Recombinants between herpes simplex virus types 1 and 2: analyses of genome structures and expression of immediate early polypeptides.

(1)

Vol. 28, No. 2 JOURNALOFVIROLOGY, Nov.1978, p.499-517

0022-538X/78/0028-0499$02.00/0

Recombinants Between

Herpes

Simplex

Virus Types 1 and 2:

Analyses of Genome Structures and Expression of Immediate

Early Polypeptides

V. G. PRESTON,* A. J. DAVISON, H. S. MARSDEN, M. C. TIMBURY, J. H. SUBAK-SHARPE, AND N. M. WILKIE

Medical Research Council Virology Unit, University of Glasgow, Glasgow Gll5JR, Scotland Received for publication 17 April 1978

Recombinants between temperature-sensitivemutantsofherpessimplexvirus

types 1(HSV-1) and2(HSV-2)wereconstructed.Using restrictionendonucleases,

weanalyzed thegenomecomposition of17intertypicrecombinants and detected

crossovers in everyregion of the genome. Thevirion DNA ofonerecombinant

appearedtobelargely "frozen" intwoof the four possiblegenomearrangements of HSV. Knowledge of the genome structures ofrecombinants enabled us to

physically map immediate early polypeptides. We present evidence that the

immediateearly polypeptideVmw IE 110of HSV-1and itsfunctionally equivalent polypeptide,Vmw IE118,of HSV-2may mapinthe repetitive sequences bounding

thelong unique region of HSV.

The genomes of bothherpes simplex viruses

type 1(HSV-1)andtype 2(HSV-2) containtwo

unique (U)sequences,ofapproximately9 x 106

daltons (theUssequence) and 68 x 106daltons

(the UL sequence), which are bounded by

ter-minal and internal invertedrepetitions (TR and IR, respectively) ofapproximately 4 x 106

dal-tons (TRs and IRs) and 6 x 106 daltons (TRL

andIRL), respectively (4, 25, 31).Themannerin

which these sequencesarearranged onthe ge-nomeisillustratedatthe bottom ofFig.3.Asa

consequence of this unusual structure, the

unique regions can invert with respect to one another, and fourmajor genome arrangements

have been identified in virion DNA(4, 6, 11,25, 26).

HSV-1 and HSV-2 share homologyin about 50% of their DNA sequences (15), and both complementation and recombination between

the two serotypes have been reported (7, 29).

Studies on virus-induced polypeptides and an-tigens confirm that true recombinants result fromgeneticcrossesbetween HSV-1 and

HSV-2 (10). The restriction endonuclease maps of HSV-1 and HSV-2 DNAs (4,5a)arevery

differ-ent, and this property allows the location of HSV-1 and HSV-2 DNA sequences in the

ge-nomesofintertypic recombinants (19, 33).

Analysis by sodium dodecyl

sulfate-polyacryl-amidegelelectrophoresis(SDS-PAGE)resolves

about50virus-inducedpolypeptides incells

in-fectedwithHSV-1 orHSV-2 (12, 17,20). Most

HSV-1-induced polypeptides can be

distin-guished from HSV-2-induced polypeptides

solely on thebasis of differences inmobilityin SDS-PAGE (21). By analysis of polypeptides inducedby recombinants of knowngenome

com-positions, it ispossibletodetermine thephysical locations of the genesfor several polypeptides.

Thestrategyisessentiallythesame asthatused

to determine thephysical map coordinates for adenovirustemperature-sensitive (ts) mutations andpolypeptides (9, 18, 24,34).

Inthiscommunication,thegenome structures

of17recombinants between HSV-1 and HSV-2, including1whichappears tobelargely"frozen" intwooutof thefourpossiblegenome

arrange-mets, are analyzed. Knowledge of the genome

structureshasenabledus todetermine themap

coordinates for polypeptides belonging to the immediateearly (IE) class (22) of HSV-induced polypeptides.SincesomeIE RNAis encodedby

therepeatregionsof HSV-1 (5),wehave

exam-ined IE polypeptides induced by recombinants containingoneHSV-1 L repeatandoneHSV-2

L repeat to elucidate the expression of genes

within theserepetitivesequences.

MATERUILS AND METHODS

Cells. BHK-21 clone 13(C13) cells(16)wereused

in allexperiments except in the isolation of certain

HSV-1/HSV-2 recombinants, in which Vero cellswere

used. BHK-21 (C13) cells were cultivated in Eagle

medium containing twice the concentration of

vita-mins and amino acids, 10% (vol/vol) tryptose phos-phatebroth, and10% (vol/vol)calfserum.Verocells

weregrownin this mediumexceptthat 10% (vol/vol)

fetal calfserum (Gibco-Biocult) wasused instead of calfserum.

499

on November 10, 2019 by guest

http://jvi.asm.org/

(2)

500 PRESTON ET AL.

Virus. Two HSV-1ts mutantsfrom strain17

(Glas-gow),tsB syn+(nonsyncytial plaque morphology) and

tsD syn (syncytial plaque morphology) (3),andone

HSV-2tsmutant,ts1,from strainHg52(28),together

with the parental wild-type viruses,wereused. Virus stocks. All virus stocksweregrownin

BHK-21 (C13) cellsasdescribedpreviously (3).

Virus titration. Virus titrationwasperformedas

describedbyMarsden etal. (17). Infected cellswere

incubatedat31°C, the permissive temperature, for3

daysor at38.5°C, the nonpermissive temperature, for

2days.

Isolation of recombinants. Recombinants 2Sa,

15 Sa, 34 Nsd, and 34 sd were isolated in BHK-21 (C13) cells from thegeneticcross(ts D synxts1)as

described byTimbury andSubak-Sharpe(29). In this and subsequent papers these recombinants will be referred to as Dx1(2), DX1(15), Dx1(34-1), and Dx1(34-2), respectively.Similarly, recombinants 2444, 2641, 2853, 3134, 3145, and R13, isolated from the

geneticcross (tsB syn+ x tsl),willnowbeknownas

Bx1(24), Bx1(26), Bx1(28), Bx1(31-1), Bx1(31-2), and Bx1(13),respectively. Note that inWilkieetal. (33) theearliernomenclature has been used.

Recombinants Bx1(24), Bx1(26), Bx1(28),

Bx1(31-1),andBx1(31-2)wereisolated in Vero cells

as described in Fig. 1. Recombinants Dx1(31),

Dx1(32), Dx1(43), Dx1(48), Dx1(51), Dxl(53),

Dx1(57), and Bx1(13)wereisolated in BHK-21(C13) cellsessentiallyasdescribedforBx1(24).

SDS-PAGE.Sampleswereanalyzed by

discontin-uous SDS-PAGE. Two types of gels were used: a

gradient gel (5.5% to 12.5% polyacrylamide with a

stacking gel of 5% polyacrylamide) as described by

Marsden et al. (17) and a single-concentration gel

(7.5%polyacrylamidewithastacking gelof4.5%).The

ratio ofN,N'-methylenebisacrylamide to acrylamide

in thelatter type ofgelwas1:40 (wt/wt). The same

buffer (0.05 M Tris,0.055 Mglycine, 0.1%SDS) was

used in both the upper and the lower buffer tanks. Afterelectrophoresis, thegelswerefixed and stained

asdescribedpreviously(17).

Autoradiography. The gelswere dried, and

au-toradiographsweremade with Kodak KodirexX-ray filmat roomtemperature.

