Anatomy of herpes simplex virus (HSV) DNA. X. Mapping of viral genes by analysis of polypeptides and functions specified by HSV-1 X HSV-2 recombinants.

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JOURNAL OFVIROLOGY, May1978, P. 389-410 Vol. 26,NO.2

0022-538X/78/0026-0389$02.00/0

Copyrighti 1978 AmericanSocietyforMicrobiology Printed in U.S.A.

Anatomy

of

Herpes Simplex Virus (HSV) DNA

X. Mapping of Viral Genes by Analysis

of

Polypeptides

and

Functions

Specified

by

HSV-1 x

HSV-2 Recombinants

LAWRENCE S.

MORSE,'

LENORE PEREIRA,' BERNARD

ROIZMAN,I*

AND PRISCILLA A.

SCHAFFER2

MarjorieB. Kovler ViralOncologyLaboratories, University of Chicago, Chicago, Illinois60637,'and

Sidney

Farber CancerInstitute,Harvard MedicalSchool,Boston,Massachusetts 021152 Received for publication 13 October 1977

In an

earlier

paper(Morse et al., J. Virol. 24:231-248, 1977) we reported on the

provenance of theDNA sequencesin26herpessimplex virustype 1 (HSV-1) x

HSV-2 recombinants as determined from analyses of their DNAs with atleast

five restrictionendonucleases. Thisreport deals with the polypeptidesspecified

by the recombinants and

by

their HSV-1 and HSV-2parents.Wehave identified

(i) the corresponding HSV-1 and HSV-2 polypeptides with molecular weights

ranging from 20,000tomorethan200,000,(ii)the polypeptides that undergorapid

post-translational processing,

and

(iii) polypeptides

that vary intratypically in

apparentmolecular

weight. By comparing

the

segregation

patterns of the

poly-peptides with those of the DNAsequenceof therecombinants,wehave mapped

the

templates

specifying

26

polypeptides

and several viral functions on the

physicalmapof HSV DNA. The data show thefollowing: (i)apolypeptidesmap

atthe terminiof theLandScomponentsofthe HSV DNA.AlthoughaICP27

mapsentirely within the reiteratedregion ofthe L component, the template for

aICP4maylie

only

inpartwithinthe reiteratedsequencesof theScomponent.

Ofnoteisthe

finding

thatcellsinfected witharecombinantthat contains both

HSV-1 and HSV-2 DNAsequencesin theS componentproduced a ICP4of both

HSV-1and HSV-2.

(ii)

Templates

specifying,8

andypolypeptidesmapinthe L

componentand appearto be

randomly

distributed. (iii) Thymidine kinase and

resistance to

phosphonoacetic

acidmapped in theL component.Inaddition,we

have takenadvantage of the rapid inhibition of host protein synthesis

character-istic of HSV-2 infections and

syncytial plaque morphology

to also map the

template(s) responsible

forthesefunctions in theL component.The

implications

of thetemplate arrangement in HSV DNAarediscussed.

In this paper we report the

location,

in the arethe

following

data:

(i)

Honess andRoizman

DNAs of herpes

simplex

virus types 1 and 2 (13)

reported

that HSV-1

specifies

in infected

(humanherpesviruses1and 2;HSV-1 and HSV- cellsatleast48

polypeptides

ranging

from 20,00

2), of the

templates

specifying

26 polypeptides to greater than 200,000 in molecular

weight.

and several viral functions. The

experimental

Several additional

polypeptides

less than20,000

design

used in these studieswassimilartothat inmolecular

weight

were

reported

by

Marsden

used in

mapping

adenovirus

templates (9, 25)

et al.

(24).

Asimilar

list

ofHSV-2

polypeptides

andconsisted of

comparing

the

segregation

pat- was

reported by

Powell and

Courtney

(30).

Stud-terns of

polypeptides specified by

HSV-1 x ies

by

Courtney

and Powell

(3),

Pereira etal.

HSV-2intertypic recombinants with their DNA (29), Gibson and Roizman

(8),

and Cassai etal.

sequence arrangements.The DNAsequencear- (2) showed thatmany HSV-1 and HSV-2 virion

rangements of the intertypic recombinants used polypeptides and infected-cell polypeptides

in these studies were reported in a previous (ICP)differ in

electrophoretic

mobility,

but

only

paper in this series (26). We nowreportonthe a few of these

polypeptides

were identified as

polypeptides producedincellsinfected with the functionally identical.

intertypicrecombinants andonthephysicallo- (ii) The

synthesis

of bothHSV-1 and HSV-2

cation in HSV DNA of thetemplates specified polypeptides is regulated. On the basis of the

bythem. temporal

pattern

andrequirementsfor their

syn-Pertinent totheresultspresented in this paper thesis,HSV-1ICPswereshownto

form

atleast

389

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390 MORSE ET AL. J. VIROL.

threegroups,designateda,

,8,

andy,whosesyn- MATERIALS AND METHODS

thesis is coordinately regulated andsequentially

ordered inacascadefashion (14, 15,32-34). Radiochemicals. L-[U-14C]isoleucine,

L-[U-_4C]-(iii) HSV-1 and HSV-2 DNAs are linear dou- leucine, andL-[U-_4C]valine (allwith specificactivities

ble-stranded molecules approximately 97 x 106 of approximately 300 mCi/mmol) were purchased

to 99 X 106 in molecularweight (21, 39). HSV-1 fromNewEngland Nuclear Corp.,Cambridge,Mass.

DNA consists oftwo covalently linked compo- Cels. HEp-2

ceUs

were grown in Eagle minimal

nents, L and S, containing 82 and 18% of DNA, essentialmediumsupplementedwith 10% calf serum,

respectively.

The terminal reiterated sequence 0.001% ferric nitrate, and 1% sodium pyruvate.

bracketing.thne

Lcomponent,designated

asequence

Viruses. The virus strains used in thisstudywere

bracketing the L component, designated as ab, (i) HSV-1 (KOS tsE6), a DNA' ts mutant, and

HSV-and its inverted repeatb'a'each contain 6.0% of 2 (186

tsB5),

a DNA- mutant described elsewhere(35,

the DNA, whereas the sequences bracketing the 36); (ii) HSV-1 (17 tsJ), a DNA- ts mutant (24, 38)

S component, designated as a'c' and ca, each kindly provided by J. Subak-Sharpe; (iii) HSV-1

contain4.3% of the viral DNA(32,33, 39). DNA (HFEMtsN102),aDNA- mutantkindly providedby

extracted from virions or from infected cells A. Buchan; (iv) HSV-1 (B2006 ts-), kindly provided

consists of fourequimolar populations differing by S. Kit (20); (v) HSV-2(186), theparent strainof

in the orientation of L and S components

(10,

HSV-2 (186 tsB5); (vi) HSV-2 (GP6), a

syn-

mutant

33

39).

StudiesofHSV-2DNA withrestriction (1)of HSV-2 (G); (vii) HSV-2(GP6

PAAN),

amutant

39).

Studies

ofdHSV-2

DNa

with res

trsictio

ofGP6

resistant

tophosphonoacetic acid (PAA),

se-endonucleases

iiaycate

that it

has

a similar lectedbyprocedures described in reference 26; (viii)

structure (G. S. Hayward, T. G. Buchman, and HSV-1 (F) and HSV-2 (G)

(32-34);

and

(ix)

24

inter-B. Roizman, manuscript in preparation). Anal- typic recombinants derived by crossing HSV-1x

HSV-yses of HSV-1 x HSV-2 intertypic recombinants 2 parental strains (26). Virus stocks were prepared

reported in the preceding paper (26) suggested fromplaque-purified seedaspreviously described (26).

that only a limited number of HSV DNA ar- Labeling ofproteins synthesized by infected

rangementsparticipatedin theformation of re- celis. Confluent HEp-2 cell monolayers in 25-cm2

combinants and by extension were capable of tissueculture flasks wereexposed to 20 PFU ofvirus

replication. The HSV DNAarrangement shown per

cell

in 1.0ml of maintenancemedium. After1hof

torbeP11at10n.e

topaicipateinte

foraementinown

o incubation of

33.5°C

with constant

agitation,

the

in-to be able in-toparticipate in the formation ofall oculum was replaced with 5.0 ml of maintenance

me-recombinants analyzed in the preceding study dium, and incubation was continued at 33.5°C. For

wasdesignated as prototype (P) (26). Thethree. labeling polypeptides, the cultures were replenished

otherarrangements were designated as Is, inver- with labeling medium containingone-tenththenormal

sion of S component; IL, inversion ofL compo- amountofleucine, isoleucine, and valine, but

supple-nent;andISL,inversion ofboth L and S compo- mented with

["4C]leucine,

-isoleucine, and -valine (2.0

nents. For the sake of simplicity, only the P uCi of each amino acid per ml of medium). For labeling

arrangements of HSV-1 andHSV-2 are shown early viralpolypeptides, HEp-2cellswereexposedto

inthispaper. virusat370C for 1 h and then labeled for 2 h at39°C.

mi

this

paper. y At the end of the labeling period, the cellswere

(iV) The location of templates specifying rinsed with ice-coldphosphate-bufferedsaline (3 x 5.0

knownviralfunctionsin HSV DNAarelargely

ml/flask)

toterminate incorporation, either harvested

unknown. Previous studies have shown that immediately (pulse) or washed, and then reincubated

viralRNAsequences accumulating in thecyto- in the absence oflabeledaminoacids(chase).

plasm in thepresence ofcycloheximide (aRNA) Preparation of samples for

electrophoresis.

hybridize

predominantly with terminal frag- The labeledcells werestripped from the dish,

dena-ments ofL and S components and to a lesser tured, and

solubilized

byheatingfor 2 to 3

min

at80°C

extentwith internalfragments intheL compo- in the presence of 2%sodiumdodecyl sulfate (SDS),

nent (19). 5',B-mercaptoethanol, and 0.05 M Tris-hydrochloride

(v)Rcentlwerportdon he usfulnes of (pH 7.0).

