A temperature-sensitive mutation in a herpes simplex virus type 1 gene required for viral DNA synthesis maps to coordinates 0.609 through 0.614 in UL.

0022-538X/88/030715-07$02.00/0

CopyrightC) 1988,American Society for Microbiology

A

Temperature-Sensitive

Mutation in a Herpes Simplex Virus Type

1

Gene Required

for Viral DNA Synthesis Maps to Coordinates

0.609

through

0.614 in

UL

MICHAEL E.

MARCHETTI,

COLTONA.

SMITH,

AND PRISCILLA A. SCHAFFER*

LaboratoryofTumor VirusGenetics, Dana-Farber Cancer Institute, and Department of Microbiology and Molecular

Genetics,

Harvard Medical

School, Boston,

Massachusetts 02115

Received 19 October1987/Accepted3December1987

ts701 is atemperature-sensitivemutantofherpes simplex virus type 1 strain KOS induced byhydroxylamine

mutagenesis (C. T. Chu, D. S. Parris, R. A. F. Dixon, F. E. Farber, and P. A. Schaffer, Virology98:168-181,

1979). In the present study, the mutation rendering ts701 temperature sensitive was mapped to coordinates 0.609 through 0.614 in the UL region of the genome. At the nonpermissive

temperature,

ts701 (i) failed to inducethesynthesis of viral DNA, (ii) exhibited a dramatically reduced ability to drive replication of a plasmid containing the herpessimplex virus origin of viral DNA synthesis,

oris,

(iii)generated no viral polypeptides of the late

(72)

kinetic class, and (iv) produced virions with electron-translucent cores. Northern (RNA) blot hybridization demonstrated that two mRNAs-one of the I kinetic class and one of the

'y

kinetic class-hybridized to a1.3-kilobase viral DNA fragment that rescued the mutation ints701. Basedon thephenotype andmapping ofts701, it is likely that its mutation lies in the gene specifying the 65,000-Mr DNA-binding protein

(65KDBp)

recently described by Marsden et al.(H. S. Marsden, M. E. M. Campbell, L. Haarr,M. C.Frame, D. S. Parris, M.Murphy, R. G. Hope, M. T. Muller, and C. M. Preston, J. Virol. 61:2428-2437, 1987).

The identification of virus-encoded proteins required for herpessimplex virus (HSV)type 1 (HSV-1)DNA synthesis has been accomplished to date by two markedly different procedures. First, studies of

temperature-sensitive

(ts) mu-tants have led to the identification of six distinct viral

proteins thatareessential for viralDNAsynthesis(27). The

functions of only two of these

proteins,

the viral DNA

polymerase (6) and the

major

DNA-binding protein ICP8 (32),have beendetermined.Recently, a meansofidentifying adenovirus proteins involved in viral DNA synthesis was

adapted forusewithHSV-1byChallberg (4).Thisprocedure utilizes clones of viral DNA fragments representing the

entire genome. These clones are assayed in transfection experiments in all possible combinations to determine whethertheproteins they encode cancollectively drivethe

replication ofa plasmid containing an HSV origin of repli-cation. With this procedure, sevengenes essential forviral

DNA

synthesis

have beenidentified andfinely mapped (M.

Challberg, personal communication). Six of these seven genes were identified and mapped previously with ts mu-tants.Todate, nospecific functionhas beenascribedtofive of the seven viral proteins, although biochemical studies

suggestthat,inadditiontothepolymeraseandmajor

DNA-binding protein, thevirus may encode its owntopoisomerase (2, 17, 21), DNA primase (W. Ruyechan, personal

commu-nication), andorigin-binding protein(11).

Inthisreport, wedescribeamutantof HSV-1 strain KOS,

ts701, which is temperature sensitive for viral DNA

synthe-sis. We have mappedthe mutation rendering ts701 temper-ature sensitive to coordinates 0.609 through 0.614. The

location ofthe ts701mutation indicatesthatit lies in thegene

fortheonly essential viral DNAsynthesis protein forwhich no ts mutation has yet been identified (M. Challberg,

per-sonal communication). Several transcripts transverse the

*Correspondingauthor.

coordinates 0.609 through 0.614 (12), but only one is the ,B

kinetic class. It is 1.44 kilobases (kb) long and encodes a

protein of64 kilodaltons, asascertainedby in vitro transla-tion (12). This 64-kilodalton product maybe the

65,000-Mr

DNA-bindingprotein

(65KDBp)

describedby Marsdenetal.