Virus-induced IEpolypeptides. A temperature

of38.5°Cwasusedthroughoutvirus induction of IE

polypeptides. Cells in30-mmpetri disheswere prein-cubatedfor 1.5h before virus infection inEagle

me-diumcontaining200pgofcycloheximideperml. Cells

were infected at amultiplicityof infection of about 20

PFU percell, washed, and incubated forafurther5h, all in the same medium. Actinomycin D was then addedto afinal concentration of2.5jug/ml.After15

min, this cycloheximide-containing medium was re-moved, and plates were washed twice with

methio-nine-reducedEagle medium containing 2% calfserum and actinomycin D (2.5pg/ml). [35S]methioninewas thenaddedat aconcentrationof 100lCi/ml,andthe cellswereincubated forafurther2h.Control cultures

were treated in thesame mannerexceptthat cyclo-heximidewasomitted.

Samples were harvested as follows. The medium

was removed from the petri dishes, and denaturing

buffer (final sample buffer [17]) (0.35 ml) wasadded

Recombinotion Test tsBsyn x ts I

I~

Tncubate infected vero cells at 31 C for 24 hours

1

Harvest cells Titrate virus in vero cells at 310Cand 38.500

1

Pick plaques at

38.50C

Titrate virus in vero cells at:

3100 38.500

Pick plaquesl

1

Pick

plaques

Four successive single plaque purifications at 310C in

vero cells

Recom binants Bx1(24)

Bxli28) Bxl(31-1)

Recombinants Bxl (26)

Bx1 (31-2)

FIG. 1. Isolation ofHSV-1/HSV-2 recombinants from thegeneticcross(ts Bsyn+xts1).

to each dish. Thedishes wereheated to 70°C for 15

min.Thecontents werethentransferred to vials and furtherheatedfor2 min at 100°Cto reduce the vis-cosity of the samples. The amount of radioactivity incorporatedintotrichloroaceticacid-precipitable ma-terialwasdeterminedsothatsamples with the same

amountofradioactivity couldbeloadedontogels.

Labeling and isolation of viralDNA. Samples

of 107 BHK cells in 10 ml ofphosphate-free Eagle J. VIROL.

http://jvi.asm.org/

[image:2.499.286.441.55.539.2]

(3)

VOL. 28, 1978

mediumsupplemented with 2% calfserum wereseeded

onto 90-mm petri dishes and left for 24 h at 370C. Thesecells werethen infectedat31°Catamultiplicity

ofinfection of about 5. After 1 hof absorption they

werewashed andincubatedat 31°C,all inthesame

medium. Two hours postabsorption, carrier-free 32p;

(Amersham)wasaddedtoafinal concentration of100

,uCi/ml, and thecells wereincubated forafurther 24

hat31°C. The culture mediumwasremoved,and

cell-released viruswaspelletedat25,000rpminaBeckman

Ti 50rotorfor1h. The DNAwasextractedfrom the

virionsaspreviouslydescribed by Wilkie (30).

Alter-natively,virions wereextracted frominfected-cell

cy-toplasm as described by Wilkie (30) and combined

with tissue culture medium containing cell-released virus. Viruswaspelleted and viral DNAwasextracted

in the same way asdescribed for cell-released virus.

Phagelambda DNA. Lambda DNAwasobtained

by phenol-SDS extraction ofCsCl-banded virus from

a heat-inducible lysogenic host strain, Escherichia

coli 805(CI 85757).

Restrictionendonuclease analysis. Restriction endonucleasesEcoRI, HindIII,Bgl II, Xba I, Hpa I, andKpnIwerepurifiedessentiallyasdescribed

pre-viously (4, 33; R. N.Yoshimori, Ph.D. thesis, Univer-sity ofCalifornia, San Francisco, 1971). One unit of

enzymeis definedasthatquantity ofenzyme neces-sary to produce a limit digest with 1 ,ug of lambda

DNA after incubation for 3 hat370C inavolume of

50

pl

of0.01 MTris-hydrochloride (pH 7.4),0.006 M B-mercaptoethanol,0.006MMgCl2,and0.1%(wt/vol) bovineserumalbumin.

Labeled HSV DNA samples containing less than

0.01 ,ugof viral DNAwere digested under the same

reaction conditions with1,ugoflambda DNA and1to 2 U of enzyme. Reactions were terminated by the

addition of 0.1 volume of 25% Ficoll-0.1 M EDTA containing bromophenol blue. DNAfragments were

separated by electrophoresis onslab gels of 0.3, 0.5,

0.75,or1.0%agaroseaspreviouslydescribed(32).The

gelsweredried, and autoradiographsweremade with

Kodirex KD54Tfilm atroomtemperature.

DNA-DNA hybridization.Unlabeled HSV DNA

was prepared essentially as described before (30).

HSV-1 and HSV-2 DNAs, further purified by rate

zonal centrifugation (32), were nick translated with

32P-labeled deoxyribonucleosidetriphosphates (Radi-ochemical Centre) by the technique described by Rigbyetal.(23). UnlabeledHSV-1 and HSV-2 DNAs

were also cleaved with Hpa I and Xba I, and the

fragmentswere purified byhydroxylapatite

chroma-tography as describedpreviously (31).Isolated

frag-mentswerealsonick translated. Thenick-translated

DNAshad specific radioactivities intherangeof4x

107to2x 108cpm/Ag.

Unlabeled DNA from Bx1(28) was cleaved with

Kpn I plusXbaI,and thefragmentswereseparated

by electrophoresis on slabgels of0.5%agarose (31).

DNAfragmentsweredenatured with alkali in situ and

transferredtosheets of nitrocellulosebythetechnique ofSouthern(27). After alkalidenaturation,

nick-trans-lated[32P]DNAwashybridizedto

DNA-fragment-con-tainingstripsfromthe nitrocellulose membrane sheets

asreportedpreviously (31). Afterwashingthe

nitro-cellulose membrane strips in 2x SSC (0.30M NaCl

HSV INTERTYPIC RECOMBINANTS 501 plus 0.03 Msodium citrate) at 55°C, DNA fragments hybridized to the32P-labeledprobewerevisualizedby

autoradiography.

RESULTS

Biological properties of recombinants. Most of therecombinants grew as well as

wild-type HSV-1 andHSV-2 viruses, but therewere some which grew poorly atthe nonpermissive temperature,withvariable and lower efficiencies of plating at 38.5°C/31°C. Table 1 shows effi-ciencies of plating at 38.5°C/31°C for the re-combinants and the controls. Recombinants Bxl(31-1) and Bxl(31-2), which are clonally related,weretsinBHK-21 (C13) cells andwere

never observed to form plaques at 38.5°C. In Vero cells, however, in which they were origi-nallyisolated, they generally formed plaques at 38.5°C and had muchlower and more variable efficiencies of plating at 38.5°/31°C than did wild-type HSV-1 and HSV-2.

Analysis of crossoverpointsin

recombi-nants. To analyze the crossover points in the

DNAof therecombinants, the physical maps for HSV-1 and HSV-2 DNA have to be aligned. Results from the hybridization of separated HSV-1 [32P]DNAfragmentstounlabeled sepa-rated HSV-2 fragments and vice versa by the

TABLE 1. Relativeefficienciesofplating(EOPs) of recombinant viruses(38.50C/31°C)

Relative EOP Temperature Virus (38.50C/310C) sensitivity'

Dxl(2)b 1.5X 10-2 Reduced

DX1(15)b 7.3x 10-3 Reduced

DX1(34_1)b 4.4 Wildtype

DX1(34-2)b 6x 10-' Wild type

Bxl(13) 1.2 Wildtype

Bxl(24) 1.3x 10-' Reduced

Bx1(26) 2.3x10-2 Reduced

Bxl(28) 9.5x 10-' Wildtype

BXl(31-1) <1.9Xlo--5 ts

BXl(31-2) <3.4X 10-6 ts

DX1(31) 1.9 Wildtype

Dxl(32) 1.5 Wildtype

Dxl(43) 1.4 Wildtype

DX1(48) 1.6 Wild type

DX1(51) 6.9x 10-1 Wildtype

Dxl(53) 2.1 Wild type

Dx1(57) 4.4 Wildtype

Hg52 5.8x 10-1 Wildtype

Strain17(syn+) 7.3x10-1 Wildtype

Strain17(syn) 1.1 Wildtype

ts 1 <1X10-6 ts

ts D (syn) <2x 10-6 ts

tsB (syn+) <5x 10-6 ts

aWildtype, EOP>2 x

10-1;

ts, EOP< 1 x10-4;

reduced,EOP<2x 10-1and>1 x10-4.

bSee reference29.

http://jvi.asm.org/

[image:3.499.252.443.395.644.2]

(4)

502 PRESTON ET AL.

Southern blottechnique (27) (N. M. Wilkie and R.Cortini,unpublishedobservations)showthat HSV-1fragmentshybridizetoHSV-2fragments with corresponding map positions on the

ge-nome,indicating thatsequencehomologyis

dis-tributed according tomap location throughout both the L and the S regions of the genome.