(v) Recently wereported on the usefulness of

Polyacrylamide

gel electrophoresis. The

elec-intertypic (HSV-1 x HSV-2) recombinantsfor trophoretic, staining, andautoradiographictechniques

mapping viral markers on the DNA (26). The were as described previously (11, 37). The

polyacryl-recombinants used in these studies were pro- amide gelelectrophoresis was done in a discontinuous

duced by crossing ts HSV-1 x tsHSV-2 or by buffersystem containing0.1% SDS. Thestacker and

crossing tsPAAr HSV-1 x HSV-2. The prove- separation gel contained 3 and 9%acrylamide,

respec-nance of the DNA sequences in the cloned re- tively, cross-linked with N,N-diallyltartardiamide

combinants were determined by mapping with (Aldrich Chemical Co., Milwaukee, Wis.) in an amount

Hpa I,

Bgl

restr,BglII,XbaI,Ection ThuI,and,ioe

II,XbaI, EcoRI,HsuI,and,insome _Thecorresponding

_{separation gel}

to 2.6% of the weight of acrylamide.

was20cmin

length.

The

proteins

instances,K~pn Irestr±ctl endonucleases. The used for molecular-weight calibration were

f,',

,B,and

nomenclatureofthe recombinants, themapping asubunits of Escherichia coli RNApolymerase,

bo-procedure,and detailedrestriction endonuclease vine serum albumin, and soybean trypsin inhibitor

maps werereportedinthepreceding paper (26). (T,) (Boehringer Mannheim, Indianapolis, Ind.) with

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VOL. 26, 1978 ANATOMY OF HSV DNA 391

molecularweights of165,000, 155,000, 39,000, 69,000, vary intratypically among HSV-1 and HSV-2

and21,000,respectively. strains, (iv) provisionally matches the

corre-sponding HSV-1 and HSV-2 polypeptides, and

RESULTS (v)describes thekinetic classto whichthe

poly-Comparison of HSV-1 and HSV-2 ICP. peptidesbelong.

Table 1 summarizes the data derived in the The experimental data relevant to the

con-courseof these studiesontheapparent molecu- structionofthistableare asfollows:

larweights and otherproperties of HSV-1 and (i) The criteria for

identifying

virus-specific

HSV-2polypeptides. The polypeptideswere des- ICP was that of Honess and Roizman (13) as

ignated accordingto Honess andRoizman (13) applied by them foridentifying HSV-1

polypep-as extended by Pereira et al. (29). Thus, the tides and byPowelland Courtney (30) for

iden-polypeptidesarenumberednumericallyin order

tifying

those of HSV-2. We have largely retained

of decreasing molecular weight (13). Polypep- the numerical designation ofHoness and

Roiz-tides undergoing rapid post-translational proc- man (13) for HSV-1 ICPs and, for simplicity,

essingareidentifiedby number and lettersa, b, assignedtoHSV-2ICPnumbers that correspond

c, etc.,

denoting

theprecursor(a) andproducts tothoseof HSV-1 ICP.

(b, c, etc.) (29). Table 1 (i) enumerates HSV-1 (ii) Identification of polypeptides whose

mo-and HSV-2 specific polypeptides in infected-cell lecular weightswerealteredas aconsequenceof

lysates, (ii) identifies the polypeptides thatun- rapidpost-translational processingwasbasedon

dergorapid post-translational processing, result- experiments involving pulse-labeling followed by

ing in an

appreciable change

in the apparent an

appropriate

chase in the absence of

radioac-molecular weight, (iii) lists the polypeptides tive precursors. An example of the results of

[image:3.509.52.451.330.637.2]

whoseapparentmolecularweightwasfoundto such anexperiment is shownin Fig. 1, where we

TABLE 1. ElectrophoreticpropertiesofHSVpolypeptidesa

Poly- Molwt(x10-3) Poly- Molwt(xlO-3)

peptide Form Group peptide Form Group

no. HSV-1(F) HSV-2(G) no. HSV-1 (F) HSV-2 (G)

1 b >221 221 Y 20 77* 78* Y

a 21 72.5 73.5*

2 b 205 >205 Y 23 71 72 Y

a 196 198 24 b 68 68 /Y

3 194 191 Y a 67.5 67

4 c 170* 180* a 25 b 64 63 y

b 165 177 a 63 62.5

a 163 172 26 b 61.5* 62 /3

5 b 151 153 .Y a 60 60

a 149 151 27 b 58 59 a

6 b 146 146* a a 56.5 58

a 143 143 28 56 57

7 139* 138* 29 b 55 54.5 a

7.5 b 132* 135 a 54.5 53

a 130 131 31 52 52

8 128 127 /3 32 51.5 50.5

-Y

9 b 122 121 Y 33 50 49

a 119 119 34 49.5 48

10 117* 122* //y 35 b 46.5 45* Y

11 114 114* Y a 45

12 111 113 36 42.5 42.5 /3

13 109 112 37 b 39 39.5* Y

14 106 107 a 38.5

15 103* 101* Y 38 37 35.5

16 101 100 39 b 36 35 /3

17 92 91* /3 a 35

18 b 88* 85* Y 40 33.7 34 A

a 85 41 32 33.5 A

19 78 79 y 43 26.5 26 Y

44 24.5 25

-Y

aThe polypeptides are numbered numerically in order ofdecreasing molecular weight. The table also

identifies thepolypeptidesthatundergo changesin theirelectrophoretic mobility,whicharedesignatedasa,b, or cforms.Polypeptides showingintratypic variabilityaremarked withanasterisk.

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392 MORSE ET AL.