(20). Since all HSV genes encoding proteins directly in-volved in viralDNA synthesisareof the,1 kinetic class(27),

it is likely that ts701 is defectivein

65KDBP.

MATERIALS ANDMETHODS

Cells and viruses. Verocells werepropagatedasdescribed previously (32). ts701 and tsA24 are derivatives of HSV-1 strainKOS (5, 27).Mutantandwild-type virusesweregrown

andassayedas describedpreviously (32).

Plasmids.Formarkerrescueanalysis,theEcoRI library of KOS derivedby Goldin etal. (13)wasused. Thefirstseven

subclones ofEcoRI-Alisted in Table 1and shown in Fig. 1

(A-2 through 4ofA-1) were cloned into pUC8 by standard techniques (18). Bal 11 was derived by linearizing1 ofA-1

withHindIII,digestingitwith Bal 31nuclease,andligatingit

with HindIII linkers. Plasmids 9, 14, and 8 ofBal 11 were

derivedin the same manner, except that Bal 11 wasusedas

theparental plasmid. Plasmids Bal11-15, Bal 11-13,andBal

11-10 werederivedbylinearizing Bal11with

SalI,

digesting

it with Bal 31 nuclease, and ligatingit with EcoRI linkers.

DNA-modifying enzymes and HindIII and EcoRI linkers were obtainedfrom New England BioLabs, Inc. (Beverly,

Mass.) and used asprescribed by themanufacturer. Nucleicacid isolation. Purified HSV-1 and bacterial

plas-mid DNAs were isolated as described previously (13, 18). Cytoplasmic RNAwas isolated fromNonidet P-40extracts ofVerocells as describedpreviously (18).

Markerrescuetest.Markerrescue experimentswere car-ried out as described byWeller et al. (32)with the

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716 MARCHETTI ET AL.

TABLE 1. Markerrescue analysis'

Marker rescue inexpt: Plasmid

1 2 3 4

pUC8 <1.1 x 10-4 <2.0 x 10-3 <9.0 X 10-4 <9.7 x 10-4

EcoRI-A 1.51

A-2 <1.1 x 1O-4

HindIII-K 9.0 X 10-5

A-1 1.01 0.36

1ofA-1 0.18

2ofA-1 <4.0 x 10-3

3 of A-1 ND

4of A-1 <3.3 x 10-3

Bal 11 0.13

9ofBal11 0.42

14ofBal 11 0.66

8 of Bal11 0.04

Bal11-15 <5.5 x 10-3

Bal 11-13 <1.1 x 10-3

Bal11-10 0.44

a Marker rescue analysis was conducted with the indicated plasmids appropriately linearized prior to transfection. The data indicate the percentage of the total transfection progeny found to be ts+. ND, Not determined.

tion protocol of Parris et al. (22), except that DNA was

precipitatedwith CaCl2for 30minand Vero cells were used. Southern and Northern (RNA) blot analyses. Restriction

enzyme-cleavedDNAsseparated by agarose gel

electropho-resis were transferred to nitrocellulose by the method of

Southern (18,28). Forthe experiment showninFig. 4, pUC8 was used as probe.

Formaldehyde gel electrophoresis ofRNAsandNorthern

blot hybridization were performed as described previously

(8). FortheexperimentshowninFig.6,8 of Bal11 was used as aprobe.

a b

E___

E

Probesfor both Southernand Northernblot analyseswere

nick translated with 32P-labeled dCTPand dGTP as

previ-ously described (18).

Polypeptideanalysis. Lysatesof radioactivelylabeled cells wereanalyzed by sodium dodecylsulfate-polyacrylamide

gel,

electrophoresis by the method of Laemmli(16) as modified

byManservigietal. (19). Cellswereinfectedat amultiplicity of infection of20 PFUpercell, incubatedat34 or 39.7°Cfor various times with 100 ,uCi of

[35S]methionine

per ml, and harvested asdescribed byDeLuca etal. (7).

