- SL

3.Okb-

*.

..:

':::::,o

.,.

lacZ

FIG. 3. Southern blotanalysisof HP66 viral DNA. Viral DNAs from wild-type KOS andmutantshrR3and HP66weredigested with BamHI, electrophoresed through 1%agarosegels, and transferred tonylon. Membraneswereprobed with either 32P-radiolabeled pol DNA(BamHIQfragment, Fig. 1)orlacZDNA.Fragment sizes(in kilobases)areindicated.

r

DP6 T Nero

, I Y 2 I E

FIG. 2. Analysis of 6C4- and7E4-infected-cell polypeptides. (A) [35S]methionine-radiolabeled proteins from Nero cells mock infected

orinfected with KOS, KOS in the presence ofphosphonoacetic

acid,or6C4 and 7E4wereelectrophoresed through

SDS-polyacryl-amidegels and exposedto film. Arrowheads indicate examples of

lateproteinspresentinKOS-infected cells and absenceorreduced

inmutant-infected cells. (B) Proteins fromNerocells infected with wild-typestrain KOS andmutants6C4 and7E4wereelctrophoresed

throughSDS-polyacrylamidegels,transferredtonitrocellulose, and reacted withantiserum (oa) specific for either Pol (left)orICP8 (right). Pol inNerocells andspecifiedaprotein that is thesamesize as ,-galactosidase in both Nero and DP6 cells (Fig. 4).

Figure 4 also shows that uninfected DP6 cells make no

detectable Polprotein.

FIG. 4. Western blot analysis of HP66-infected-cell proteins.

Protein lysates from mock-, wild-type strain KOS-, and mutant HP66-infected Nero and DP6 cells were electrophoresed through

polyacrylamide gels and transferred to nitrocellulose. The

mem-braneswereprobedwithanantiserumspecificfora

Pol-,-galactosi-dase fusion protein (49). The leftmost lane contains a set of

molecularweight standards, ofwhich P-galactosidase isa compo-nent.

A <

0-

Pol-_ -- -IGP8

pol AICP8

-Pol -9-gal

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m

x

---X Qa

T-(-><

C

ao

U)

'-00

Cl) c0Q

1> C)O O

< C] y >< xoI'll 0

<: < Y- 2

205-

Pol-

116-a..

97- d

Ut

- 3.3 kb

65--3.3kb

q0,..NPp46m

E1: -2.1 kb -1.3kb

[image:6.612.74.287.78.383.2]

45-A PoI Tk

FIG. 5. Southern blot analysisofdeletion mutantviral DNAs. ViralDNAs from AS1, AX14, and AX17 andplasmidDNAsfrom pDP1,pDPtkBG, pDPRS, andpDPAX weredigested withBamHI

andelectrophoresed through 1%agarosegels. TheDNAfragments

were transferredto nylon andprobed with radiolabeled polDNA (BamHI Q fragment, Fig. 1). Fragment sizes (in kilobases) are

indicated.

Recovery ofpol host-range deletionmutants.WeusedHP66 to obtainpol deletion mutants. Infectious HP66 DNA was

cotransfected withpDPAS orpDPAX (Fig. 1), theprogeny

from the transfections were plated onto DP6 cells and

stained with X-Gal, and the white plaques were picked.

Small stocks of virus werederived from each plaque pick,

andthe viral DNAwasrecovered andanalyzed by Southern blotting. Approximately 80%of the white plaquesfrom the

pDPAS and pDPAX transfections contained the expected

2.0- and 1.2-kilobase pol deletions, examples of which are

provided (Fig. 5). Two mutants that contained the XhoI deletion(AX14andAX17)andonemutantthat contained the

SalI deletion (AS1) were plaque purified, amplified and

selected, and analyzed further. All three mutants grew

efficientlyonDP6cellsandinefficientlyonVero cells (Table

2). Stocks ofAX14, AX17, and HP66 contained low levels

(approximately 1 in 105viruses) of virusthathadwild-type growth characteristics. Presumably, thesewild-typeviruses

arose by recombination between the pol gene resident in

DP6cellsandeach deletionmutant.