J. VIROL.

HSV-1

CYCLO

H SV- 2

3

4

C

~~~~~~~~~~4

a

)4 - 4 4

5 5we

m

5O~54

S

v~~

S6_I6

a

US 61S

j

nn

0

bl10

9

afb

9

11 ab I19'1 '- b1

15

;8 17

24 C 24

25

-26

nU.AJW7

~

427--

- 7 _

-4

-,

22UU7

JUl

32 32

34

344 4W

153

,,W,,

U_'151

S

j

35-o-

e

ae

- me

-37 a

38

39

ekf

cut

0.

5m

41 P

C

P

C

P

C

P

C

PC

P

C

3 hr

4 hr

5hr

3 hr

4hr

5Shr

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VOL. 26,1978 ANATOMY OF HSV DNA 393

show the polypeptides undergoing rapid post- peakrates ofsynthesis between 5 and 7 h

post-translational cleavages relativelyearlyin infec- infection and were identified on that basis.

Iden-tion. Some of these (e.g., ICP4and6) have been tification of y polypeptides was based on the

previously showntobeprocessed after synthesis. observation that they are made at increasing

Table 1lists 16polypeptides thatwerefound to ratesuntil12 to 15hpostinfection.

besignificantly alteredinelectrophoreticmobil- (v) Identification of corresponding HSV-1 and

ity after synthesis. This is a minimal estimate HSV-2 ICPswasbasedonthe following

ration-becausepost-translational modifications that do ale. Comparison of the twosetsof ICPs showed

notresult inaperceptible change in electropho- that they fall into two classes. The first

com-retic mobility wouldnotbe detected. Two points prised HSV-1 and HSV-2 ICPs that fell into

are noteworthy concerning the processing of identical kinetic classes and didnotdiffer with

polypeptides. First, with onlyoneexception,all respect to electrophoretic mobility. Although

processed forms of the polypeptides showed a they are not central to the objectives of this

decrease in

electrophoretic

mobility. Secondly, study, for the purpose of identification we

as-weresolved in these experimentstwobands for sumed that they have identical functions and

the major capsid protein, ICP 5. Pulse-chase numbered them accordingly. The second set

experiments of the kind described in the legend comprises HSV-1 and HSV-2 ICPs that could

to Fig. 1 failed to show a product-precursor notbematched withrespect toelectrophoretic

relationship. One possible interpretation of our mobility. Examples of such are ICP 4 and 27,

observations is that of a fraction of ICP 5 is whichfallintothea group,andICP5,member

altered during assembly of the HSV capsids. of the y group(14). These polypeptidesare

cen-Thedesignations5aand5b are arbitrary. tral to theobjectivesof thisstudy because

iden-(iii) Intratypic variability of structural poly- tification of the HSV-1 andHSV-2 polypeptides

peptides among HSV-1 strains has been re- specified by therecombinantswasbased inpart

portedpreviously (28). Comparisonofthe paren- ontheirelectrophoretic mobilities. Thepairing

tal strains usedto construct the recombinants of thecorrespondingpolypeptideswasbasedon

revealed numerous ICPs that varied among analysesofICPs specifiedby therecombinants

HSV-1 andamongHSV-2 strains. The dataare andrestedon twocriteria. First,withone

excep-basedonelectrophoreticmobility of the labeled tion identified later in thetext, thepresence in

ICPs on the same gels. Some of the data are thecell lysate ofapolypeptide specified byone

shown in

Fig.

2through 10 andsummarized in serotype excluded the presence of the other.

Table 1. Aninteresting example ofanonstruc- Second,eachICP specifiedbyHSV-1orHSV-2

tural

polypeptide

thatappeared to be variable could be mappedindependently as either

pres-wasICP 4;asshowninFig. 2, the fully processed ent or absent, and in each instance the paired

form (ICP 4c) of HSV-2 (G) migrated more ICPsmappedinthesamelocation.

slowly

than the corresponding polypeptide of Mapping of HSV polypeptides. In

princi-HSV-2 (186). ple, by correlating the segregation patterns of

(iv) The identificationof anICP as a,

f,,

or y the polypetides specified by the recombinant

wasbasedonthefollowing operational definition viruses with the DNAsequence arrangement of

andonthebasis of the kinetics oftheirsynthesis the recombinant virus, it should be possible to

(14, 15).Bydefinition, apolypeptidesaremade map the physicallocation of the templates

spec-immediately

after withdrawal ofcycloheximide ifying viral polypeptides on the HSV genome.

presentin the mediumduring infection andfor The experimental procedure was as follows.

several hours thereafter (14, 15). An example of Cells infected with parental strains or with

re-a cycloheximide withdrawal experiment is combinants were pulse-labeled at three time

in-shown in

Fig.

1.Inthat

experiment,

only ICP4 tervals after infection as described in Materials

and27 weremade inHSV-1-infectedcells after andMethods and in the legends to Fig. 2 through

withdrawal of the drug.a Polypeptidesreached 10. Table 2 shows thesegregationpattemofthe

peakratesofsynthesis between2and4hpost- paired HSV-1-HSV-2polypeptides whose

tem-infection. In contrast, ,B polypeptides reached plateswere mapped. Figure 11 shows the DNA

FIG. 1. Autoradiographic images of electrophoretically separatedpolypeptides from HSV-1- and

HSV-2-infectedcells.HEp-2cellswereinfectedwitheitherHSV-1(F)orHSV-2(G)strains and thenlabeledfor15

minwith

"4C-amino

acidsat the times indicated. At the endof the pulse-labeling period, the inoculated cultureswereeither harvestedimmediately (P)orchasedby incubation inunlabeled maintenancemediumfor anadditionalhour(C). Thecycloheximidewithdrawalexperimentwasperformedby incubating HEp-2cells

infectedwithHSV-I(F)in thepresenceof50 pgof cycloheximidepermlofmedium.After 7h,thecycloheximide

waswashedoutandreplaced with"C-aminoacidlabeling medium. The cellswereharvestedafter 15 minof incubation.

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394

MORSE ET AL. J. VIROL.

Series A and B

4 44 4

4W

4 4 4 4 4 4 165

155

cre5_

WP5 5;

_17 7 74117 7 7J7 7 7 7 7 7

7e7

t 8 8S8 8 84_8 8 8 8 8 8 8S_8

10 tO

~~~~~~~~~~~~~~~101

10

ITse -w

1S 15 15 15

_ow7aA 17

-

18s

i8

.\4A. .S A 20 20

P't.

24~~~~~~~~~~~~~~2

: 25

26

__ _

534 oiLL-i

-_

_SIO

O

eO

e

4

39

[image:6.509.70.456.69.586.2]

G tsJ

A1EJ

GP6 A7D

A1G3

GP6 A5C A2DA4D tsJ A6D Bl E 186 mock

FIG. 2. Autoradiographic images of electrophoretically separated polypeptideslabeledwith

"4'C-amino

acid

fr-om1to3hpostinfection.ParentalstrainsforseriesAintertypicrecombinantswereHSV-1(tsJ)and

HSV-2(0P6). TheparentalstrainsforseriesBwereHSV-1(tsej andHSV-2(186).

sequence arrangementofthe recombinants de-

fying

IOP

5 illustrates the

mapping procedure.

termined

previously

(26).

Figure

12 shows the

Thus,

examination of the

IOP

5

specified by

physical

location of the

templates

mapped

in recombinants C4D and

A6D (Table

2;

Fig.

4, 7, this

study.

The

mapping

of the

template speci-

and10) indicatesthat the

template (Fig.

11) is

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VOL. 26, 1978 ANATOMY OF HSV DNA

395

Series A and B

4 4 4 4 4 4 4 4

6a,4

_6_s z4hi- -.q.5

S F5emw<m5-

--S

JWJ*4

aB._nri

5n&

5

_1

le I

AD 7DulO.0aw-Of7

1St' 41 ~ 4 ...4.s.4n141Wj1

GP6AlEl A1G3 tsJA7

A2D

TIA4DOG6SIADAE

with~~~~zseriesjrA in*teryi reobiat frm . to 6 h potnecin

~~~~~~~~~~~~~~~~~~~~~~~~~~~-18

4 4Do-.4 "OW0-rM ~, do*-_ 4

41 4 - 41 41

-.4

4

43

43Vp,

43 4

.4

4Q

3

'43|

--,e,3 A

44 44 44. 44 44 44 44 4-4 44 44 4

[image:7.509.106.389.66.621.2]

GP6

AlEl

A1G3

tsJ A7D A2D

A4D

GP6A5C

A6D A1E2

FIG. 3. Autoradiographic images of electrophoretically separated polypeptides labeled in cells infected

with series Aintertypicrecombinants

firom

4.5to6hpostinfection.

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Series

A

and

B

4 4

w7

7mr=r

=

18 f8

*w.

4;

8 *b..8

8

7}t?@i

8*#$-8

'

8

a

8 8

.st

"8

9

99999 9 9 9

10 0 ?.1

4MW _

fl

4S#

PSI

_1M1

1

8

_

_ 18 -;

18*P'84+

'18

18~

- ss 1 8 '

18*Lr18

1 -eA 18

w I

___.'

I_

'3

_{3_:23;}

72

_223:.0

23

2

4t2wmW*..24~lY E:=4 wQ4

_24,

...~~~~~~~~~~~~~~2 . w.WaoQ 27 - 2727

35

-~~"40~ 40'

-t394~39_

39 df9l39

*4

_GP6

AlE)AlG3tsJ1

41

4 ₇

4D

n40

4Au11

_As

.4_t40 44 44.0

_E18A

_E16

4

41

4

[image:8.509.82.453.45.645.2]

41~

~~~~~~

41a

'4

~

~~~~~~~~~F

416IMW-T

FIG. 4.

AutoradiograpI

iaeo et

s

infected

wIth seriesAuinter

gryphicrcmbnans

ofro

9etro1hposticy

e p infectl e in cl

withseries A

intertypi

recomb ts

fm

9to po 40

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(9)

Series C

UF"

fl4IIIS4

4_

*

t44

= S4

fl4

a_ 5i _5a 5

SE7w-7aiOSz

.7inZ

~7

_

7'

7

7fl

7

.., ..10

eI fi aa4Taf_ 8_ *~~1 8w am4

-15

:-

15

1 5,-15-1 -

-.S-15

auL _18a-15_-Ia

-@-20;)2D

a2O

20

20 20

-~

~~~~~~~~~~

4-4*tU C.