Viral DNAphenotypes. The viral DNA phenotype of ts701

b'

a'cloris

oris c a 4---11----471

E -

-L

H

EcoRIA (21.2) A-2 (5.46)

H

H

H S

i I

S SS s

S E

l

-H S

H4 H

F II

H S

H S

H E

I

H E

i

H E

I

HindIII K (9.26)

E

-4

A-I

(6.49)

of A-l (3.8)

3,4 of A-I (0.8)

2 of A-I (1.9) BaI I I (3.3)

9 of Bal I (2.9) 14 of Bal I (2.0)

8 of Bal I1 (1.3) Bal 1 1-15 (1.1)

Bal 1 1-13 (2.7)

Bal I 1-10 (3.2) FIG. 1. Subclones ofEcoRI-A used inmarkerrescuetestswith ts701. At thetopof thefigure isadiagram of the HSV-1genomeillustrating

thelocationof the EcoRI A fragment. Thelocation of the diploidorisin thecrepeatsis alsoshown(oris). Beneath the expandedEcoRIA

fragmentareshown thesubclones used inmarkerrescuetests.E, EcoRI; H,HindIII;S, Sall. Numbers of parenthesestothe rightof plasmid designations aresizes of thefragmentsinkilobases.

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MAPPING OF HSV-1 KOS MUTANT ts701 717 was determined in Vero cells infected at a multiplicity of

infectionof20 PFU percellat34or39.7°C by the method of

Aron etal. (1).

oris

replication assay. Vero cells (3 x 106) in 100-mm2

plates were transfected with 0.1 mg of pUCori (a pUC8 plasmid bearing

oris

as a BamHI fragment) and 18 mg of salmontestis DNA by the procedure of Weller etal. (32).

After 1 hofincubation of 37°C,the cells wereinfectedwith 20 PFUofmutant orwild-type viruspercell andincubated for anadditional 18 h atvarious temperatures. TotalDNA was then extracted and cleaved withBamHIand DpnI, and

fragmentswereresolvedon an0.8%agarosegelasdescribed

previously (18). The gelwas blotted ontonitrocellulose and probedwithpUC8 DNA asdescribed previously (28).

Electron microscopy. Vero cells wereinfectedwith 5 PFU

of eitherts701 orKOSpercellandincubatedat34 or39.7°C for 36 h. At this time, the monolayers were washed twice

with cold Tris-buffered salineand fixed with2.5%

glutaral-dehyde. Monolayerswereprocessed for thin sectioning and examined by electron microscopy as described

previously

(26).

the

-Yl

polypeptide ICP5. ts701 and tsA24 generated the same amounts of the early polypeptide thymidine kinase(ICP36)

asdid KOS at 39.7°C.

Viral DNA synthesis. To date, all HSV ts mutants that fail toinducethesynthesis of

Y2

polypeptides at the

nonpermis-sivetemperaturehave been defectivein viral DNAsynthesis

atthistemperature (27). Wethusdeterminedthe viral DNA phenotype of ts701 at 39.7°C. ts701 failed to induce the

synthesis of detectable viral DNA at 39.7°C, whereas it induced -85% of wild-typelevels at340C (Fig. 3).ts701 was as efficient as wild-type virus in inhibiting cellular DNA

synthesisatboth temperatures.

Tofurtherassess theability ofts701 toinduce viralDNA

synthesis, we tested its ability to drive the replication ofa

340C

0 N

4 N u

39.7 5C

X XN o O

RESULTS

Marker rescue analysis. Using gel-purified EcoRI

frag-mentsin markerrescuetests, Chuetal. showed previously

that the mutation in ts701 lies within EcoRI-A (5). We

confirmed this finding by using cloned fragments; only

EcoRI-A yielded ts+ recombinants (data not shown). Sub-fragments ofEcoRI-A were thencloned and used in

subse-quent marker rescue experiments (Fig. 1 and Table 1).