We expected mutants containing the XhoI deletion to specifyatruncatedpolypeptideboth because of the deletion andbecause the deletion results inaframeshift. This

possi-bilitywas examined byimmunoprecipitating Pol from [355]

methionine-radiolabeled extracts of mock-, KOS-, AX14-,

and AX17-infected cells (Fig. 6). The predicted molecular

FIG. 6. Immunoprecipitation analysis of Pol proteins Synthe-sized by pol deletion mutants. Antisera specific for Pol(left) and thymidine kinase(right)were used toimmunoprecipitate 35S-radio-labeledproteins frommock-,AX14-, and AX17-infectedVerocells. The immunoprecipitated proteinswere separated ineithera7.5% (left) or a10%(right)SDS-polyacrylamide gel. Positions of molec-ularweight markers (inthousands) are indicated for the gel

contain-ing immunoprecipitated Pol.

weight of the truncated Pol specified by these deletion

mutantsisapproximately 65,000 (65Kpolypeptide),due to a

termination codon inthe newreading frame after the XhoI

site. Virus AX14 specifies a 96K Pol-related polypeptide. This sizeproteinisconsistent withanadditionalmutationin

AX14 which causes the reading frame to be brought back into register, allowing expression ofa largerprotein. Virus

AX17 specifiedno protein detectable with the Pol antisera

butproduced wild-type levels ofthymidine kinase, indicat-ing that the cells were efficiently infected. Analysis of

proteins in AS1-infected cells indicated that this mutant

specifies atruncatedPol consistent with the deletion muta-tion (49).

Recovery of a pol deletion mutant lacking upstream

se-quences. Since the marker transfer techniquewith the lacZ insertion mutant was useful in obtainingpol recombinants

containing mutations in the majorpol open reading frame,

we employed the same procedure to obtain a mutation

outside of theregion substituted inHP66. Previous

experi-ments torecover HSV tkrecombinants had indicated that

sequencesasfar as1,000bp awayfrom the selected marker could be transferred into the virus(2,11, 26).To examinethe

role in HSV infections of the short upstream open reading

frame (Fig. 1, SORF) which encodes a decapeptide, we

wishedtoobtainamutantlacking these sequences. Plasmid pDPAB (Fig. 1) contains twodeletions; the first

deletionisapproximately 62bpwithinoriL. Such deletions are commonwhen

oriL-containing

sequencesare cloned in

bacterialvectors(38,47, 48). The second deletionremoves

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[image:6.612.319.555.83.357.2]

BstII Sfil LL O

CI

LL °

Cf)iU'C0 wa'

-'

-it

.

SmaI

U-0C:

C OU 0 LL

oY

m

Y a

1

-4.2

-3.6

-3.0

-1.7 -1.5 _ _

~~~~-

1.3

1.4

16-d-.79

_R ~~-.6586

.11~~~ -"l .566

-.45

FIG. 7. Restriction analysis of

AB10

viral DNA. Viral DNA samples from wild-type strain KOS, mutant AB10, and plasmid pKEF weredigested withBstII,Sfil, orSmaI.The fragmentswere separated in a 1.5% agarose gel, transferred to nylon, and then probed with 32P-radiolabeled BamHI Q-fragment DNA (Fig. 1). Dots adjacentto bands highlight fragments discussed in the text. Positions of molecular size

mark~ers

(inkilobases) electrophoresed in parallel are indicatedattheright.

the144bpto theright oftheBamHI site inthe 5' leader of

pol mRNA, which includes the short open reading frame.

InfectiousHP66 DNAandpDPABDNA werecotransfected

intoDP6cells, and theprogeny wereplatedontoboth Vero

andDP6cells.ViralDNA wasprepared from whiteplaques

and compared by

Southern

analysis. BamHI digestion of

DNA from oneplaque isolate, AB10, resulted in a5.4-kbp

BamHI fragment hybridizing with radiolabeled BamHI Q

BgU

StlI

BamHl

Sm!a

I t

Sma! Bu!

If

B

U

fragment (results not shown). This size is consistent both

with the predicted loss of the BamHI site separating the

3.3-kbpBamHI Q and 2.3-kbp BamHIVfragmentsandwith

thedeletion of100 to 200bp.

AB10wasplaquepurified, amplified, andanalyzed further

by restriction

digestion

and

hybridization

with radiolabeled

BamHI Q fragment (Fig. 7). The relevant restriction sites

andthededuced site oftheAB10deletionsareshown in

Fig.

8. The presence ofthe 144-bp deletion removingthe short

openreading frame was verifiedby the loss of the

1.2-kbp

SfiI

fragment found in KOS and

pKEF-P4

DNA and its

replacement with a fragment about 150 bp smaller, by the

loss ofa 0.87-kbpBstEII fragment in KOS andpKEF-P4

DNA and its replacement with a fragment about 150 bp

smaller, and by the loss ofa0.87-kbp BstEII fragment in

KOS and pKEF-P4 and its replacement with a

0.72-kbp

fragment in AB10. Similarly, the 0.82-kbp SmaI fragment

whichcontainstheshortopenreading framewasmissing in

AB10 and replaced by a 1.1-kbp fragment, consistent with

the predicted loss of a SmaI site, fusing the

0.82-kbp

fragment to a0.45-kbp fragment (which lies mainly outside

the

BamHI

Q fragment), and the144-bp deletion.

The analysis was notconsistent with the presence ofthe

orideletion in AB10. Forexample, the BstEII fragment in

AB10 was 0.72 kbp, not the 0.66 kbp expected if the ori

deletion was present. Similar results were obtained with a

variety of otherrestriction enzymes (not shown).

Virus AB10 formed plaques on Vero cells as well as on

DP6cells(Table 2), demonstratingthatthe144-bp

fragment

containingthe smallopenreading frame is notessential for

virusgrowth in cell culture.

DISCUSSION

Inthisreport, wedescribetheisolation ofacell line that

containsthe HSVpolgeneand itsuseintherecoveryofnew

spontaneous andconstructed HSVpolmutants.

Isolation of a cell line thatcomplementspol mutants. The

DP6cell line complementsavariety ofpolmutants,

includ-ing ts and deletion mutants. Plaque formation of pol ts

mutants at 39°C is more than 500-foldgreater on DP6 cells

than on Verocells andranges as

high

as

50,000-fold

greater.

Muchlower levelsof

complementation

havebeenobserved

withpol mutant tsC7 in a transient assay using a

plasmid

containing the pol and UL31 genes (15). The greater

effi-ciency of

complementation by

DP6cellsmay be duetothe

use ofa stable cell line and/or thepol

plasmid

construct

used. DP6cells

complemented

tsC4 andtsC7 less wellthan

tsD9;such

allele-specific

differences in

complementing

abil-ity by cells expressing a wild-type gene product have also

been observed with the UL5 gene (S. Weller,

personal

But! Sma! BglSf1!

m

-ICP8

orlL

pKEFAII

D

SOR?

DNA POLR

ATG

O.O5Kbp

, ABIOSO

FIG. 8. Enlarged view ofsequences removed from

AB10.

Thelocation of the pol transcript is indicatedO-),and openreading frames are shown (=). Plasmid pKEFP4 lacks the 62 bp from oriL, and virus AB10 lacks the 144 bp between the BamHI site and 11 bp upstream of theATG for thepol longopen reading frame. A bracket over the transcript designates a region predicted to form a stable secondary structure (22). SORF, Short upstream reading frame.

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[image:7.612.86.277.76.405.2] [image:7.612.76.552.598.690.2]

communication). Regardless, DP6 cells appear well suited

for the isolation ofnewpol mutants. Moreover, because DP6

cells were constructed by using a plasmid that contains no other intact genes besides pol, the complementation of mutantsby DP6 cells effectively maps these mutations to the pol gene.

Complementation of ts and host-rangepolmutants. Previ-ous work with pol ts mutants has shown that