__*-r

4U

'

PM-~a

_ _ 334 34

_O O4ine 0ta

G

N102 C2D

186 C3DI

C4D

N102

C5D C6D C7D 186

mock

FIG. 5. Autoradiographic images of electrophoretically separated polypeptides labeled in cellsinfected

with series Cintertypic recombinantsfr-om 1 to3hpostinfection as described inMaterials andMethods. Parental strainsforseriesCintertypic recombinantswereHSV-1asNlo2 andHSV-2(186).

.b_i~~ ~~~~~9

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Series C

4

4 4 44 4

4 -,a4 4

5-5Amm5

5-ma. 5A 5-. 5__5_5 50oo5

-7esLUIf eW b f7~7u7fl.0#Fa

8 cm

N3

eu> 8s aa m a n.. .

XW5th_ e

18

4000

a

-4 2 0 2Q

~~~~~~20

20

a~*}.

->s.ti

_

1 jje_~

?

__*~n3~

3__

4_07

7 WI_

_fS

35 |l35

5 |

5 SX

35 3535

4 * e 4 35 -G

40

;F41

40 40,;00A

|-m41 M41_

00-743 43 -4 3 4- .& 43_

44 44 44 4 44nA444 44 44

F

G

N102

ClD

C2D

186 C3D10NI2

C5D C6D

186

FIG. 6. Autoradiographic

images of

electrophoretically separated

polypeptides

labeledin cells

infected

with seriesCintertypicrecombinants

from

4.5to6h

postinfection.

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Series

C

4 4 4

-~~~-

*

cIinsSSin5-amIgI7aa~glom

7004WZM7&4aas.tmo7flO

7007 ILh4

_9

^~~~00

99

9

j"n_49

a"1

ORa

_

__

.v

24

*24

..

i36-~~~~~~~~~~~~~~3

F

0 N102

CID

C2D

186 C31D1

C5D

N1O2

C6Dl86

mock

FIG. 7. Autoradiographic images of electrophoretically separated polypeptides labeled in cells infected

with series Cintertypicrecombinantsfrom9to10hpostinfection.

o399

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[image:11.509.92.415.46.640.2]

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Series

D

4 4!;4r44 4 4

S-W~~~

4

~~~~~~~~~~~~~~~~

4S

qw

4~~~~~~~~~

=4 4 4

v 5 5

.ub7? 7uIUht_ _ 7 7a1t *7* .7 7 7 7 7

CP8- faS^Uh8iB _ usu8e.e a8 8 8 8 8

10

15 .15

-17t-- *< <

18B

58a

20

2~~~~~~4

20

* teU 4ibP~'W 'f

- 4j -D --,¢t.

_.

a

a aaa

a

SS

.40~~ ~ ~~~~0

-_9F~~~~~~~~~~~~~~~~~~~~~~~~~~~~~v

gR.7

G sE

DEIAE

4E DE D E tE D4E

D5E

D5Emock:re

lPW}; FI. Auordogapi image of_ elcrohrtial seaaedoyepie labeled_ in celsifete

witseie D-inerypi reobiat fro t3hpoinein. Paena stais fo eisDitryi

recombinantaweeHV1(O tWsE6 an HS-16tB

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Series

D

4 4 4 4 4 4

4 4 4

5 5 5s-,.6 5 5 5 5 5

Mpw

7 0*wA7 IM7Ul7IU77

717

77 7W?

7

7 n-7

_ w8-.-4 j E sm8 88 3 8e8_naB

ugah

_nw9

nub

Zll _11tUns_

l W*A6.RJl 11

9'el

18 8-- IL 18b

l8b

- 18b yjj s

*-w^1*}Y-1856 *118b8a*riWu) l18a Ila 185 ]L.Z.

40 40 40

40

41 41 41 41

41-D3E2

tsE6

A8E D4E3

D1E4

186 DIES D4E1

D4E2

tSE6

D5E1

DSE2

FIG. 9. Autoradiographic images ofelectrophoretically separated polypeptides labeled in cells infected

with series Dintertypicrecombinantsfrom4.5 to 6hpostinfection.

-;t ~~~~~40

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[image:13.509.87.413.44.645.2]

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Series

D

4 4

_t_555 4_5e.e5eI 5R u5e5 5

54n5

a7ain7 7 7 7 7 7

d-777

7

.7

7

080__-8 8 8ee..--8--8

ewwin8

ea e e8m 8 a A 8

99 9 9!.1992-9

10 lO 10O T10 10 10IL 10 10 o 10

IO.b~~~

1 18b 1 #9

b_1ib

18b 18ba j I_

;iga

wb 18a 18a 18a i

Tala

18a ISa

188'

17

20 20 20m2O--20 20- 20

-.23

~~~~~24

23 ---23 ;-23 23 23_1 -0-23 23 23 23 23 23 23

24_3

24. 24 244224 21 24 24 24

25 2 Oa~

.27

27,iv

?.2Mp7

27--- 2 v 27 27

29*fl629 29 e _2g

32_32 32 32 _ 32 32 32 32 32 32

_

'333m34_

_ ; s 3;3 ;0

33 3-_

t~~~~~~~~~~~~~~~~~~35

_,35zs

E % ~~~~~--n660U 3 6*# 3 36 36

@

_~7

iulwZ 37 43 37 37 37

"_

9 339133139Si3939

301P4W39

'

39_'U39

39

_I 40 40 40 40 40

_I_ L 4

~~~~~41-RW,41

41 +_ 41 41 41 41 41

;2Sa4S3

~~Ua43W31m_

_ 43m43 43

winj3

-v

44 44 4-

444

4

a4E4

44e44_ 44e 4 44 44 4

tsB5

DlEl

D3E2 tsE6

A8E

D4E3

D1E4

tsB5

D1E5

D4E1 D4E2

tsE6

D5E1

D5E2

FIG. 10. Autoradiographic imagesofelectrophoretically separatedpolypeptides labeled in cellsinfected

with series Dintertypic recombinantsfrom9to10hpostinfection.

402

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[image:15.509.45.444.80.358.2]

VOL. 26, 1978 ANATOMY OF HSV DNA 403 TABLE 2. HSV-1- and HSV-2-infectedcellpolypeptidesa

Intertypicre- Polypeptideno.

combinant 2 4 5 7 8 9 10 11 15 18 19 20 23 24 25 27 29 32 33 35 36 39 40 41 43 44 DlEl 2 1.2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

AlEl 1 2 1 1 1 1 11 1 1 1 1 1 1 1 2 2 1 1 1 11 1 1 1 1

A1G3 1 2 1 1 1 1 1 1 1 11 1 11 2 2 111 1 11 1 1 1 1

A7D 1 2 1 1 1 1 1 2* 1 1 1 1 1 1 1 1 2 1 1 A 1 1 1 1 1 1

A1E2 1 2 1 1 1 1 11 1 1 1 1 1 1 1 2 2 1 1 1 11 1 1 1 1

A2D 1 2 1 1 1 2* 111 1 1 1 1 1 1 2 2 1 1 1 1 2* 1 2* 1 1

A4D 1 2 1 1 1 1 11 1 1 2 A 1 1 1 2 2 1 1 111 1 1 1 1

A5C 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 111 1 1 1 1

D3E2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2

ClD 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 1

C2D 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 2 2 2 2 2 2 1 1

C3D1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 2 2 2 2 2 2 1 1

A8E 1 2 2 1 2 2 2 2 2A 2 2 2 2 2 2 2 2 1 2 2 2 2 2 2 2 1

C4D 1 2 2 1 1 2A 1 1 2 2 2 2 2 2 1 2 2 A 2 1 1 2 2 2 1 1

A6D 1 2 1 1 1 2 1 1 1 222211 2 1 2 1 1 1A 1 2 1 1

BlE 1 2 1112 1 1 1 2 1 1 1 1 1 2 2 1 2 1 1 1 1 2 1 1

D3E1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1

D4E3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1

DlE4 1 2 A 1 2 2 1 2 2A 2 2 2 2 2 1 2 2 1 2 2 2 A 1 2 2 1

DiE5 1 2 A 1 2 2 1 2 2A 2 2 2 2 2 1 2 2 1 2 2 2 A 1 2 2 1

D4E1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2A 1 1 1 1 1 1 1

D4E2 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 2A 1 1 1 1 1 1 1

C5D 1 2 1 2 1 2 2 1 2 2 2 2 2 1 2 2 2 2 2 1 1 2 2 2 2 2

C6D 1 2 12 1 2 1 1 1 1 2 2 2 1 2 2 2 1 1 1 11 1 1 1 1

D5E1 1 2 1 1 2 2 1 2 1 2 1 1 1 1 1 1 2 1 2 2 1 1 1 1* 2 2

D5E1 1 2 1 1 2 2 1 2 1 2 2 2 1 1 1 2 2 1 2 2 1 1 1A 2 1 1

aPolypeptides identifiedasHSV-1 arelabeledas1;those of HSV-2arelabeled as 2. The asterisk identifies

the few instances in which the polypeptides produced by thecells infected with the recombinants did not

cosegregatewith theDNA sequencespredicted byotherrecombinants.TheletterAdenotespolypeptideswith

anaberrantelectrophoreticmobility.

tothe leftof theleftcrossoversiteofC4D(0.30 a few instances inwhichthe

polypeptides

pro-mapunit) buttothe right of theleft crossover duced by the cells infected with the

recombi-site ofA6D (0.22 mapunit). The map position nants did not cosegregate with the DNA

se-oftheICP5template ismoreprecisely defined quences

predicted by

other recombinants.