Subclonesthatrescued thets701mutation included A-1;1of

A-i;

Bal 11; 9, 14, and 8 of Bal 11; and Bal 11-10. 8 of Bal11

proved to be the smallest rescuing fragment. This latter result, combined with the fact that Bal 11-10 alsorescued, localized the ts701 mutation to a 0.7-kb length of DNA

betweencoordinates0.609and0.614 on thephysicalmapof HSV-1.

Two ts+ recombinants derived from the marker rescue

experiment with 8 of Bal11 were analyzed with respect to

plating efficiency inVero cellsatthepermissive (34°C) and nonpermissive (39.7°C) temperatures. The platingefficiency (PFU per milliliter at 39.7°C/PFU per milliliter at 34°C) of KOS was 0.67, whereas the ts+ recombinants exhibited plating efficiencies of0.40 and 0.43. By contrast,theplating

efficiency

ofts701was 2.2 x

10-'.

Polypeptideprofileof ts701 at39.7°C. We nextdetermined the spectrum of viral polypeptides induced by ts701 at the

permissiveandnonpermissivetemperatures. tsA24, a

DNA-negative ts mutant defective in ICP8 (complementation

group 1-1), andKOS, thewild-type virus, were included as

controls in these experiments. The three viruses exhibited virtually indistinguishable polypeptide profiles at 34°C (Fig. 2).

At39.7°C, ts701,liketsA24, (i) failedtoinducedetectable

levels of late polypeptides of the _Y2 class

(polypeptides

whose synthesis is absolutely dependent upon viral DNA

synthesis) and (ii) induced markedly reduced levels of late

polypeptides ofthe

-Yl

class(polypeptidessynthesized in the

absence ofviral DNAsynthesisbut whosesynthesisreaches maximum ratesonly whenviral DNA replication is allowed to occur). This phenotypeis exemplifiedby the absenceof ICP19 and ICP20(Y2)andbythereduced levels of gB, ICP5,

andICP25

(-Yl)

in mutant-infectedcells. Although theprofiles

ofts701 and tsA24 werequalitativelysimilar at39.7°C,ts701

induced higherlevels ofthe early polypeptide ICP8 and of

-mmW _-_

- mo

5

6

gB

19/20

25

36

-do-W. _ - d

u-|

_W

_.

OM

- 41

FIG. 2. Polypeptideprofile ofts701. Verocellswereinfectedat 34or39.7°Cwith the indicatedvirusesat amultiplicityof infection of20 PFUpercell,labeledwith100,uCiof[35S]methionineper ml from 5 to 18 h postinfection, and processed for sodium dodecyl sulfate-polyacrylamidegelelectrophoresis. Thenumbersattheright indicateinfected-cellpolypeptides.

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718 MARCHETTI ET AL.

thickening of the nuclear membrane (Fig. 5D), a character-istic of KOS-infected cells (Fig. 5A). These findings are consistent with theDNA-negative phenotype and

polypep-tide profile ofts701-infected cells at 39.7°C (Fig. 2 and 3)

(26).

mRNAs that map to coordinates 0.609 through 0.614. To determine the sizes and kinetic classes of transcripts en-codedby sequences that contain the mutation in ts701, we extracted cytoplasmic RNA at 6 hpostinfection from

cyclo-co heximide- and phosphonoacetic acid (PAA)-treated Vero

C:

o cells infected with HSV-1 KOS and at 11 h

postinfection

o from untreated Vero cells also infected with HSV-1 KOS. RNAs weresubjected to Northern blot analysis by using as

C aprobe the smallest fragment that rescued the mutation in

ts701,

the 1.3-kb

fragment,

8 of Bal 11

(Fig. 6).

Two

unique

,< mRNAs wereidentifiedinKOS-infected cellsintheabsence ofdrug. The largertranscript (-2.7kb) was not synthesized 3 inKOS-infected cells treated with either the DNA synthesis o inhibitorPAAor aninhibitor of

protein synthesis,

cyclohex-3 imide,indicating that this transcript is of the -y kinetic class. The smaller transcript (-1.4 kb) was synthesized in the presence of PAA but not in the presence ofcycloheximide,

indicatingthat itis of the 1 kinetic class.SinceallHSV genes

encoding proteins directly involved inDNAsynthesisareof the 1 kinetic class(27), itisperhaps morelikelythat the ts mutationin ts701 lies in thegenespecifying the1 transcript.

