Characterization of the herpes simplex virion-associated factor responsible for the induction of alpha genes.

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0022-538X/83/050371-07$02.00/0

CopyrightC1983,American Society for Microbiology

Characterization of the Herpes

Simplex

Virion-Associated

Factor

Responsible for the Induction

of

ot

Genes

WILLIAMBATTERSONANDBERNARDROIZMAN*

MarjorieB.KovlerViralOncology Laboratories, TheUniversityof Chicago, Chicago, Illinois 60637 Received27October1982/Accepted 25 January 1983

Herpes simplex virus (HSV) genes form three groups, a.,

(3,

and -y, whose

synthesis

is

coordinately

regulated and

sequentially

ordered inacascadefashion.

Earlier studies by Post et al. (Cell 24:555-565, 1981) have shown that chimeric

genesconstructedby fusion of 5' noncoding leader andupstreamsequences ofa

genes tothe 5'noncoding leader and structural sequencesof the viralthymidine kinase (TK),a

1B

gene, areregulatedasagenes uponrecombination into the viral

genome. In cells converted from TK- to TK+ phenotype, these chimeric genes are induced by infection with homologous TK- virus. The induction of the resident chimericgenedoesnotrequire viral protein synthesis and isindependent of the presence of functional at gene 4product

required

forthe

expression

of,B genes. In this paper, we report on the

properties

of the a-TK gene chimera resident in converted TK+ murine (L316) and human

(1316)

cells. Our resultswere asfollows.

(i)

Thepatternof induction of L316 cells

exposed

to0.1, 1.0, and 10

PFUpercellsuggested thatexposure to competentvirus is

required

forinduction and that in untreated preparations this virus correspondsto infectious virus. (ii)

UV

light-irradiated

viruswas

just

aseffectiveasuntreatedvirus in

inducing

a-TK

chimeras.

(iii) HSV-1(HFEM)tsB7

induced the a-TK gene chimeras at the

nonpermissive (39°C) temperature; at

39°C

the

parental HSV-1(HFEM)tsB7

capsids

accumulateatnuclearporesand donotreleaseviralDNA.

(iv)

Thea-TK

genechimeras were notinduced by infection withspontaneous TK- mutants of pseudorabies virus and bovine mammillitis virusorwith human

cytomegalovirus

oradenovirustype 2 or

by

exposure to

lysates

ofHSV-1-infected cells from which

thevirus was removed by centrifugation. These results indicate that theagene

inducer is a virion component located outside the capsid and that its function might beto stimulate thetranscription ofagenesby recognizing regulatory sites

on viral DNAsorhost cellproducts orboth.

A characteristic of the reproductive cycle of herpes simplex virus 1 (human herpesvirus 1,

HSV-1) is that its gene expression is tightly regulated.

Specifically,

the HSV-1 genes form

three groups, a,

1,

and y, whose synthesis is coordinately regulated and sequentiallyordered

ina cascadefashion(10, 11). The five known a

genes are expressed first, and at least one, a

gene 4 (6, 16, 31), is required for the expression

of 1 genes. The1 genesturn offthe synthesis of

a genes and enable the expression ofy genes. On thebasis of studieson viral gene expression

in sensitive and resistant cells exposed to

a-amanitin, Costanzo et al. (5) concluded that cellular RNA polymerase II is involved in the

transcription of viralDNAthroughout the repro-ductive cycle. Central to our understanding of

the regulation of HSV gene expression is the

mechanism by which the cell transcriptional

machinery differentiates between theot,

1,

and-y genes.