Pos-by the crucial recombinants D1E4 and

DiE5,

sible

explanations

forthese inconsistencies are:

each of which

specifies

anICP5withanelectro- (i) intragenic recombinationevents resultingin

phoreticmobility intermediatebetween those of a recombinant gene

specifying

a polypeptide

HSV-1 andHSV-2. Thissuggeststhepossibility with an electrophoretic

mobility

characteristic

ofan

intragenic

recombinationevent. Compari- ofoneof the

parental

types, as illustrated

dia-son with the ICP 5

specified

by D4E1, D4E3,

grammatically

in

Fig. 13A; (ii)

DNA

heterodu-andC5D indicates that the left

boundary

ofthe plex mismatch

repair;

and

(iii)

undetected

dou-template

for ICP 5does notextend tothe left ble-crossovereventsin

regions

of the DNA

lack-beyond

0.23map

unit,

which indicates thatit is

ing

restrictionenzymesites. We noted

previously

the

right

crossoversite ofD1E4 and DlE5 that

(26)

that double-crossoverevents

encompassing

ismost

likely

responsible

fortheaberrant elec- small

regions

of the DNA would be detected

trophoretic properties

of ICP 5 produced by

only

if

they

bracketed a restriction enzyme

these recombinants. The map

position

of the

cleavage

site. The secondset, identified with the

template forICP 5 was thus determined to be letterA,denotespolypeptideswithan aberrant

between 0.23 and 0.30 map units. The DNA

electrophoretic

mobility.

Insomeinstances

(e.g.,

sequencearrangements andtheICP5specified ICP 5 of D1E4 and

DlE5),

the

electrophoretic

by all other recombinants are consistent with

mobility

was intermediate between that of the

thisconclusion. parental types. In other

instances,

the

poly-The segregation patterns of thepolypeptides peptidemigratedfaster than thecorresponding

mappedin thisstudy aresummarizedin Table parental

polypeptides.

Possibleexplanationsfor

2, which identifies two sets of inconsistencies. the emergence of

polypeptides

with aberrant

Thefirst, identified withanasterisk, comprises

electrophoretic mobility

are:

(i)

arecombination

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:E.

DiE

matically

in

Fig.

13A and C maynothave been

_ AlEl processedproperly.

__________________________ A From

Fig.

11and

12,

itbecomesapparentthat

a A somepolypeptides are located in cluster groups

_______AID

A7D with

overlapping

boundaries. These

polypep-A=E2

tides can,

however,

be ordered

by analysis

of

- A

^2D

their

segregation pattern

among the

recombi-- D~ nants. For

example,

ICP 10 must lie to the left

____________, ,,,of the ICP 7 and 25, since recombinant C6D

--

St specifies HSV-1 ICP 10 but HSV-2 ICP 7 and

- _____________ D3E2 25.Accordingly, a correlation of this segregation

(D1

pattern with the DNA map of C6DplacesICP

C2D

10totheleft ofICP 7 and 25. The linear order

_____,*L-< C3DI ofthe polypeptides deduced in this fashion

is,

301

**;from leftto

right,

9, 41, 18, 33(15, 39),40, 5, 24,

'3

Al[

36(35, 11),8, 43, 44, 32, 2, 10(7, 25),23, 20,27,

C(4

29,

4.

-LP--

9ZASD

The map positions of severalpolypeptidesare

__ ____ of

special

interest andaredealt with below.

ICP4.Thesegregation

patterns

ofICP4(Fig.

_

-~D3EI

2,3, 5, 6,8,9, 11, and 12)

indicate

that thegene

__ _____________ D4E3 forthat polypeptide is located to the right of

=~

D1E4

0.787 map unitasdefined

by

recombinants

AIE1,

DIES

AI63,

A7D,

and

DIEL.

Several observations

U64 made in this and otherstudies define the

loca-4D4E

tion of this

template

more

precisely.

Specificaly:

- f D4E2 (i) Recombinant DIE1 specifies in infected

(sD

cells

(Fig.

8 and

9)

the

fully processed

fonn of

,t_________t_.

_________ HSV-2

ICP

4

(ICP

4e)

andthe

underprocessed

7 7t(tD

forms

ofHSV-1 ICP 4

(ICP 4a).

The

only

de-==

OSEl

tectedHSV-1sequencesinDIE1arelocatedat

c

==:~

_0~ :--n%

D5E2

map positions 0.93 to 1.00, indicating that the

template

forHSV-1ICP4is in theS

component

0 12 0 is and,

by

extension,

that the

template

for HSV-2

FIG. 11. Provenance of the DNA sequences in the ICP4mustalso be in the S component. It would

intertypic recombinantsused in thisstudy.Theupper follow, therefore, that in DIE1 HSV-2 ICP 4

andlowerlinesof each doubletrepresent HSV-1 and maps between 0.83 and 0.93, whereas HSV-1

HSV-2DNA, respectively. Theheavy line identifies

ICP

4mapsbetween 0.93 and 1.0. In Fig.

12,

the the DNA sequences presentintherecombinant virus. * * H *

Thediagonallinespanstheboundariesofthecross- map

position

ofHSV-1 LOP4is

shown

as

deter-over sites asdefined bythefive restrictionendonucle-

mined

from analyses of recombinants DIEl1 ases.Hatch marks at or near thediagonallineiden- There is evidence,however, that HSV-1 DNA is tifycriticalendonuclease cleavage sites thatdefine

diploid

for theICP4gene and thatDIE1isone

the boundaries of the crossover event. The hatch

example

ofanICP4

heterodiploid

recombinant;

marks with aknob identify the restriction sitesre- this conclusion emerges from the observation

tainedinrecombinantDNA,whereasthose without thatrecombinants formed between intact

HSV-knobsrepresent parentalcleavagesites thatareab- 1 DNA and Xba-1 fragment I of HSV-2 DNA sent in therecombinant DNA. The boxes on themap also specify both HSV-1 and HSV-2

ICP

4. In

unitlinerepresent thereiteratedsequence ofLand theserecombinants the HSV-2 sequencesarein

Scomponents of HSV

DNA.teercmmnsteHV2sqecsaei

*

componentsofHSVDNA. mappositions0.945 to1.000;i.e., theyare

recip-rocal to those of the DIE1 recombinant (D.

eventmayhaveoccurred within a gene, resulting Knipe,W. Ruyechen, B.Roizman,and I.

Halli-in premature termination of transcription or burton,submitted forpublication).

translation(Fig.13B);(ii)arecombinationevent (ii) A possiblecomplicationthat might affect

mayhave occurred within ageneonly partially the mappositions of the genesspecifyingICP4

aligned,

resulting

in a recombinant gene speci- stemsfrom the observation thatalthoughviral

fying

a

polypeptide

withanapparentmolecular RNA extracted frompolyribosomes primedfor

weight

intermediate

betweenthat of the paren- synthesis ofapolypeptideshybridizesto

restric-tal types (Fig. 13C); or (iii) the product of a tion endonuclease fragments of the S

compo-recombinant gene such as

illustrated

diagram- nent, the amount of the reiterated sequences

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(17)

VOL. 26,1978 ANATOMY OF HSV DNA 405

Component . -l

s-

s->

Sequenceurnumgmuns.t US UL US C

Hybridintion

ofaRNA T

Origin

P~pelde

of

defetive DNA

4Il I|

mber

I

a

: . I

II

41

JJ3I

2

MFrIG.n funLcations of tepae spe TKnHS makes fucin,adplppidsdtrierma

analysisofHSV-I44V-2

royepresentedbin a'i RN wa to smll by a

I20

factr1

22

1

~

7

Fr(tionowever,_Fratioal

itoesumofthesequ

_estnce0

_0.1 _0.2 s

_0.30.4

i 44e_0.5 _0.6 19I_0.7 _0.8 _0.9 _1.0

FIG. 12. Locationof templatesspecifyingHSVmarkers,futnctions, and

polypeptides

determinedfr-oman

analysis ofHSV-1 xHSV-2 recombinants.

represented

in this RNA was too

small,

by

a A

factor of

2,

to contain the entireICP4

template

Iv-1

(19).