DISCUSSION

In vitro replication assays (4; M. Challberg, personal

communication)havepredictedtheexistence ofsevengenes Fraction Number

FIG. 3. Viral DNAphenotypeof ts701. Cesium chloridedensity gradient profiles of ts701- and KOS-infected cell lysates were

obtained as described previously (1). The peak of viral DNA occurred at a density of 1.727 g/cm3, and that the cellular DNA occurredat 1.699g/cm3.

plasmid bearing the HSV origin ofreplication, ori5 (4).oris

acts in cis when the appropriate HSV gene products are

provided in trans(29, 30). Vero cells weretransfected with

pUCori, a plasmid consisting of pUC8 into whose BamHI

site

oris

hasbeen cloned(N. DeLuca, personal communica-tion)(Fig. 4). Cellsweretheninfected with virusesatvarious temperaturesand harvestedat18 hpostinfection.The DNAs were then extracted and cleaved with BamHI and DpnI.

DpnIdigestionwasusedtodifferentiate between unreplicat-ed and replicated pUCori DNAs (23). The digested DNAs

were then separated in agarose gels, blotted, and probed

with pUC8. At 34°C, KOS, ts701, and tsA24 drove the replication of pUCori efficiently. By contrast, at 39.7°C, KOS drove the replication of pUCori efficiently, whereas tsA24 and ts701 did not. Thus, as shownby twomethods, ts701 is defective in itsabilitytoinduce thesynthesis of viral DNA at39.7°C.

Electron microscopy. Electron photomicrographs ofthin sections ofmutant-infected cells after 36hof incubation of 34°C were indistinguishable from those of thin sections of

wild-type virus-infected cells at this temperature (data not shown). At 39.7°C, however, infection with ts701 did not lead tothe production ofmature, enveloped, extracellular particles orintracellular virions with electron-dense cores,

as did infection with KOS (Fig. 5A, B, and C); only

translucent, DNA-less, incomplete nucleocapsids were seen

innuclei(Fig. 5DandE). Additionally, ts701failedtoinduce

340

0

11-.

z i

o 0 o f4

v E Y b <

PUC 8

39.70

0

I-z

0 u

0

:E _

0 o0 Y P% <

I..

I

FIG. 4. Southern blotanalysisof theorisreplicationassay.Cells

were transfected with pUCori (a pUC8 plasmid bearing oris as a

BamHI fragment),infected at theindicated temperatureswith the

indicated virusesatamultiplicityof infection of 20 PFUpercell,and harvestedat18 hpostinfection.Total DNAwasextracted,cutwith

BamHI andDpnI, runon anagarosegel, blotted,andprobedwith pUC8.ThesequencesdetectedarethepUC8sequencesofpUCori.

0-I2

0

%._

E

Ur

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MAPPING OF HSV-1 KOS MUTANT ts701

.-_.KX

-. E

0~~

ytr:'_* RI ,-~~~~~~~~~~~~~~~~

_',-9.I I

- K-*~

r}

. .

./

aov

Ar V

,.. 6 t~~~~~~'

W\f*)t91',t4 S*

*-~~~a

* \ .

:-'4

0--X

Vs

- .1

I. A" Q.,Gv

i.

..4.

.A ....

~~c~...

FIG. 5. Electronphotomicrographs of thin sections of Vero cells infectedwith 5 PFU of KOS(A, B,andC)orts701(DandE)percell

for 36 hat39.7°C. Mature, enveloped, extracellular particles with dense (full)cores(-*)canbeseeninpanel A,ascan aregionofthickening

of the nuclear membrane (-*). Full(DNA-containing) particles (-*)andparticles containing internalstructures butlackingdensecores

(partial, DNA-less particles) (-) can be seen in panels B and C. A particle withno internal structures (->) can be seen in panel B.