In an attempt to deal with this

question,

we mapped the site of initiation of

transcription

of fourax genes,

i.e., aO,

a4,

oa22,

andat27 (19), and constructed chimeric genes

consisting

ofa

por-tion ofthe 5'

noncoding

leader sequences and

the sequences upstream from the initiation of transcriptionofagenes fused to the 5'

noncod-ingsequence and the structural sequences of the HSV-1 thymidinekinase (TK), a13 gene(19-22, 26).Theo4-TKgenechimera,whenrecombined into the viral genome, was regulated like an a

gene (26). Ofparticular interest, however, was the observation that in cells converted from

TK- toTK+phenotype, the a4-TK gene

chime-ra was inducedby infectionwith HSV-1 TK- in the presence of cycloheximide and at 39°C in cells infected withtemperature-sensitive (ts)

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ruses with mutations in a gene 4 (26). These

observations suggested that unlike the induction of the natural

P-TK

gene(13, 17), the induction

of the chimeric a-TK gene can occur in the

absence of viral protein synthesis and specifical-ly does notrequire thepresence ofafunctional

a4 gene product. Similar results were obtained

with cellsconvertedtotheTK+ phenotype with aO- and a27-TK chimeras (21, 22).

In this paper, we reportthat theinduction of the chimeric a-TKgeneinTK+ converted cells requires exposure of the cellsto virus and that the chimericgeneis inducedby UV

light-irradi-ated virus and by ats mutant defective in the release of viral DNA from capsidsatthe

nonper-missive temperature, but not by several other herpesviruses orby adenovirus type2 (Ad-2).

MATERIALSANDMETHODS

Virusand cells.Theproceduresforthepropagation of virus and cellsand thecomposition ofgrowth and

maintenancemediahave beenpublishedelsewhere(7, 29). The construction and properties of

HSV-1(F)ts502A305, temperature sensitive inthe a4 gene andcontaininga700-basepairdeletion in the TKgene,

have been previously described (26).

HSV-1(HFEM)tsB7 syn+ wasisolated by

bromodeoxyuri-dine mutagenesis of HSV-1(HFEM)-infected cells

(14).Previous studieshave shown that incells infected withHSV-1(HFEM)tsB7 and maintainedat39°C, viral capsids accumulateatthe nuclearmembrane andno

viralgene expression can bedetected (1). Ad-2 was

obtained from M.Green, Institute forMolecular

Virol-ogy, St.Louis, Mo. Humancytomegalovirus (human herpesvirus5,CMV), strain AD 169,wasprovided by

F. Rapp, The Pennsylvania State University,

Her-shey. HSV-2(333-B3) TK- stock wasobtained from

R. B.Tenser, The PennsylvaniaStateUniversity.

TK-negative variants of bovine mammilitis virus (bovine herpesvirus-2, BMV) and ofpseudorabiesvirus(Suid herpesvirus-1, PRV)were selected bypreviously

de-scribed methods(26).

Ltk-cells(12)wereobtained from S. Kit,Medical

College of Baylor University, Houston, Tex. The

143(TK-) humancell line (4) was obtained from C.

Croce, The Wistar Institute, Philadelphia, Pa. L316 cells and 1316 cellswereprepared byconverting

Ltk-and143(TK-) cells, respectively,toaTK+ phenotype

withpRB316,which contains DNAsequences

encod-ing the a4 gene promoter-regulator fused to DNA

sequencescoding for the structuralportionof the TK

gene(19-22, 26). L103 cellsprepared bytransfection of Ltk- cells with theHSV-1(F) BamHI Q fragment

were aspreviously described(26).

TK+cellswereselected and maintained inmedium

containing10-5Mhypoxanthine,1.6 x10-5M

thymi-dine,and 4.4x 10-7M methotrexate.

TK assays. Procedures forsuperinfection of

trans-formedTK+cells and for extraction andassayofTK activity in these cells were as previously described

(19-22, 26).

Preparationofsupernatant fluid from HSV-1-infected

cell lysates. The preparation of infected cell lysate

stocks has been described elsewhere in detail(7, 29).