However,

thesum of thesequencesinthe N

reiterated and unique sequences that are

pre-sented inthe a RNA could contain the ICP 4 flV-2

template. Additional evidence derivesfrom

stud-ies (5, 6)on defective virus generatedby serial B

passage of HSV-1 (Justin). Cells infected with l

certain populations containing defective

parti-cles

overproduce

ICP4.The defectiveDNA was

showntoconsist ofareiteratedsegmentarising

from the ac and

Us

sequences of S located C

between map positions 0.939 and 1.000 in the

arrangement ofHSV DNA shown in Fig. 11(6).

Analyses of thesequences of the defective DNA L

that hybridize witha RNA (H. Locker andN.

Frenkel, in Proceedings ofthe Symposium on FIG. 13.

Hypothetical

models of recombinational

Herpesvirus and Oncogenesis, in press) also eventsgivingrise to inconsistent or aberrant

polypep-show that the amounts of reiterated ac se- tidesspecifiedbysomeofthe HSV-1 xHSV-2

recom-quences of the S component represented in a binants. (A) Intragenic recombinational event

be-RNA are not sufficient to specifyICP 4.

Thus,

tweengenes in whichonly the

5'

endsare aligned.(B)

although the evidence clearly indicates that

Intragenic

recombinational event

resulting

in a

pre-least partially diploid for ..OP.4 ge, mature termination oftranscription ortranslation.

HSaV

iSatleast

partially

diploid

for ICP4gene,

HSV s at .

(C)

Intragenic recombinational

eventbetween genes

we cannot at this time determine whzether the only partially aligned resulting in a recombinant

putative

remainder ofICP 4gene extends into genespecifyingpolypeptideswith an apparent

molec-theuniquesequencesof Sorinto the Lcompo- ularweightintermediatebetweenthose specifiedby

nent. HSV-1 andHSV-2parents. The heavy line represents

ICP

27.Because

A7D, A5C,

D5E1, D4E1,and the

corresponding

polypeptide template. The dashed

D4E2allspecifiedICP27ofHSV-1 and because linerepresents the DNA sequences in the

recombi-the right crossover site in A7D and A5C near nant.

theL-Sjunctionhad to be to theleftof 0.84 map

unitasdeterminedbyKpn Idigestion(26), the the

right-hand

end of the Lcomponent between

template specifyingthispolypeptide mustmap 0.787 and 0.83 map unit. In support of this

within the Lcomponent of the DNA. By similar location for the

template

specifying IOP

27 is

reasoning, analyses of recombinants AlEl, theobservationthatthe

parental

strainsusedto

A1G3,and A2Dsuggest that ICP 27 maps within construct the recombinants in series Awere a

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(18)

PAA' mutant of 17 tsJ (HSV-1) and wild-type pcU

HSV-2 (GP6) and that the selection was for Recombinant Boundaries 27 33.5

PAArts+progeny. From thoseexperiments, we L S

previously concluded that tsJ maps to the right A HSV-1 HoI EcoRI

ofmapposition0.78 (26). Marker rescue exper- 3 1.32

iments(Knipe, Ruyechan, and Roizman, unpub- SV;2.

lished data) showed that the

temperature-sen-sitive lesion of tsJ is located between mapposi- L2

tions0.70and0.83and therefore that the selec- B . 1.0

tion is consistent with a requirement for replace- -_

_________2

1.0

ment of HSV-1 sequences with HSV-2

se-quences.

Threecommentsconcerning ICP27shouldbe C 12

made. First, ICP 27 is an a polypeptide. It is.1 (0

thereforeof interest to note that the DNA se- - -s --- - .

quencesarising from the region containing the

template forICP 27 are represented in a RNA 0oy"7 o08

of

(Fig. 12). Second,ourdataplace ICP27within

theregions of the reiteratedsequenceab ofthe

map

units

Lcomponent. However, recombinants contain- FIG. 14. Possible location of crossover sites near

ingheterologous ab sequences (e.g., A4D,

A1G3,

thejunctionsof L and Scomponentsin theDNAsof

A2D, A1E2, and D5E2) do not produce both recombinants

AlEl,

A5C, and A7D. (A) The

bound-HSV-1 and HSV-2 ICP 27.Third,ICP 27 can be aries of the crossover sitesbased on restriction

en-used to better define the position ofcrossover donucleasemapsoftherecombinant

DNAs.

As

indi-catedin the text, andpreviously reported, the

recom-events in the DNA

ot

several

recombinants.

binational event occurredbetween the

Hpa

I cleavage

Thus, because of the absence of known restric- site inHSV-1 and the Kpn I site in HSV-2. (B) The

tion endonuclease cleavage sites between map probable location ofthe crossover site inrecombinant

positions0.78and 0.83 for HSV-1 and 0.76 and AlEl defined by the electrophoretic mobility of ICP

0.83 for HSV-2, the crossover events near the 27 andefficiency ofplating. (C) Theprobablelocation

junctionof the L andScomponents inthe DNA ofthe crossoversite inrecombinants A5C andA7D

of recombinants D5E1, A5C, A1G3, AlEl, and based onthe electrophoretic mobility of ICP27and

A7D are shown as occurring within a broad partialtemperature sensitivity of the recombinants.

regionsanningthejunction.Comp s of In(A)and(B),the

heavy

linerepresentsthetemplate

region

spannmng

the

junction. Comparisons

orfo IC 7 h ahdlnerpeet h N

the DNA sequence arrangements, polypeptides, for ICP 27.

The dashed line

represents the DNA

andbiological propertiesof recombinants shown sequencesin the recombinant.

in Fig. 14 suggest that in AlEl the crossover

event is nearer the map position 0.78 than in Mapping of specific viral functions. (i)

A5C and in A7D. The conclusionis based on the HSV-1

(HFEM)

yn- locus. The biological

observation that AlEl

specifies

the ICP 27 of properties of the intertypic recombinants were

HSV-2,whereasthe othersspecify the ICP27of

reported previously (26).

In that report it was

HSV-1 andare

partially

temperature sensitive. observed that

intertypic

recombinants (C3D2,

ICP6. Wesuspectthat the broad band des-

C4D, C5D, C6D,

andC7D) expressed the

syn-ignatedasICP6andICP7containsatleasttwo

plaque morphology phenotype

characterizedby

polypeptides. In earlier studies (29), it was fusion of cells intopolykaryocytes. Inasmuch as

shown thatasingle bandappears inthatregion series Crecombinants werederivedfromacross

during a

pulse

andthat onlya portion of that ofHSV-1

(HFEM

tsN102 syn-) x HSV-2 (186

band is then chased to a higher molecular syn+), those recombinants expressing the

syn-weight.Current data indicate that early ininfec-

phenotype

most

likely

contain HFEM DNA

se-tion, thepulse-labeled bandsimplybroadens to quences

specifying

thisfunction. Analysisof

re-a higher apparent molecular weight during the combinant DNAs shows that the template

re-chase (Fig. 1). WehavetentativelydefinedICP

sponsible

for thesyn-

phenotype

mustmap to

6 as that

polypeptide

which shifts to a higher the

right

oftheleftcrossoversite ofC4D (0.30)

apparent molecular weight and ICP 7 as that andtothe left of therightcrossoversite ofC5D

polypeptidewhich does not becomeappreciably (0.42).

alteredduringthe chase. Wehave not been able

PAAr.

PAA has been shown to

selectively

to map ICP 6 because theelectrophoretic mo- inhibit the HSV DNA

polymerase (23, 27).

Re-bility ofHSV-1 ICP 6 is not different enough sistancetoPAAwasused for selection of

recom-fromthat of HSV-2 ICP 6 to obtain a reliable binantprogeny in seriesA,B,and C.

Previously

segregationpattern. we

reported

that the PAA resistance marker

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mapsbetween0.43and0.52mapunit basedon be 44,000andbyCourtneyetal. (4) to be 43,000.