ts701-infectedcells(panels DandE) characteristicallycontainednofullparticles. Inaddition, nomature,enveloped particleswere seenin

the extracellularspace. Nuclei, however, contained smalltomoderate numbers ofpartial nucleocapids ().

whose productsareessential for viral DNA synthesis. Of the

seven, onlytwohave been characterizedextensively, anda third has beencharacterizedtoalesserextent.These include the DNA polymerase (6), the major DNA-binding protein ICP8(32),and65KDBP(20), although other proteins directly involved in viral DNA synthesis have been reported to be inducedfollowingHSV infection. The latterproteinsinclude

atopoisomerase (2, 17, 21),aDNAprimase (W. Ruyechan,

personal communication), andanHSV-induced proteinthat binds specificallyto

oris

(11).

HSV DNAalso encodes severalenzymes involvedin the

generation of DNA precursor molecules. These include a

thymidine kinase(9),analkaline nuclease(15, 24),a ribonu-cleotide reductase (10),auracylDNAglycosylase (3),anda

dCMP deaminase (25). Deletion of thegenes for the former threeenzymesdoesnotaffect theviabilityof the virus in cell cultures (S. Weller, personal communication).

Prior to this report, ts mutations had been described in only six of the seven essential DNA replication genes identified by the in vitro replication assay. The characteri-zation of ts701 now brings this number to seven. ts701 is temperature sensitive for viral DNA synthesis, and its

mu-tation maps to coordinates 0.609 through 0.614. Northern blot analysis indicates that at least two mRNA transcripts

A

:4

I

0

p

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720 MARCHETTI ET AL.

0~~~

o

~~~v

4

0 0 0

Fw

28SSt

18

S*-FIG. 6. Northern blot analysis of KOS-infected cells with the

smallestfragment (8 of Bal 11) thatrescuedthemutationin ts701as a probe. The larger transcript (y) was not synthesized in KOS-infectedcells treated with either theDNA synthesisinhibitor PAA or aninhibitor ofprotein synthesis, cycloheximide (CYCLO.). The

smaller (1) transcript was synthesized in the presence of PAA but not cycloheximide.

traverse these coordinates (Fig. 6). One of these is a y

transcript which probably corresponds to the late

tran-script(s) mapped between coordinates 0.610 and 0.633 by Frinketal. (12). It isunlikely thatthis transcript encodes a

protein involved in DNA synthesis, since viral DNA repli-cation isa prerequisite for its production (14) (Fig. 6). The

othertranscripttraversingcoordinates 0.609 through 0.614 is ofthe kinetic class and probablycorrespondstothe 1.4-kb early transcript mapped by Frink et al. (12). By in vitro translation, it was found that this transcript encodes a

64-kilodalton protein which likely corresponds to 65KDBP described by Marsden et al. (20). It has been shown that

65KDBP

specifically associates with the HSV-encoded

poly-merase, asdemonstrated by theobservation that itcopurifies

with the polymerase duringcolumn chromatography andon

immunoaffinity columns (31; M. Gallo, M. Muller, H. Mars-den, and D. Parris, personal communication). Using interty-pic HSV-1 x HSV type 2 recombinants and type-specific antibodies, Marsden et al. mapped the gene encoding

65KDBP

to coordinates 0.574 through 0.682 (20).

These coordinates encompass the area to which we have

mapped the mutation in ts701. What function 65KDBP per-formsisunknown,but thisreport suggeststhatitis essential for viral replication. However, its amino acidsequence has been reported to share homology with those ofeucaryotic primases (H. Marsden, personal communication). We are currently constructing mutants containing deletion muta-tions in the gene which is mutated ints701 and which most likely encodes

65KDBp.

Thesemutants should prove useful in elucidating the role of 65KDBP inthe synthesis of HSV DNA.

ACKNOWLEDGMENTS

We thank N. DeLucafor plasmids and for valuable discussions, R. Byrn, N. DeLuca, and W. Sacks forassistance in many of the experiments, and M. Kaveny formanuscript preparation.

This investigation was supported by Public Health Service grant CA-21082 from the NationalCancerInstitute. C.A.S. was supported by National Science Foundation Graduate Fellowship RCF-84-50074.

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