Specifically, Vero cells infected with

HSV-1(F)ts502A305andmaintained at 33°C were disrupted in a Dounce homogenizer and then sonicated at the maximum intensity with a Branson Cell Disruptor (model 200). The infected cell lysate was cleared of cell debris bycentrifugation, the titer was determined tobe 1.9 x 109PFU/ml in Vero cells, and thelysate

wasstored at-70°C. The virus stocks were diluted to concentrations of4 x 107 PFU/1.5 ml and 4 x 108 PFU/1.5 ml withphosphate-buffered saline containing 1% (wt/wt) glucose and centrifuged in a Beckman SW41 rotor for 90 minat30,000 rpm and4°C to pellet thevirions. Thesupernatantfluids were then exposed to L316cells as described above.

UV irradiationofvirus. Stock HSV-1(F)ts502A305

virus was suspended in phosphate-buffered saline con-taining 1% glucose to yieldaconcentration of 2 x 107

PFU/1.5 ml. A2-ml amount of virus suspension in a

10-cm-diameter petri dish was shaken on a rotary

platform and exposed to a germicidal lamp (wave-length, 254 nm). A total dose of 1,000ergs/mm2 was deliveredatthe rate of 15ergs/s permm2tothesurface of the fluid. Samples (1.5 ml) of irradiated virus were thenused to infect L316 cells. A portion of the UV light-irradiated virus was assayed for infectivity.

RESULTS

Effect of

multiplicity

ofinfectionon induction of the a-TK chimeric gene in L316cells. The pur-poseof this series ofexperiments wasto deter-mine theeffect of the multiplicityof infectionon

theinduction of thea-TKchimericgeneresident in L316 cells.

In these experiments, replicate cultures of

L316cellswere exposedtoHSV-1(F)ts502A305

at a multiplicity of infection of 0.1, 1, and 10

PFU per cell for 2 h. Viral inocula were then removed, and the infected monolayers were

washed, replenished with fresh medium, and

additionally

incubated at either

permissive

(33°C)ornonpermissive (39°C)temperatures. At

appropriate

times,

the cultures were harvested, and cell extracts prepared from the infected cultures were assayed for TK activity. The

results of these experiments (Fig. 1)

demon-stratethat atbothpermissiveandnonpermissive

temperatures, inductionrequires infection of the

cellcarryingtheresidenta-TKgene chimera.As

has been previously observed, the extent of

a-TKinductionwasgreater at39°Cthanat33°C in L316 cells infected with HSV-1(F)ts502A305

(26).

Theseresults thusaffirmand extendprevious observations that induction ofthe a-TK

chime-ric gene residentin L316cells isaconsequence

ofviralinfection.

Ability of UV light-irradiated virus to induce the a-TK gene. Inasmuch as induction of

tran-scription of thea-TKgene can takeplaceincells infected in the presence of

cyclohexmide

(26),

thepurpose of these experiments was to deter-mine whether UVlight-irradiated virus retained its abilitytoinduce TK activity inL316 cells.

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O,,o,39° 010 L316 cells were infected with 5 PFU of HSV-50 -

*-330

1(HFEM)tsB7,

or of

HSV-1(F)ts502A305,

per

l. .UJ./cell or were mock

infected,

and the cultures

40- I were then incubated at the permissive (33°C) or

nonpermissive (39°C) temperature. At various

timesafter infection, the cultures were harvest-ed,and cellextracts wereassayed for TK

activi--3/

/ ty. The results (Fig. 3A) indicate that at 390C,

HSV-1(HFEM)tsB7was ascapable ofinducing

10 a-TK activity as was

HSV-1(F)ts502A305

and

20 - thatat

330C,

HSV-1(HFEM)tsB7

induced

some-1 what more a-TK

activity

than did

HSV-01

1(F)ts502A305. Since HSV-1(HFEM)tsB7 isnot

10- 0.1 a TK- mutant, its ability to express TK activity

--k

was assayed in 143(TK-) cells (Fig. 3C). The

results of this experiment showed that

HSV-, , , . , 1(HFEM)tsB7 expressed a small amount of TK

0 2 4 6 8 10 12 activity at 330C, but none at 390C.

hours These results indicate that the a-TK gene

chimera can be induced at the nonpermissive FIG. 1. Effect ofmultiplicity ofviral infection on temperature by

HSV-1(HFEM)tsB7.