DNAsequence arrangementsofClD, C2D, and ICP 35 (molecular weight 42,000) mapped

be-C3D1. tween 0.27and0.35 map unit, whereas ICP36,

Shutoff of host protein synthesis. HSV-1 another candidate with a molecular weight of

and HSV-2shut off hostprotein synthesis within 47,000, mappedbetween 0.31 and 0.38 map unit.

a few hours after infection. Pereira et al. (29) Theseresults are also consistent with a recent

and Powell andCourtney(30) noted that HSV- reportthat theHpa I DNA fragment contained

2 shuts off host protein synthesis much more the geneticinformation necessary to

biochemi-rapidly and efficiently than HSV-1. We have cally transform tk- cells to the tk+ phenotype

used this differentialproperty to mapthe func- (41;S. Silverstein, personal communication).

tionresponsible for the accelerated inhibition of

hostprotein synthesis. DISCUSSION

Analysis of HSV-infected HEp-2 cells labeled In this paper we have (i) enumerated and

from 1 to 3 h postinfection (Fig. 2, 5, and 8) provisionally matched HSV-1 and HSV-2

poly-clearly demonstrated that five recombinants

peptides

with molecular weights of 20,000 to

(C2D,C3D1, DlEl, C5D, and C6D) rapidly and more than 200,000, (ii) identified polypeptides

efficiently shut off host protein synthesis in a that vary in apparent molecular weight from

mannercharacteristic of HSV-2. Correlation of strain to strain and those that undergo rapid

thisphenotype with the

physical

DNA mapof post-translationalprocessing,

resulting

in an

ap-the recombinants suggests that function in- preciable change in the apparent molecular

volvedinthe accelerated inhibitionofhostpro- weight, and (iii) mapped the approximate

loca-tein

synthesis

mustmapbetween0.52 and 0.59 tionof the

templates

specifying

26polypeptides

mapunit. Itmaybe

significant

that this

pheno-

and several viral functions. Several aspects of

typecorrelated with thepresenceof HSV-2ICP thepresentdata meritdiscussion.

7and27.Contrarytoexpectations, ClDdid not limitations of the

mapping

procedures.

exhibit this phenotype,

possibly

because the The HSV-1 and HSV-2 genes that were

ame-gene functionwas inactivated

by

the recombi- nabletomapping by the procedure used in this

nationeventin thatregion. Sincewe weremap- paper arethose thatdiffer in theapparent

mo-ping accelerated inhibition of host protein syn- lecularweight oftheirproduct. Thegenes whose

thesis, these observations should not be con- products do not differ in apparent molecular

struedtoindicate that the number and locations

weight

orinimmunological

specificity

will have

of all genes responsible for inhibition of host tobemapped by other

procedures.

functionsareknown. The

precision

of the

mapping procedure

used

Thymidine kinase. Several linesofevidence in this study is

governed

by two factors: the

suggestthatthe gene

specifying

the thymidine number and distribution ofcrossoversites and

kinasemapsbetween0.27and0.35mapunit. the numberofrestrictionenzyme cleavage sites

(i) HSV-1 x HSV-2 intertypic recombinants available topinpoint the exact location of

cross-were constructed from acrossbetween HSV-1 over sites. There were no crossover sites between

(B2006

tk-)

xHSV-2(GP6PAAr). Progeny were map positions 0 and 10, and only one

recombi-selected that were PAAr tk- by plating in the nant contained a crossover site in the S

compo-presenceof100 ,ugof PAA(17, 23) and 100

,ug

of nent suitable for mappingpurposes. In addition,

thymidine arabinoside (ara-T) (7) permlof me- the exact position of crossover sites was

deter-dium. The only recombinant obtained in this mined (26) by a total of 56 restriction enzyme

crosscontained HSV-2sequencesbetweenmap cleavage sites in HSV-1 DNA and 40 sites in

positions 0.38 and 0.44, suggesting that the tk HSV-2 DNA. In consequence, no polypeptides

gene maps totheleftof 0.38 map unit or tothe were mapped in mappositions 0 to 10, only one

right of0.44mapunit. polypeptide wasunambiguously shown to arise

(ii) Theparental HSV-1 mutant (tsE6) used from a template in the S component, and, as

in the construction of the Dseries of recombi- shown inFig. 11, the boundaries for some ofthe

nantsistk- (4). Wehaveanalyzed the abilityof polypeptide locationsare quite broad and may

recombinantsD1E4,

DiE5,

D5E1,and D5E2 to have to beredefined byothertechniques.

form plaques in the presence of 20

,ug

of ara-T Considering the

complexity

of the genome,

per ml of medium. Only D5E1 and D5E2 were the

correspondence

between the

segregation

abletoplaqueinthepresence of ara-T and were patternsof the DNA sequences and that of the

therefore tk-. This suggests that the tk- gene

polypeptides specified

by

the recombinantswas

maps between 0.27 and 0.35 map unit. quite

good.

The number ofinconsistencies was

(iii) The molecular weight of the

HSV-1-in-

small

(Table

2) and could be ascribedtoone or

ducedthymidinekinasepolypeptide in SDS-gels moreofseveral

possibilities

asdescribed earlier

was

determined

by Honess and Watson (16) to inthetext.

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Apparent gene arrangement in HSV double-strandedRNA. Wehaveinfactreported

DNA. Several aspects of the arrangement of that nuclear transcripts are many times larger

HSVgenetemplatesareofinterest.First, there than

polyribosomal

viral RNA

(18,

40) and that

appears to be a clearsegregation of templates avery

large

fraction of the DNA is transcribed

specifying a polypeptides. Thus both a tem-

symmetrically (17, 22).

platesmap atthe terminiof L andScomponents Presence ofgene

templates

inreiterated

atleastpartly within the reiterated regions. The sequencesof viral DNA. As indicated in

Re-position ofa templates coincides with themap sults, ICP27maps

entirely

within the ab

region

position ofsome aRNAsequences asreported of theDNA, whereas ICP4mapsatleast inpart

previously. Theproximity ofatemplatestothe in the reiteratedacsequencesof the S

compo-termini of theLandScomponents suggeststhat nent

(Fig. 12).

These observations

would

indi-the promoterfortranscriptionofthesefunctions catethatHSV isdiploid for ICP27andatleast

maybeatthe termini.Second, althoughno

fl

or partially diploid for ICP 4. However, whereas

y polypeptides were mapped in the S compo- ICP4heterodiploidywasreadily demonstrated

nent,thoseintheLcomponent appeared to be inrecombinants withheterotypic acsequences

intermixed. It is also interesting to note that (e.g.,DIE1), itwas notdemonstrable for ICP27

viral functionsmostlikely involving

,8

polypep- inrecombinants with

heterotypic

absequences

tides (e.g.,tk, PAAr, and accelerated shutoffof (AIG3, AIE2,A2D, A4D, and

D5E2).

Two

pos-hostmacromolecularsynthesis)wereintermixed sible

explanations

may accountfor this

obser-withyfunctions(syncytial plaque morphology). vation. First, it is conceivable that an ICP 27

These observations have several implications. template is located in the left end of the L

The apparent random distribution of tem- component,butthat it lacksapromotersite and

plates of

/8

andy

polypeptides

is ofspecial inter- is nottranscribed.Itshould be noted that if the

estin view ofthe evidence

indicating

that the promoterfor

transcription

ofICP 27werein

S,

sequential switch-on of the

coordinately

regu- asconsideredinthe

preceding

section,

thenboth

lated ,B and y

polypeptide

syntheses is deter- theright and left

templates

ofICP27would be

minedatthe

transcriptional

andpost-transcrip- transcribed off the P and IL

arrangements

of

tional levels (14, 15,32,34). Onepossible expla- HSV DNA, respectively, and both HSV-1 and

nation for the data is that

,B

and ytemplatesare HSV-2 ICP 27wouldbe made.The second

hy-segregatedonopposite strands.Toillustrate this pothesisis thatatleast

portions

of the terminal

hypothesis further, it could be that (i) thepro- inverted sequences must be identical but

in-moter for a template transcription is in the verted and that the ICP 27 gene lies entirely

reiterated sequences of the S component and within suchsequences, whereastheICP4gene

allows

transcription

from

right

toleftacrossthe does not. Thishypothesis arises from the notion,

function, (ii)

the promoter for

,8 polypeptides

in tobedetailed elsewhere

(manuscript

in

prepa-the L component istothe left ofmapposition ration), that the inversion of L and S

compo-0.78andallowstranscriptiontotheleft, and (iii) nents relative to each other arises as a

conse-atleastone promoter fory

templates

isto the quenceofan

obligatory

post-synthesis eventin

leftofmapposition0.12andpromotestranscrip- which the terminal ab and ac sequences are

tion tothe

right.

In support ofthis hypothesis repaired or regenerated, using the reiterated are the

following

data.

(i)

Trace amounts ofa baacjunction as a template. To evaluate this RNA

hybridize

tosequencesoccupied by

,8

and hypothesis, wereexamined the data indicating y

templates

(19). Consistentwiththis

finding,

in thattherecombinantsA1G3,

A1E2,

A2D, A4D,

manyexperimentsinwhichattempts weremade and D5E2 have heterotypic ab regions. The

torestrict viral

protein

synthesistoa

polypep-

conclusionsarebasedonanalyses ofthe DNAs

tides,

traceamountsofasubset

of/8

polypeptides

ofrecombinantviruses withthe restriction

en-designated as /3' (ICP 6, 8, 17, 29, 39, and 41) donuclease Hpa I which cleaves HSV-1 DNA

were also made (29; Fig. 2, 5, and 8).