Thisinforination allowsalignment of the

HSV-1 and HSV-2 physicalmaps asshown in Fig.3

for sixrestriction endonucleases. Itcanbeseen from Fig. 3 that the restriction endonuclease

mapsof HSV-1 and HSV-2 differradicallyfrom each other.

The analyses of the DNAs from two

recom-binants, Dx1(53) andBx1(28),aregivenin de-tail toillustrate how the locations ofcrossover sites were determined. Figure 2 shows the re-striction endonucleasepatternsof DNAs fromts D,ts1,andrecombinant Dx1(53) isolated from (ts D x ts 1). Analysis depended on observing which DNA fragments from the recombinant DNA comigratedwith fragments ofone orthe other of theparentaltypes.Whenarecombinant fragment comigratedwith aparentalfragment, itwasassumed that the restriction sites which delimit the parental fragment were present in this recombinant DNA. In some cases novel fragments, which corresponded toneither

par-ent, were observed. Such fragments consisted

partly of HSV-1 and partly of HSV-2 DNA

sequences and hence contained the crossover

sites.Thus,acombinationmapofparental-type restriction endonuclease sites could be

con-structedfor the recombinant genomewhich al-lowedanestimate of thecrossoverpoints in the recombinant DNA.

Analysis ofDx1(53)DNA.HpaIfragments ofDx1(53) DNAcomigratedwith allthe

HSV-2fragments excepte. Fragments corresponding

to HSV-1 eandqwere alsopresent.The band inthe recombinantprofile correspondingtothe

1 M band q of HSV-1 was, however, 2 M in relativeamount,suggestingthat therewere two

fragmentsof this size in theHpaIdigestof the recombinant genome. The additional fragment

the size ofq can be accounted forbya crossover

eventwithin HSV-1 sandHSV-2 e.Therewas

also another novel fragment, which had a

slightlygreatermolecularweightthan HSV-1m.

Thisnovelfragmentcanbeexplained bya

cross-over eventwithin HSV-1fand HSV-2 e (refer

tophysicalmaps[Fig. 3]).

The region of the recombinant genome in

whichtheparentalnatureoftheDNA was

un-certain isrepresentedinFig.3bycrosshatched

lines. The sites which delimit this region give

the map coordinates between whicha crossover

has occurred. Thus, from the Hpa I analysis,

DX1(53) consisted ofHSV-2 DNAwith an

in-sertion of HSV-1 DNA with maximum map

coordinates of0.56 to 0.75 and minimum map

coordinates of0.60 to0.72.

The results of all the other enzyme digests

wereinagreementwith thisanalysis and further

defined the estimates of the map coordinates for

the crossovers. By combining all theresults, it

could be concluded that Dx1(53) contained an

insertion of HSV-1 DNA with maximum map

coordinates from 0.58to 0.74and minimummap

coordinates from 0.60 to 0.72.As Dx1(53) was

wildtypeingrowth properties,weassumed that

thetslesionts1hadbeenreplaced by wild-type information from HSV-1 DNA, and hence that

ts 1lay within themapcoordinates0.58 to 0.74.

Analysisof the DNA of Bx1(28).The DNA restriction endonuclease fragment patterns of Bx1(28)weremuchmorecomplex than those of Dx1(53). Figure 4 shows autoradiograms of Bx1(28) [32P]DNAdigested with EcoRI, BglII,

HpaI,HindIIIplusHpa I, and KpnIplus Xba I.Individual fragmentswereidentified bya sim-ilar analysistothatgiven for Dx1(53). It could be deduced that Bx1(28) contained HSV-2

se-quencesfrommapcoordinates0tobetween0.49

and0.52,where therewas acrossover toHSV-1

sequences;theHSV-1sequencescontinuedto a

point in IRs (or

TRs)

andthen crossed backto

HSV-2 sequences. Most of the S region

con-tained HSV-2 DNA. Inaddition, the KpnI/Xba Idouble-digestdata strongly suggested that the population of DNAwasheterogeneous inthe S

region, and this will be considered inmoredetail

below. Figure 5 summarizes the restriction

en-donuclease analysis of the crossover points in

Bx1(28).

Previous studies (4, 11, 26) have shown that,

as a consequence of inversions of UL and Us,

restriction enzymes which cleave HSV DNA

only in Us and UL produce four terninal

frag-ments, with relative molarities of0.5, and four

fragments which span the "joint" region, with

relative molarities of0.25. Figure 4 showsthat

the terminalfragmentHSV-2EcoRI-f appeared

tobe overrepresentedin the DNA ofBx1(28). Conversely, thejoint-spanning fragments

HSV-2 EcoRI-b and -d, which contained EcoRI-f, appeared tobe underrepresentedin the digest.

In the Bgl II digest, joint-spanning fragments

HSV-2aand b werealsounderrepresented,and

these also contained the HSV-2 EcoRI-f

se-quences. The HSV-1Bgl II-ffragment,from the

opposite terminus toHSV-1 EcoRI-f, was also clearly underrepresented.Noinformation could

be obtainedonthe HSV-2 terminalBglII

frag-mentd,asthisfragment comigratedwith

HSV-2 fragments c and e. The submolar fragments J. VIROL.

http://jvi.asm.org/

(5)

XbaI

Co x

ch

x

I -A

)

I OM

S (I)

IC

N)

Hind

S

I -&

-S CA IC

Eco

RI

I O

< -A

(A)

S

U)

IC

N)

ab.

cw.

de.

a.#o_O abc

b_ od

C .

Owe

.1

de .

f. = :.JO

st_ o9

9.

~ ~ O

m.i °h

9i

a

Oi

k.°

j

k

I.9

*Al

°

Me qb -A low& OM

Qi

_

.-_

°

i

o

ofn

no.

os .

.0

nfl

0s0a

[image:5.499.50.440.46.636.2]

oO

FIG. 2. Autoradiograms of [3PJDNAsof recombinant Dxl(53)and parental virusests1(HSV-2)andtsD

(HSV-1).TheDNAsamplesweredigested with restrictionendonucleases Xba I,HindIII,EcoRI,BglII, Hpa

I, andKpnL Theletters refertospecific HSV-1 orHSV-2 DNAfragments, thephysicalmap locationsof

which aregiven in Fig. 3. Symbols: U, HSV-1 DNA fragment; 0, HSV-2 DNA fragment, *, novel DNA

fragmentproduceduponrestrictionendonucleasedigestion of the recombinant DNA. 503

%*w

bc

ab. _ d

_t~ e

defga

h.

ia

%wo

_°m

on

k.

d

I .%%W

69.

.;...

http://jvi.asm.org/

(6)

504 PRESTON ET AL.

BgI

II

2: O h

cI

<

J. VIROL.

Hpa

I

0 x

Kpn

I

:c =:

I wwl I

C-& %& Ct

oa 0e

cdo

f40~~0

~:f l_

opq .t

S.~~~~~~~~~~~~._

a

to

cq

V.

or

bcd

abcd

f

efg

*

.h:

!

a

k a 00

me

no

.