16cells. Replicatecell cultureswereinfected with

,1.0,or10.0PFU of

HSV-1(F)ts502A305

virusper HSV-1(HFEM)tsB7 parental

virus, capsids

ac-ll, and TK activity was measured as previously cumulate at

390C

at the nuclear membrane and scribed (20-22) in cells harvested at the times do not release the

viral

DNA into the nucleus )wn. TK activity is expressed as picomolesof TMP until thetemperature is shifted to the permissive

medpermicrogram of protein. After adsorptionof range. Consistent with this

observation,

noviral us to cells at 100C, the cells were transferred to gene expression was detected at 39°C (1, 14). her 33 or 390C. Time 0 representsthe time at which Thus,the data presented here demonstrate that lls were placed at 33 or 390C. theinduction of the chimeric a-TK gene in L316

cellsmustbemediated byanextracapsid virion

component.

L316cellswereinfected with5PFUof HSV- Effect of supernatant fluids of infected cell

7)ts502A305 percellor withanequalamount lysateson the a-TK gene. To test the hypothesis

of virions that had been exposed to UV-light irradiation as described above. After 2 h of

adsorption,

the cells were treated as described

above. The UV-light dosage was sufficient to

reduce the effective input multiplicities of the inocula from 5 PFU per cell in the untreated viral suspensions to 0.002 PFU per cell in the

UV

light-irradiated

samples. The L316 cells

wereextractedand assayedforTKactivityat 2, 6, and 12 h afterexposure to untreated or UV light-irradiated virus.

The results (Fig. 2) demonstrate that virus exposedto

UV-light

irradiation retained its

abili-ty to induce TK activity in L316 cells atlevels comparabletothose inducedby untreated virus, notwithstanding the loss in infectivity. These results are consistent with the conclusion that thefactor responsible for induction ofTK activi-ty inL316 cells is a structuralcomponentofthe

virion.

Effect ofHSV-1(HFEM)tsB7 on TKactivityin L316cells. The purpose ofthis seriesof

experi-ments was to determine whether

HSV-l(HFEM)tsB7 could induce the resident a-TK

chimeric genes at the nonpermissive tempera-ture.

In these experiments, replicate cultures of

50r-c

0

h.

06

.2

40?-30h

20H

lo0

___ _ --.A

0 2 4 6 8 10 12

hours

FIG. 2. Induction ofa4-TKgenechimeras inL316 cellsby UV-irradiated virus. Cellswereinfected with

5PFUofHSV-1(F)ts502A305 viruspercellorwithan

equivalent amount of HSV-1(F)ts502A305 virus that

had been UV irradiated as described in the text. Infectedcultureswereincubated atthetemperatures

andforthetimesshown.

c 0 0. 0

A6

06 E 0.

nd L3 0.1 cel des shc for vir

eiti

cel

1(F

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BATTERSON

observation that induction of TK activity is

demonstrable after exposure of L316cells to 1 tsB7 PFU per cell (Fig. 1), thedataindicate that the

ts502A305

supernatantfluidsaredevoid of

inducing

activi-tyand that the inducing activity partitions with the virus.

Effect of infection withHSV-2(333-B3)TK-on

the a-TK gene. In this series of experiments, ftB7 L316 cells were infected with HSV-2(333-B3) TK- virus at a multiplicity of infection of10

PFUper cell. The results (not shown) indicate that HSV-2 induces the HSV-1 a-TKgene chi-meras in the same fashion as the homologous

hS02A305

virus. Inasmuch as the studies on HSV-1 x

HSV-2 recombinants failed to reveal forbidden

juxtapositions of HSV-1 and HSV-2 DNA se-quences(23-25), the resultswere not unexpect-ed.