Normal

within the ab sequences at mappositions0.037

synthesisof

/31

polypeptides, however, required and 0.787. The ICP 27 template was mapped

transcription in the presence of functional /3 betweenmap positions 0.787 and 0.83. Since we

polypeptides (29). One possible explanation is have norestriction enzymecleavagesites in the

that the trace amounts of/3' polypeptides and ab sequences between 0.000 and 0.037 and

be-corresponding RNA represent read-through tween 0.787 and 0.83, we cannot exclude the

from right to left throughtheputative

/3,

pro- possibility that these regions are in

fact

homo-moter.(ii)Thehypothesispredictsnuclear tran- typic, andconsequently onlyoneICP 27 would

scripts considerably greater inlength than the be expected.

polyribosomal mRNA as

well

asthe accumula- Contribution of the recombinants to the

tion oflargeamountsof RNA transcribed from study of the evolutionary divergence of

both strands andcapable ofannealingtoform HSV-1 and HSV-2. Numerous studies have

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shown the close genetic relationship between Proceedingsof the Symposium on Herpesvirusesand

HSV-1 and HSV-2, and it has been suggested Oncogenesis.I.A.R.C., Lyon.

that their current evolutionary divergence re- 4. Courtney,R. J., P. A. Schaffer, and K.Powell. 1976.

flectsacquisitionofaffinityfor differentecolog- Synthesisof virusspecific polypeptides by temperature-sensitivemutantsofherpes simplex type 1.

Virology

ical nichesas aconsequence ofdifferent modes 75:306-318.

oftransmission (31).Implicitinthis hypothesis 5. Frenkel,N., R. J.Jacob, R. W. Honess, G. S.

Hay-is the heurHay-isticconceptthat the viruscarriestwo ward, H. Locker,and B. Roizman. 1975.Anatomyof

offunctions: those that pertaintothe inter- herpessimplexvirus DNA.III.Characterizationof

de-setsottunctlons: t lose t zat pertam to t le mter- fective DNA molecules and biological properties of

action of viral macromolecules among them- virus populations containingthem. J. Virol. 16:153-167.

selves and those that involve interaction of viral 6. Frenkel, N., H.Locker,W.Batterson, G. Hayward,

and hostmacromolecules (31). Analyses of the and B. Roizman. 1976. Anatomy of herpes simplex

DNAsof therecombinantvirusesreportedpre- DNA. VI. Defective DNAoriginatesfrom the S

com-ponent. J. Virol.20:527-531.

viously (26) led to the conclusion that HSV-1 7. Gentry, G. A., and J. A. Aswell. 1975. Inhibition of

and HSV-2 DNAsare atleastgrossly colinear, herpes simplex virusreplication by Ara-T. Virology

i.e., that the arrangement of the genes was not 65:294-296.

alere

drat

the courseof the

.evolution

8. Gibson, W.,and B. Roizman. 1972.

Proteins

specified

altered

drastically

inthecourseoftheevolution- by herpes

simplex

virus. VIII. Characterization and

arydivergence. This study contributedanaddi- compositionof multiple capsid forms ofsubtypes1 and

tional conclusion based on the finding that at 2. J. Virol.10:1044-1052.

leastsomeof therecombinantsspecifyanearly 9. Grodzicker, T., J.Williams, P.Sharp,.and J.

Sam-equal

number of HSV-1 and HSV-2 polypep- brook. 1974. Physical mapping of

temperature-sensi-tive mutations ofadenoviruses. Cold Spring HArbor

tides,implyingthatHSV-1andHSV-2 polypep- Symp. Quant. Biol. 39:430-446.

tides arecapable of interacting. Althoughnot all 10.Hayward,G.S.,R.J.Jacob, S. C. Wadsworth, and

permutations have been observed, and therefore B. Roizman. 1975. Anatomy of herpes simplexvirus

wedo notknowwhetherprohibited, i.e.,lethal, DNA: evidence for fourpopulationsofmolecules that

wedonotknoweterproibte,.differin therelative orientationsoftheirlongandshort

genecombinations exist,the dataare adequate segments. Proc. Natl. Acad.Sci.U.S.A.72:4243-4247.

toconclude that the sitesonviralpolypeptides 11. Heine, J. W., R. W.Honess, E. Cassai,and B.

Roiz-specifically

definingtheinteractionof viralmac- man.1974. Proteins specified by herpessimplex virus.

romolecules_romoleculesamong_amongthemselves have beencon- XII. The virionpolypeptides of type1strains.J.Virol.

~~~~~~~~~14:640-651.

served. The conclusions are consistent with 12.

Hilliker,g;.,

and D. Botstein. 1976. Specificity of genetic

those ofHillikerand Botstein (12) fromstudies elements controlling regulation ofearly functions in

on bacteriophage lambda that evolution pro- temperate bacteriophages. J. Mol. Biol. 106:537-566.

ceededsuch that sequence

divergence

occurred

13-

Honess,R. W., and B.Roizman.1973.Proteinsspecified

by herpes simplexvirus.XI.Identificationandrelative

between recombinable segments. molar rates of synthesis of structural andnon-structural

herpesvirus polypeptides in infected cells. J. Virol.

ACKNOWLEDIGMENTS 12:1346-1365.

WegratefulyacknowledgethehelpofIanHalliburtonin 14. Honess, R.W.,and B. Roizman. 1974.Regulation of

settingupthenewconditions forpolyacrylamide gelelectro- herpesvirusmacromolecularsynthesis.I.Cascade

reg-shorettingsup.the

new conditionsfor polyacrylamide geleectro ulationofthe

synthesis

of threegroupsofviralproteins.

phoresis. J. Virol. 14:8-19. I

The studiesconductedattheUniversity of Chicago were 15. Honess,R.

W.,

and B.Roizman. 1975. Regulation of

aidedbygrantsfromthe National Cancer Institute, Public hersu macromolecula

tion-Health Service (CA 08494 and CA 19264), the American

herpesvirus

macromolecularsynthesis: sequential tran-CancerSociety (VC103L), and theNationalScience Foun- sition ofpolypeptide synthesis requires functional vi-dation(PCM 76-06254). The studiesatSidney FarberCancer ral polypeptides. Proc. Natl. Acad. Sci. U.S.A. Center,HarvArdMedicalSchool,wereaided by grants from 16.

72:276128.

theatinalCanernsttut, Pbli HelthSerice(CA

16-Honess,

R. W., and D. H. Watson. 1974. Absence of a the National Cancer Institute, Public Health Service (CA requirement forhost polypeptidesin the herpesvirus

10893and CA 20260). L.S.M. is apredoctoral trainee sup-

requinement

J.Gen.

poly

22

th7

er185

u

ported by Public Health Service training grant 5-T32 17. tymidine kinase. J. Gen. Virol. 22:171-185.

GM07183 from the National Institute of General Medical 1-Jacquemont,B., and B. Roizman. 1975.RNAsynthesis

Sciences.L.P.isapostdoctoral trainee supportedbyPublic in cells infected with herpes simplex virus. X. Properties

Sciences.Servis a grantrAInee frte bytional of viral symmetric transcripts and ofdouble-stranded

Health Service training grant AI-00184 from the National RNApreparedfromthem.J. Virol.15:707-713. Institute ofAllergyand Infectious Diseases. 18.Jacquemont, B., and B. Roizman. 1975. Ribonucleic

LITERATURE CITED acid synthesis in cells infected with herpes simplex virus: characterization of viralhigh molecular weight 1. Cassai,E., R. Manservigi, A. Corallin,and M. Terni. nuclear RNA. J.Gen. Virol. 29:155-165. 1

1976. Plaque dissociation ofherpes simplex viruses: 19. Jones, P. C., G. S. Hayward, and B. Roizman. 1977. biochemical andbiological charactersof the viral var- Anatomy of herpes simplex virus DNA.VII. a RNA is

iants.Intervirology6:212-223. homologous tononcontinuous sites in both the L and S 2. Cassai,E.N., M.Sarmiento,and P. G. Spear. 1975. components of viral DNA. J. Virol.21:268-276.

Comparison of the virion proteins specified by HSV 20. Kit, S., and D. Dubbs. 1963. A non-functional thymidine types1and2.J.Virol.16:1327-1331. kinase cistron inbromodeoxyuridine resistant strains of 3. Courtney,R.J.,and K. L.Powell.1974.Immunological herpes simplex virus. Biochem. Biophys. Res. Commun.

and biochemical characterization ofpolypeptides in- 13:500-504.

ducedbyherpes simplexvirus types 1 and 2, p. 63-73. 21. Kieff, E. D.,S. L. Bachenheimer, and B.Roizman. InG. deThe,M. A.Epstein, and H. Zur Hausen (ed.), 1971.Size,composition, and structure of the DNA of