complementa-tion with these mutants is complex and variable and that intragenic complementation is frequently observed (6, 39, 46). We found (Table 3) that tsD9 and tsC4 complemented each other well, as previously reported (39, 46), and that theyinturn could be complemented by one of the host-range mutants (6C4) which expresses full-length Pol polypeptide (Fig. 2). On the other hand, we detected virtually no com-plementation with host-range mutant 7E4 and the ts mutants, while 7E4 complemented 6C4 well. Given the ability of 7E4

to complement 6C4, it was somewhat surprising that we

were unable to detect full-length Pol polypeptide in

7E4-infected cells. This could be due to the failure of the Pol

antisera to recognize an altered polypeptide. Alternatively,

the 7E4 complementing activity might require only low levels of either full-length or truncated Pol polypeptide.

Ourfindings of intrageniccomplementation with the

host-range mutants areconsistent with previous findings with pol

tsmutantsand may be areflection of the numerous activities

specified by pol. Purified polymerase has been associated

with3'-5'-exonuclease activity and RNase H activity (12, 28, 33; A. I. Marcy, P. D. Olivo, M. D. Challberg, and D. M.

Coen, Nucleic AcidsRes.,inpress).The maplocationof the

tsD9 mutation (complementation group 1-4) andits altered

drug sensitivity phenotypes (17, 18, 36) suggest that it identifies the polymerase activity; perhaps the

complemen-tation group 1-3 mutations and 6C4 identify the two other

functions.The presenceof multiple complementationgroups

forone protein may also imply that the protein is active at

least in part as amultimer (24, 25). Biochemical analysis of

Polindicates that the enzyme activity sediments as a

mono-mer (28, 33) but leaves open the possibility that multimers

exist invivo.

Removal of the small upstream open reading frame. The

deletion in AB10 removes sequences that could code for a

small decapeptide. Ourinterestin apotentialfunctional role

intissueculturefor this open reading framewasinitiatedby

the observation that it shares partial homology with

sub-stratesfor cAMP-dependent proteinkinase (18). Inaddition, recentwork (H. Bludau, K. Weisshart, U. K. Freese, C. W.

Knopf, and H. Zur Hausen, Abstr. 14th Int. Herpesvirus

Conf. 1989, abstr. no. 161, p. 161) indicates that in asmall

percentageof poltranscripts the short open reading frame is

splicedinto the pol open reading frame. The deletion in AB10

would also prevent formation of these spliced transcripts by removing the splice donor site. Since this virus grows as well

on Vero cells as on the DP6 cell line, which also contains sequences for this open reading frame (Table 2), we

con-clude that the peptide and the spliced transcripts are not

essential for growth in tissue culture. It remains to be

demonstrated whether this open reading frame is also

dis-pensable inanimal models of HSV infection.

The short open reading frame is downstream from the

transcriptional start site for pol (48) and we expect it to be

translatedfrom the same mRNA as pol, but it is not known whether this protein is synthesized in infected cells.

Expres-sion of this short open reading frame would imply that the pol mRNAisbicistronic. Inthat case, polexpression could be duetotranslational reinitiation, as can occur in the HSV

thymidine kinase mRNA (26), or, less likely, due to initiation at aninternal AUG codon (29, 30).

Recovery ofnewpolmutants. The selection method

pro-vided by the lacZ insertion mutants has proven to be a useful

method for obtaining viruses containing site-specific

muta-tions (3, 4, 21, 34; this report). Our results, in which we

recovered several host-range mutants that either did not

containtheexpected mutationsorcontained more than one

mutation, emphasize the importance of demonstrating that

theengineered changeis in fact theonly relevant alteration.

We expect that the deletion mutants described here and point mutants currently being isolated (J. S. Gibbs, A. I. Marcy, and D. M. Coen, unpublished results) will be critical forefforts to separate domains of Pol that contributeto its variousenzymatic activities andbiological activities, suchas

mutation rate, antiviral drug sensitivity, and intracellular localization.

ACKNOWLEDGMENTS

We thank N. DeLuca and D.Panicali forplasmids, S. Weller for mutanthrR3,D.Knipeand W. P.Summersforantisera,J.Miller for Dam-bacteria,K.Kerns andK.Hicksfor technicalassistance, and P.Schaffer foracriticalreading of the manuscript.

A.I.M. was the recipient ofa postdoctoral fellowship fromthe American Cancer Society, and D.R.Y. was the recipient of a

postdoctoral fellowship from the National Institutes of Health. This workwassupported by Public Health ServicegrantsAI19838 and BRSGS07RR05381 from the National Institutes of Health.

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