Effect of other viruses on the a-TKgene. The purposeof these experiments was to determine

whether otherviruses, particularly PRV,BMV, CMV, and Ad-2, were able to induce resident a-PlRv TK gene chimeras in transformed cells. Since

iBMV

BMVandPRVeachspecifyaTK, we selected

~MOCKCS

mutants deficient in this

function,

asdescribed above.

The experimental proceduresfor infection and maintenance of cells withtheseviruseswerethe

same as those described above. The

multiplic-ities ofinfectionwere10 PFUof PRV andBMV

ftB7

,percell, 100PFUof Ad-2 per

cell,

and 50 PFU of CMV percell. The results (Fig. 3) show the

DtsB7 following.

GuPWV,BMV

[image:4.490.54.246.54.444.2]

hours ofter absorption

FIG. 3. Induction of TKactivity byTK- HSV-1, BMV,and PRVherpesviruses. Theexperimental

pro-tocols are described in the text. The reference box

symbolsin A denote thetemperatureofincubation and

typeof virus usedin theexperiments illustratedinA, B, and C. (A) Inductionof TKactivityin L316 cells

infected with HSV-1(HFEM)tsB7 and by

HSV-1(F)ts502A305. (B) Induction of TKactivity in L316 cellsbyTK-mutantsof BMV and PRV. Selection of these TK- viruses is described in the text. (C) TK

activity in 143(TK-) cells infected with

HSV-1(HFEM)tsB7, BMV(TK-), or PRV(TK-) viruses. Note thatHSV-1(F)ts502A305 carriesa700-basepair

deletion inthe TKgene(26).

thatthe a-TKgeneinL316 cellsis inducedbya

nonvirion-associated factor accumulating in in-fected cell lysates, L316 cells were exposed to

supernatantfluids obtainedafterhigh-speed

cen-trifugation oflysates of Vero cells infected with HSV-1(F)ts502A305 as described above. The

L316 cells were notinduced after exposure to

supernatant fluids containing the equivalent of

10or 100 PFUpercell(Table1). Inlight of the

TABLE 1. TKactivity of L316 cells exposed to supernatantfluids oflysates of Vero cells infected

with HSV-1(F)ts502A305 at 33°C

Hourspost- pmol ofthymidylateper

p.g

ofproteinin:

exposure or Cellsex-

Mock--infection' Infected posed to Mock-cells supernatant

ifctedl

fluidcel

Expt1

2 9.7 7.1 6.3

6 26.2 5.2 7.4

12 49.4 6.9 6.5

Expt2

2 NDb 5.3 5.8

6 ND 6.1 4.9

a The

supernatant

fluids were adjusted to yield a

virus-equivalent concentration of the cell lysateof 10 PFU (experiment 1) or100 PFU (experiment 2) per cell. The procedure forpreparation ofthesupernatant fluid is described inthe text.

bND, Not done (preliminary experiments have

es-tablishedthat at highmultiplicities of infection [e.g.,

100 PFUpercell], theinduction of the oa-TK gene in

L316 cells wasgenerally not as highas atlower (10

PFU percell) multiplicities ofinfection).

7or

A

60

50

40

OA, 390

OA, 330

0,0,370

30-c

* 20

a

0.6 10 0 10 co 5

10

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INDUCTION OF a GENES BY HERPES SIMPLEX VIRUS

(i) None of the viruses

selected for

this study induced TK activity in human 143(TK-) cells. Theresultsobtained for BMV and PRVmutants areshowninFig. 3C.

(ii) Neither BMV nor PRV significantly in-duced the resident a-TKgene chimerain L316 cells(Fig. 3B).

(iii) NeitherCMV norAd-2induced the resi-dent a-TK gene chimera in I316 cells

(Fig.