401

P m

-q _

r.

t a

u.

np.. oh

WX*

yZ a

a'.

b

c."

abc

Q_o _od

o ef

ltb

0h

O j

f

o

0k

Im

Io.

.'#

*vo

nop

O q

0 o rs

[image:6.499.69.459.58.646.2]

ot

FIG. 2-Continued.

a a

bc

: d

*

I

e a

f

*ai

gh

:

i

4 i

a.

k. I

I.

m.a

nfl

0.

P

"W

;.

Go

ab ocdE of

g

o l.

o J

ok

oI om

on

- 00

°o ....liE 0

I.

b

http://jvi.asm.org/

(7)

HSV INTERTYPIC RECOMBINANTS 505 Dxl

(53

)

9

Xba I

Eco RI

Hind III

Bgl H I

d

Hpa I

c f e d IS)

D ~

~~~~~~~~

|

c d g h j

j d g n f mo a

r

e k h k

11 11 I

11 11

f i g a p I h k n om

io h j a k II d im n g

II I

K\NWl

==:El

b n h e

k o pn m d

I I III

l l XXN

I11

rp g j 0

m o I p ku j n t b

I I 11

g a h

C

h

I

d r b y f qw lmtn pva'x c (

Kp 11UI1 11111

KpnfI

lI

f klIqnh op j mt g c

Summary: TRL

11

o J n I I k

g f h I

I. I1

n h im q I k

f e qsvrm, c g

11111

II e

I~f

If

ISl

S

cs d zu o g r: j h k

I I 11 I

.4JlZ

II I I

s c ,r a r

'joint"

4. S -IRL IR Us TRS

I -i

.L

z1.

[image:7.499.101.387.61.409.2]

O 0.1 0.2 03 0.4 0.5 0.6 0.7 0.8 0.9 1.0 fractional genome length

FIG. 3. Analysisofthe crossoverpointsinrecombinantDxl(53), usingsix restrictionendonucleases.

HSV-1physicalmaps have beenalignedwith thoseofHSV-2. Only oneorientation of ULand Usisshown, the

orientationof UL beingthat whichpredominatesinBx1(28)virionDNA. The HSV-1physicalmapsareshown

above, whereas thoseoftheHSV-2areshown below, the genomestructureofthe recombinant. TheHSV-1

HindIII,XbaI,EcoRI, Bgl II,andHpa Imapsaretakenfrom publisheddata(5,26, 31),andthecorresponding

mapsofHSV-2arefrom Cortini and Wilkie (5a). Kpn ImapsofHSV-1 and HSV-2are takenfromthe

unpublisheddataofA.J. Davison andfromMorseetal.(19).The solidregionsofthegenomereferto

HSV-1DNA sequences, and the openregionsrefertoHSV-2 DNA sequences. The crosshatchedregionsindicate DNA sequences in whicha crossoverhasoccurred. Thearrowabove the HSV-1 cleavagesite in theHSV-1 HindIIIphysicalmapreferstothesingleHSV-1HindIIIcleavagesitepresent in the recombinant DNA.

from the S region were present in normal

amounts. These observations suggest that Us

inverted normallyinBxl(28),but the Lregion

appearedtobelargelyfrozen inoneorientation,

the same asthatdepictedin Fig.5. TheHpaI

fragmentpattern ofBxl(28)showed that

HSV-1HpaI-rwaspresent.Thus,theHpaIsite which

delimits Hpa I-mmust also have beenpresent

(Fig. 5). However, HSV-1Hpa I-mwasmissing

from thedigest, and this HSV-1site must

there-fore have remained largely within the IRL

se-quence, in keepingwith observations fromBgl

II andEcoRIdigests. Figure5 shows that inthe

HindIII/Hpa

I double digest the S region of Bxl(28) wascleaved in two places,as wasthe HSV-2 S region, unlike the HSV-1 S region, which was cleaved in three places. If Bxl(28) contained theHpaIcleavagesite whichdelimits HSV-1 Hpa I-m (fragment 12 in the

HindIII/Hpa I digest of Fig. 5), then the

HindIII/Hpa Idigest ofBxl(28) DNA should have generated two submolarfragments, of10

x 106and 8.7x

106

daltons(HSV-1 fragment12

plus HSV-2fragment8 [12,+82] and12, + 132 in Fig.5). Figure4 shows thepresence of these two fragments, marked with asterisks.

More-,.

_

VOL. 28,1978

_;v

I on November 10, 2019 by guest

http://jvi.asm.org/

(8)

506 PRESTON ET AL.

Eco

RI

I w -A >c

_ I1,

N)

_0

_ a

bc

ab. 4J6P ID V ."

. t

h

° -.m

a.

bc:

e

0

tI%*_.

a

b

cde f .9 h

.;O _A

1*X O'AaK k

o m

., n

n. _

0

....

...

k. ___

... 0 ..- n

p

a.

b-cde.

f.

,;Stiabcd

we

_

la .00

Om '%00

gh. a11 ;.UIf

n. _e

k

_i

Mn.

_Wf

op q

F. m.

.0.,

n.

oh

t.

U

V.

FIG. 4. Autoradiograms of [32P]DNAs ofrecombinantBxl(28)andparentalviruses ts1 (HSV-2)andts B

(HSV-1). The DNAsamplesweredigestedwith restriction endonucleasesEcoRI,BglII,HpaI,HindIIIplus

Hpa I, andKpnIplusXbaI. Theletters inthesingle digestsorthe numbers in the doubledigests referto

specificHSV-1 orHSV-2 DNAfragments,thephysicalmaplocationsofwhich aregiveninFig.5.Symbols:

U, HSV-1 DNA fragment; 0, HSV-2 DNA fragment; *, novel DNA fragmentproduced upon restriction

endonucleasedigestion ofthe recombinantDNA.

J. VIROL.

Bgl

11

I a, I

-r x cn

c: _l c

Hpa I

I W I

ozx n

_ m m

0

-Y i4jAW.

http://jvi.asm.org/

[image:8.499.69.456.75.593.2]

(9)

HSV INTERTYPIC RECOMBINANTS 507

Kpn

I/Xba I

I W X

(n X u)

'C <h

_

- CO N)

*

* _ 1

$ 2.3

04.5

1.

2334. 6

91 o.

12 _ 13.14

7

13. olJ_1

151617 I

19.

20.21:

22.

23*

24.

25. 26,27.

2829.

30a

C17

n18 19

_

o20

I 21

o22

023

o24

I CC

U) X <

-_Lr

1. 2

-3.

4.5 1

6,7:

8 a

9,10:

111

12*

I N

01

02

°3,4,5,6.7

10,101,12

013

1c.14

_°b O15

13 __a

14

of

15* 16.17.18:

19,201

-. 21,22 :

23

24. w0o. 16

JA o* - o17

25 *a U 4* o18

[image:9.499.106.396.71.554.2]

o 19

FIG. 4-Continued.

over,they comigratedwithtwofragments

pres-entinHindIII/HpaIdigestsoftwoother

recom-binants, Dxl(43) and Dxl(51), knowntohave

theHSV-1 HpaI-mcleavagesiteandan

HSV-2 Sregion (data notshown, but genome struc-ture isdepictedinFig. 10).Takentogether,the data were consistent with the conclusion that

the virion DNA ofBx1(28)wasoverwhelmingly

in oneorientation ofUL,whereasboth

orienta-tions of the Us region were present in equal

amounts.

Identification of submolar fragments in

Kpn

I/Hpa

I doubledigestsofBx1(28).An

unexpected featureof theKpn I/HpaI double digest ofBx1(28) was the excessive number of

submolar fragments present (Fig. 4). Figure 6

shows how thesesubmolarfragmentscouldhave

beengeneratedby recombination,assumingthat

Hind

III

/Hpa

I

VOL. 28,1978

http://jvi.asm.org/

(10)

508 PRESTON ET AL.

Bxl(28)

d 9 n f mo a

I1I1 111

j k o p n m

_

_11I

d rp g j a

e k h k

't1\%'\\\ \

F71

11 I 11

9 a p h k no m

d 9 f h

I _ _

11

II~~~~~~~~~~~~~~~~~~~~~~~~~~~

c n Ih mq k

j

mo p ku j n it b

II II

II

h 9

1217251811 10 15 826

,,- I, I II

h f e q svrm' c g

11111

_x\\\f

d e f ISI

28

1 5 7 1613 6 1921A201g 9 1422

'_I 4

E I I

10 19 1 16151712 5 14 2 16 9: 13 11 8 31 22 2530 27 32 29

2011 1328 3 19267S17 1518)1fl 5 1128211014X16 4 20 8 6 9

II I Eli111 111 1'1 1. ,1

5 11 1321187 1920914 16 15 1 8 1 3 :22 6 12 1022

24 23

[image:10.499.90.408.62.486.2]

0 0.1 0.2

0.3 Q4

0.5 0.6 0.7 0.8 0.9 1.0

FIG. 5. Analysis ofcrossoverpoints in the recombinantBxl(28), using restriction endonucleases EcoRI,

BglII, Hpa I,HindIII plus Hpa I, and Kpn I plus XbaLHSV-1 physicalmaps areshown above, whereas