4). Attempts to induce the enzyme in L316 cells with these viruses alsofailed.

DISCUSSION

Inthispaper, weaffirm and expand the

previ-ousreport (26) from this laboratory that

(i)

a4-TK gene chimeras are induced in

converted,

TK+ cells by infection withTK- virus, (ii) the induction isindependent of the synthesis ofa4 gene product, and

(iii)

the induction does not

require the synthesis of viral proteins directed by the DNA

of

infecting

virus. The results presented in thisreport indicate that the induc-tion is

dependent

onexposure to

virus,

that UV

light-inactivated

virusisaseffectiveasuntreated

virus, and that the chimeric gene is induced at

the

nonpermissive

temperature

by

anHSV-1 ts

mutant defective in release of viral DNA from

capsids,

but not

by

other

herpesviruses

nor

by

Ad-2.

The discovery that a4-TK genechimeras are inducibleby infection (26)wasunexpected inas-much as the infectivity of the

deproteinized

DNAsuggested that the introduction of compe-tent viral DNA is all that is

required

for a

programmatic

execution of the reproductive

cy-clein

permissive

cells. The observations that

a-TK gene chimeras are

inducible

(26), that the inducibility is conferred by amovable

"regula-tory" sequence locatedatleast 100nucleotides

upstreamfrom the site of initiation of

transcrip-tion ofagenes

(20),

and that alla genescontain within the regulatory regions homologs of a consensus sequence (22) suggest that a genes areindeedinduced andraise questionsregarding thenatureof theageneinducer and itsfunction in the

reproductive

cycle of the virus.

Signal for induction ofa genes. The observa-tions reported in this andprevious publications indicate that the

signal

for the induction of a genes is HSV-1 and HSV-2 specific and most

likelyastructuralcomponentlocatedoutside the

capsid.

Specifically,

theeffect ofthemultiplicity of infectionsuggests thatinduction ofa-TKgene

chimeras

requires

exposure to competent virus

and that the concentration of this virus in the inoculum approximates that of the infectious

virus. However, theexperimentwith UV light-irradiated virus as well as the results of induc-tion ofthe a4-TK genechimera inthepresence

of inhibitory concentrations of cycloheximide

*ts502A305,390

OtS5024305,330

ACMV, 370 OAD-2,370 XMOCK,370

c 0

a.

0

0 2 4 6 8 10

hours

FIG. 4. Induction of TK activity in 1316 cells

1143(TK-)cells transformedtoTK+ phenotypewith anoa-TK gene chimera contained inplasmid pRB316]

(26)byHSV-1(F)ts502A305, Ad-2,andCMV.

(26)

indicate thatthe

synthesis

of viral

proteins

by

the

infecting

virus isnot

required.

The stud-ies with the

HSV-1(HFEM)tsB7

mutantare

con-sistent withthisconclusionandsuggestthat the

structuralcomponent

responsible

forthe induc-tionofthe virusis located outsidethe

capsid.

It

is of interest to note that the HSV-1 virion containsatleast 24 structural

proteins

(9, 27),

of whichnomorethanhalfareeitheratthesurface

ofthe

envelope

orin the

capsid.

Thefunctionof

the

noncapsid,

nonenvelope

proteins

is un-known.

Conceivably,

among these

proteins

are

those

responsible

for the induction ofaL genes

and forotherfunctions suchasthe virion-associ-ated shutoff of host macromolecular

synthesis

(8)

and the

putative

virion

protein

kinase

(18).

Function ofageneinducer. Inasmuchasviral

mutants

lacking

aL

regulatory

regions

have not

been

constructed,

the functionofthe

regulatory

regions

and the

requirement

forinduction of the

agenes afterinfection havenotbeen

explored.

Itisconceivable that the function of the oLgene inducer is to enhance

transcription

of the a

genes

by

binding

eithertotheviral genomeorto

host gene

products.