thoseofHSV-2are shownbelow, thegenomestructureof Bxl(28). The solid regions depict known HSV-1 DNA sequences, and the open regions depict HSV-2 sequences. The crosshatched regions indicate DNA

sequencesinwhichacrossoverhasoccurred. Inthe double digests, specific fragmentsaredenoted by numbers

insteadofletters. The shortvertical dottedlines in the HindIII/Hpa I digest refertoHindIIIcleavage sites.

Similarly,those inKpnI/Xba I digests refertocleavage sites ofXbaI.The orientations of ULand Usarethe

same asinFig.3.

(i) the L region was inverted occasionally (as

already discussed) and (ii) that there was an

inequalityintherepetitive regions bounding Us

such thatone of the HSV-2 Kpn I sites which

normally delimited fragment 22 was missing

(thereweretwoof thesecleavage sites in

HSV-2DNA.) Table2lists the13submolar fragments

generated by this scheme (fragments A to M)

and their derivations, together with their

frag-ment compositions and expected molecular

weights. Some fragments concordant with the

scheme could beseeninthedigest shown in Fig.

4. To ascertain the identity of these submolar

fragments, 32P-labeled HSV-1 Hpa I-m and

HSV-2 Xba I-h and -iwerehybridizedto

unla-beledKpn I/XbaIdouble-digest-separated

frag-ments of Bx1(28) DNA, using the Southern

technique (27). Figure 7 shows the results and

thecontrol [amixture ofuncleaved HSV-1 and

HSV-2 [32P]DNAs hybridizedtounlabeled Kpn

i

Eco RI

Bgl II

Hpa I

HindIII/Hpa:

KpnI/Xba I

Summary:

rlxxx

*\.1 \ l V

=

J. VIROL.

-i

L--V

http://jvi.asm.org/

(11)

5 20+22+6 12 10 22

IlL

_4~~~~~~~~~~~~~

5 ,+ 10 12 6+22

20 5+22} 10 12 6+22*

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

b I t-.C

20 5+22+6 12 10 22

5 20+2246 12 10 22

dZ

5 20+22+6 12 10 22

2 12 6 22

20 5+ 22+10 12 6 22

f 4

20 4415 + 6 12 10+22

9

[image:11.499.49.447.52.316.2]

h L

FIG. 6. Explanation forsubmolarfragmentspresent in theKpn I/XbaI doubledigestof Bx1(28) virion DNA. Thegenome structureofBx1(28),showing KpnI andXba Icleavagesites in S and those in L which producetheterminalfragments, isdrawn induplicate atthetopofthefigure.Not allofL is drawn. Two

intramolecular recombinationevents canoccur, andthesearerepresentedbydotted lines. Event1shows that

intramolecular recombinationtotherightofthecleavagesite inTRswhichgenerates the HSV-2fragment22

inverts Usandthiscleavage site.Moleculeais thusgenerated,with this HSV-2cleavagesitenowpresentin IRs. Event 2 shows that intramolecular recombination totheleftofthecleavagesitegeneratingtheHSV-2 fiagment22inverts Usbutnotthiscleavagesite,which remains within TRs. The DNA moleculeeisthus

generated. By subsequent inversion ofL orSorboth regions in molecules a ande, eight different DNA

[image:11.499.46.240.426.604.2]

moleculesareproduced.

TABLE 2. Composition of submolar fragments from

Bx1(28)DNAdigestedwithKpn Iplus Xba I

Submo- Approx

larfrag- Composition molwt Originb

mentsa (x106)

A 52+ 222+ 62 15.7 c

B 52+ 222+102 13.0 f

C 52+ 222 11.4 b,g

D 201+222 +62 10.5 d

E 52 10.2 a,d,e,h

F 62+222 8.0 a,b

G 201+222 +102 8.1 e

H 62 6.8 e,f,g,h

I 102+ 222 5.6 g, h

J 10% 4.4 a,b,c,d

K 201+222 3.7 a,h

L 20, 2.5 b,c,f, g

M 222 1.2 C,d, e, f

a Theseareidentified inFig.7(exceptforL).

bLettersrefertoDNAmolecules inFig.6.

I/Xba I

double-digest-separated

fragments

of

Bx1(28) DNA] (slots 1 and 5). In the control

tracks, submolar fragments A, B, and C were very faint. Table2 shows thatthese fragments

contain the invertedformof the terminal

frag-ment52(seeFig. 6), again supportingthe

conclu-sion that the Lregion of BX1(28) is present in

mainly one orientation. Xba I-h (double-digest

fragments 62 + 222) hybridized more to A than to B, as would be expected from Table 2. As

expected,XbaI-h alsohybridizedstronglytoD,

F, and H, which contain 62, andto fragment I

andafragment(s) inan areaofthegelwhere K should have been present. Both I and K have

sequences from 222. Similarly,Xba I-i

(double-digest fragments 102 + 222) hybridized more to

fragmentBthantoA. Italsohybridized strongly

toG, I,andJ,allofwhich contain102,andtoD, F, H, andpossibly M, which all havesequences

from 222. Some hybridization to E (contains

sequences within IRL and TRL) was observed.

This was expected, as there are sequences in

common between the repetitive regions

bound-ingUL andthosebounding Us (8).Hpa I-m(Kpn

I/XbaIdouble-digest fragment-20, and41

con-tain sequences present in Hpa I-m) hybridized

strongly to D (20, + 222 + 62), G (20, + 222 +

102), and the Kregion(20 +222).Italso

hybrid-izedto amolarfragment(41in the double digest)

(Fig. 5). All of these fragments have sequences

VOL. 28,1978

http://jvi.asm.org/

(12)

510 PRESTON ET AL.

Kpn I/Xba I DNA

fragments

HSV-2

HSV-w x x =

X cr er X0

-C

6 fo

7

1 2'

42

3

&44.A

9._

10,11!?1 *1 J

18,190.

. 3

i

.-i.

20,21o4

22?,23o* *M ?

240

in common with Hpa I-m (Fig. 5). Thus, the

results of these hybridizations identified the sub-molarfragments. As predicted from the scheme,

-1 fragment 20 in the double digest was not de-tectable inFig. 7. Thisagainsupports the con-ceptthatBx1(28)DNA ispredominately inone

orientationofUL.

Analysis

of IE

polypeptides

induced in

selected recombinants. IE polypeptides are defined in this paper asthose earlyviral

poly-5 peptides whose mRNA's are synthesized in

in-fected cellsevenwhen cycloheximide has been

presentthroughout the time of infection. Upon

removal of cycloheximide, these mRNA's are

A translated in the presence of

actinomycin

D,

I

which prevents

further

RNA synthesis.

D Figure8showsSDS-PAGE of IEpolypeptides

E inducedby recombinants from thegeneticcross

t . (ts Bxts 1).The data are summarized in Table 1: 3,whichcomparestheIEpolypeptidesof HSV-4 1and HSV-2 with those inducedby the

recom-binants.Polypeptides VmwIE 175,110, and63of

. HSV-1probably correspondtoICPs4,0, and 27, respectively, describedbyHoness and Roizman

10 (13).

12 Recombinant Bx1(24) induced the Vmw IE

182 of HSV-2, where Bx1(26), Bx1(31-1), and

Bx(31-2) induced the HSV-1 Vmw IE 175.

14

Bx1(28),

however, induced a band containing

1.

components whichcomigrated with theVmw IE

16 182 of HSV-2 and what appeared to be further

componentswitha greater apparent molecular

weight than Vmw IE 182.

The

Vmw

IE 118/110

region

wasof

particular

21 interest. Recombinants

Bx1(26)

and

Bx1(31-1)