We cannotdifferentiate

be-tween these alternatives. However, BMV and

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PRV share many biological properties with

HSV-1 and HSV-2 and belong to the same subfamilyof herpesviruses (28); the observation thatneitherPRVnorBMV, aherpesvirus struc-turally and genetically related to HSV-1 and HSV-2 (2, 3, 30),induced the a-TKgene chime-rasuggeststhat theageneinducer hasa recogni-tion site for specific sites in the viral DNA in addition to any putative recognition sites for

hostgeneproducts. The observation that the

a-TKgene chimerasare induced atahigher level

at39 thanat33°C isof considerable interest and

potential helpindetermining thefunctionofthe inducer but does not differentiate between the

alternatives listed above.

A central question is whether, in the absence of the a gene inducer or in the absence of

regulatory regions, the agenes contained in the viralDNA would betranscribed atalevel suffi-ciently high to sustain viral multiplication. The observation that a-TK gene chimeras are

tran-scribed in the absence of the a gene inducer is

notcompelling evidence that they wouldbe

tran-scribed as partofthe DNA in the infected

cell;

thus, in the absence of functional a4 gene

prod-uct, thenatural ,-TKgeneistranscribedwhenit

is a resident gene in cells converted to TK+

phenotypebut notwhen it ispartofthegenome

ofan infecting virus.

Our results would havepredicted that

depro-teinized HSV-1 DNA wouldnotbe

infectious;

in fact,thespecific infectivityofHSV-1 DNA isof

theorder of106 moleculesperPFU(15). Among

the possible explanations are that (i) the

repro-ductive cycle initiated by deproteinized DNA

depends on the participation offunctional host

analogs of a proteins and is therefore very

inefficient or (ii) the reproductive cycle begins

with the synthesis of the a gene inducer and

continueswith aproteinsratherthanwith thea

proteins asisthe casein thereproductivecycle

initiated by virions. Additional studies should

reveal thenatureandfunction of the inducerand

its siteof action.

ACKNOWLEDGMENTS

Thesestudies wereaided byPublicHealth Servicegrants

CA-08494 and CA-19264 from the National Cancer Institute andby American CancerSocietygrantMV-2R.W.B. was a

PublicHealthServicepostdoctoral trainee(grant 5 T32 PHS

AI07182).

LITERATURECITED

1. Batterson, W., D. Furlong, and B. Roizrnan. 1983.

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release ofviral DNA and inother stages ofthe viral

reproductive cycle.J.Virol. 45:397-407.

2. Buchman, T. G., and B. Roizman. 1978. Anatomy of

bovine mammillitis DNA. I. Restriction endonuclease

maps of fourpopulations ofmoleculesthatdiffer inthe

relativeorientation of their long andshortcomponents.J. Virol. 25:395-407.

3. Buchman, T. G., and B. Roizman. 1978. Anatomy of bovinemammillitis DNA. II.Size andarrangementsofthe deoxynucleotidesequences. J. Virol. 27:239-254. 4. Campione-Piccardo, J., W. E. Rawls, and S. Bacchetti.

1979. Selective assay for herpes simplexviruses express-ingthymidinekinase. J. Virol. 31:281-287.

5. Costanzo, F., G. Campadefli-Fiume, L.FoA-Tomasi, and E.Cassai. 1977.Evidence that herpes simplex virus DNA is transcribed by cellular RNA polymerase B. J. Virol. 21:996-1001.

6. Dixon, R.A. F., and P. A. Schaffer. 1980. Fine-structure mappingand functionalanalysis of temperature-sensitive

mutants in the gene encoding the herpes simplex virus

type1 immediate early protein VP175. J. Virol. 36:189-203.

7. Ejercito, P. M., E. D. Kieff, and B. Roizman. 1968. Characterization ofherpes simplex virus strains differing intheir effectsonsocial behavior of infectedcells. J.Gen. Virol. 2:357-364.

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