induced the HSV-1VmwIE 110,whereas

recom-I

binant

Bx1(24)

induced the HSV-2

Vmw

IE

118;

but the two recombinants Bx1(31-2) and 23

Bx1(28)

appeared to induce both the Vmw IE 110 and the Vm,, IE 118. This observation

de-pendedonband identification in the gel,and an

4 alternative explanation couldbe that these two

recombinants synthesized as anIEpolypeptide

a polypeptide normally made later in infection whichcomigrated with the Vmw IE118 of

HSV-29 2 (the glycopolypeptide

Vmw

118 ofHSV-2,seen

usuallyat 5hpostabsorption).Acomparisonof IE polypeptides induced by the recombinants

FIG. 7. Composition of submolar fragments in the

KpnI/XbaIdouble digest ofBx1(28) virion DNA.

Unlabeled separated KpnI/XbaIdouble digest of

Bx1(28)DNAwasblottedonto nitrocellulose filters

andhybridizedtothefollowing by the Southern tech-nique: equal amounts of HSV-2 and HSV-1 [2JP]

DNAs (slots 1 and 5), 32P-labeled HSV-2 fragment

Xba I-h(slot 2), 32P-labeledHSV-2 fragment Xba I-i * (slot 3), and 32P-labeled HSV-1 fragment Hpa I-m

(slot 4).

J. VIROL.

http://jvi.asm.org/

[image:12.499.64.250.49.650.2]

(13)

1 2 3 4 5 6 7 8 9 10 11 12 13

0(0

C4 CY CY

_

_T

_ m

v-x c, x IA x

mC_ m -w m

U,)

co

un

HSV- 2 2 APPARENT

MW X 1

.

*

_182

.i- 157 A -138(144)

.134,132

-118

-67

-64

FIG. 8. IEpolypeptides induced by recombinants fromthe geneticcross (ts Bx ts1).Slots 1 and13are

mock-infected cellpolypeptide profiles. Symbols: U, HSV-1-induced polypeptide; 0, HSV-2-induced

poly-peptide;*,alteredpolypeptide.Allmolecularweightsareapproximateand basedonestimates made by using

gradient concentration SDS-PAGE. If the apparent molecularweightdiffered when single-concentration

SDS-PAGE was used, then an estimate of the molecular weight based on this gel system is given in parentheses.

with those labeledat5h postabsorption (Fig. 9)

excluded thispossibility,sincenodetectable late

polypeptide comigrated with Vmw IE 118.

More-over,proteolytic cleavagemapsofthe

polypep-tides Vmw 110 and 118ofBx1(28) are identical

tomapsof the VmwIE 110 ofHSV-1 andtheVmw

IE 118 ofHSV-2, respectively (C. M. Preston,

personal communications). This interesting

find-ingis discussed inmoredetaillater.

Knowledge ofthe genome structures of the

recombinants allowsthese IEpolypeptidestobe

mappedonto the genome, ascan beillustrated

with the Vmw IE 63 of HSV-1 and its HSV-2

counterpart polypeptide, Vmw IE 64. All the

re-combinants shown inFig.8induced theHSV-1

polypeptide, which thereforemust map within

the HSV-1 sequences of the recombinant

ge-nomes. Bx1(24) contained the smallest HSV-1

sequenceincommonwith theHSV-1sequences

ofthe otherrecombinants andthus located the

type-specific region ofthis polypeptide to the

map coordinates from 0.56 to 0.79. Figure 13

shows the physical map locations of

polypep-tidesVmw IE175, 110, 87, 68, and 63,derived in

asimilarmanner.

DISCUSSION

Analysis with restriction endonucleases has

demonstrated that 15outof17putative

recom-binants whichwereisolated fromtwocrossesof

VOL. 28, 1978

N _

I

X (A _( U,

:

z

ua

a

(A

i.

4i~~

nz

*t ^

iI, lr 1

HSV-1 APPARENT MW X 10 3

175w

136(143). 110

-87

-68_

63W

http://jvi.asm.org/

[image:13.499.49.443.49.438.2]

(14)

[image:14.499.62.462.77.264.2]

TABLE 3. Summaryof Data on IEpolypeptides

polypeptide HSV-2 Recombinant

polypeptide"

Apparent Apparent

molwt Intensity' molwt Intensity' Bxl(24) Bxl(26) Bxl(28) Bxl(31-1) Bx1(31-2)

(X103)b (X103)b

175 + 182 + 2 1 (a) 1 1

136 (143) V 138 + NI 1 NI 1 NI

- 134/132 + NI ND NI ND NI

110 + 118 + 2 1 1+2d 1 1+2

87 V - 1 1 1 1 1

68 V 67 V 2 1 2 1 1

63 + 64 + 1 1 1 1 1

- 42.5e V 2 ND 2 ND 2

a1, HSV-1 polypeptide; 2, HSV-2 polypeptide; NI, no information; ND, no detectable band; A, altered

polypeptide.

b_,

Noequivalent polypeptidedetectable.

+,Intense bandalways present; V, band of variableintensity whichwassometimesnotdetectable.

dSynthesized IE

Vmw

110and IEVmw118.

eThe

Vmw

IE 42.5 ofHSV-2probably corresponds to the

Vmw

43 ofHSV-1, which is seen onlylater in infection.

HSV-1 and HSV-2 ts mutants contain both

HSV-1 and HSV-2 sequencesin their genomes

(Fig. 10). Thetwoother recombinants, Dx1(15)

andBx1(26), have identical restriction

endonu-clease maps (with the six enzymes tested) to

thatoftheDNA of HSV-1 and therefore either

are revertants ofts D or tsB, respectively, or

are true recombinants with undetected small

HSV-2 DNAsequences.

Crossovereventshave beenidentifiedinevery

region of thegenome. Mostrecombinantsmust

have been formedbymore than one crossover

event,the greatest number detected in this study

being four[recombinantDx1(51)].

Odd numbers ofcrossovers in UL or Us will

produce recombinants with an HSV-1 and an

HSV-2 repeat bounding the unique region in

whichthecrossovershave occurred.The

recom-binantBx1(31-2), for example, appearstohave

oneHSV-1(L)repeatandoneHSV-2(L)repeat.

Ifthe dataforDx1(2) are included, five of the

recombinants shown in Fig. 10 have odd

num-bersofcrossoversinL, S,orL andS.Figure11

showsthatwherewehavedetectedanodd

num-ber ofcrossovers in one orin both unique

re-gions, then the actual orientation of UL and Us

can alter the minimum number ofcrossovers

necessarytogenerate therecombinant,

assum-ing that the entire LorSorbothregions invert.

However, ifthere are, in addition, crossovers

within the repeat regions, then, depending on

the position of the crossover(s) and the site of

intra- or intermolecular recombination which

leadstoinversion of theuniqueregion, the

min-imumnumberofcrossoversmay or maynotbe

affectedby the orientation of ULorUs,as

illus-trated in Fig. 12. It should also be noted that

intra-orintermolecularrecombinationeventsin such recombinantscangenerateDNAmolecules with differentarrangementsof1 and

HSV-2sequencesfrom that of theparental

recombi-nantDNA(refertoFig. 12).

The virion DNA ofonerecombinant,Bx1(28),

appears tocontainmainlyoneorientation of the

ULregion, althoughboth orientations of theUs regionarerepresentedinequalamounts. Assum-ing that the recombinant was cloned from a

single virus particle, Bx1(28) was formedby a

crossover event in the UL region and another

withinoneof therepetitivesequencesbounding Us. As a consequence, it appears to have one

HSV-1 LrepeatandoneHSV-2Lrepeat bound-ing UL (heterotypic for the L repeat region), whereas those bounding Us appear to be

par-tially heterotypic. After the initial

recombina-tionevents,sequencesinTRs which contain the

HSV-2 KpnI cleavage siteappear to be inter-changeable with the equivalent HSV-1

se-quencesinIRs,whichlackaKpnIcleavage site,

resulting in amixed population ofDNA mole-cules. The result could have arisen if the Sregion

inverts by intra- orintermolecular

recombina-tion on either sideof theKpnIcleavage site (see

Fig. 6).Alternatively,sequencescouldexchange

between TRs and IRs by a double crossover

eventwithintherepetitive region boundingUs.

Itis also feasible that intermolecular recombi-nation couldgenerate molecules which contain

theKpnIcleavagesite in bothTRsandIRsand

molecules which lack thecleavage site in both

TRs and IRs. Further tests on subclones of

Bx1(28)

are currently being carried out inour

laboratory.

Itis not known whythe DNA ofBxl(28) is

J. VIROL.

512 PRESTON ET AL.

http://jvi.asm.org/

(15)

1 2 3 4 5 6 7 8 9 10 11 12 13 14

HSV -2

APPARENT

MW X 10-3

IE 182

-IE 138(144) I E 134/132 =

118

-IE 118

IE 64

-IE

42-5-LU LU w LU LU Lu

_- z _-l

N N CV CI Cs z _ _U- o a x x x x

2 2 _- _. =: x (a ax ss to u

.,,w }

,., . A4

_04 -011 a*

FIG. 9. Comparison ofIEpolypeptideswith those made laterininfection (5hpostabsorption)in the absence

of cycloheximide. IEpolypeptidesinducedbyrecombinantsBx1(28) (slot 7)andBx1(31-2) (slot 9), by parental

viruses(slots3and13)andby wild-typeHSV-2 and HSV-1 (slots5and11)areshown. Slot1showsa

mock-infected-cellIEpolypeptide profile. Theremainingslots showpolypeptidesinducedat5hpostabsorption of

virus in the absenceofcycloheximideexceptslot2, which shows themock-infected-cellprofile.

found predominantly in only one arrangement

ofUL. EitheronearrangementofUL is generated

only atagreatly reduced frequency during the

normal replication cycle of Bxl(28) or is

strongly selected against,orthere isa

combina-tion of both possibilities. The DNA we have

analyzed in thisreportwasderived from virions,

and it is conceivable thatboth orientations of

ULwerepresentinnornalamounts in the nuclei

of infectedcellsbut thatonlyonearrangement

waspackaged.

Recombinant Bxl(31-2) also appears to be

heterotypic for the repeats bounding UL, but

with all four genome arrangements present in

virion DNA in equimolar amounts. Both

Bxl(31-2) and Bx1(28) show the minimum

number ofcrossovers when drawnin thesame

orientation of UL in which Bxl(28) is largely

frozen.Morseetal. (19) have also reported that

HSV-1/HSV-2 recombinants formed by odd

numbers of crossovers in UL also showed the

minimumnumberofcrossoverswhen drawn in

the same orientation of UL. This led them to

conclude that their "data were consistent with

thehypothesis that onlyonearrangementofthe

parental DNA participates in the generation of

ul

r-z

cr

I-U,

co m

U,, (A 4-. *-'

HSV-1 APPARENT MW X 10

- 175

- 13fi(143)

- 117

- 110

- 87

- 68

- 63 VOL. 28,1978

'w*x-,4... ,map.

If.

http://jvi.asm.org/

[image:15.499.35.431.65.461.2]

(16)

514 PRESTON ET AL.

Dxl(2) T L

Dxl(34-1)

Dxl(34-2), L-' 11~~~

Bxl(24) ---~~~

B 1( )

-l

Bxl(31-2)

Bx1(31-2) . i

l---Bxl1(13) ~

Dxl(31)

Dxl(32)

Dxl(43)

Dxl(48)

Dxl(51)

Dx1(53)

--- --- - ---

[image:16.499.66.261.46.283.2]

---o 0.1 02 03 h CB 06 07 G s 1.0 fractional genome length

FIG. 10. Summary ofthegenome structuresof15 HSV-1/HSV-2 recombinants based on datafrom di-gestsofsix restrictionendonucleases. Vertical dotted lines correspond to the endsofthe L andS repeat sequences. The sequences inarecombinant derived from HSV-1 andHSV-2 parents are shown by a thick continuous horizontal linesuperimposedontheupper andlower,respectively, oftwohorizontal dotted lines. A crossover region is shown by two vertical lines between the thick continuoushorizontal lines. The distance between the twovertical lines indicates the region in which the parental nature of the DNA is uncertain. Where the uncertainty is small, the

cross-overregionappears as a singleverticalline. In the

Dxl(2)genomestructure, the crossover in the L

re-peat sequences isquestionable. There are two inter-pretations ofthe data on this recombinant. This crossovermayormay not be present.

recombinants." Although Bxl(28) is partially fixedinthesameorientation ofUL,wefeel that thehypothesismustcontinuetobe treated with

somereservation for thefollowingreasons.

(i) Onlyasmall number of recombinants with odd numbers ofcrossovers in L or S or both regionshave been isolated. Combiningthe data

from Morseetal. (19) and from ourlaboratory,

a minimum number of 7 of 46 recombinants showanodd numberofcrossoversinL. If

recom-binantswithcrossovers close to, orwithin, the

jointregionareincluded, this givesafigureof 14 of 46 recombinants.

(ii) Thegenetic cross used toselect a

partic-ular recombinantmayinfluencethe genome

ar-rangement of theparental virusparticipatingin

the recombinationevent, andtodateonlyavery

limited number of genetic crosses have been

investigated.

(iii) Fine-structure physicalmaps have notyet been made of theIRL andTRL regions of these recombinants. It ispossible theremaybe

unde-tected crossovers within these regions which

causethe number ofcrossovers tobe unaffected

by the orientationofUL (refertoFig. 12). This could explainwhy theBx1(31-2) genome,which

appears to beheterotypic for therepetitive

se-quences bounding UL, unlike Bx1(28), inverts

UL normallytogivefourarrangementsof DNA molecules.

Using information about the physical struc-tureofwild-typerecombinants,tsmutationscan

be located onthegenome.Forinstance,

recom-binants derived from the crosses involving ts 1 allhave incommonthesamesequencesof

HSV-1DNA, and thesemapbetween coordinates0.69

HSV-1 and0.71. We thereforepredictthat this defines

--HSV-2 themaplocation of thetslesion ints 1.Similarly,

tsDlieswithinman coordinates0.83tO0.86and

0.96to0.99 orwithin theregion0.94 to 0.96.The

- L -4 S

TRI IRL IRs TRs

Onecross- _-over

Inversion of L

Two cross- m overs

Two cross

-overs

Inversion of L

4,

Three overs

cross

-Inversion of S

Two cross-overs

Inversion of L

Three cross--_=

[image:16.499.269.461.270.593.2]

overs

FIG. 11. Diagrammaticrepresentation of how in-versionof the whole ofLand/orSofHSVDNA could affect the apparent number of crossovers required for thegeneration of a recombinant. Thesolid regions of thegenome refer to HSV-1 DNAsequences, and the open regions refer to HSV-2 DNA sequences.

'JJ

-i ., w---4U

:_ 4--- 7T -! -4 .. -1.

i 11-i

J. VIROL.

.I

Dxl(57)

http://jvi.asm.org/

(17)

VOL. 28, 1978

Three crossovers

Threecrossovers

Four crossovers

Threecrossovers

Three crossovers

Four crossovers

4-El-~~~~~~

b

T hreecrossovers

FIG. 12. Diagrammatic representation of how in-version of the whole of L (a)orinversion bya cross-over within intratypic homologouw regions of the L

repeat(b) could affect theapparentnumber of cross-oversgenerating the recombinant and thestructure

of the recombinant. The solid regions of thegenome

refertoHSV-1 DNAsequences,andtheopenregions

refertoHSV-2 DNAsequences. The horizontal ar-rowsrefertothe orientationof UL.

physicalmaplocations ofthesetwo tsmutations

confirm results frommarker rescuestudies (N.

D. Stowetal. [submitted for publication], who unambiguouslylocatethetsDmutation inTRs

and IRs; P. Chartrand, personal communica-tion). Crossovers alsooccurwhichareunrelated

to events which remove the ts lesion. For

ex-ample,recombinantsDx1(51) andDx1(43)are

formed by crossover events which affect the

short region, but DX1(51), in addition, has a

double crossover in the UL region. From the

genome structures ofother recombinants, this

double crossoverisunlikelytoberelatedtothe

positionof the lesion intsD.

Most recombinantsgrowas well aswild-type

HSV-1 and HSV-2 viruses atboththe

permis-sive and thenonpermissivetemperatures.Thus,

polypeptides fromone serotypeare able to

re-place the functions of polypeptides from the

other withoutimpairing growthof the

recombi-nant virus. Some recombinants, however [for example,Bx1(24)],donotgrowas well as

HSV-1 or HSV-2 atthe nonpermissive temperature.

In such cases, the interaction of polypeptides

fromonetypeofviruswith those from the other

type (or its nucleic acid) maybeimpaired.

Al-ternatively, thecrossovereventitselfmayhave

altered the genome ofthe recombinant such that analteredpolypeptide, differing fromeitherthe HSV-1 or the HSV-2 counterpart, is produced which functions lessefficiently. A further possi-bility is that the recombinant genome itself may interact less efficiently with HSV-1 or HSV-2 polypeptides.

Analysis of the IE polypeptides induced by the intertypic recombinants permits physical map coordinates to be assigned to those poly-peptides by correlation with the genome struc-tures. This approach is outlined inmoredetail in anotherpaper(H. S. Marsden et al., J.

Virol.,

in press). It is implicit that HSV-1 and HSV-2 counterpartpolypeptidesarefunctionally equiv-alent. The assumption ofequivalence is based onsimilarityofmobilityofpolypeptidesin SDS-PAGE, kinetics ofsynthesis,amountmade, and post-translational processing of the polypep-tides. Moreover, in the case ofthose polypep-tideswhichundergoposttranslational modifica-tion,it ispossiblethat the

modifying

gene,and not the structural gene, is in fact mapped. It should be noted thatwedonotclaimtomap the entirecoding regionof anypolypeptide,butonly that part whichcauses achangeinmobilityon

SDS-PAGE. The possibility that gene splicing takesplaceasinadenovirus and simian virus40

(1, 2) has innoway beenexcludedbyourresults. The summary of map locations of polypep-tides

Vmw

IE 175, 110,87, 68,and63is shown in Fig. 13. Thepolypeptidesmap in regions ofthe genome which contain relatively abundant classes ofIE RNAtranscripts (5, 14; R. J.

Wat-son and J. B. Clements, submitted for publica-tion). We conclude that both the L and the S regions of the genome code for IE polypeptides and, at least in the case of

Vmw

IE 182, that the Srepeatregionhasbeenimplicated.Marsdenet

al. (in press)have shown that late

polypeptides

arealso made in L and S;thus, neither IEnor

late polypeptides are restricted to the S or L region alone.

In somecases wehave found that the mobili-ties ofpolypeptides specified by recombinants do not correspond to those of either of the parental forms. Forexample, BX1(28) specifies

an IE polypeptide with apparent molecular weightgreater than IEVmw182ofHSV-2aswell

as components which migrate with the upper component of HSV-2IEVmw182.Evidence from

recombinants generated by intertypic marker

rescue(Marsdenetal., in press) suggests thatat

leastpartof thispolypeptide mapswithinTRs and/orIRs. SinceBx1(28)containsa crossover

in IRs (and TRs [see discussion above]) the

sequence ofthe altered polypeptide may con-form in part to HSV-1 and inpart to HSV-2.

http://jvi.asm.org/

[image:17.499.41.235.68.288.2]

(18)

516 PRESTON ET AL.

liii

*

IlIllillhllIll liii

milniu

I

l

w

*

C'

'~~~~~~~~

1175 175:

'110 _

l

!

:87:

68

.,

₆₃

.~~~~~I

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

J. VIROL.

immediate-early transcripts

immediate-early

polypeptides

genome

[image:18.499.118.414.72.236.2]

fractional length

FIG. 13. Summary of physicalmap locationsofIEpolypeptides, togetherwitha comparison ofIE RNA HSV-1transcripts.Thesolidareasofthe IE HSV-1transcripts refertoabundanttranscripts,and the hatched areasrefertoscarcetranscripts.

Alternatively, the polypeptide's mobility could havechanged as aresult ofdifferent processing

ortermination.

Recombinants Bx1(28) and Bx1(31-2)

syn-thesize both theVmw IE 110 of HSV-1 and its HSV-2counterpart, Vmw IE 118.Comparison of IEpolypeptideswith those made later in infec-tiongivesnosupport tothepossibilitythatone

of these isnot a true IEpolypeptide,but isone

normallysynthesized later in infection. This cir-cumstantial evidence isconfirmed by results of proteolytic cleavage ofpolypeptides Vmw IE 110

and 118 induced by the recombinant Bxl(28) (C. M.Preston, manuscriptinpreparation). The simplest explanation is that the polypeptides

map in IRL and TRL, since Bx1(28) is hetero-typic for these repetitive sequences bounding UL.Thisexplanation dependsonthe assumption that the HSV-1 and HSV-2 counterpart

poly-peptides are functionally equivalent. Bx1(24) synthesized only the Vmw IE 118, and, as this recombinant hasoneHSV-2L repeatandone L

repeat which contains HSV-1 and HSV-2

se-quences, this suggests that at least the

type-specific regionsof thepolypeptides Vmw IE 110

and 118map between map coordinates 0.78 to

0.82and0 to 0.04.

We conclude from these results that both repetitive sequences bounding UL can be

ex-pressed. As Bx1(28) is predominantly in one

arrangement of UL, it is possible that genes

withinthese sequences can beexpressed in both

IRLandTRLarrangements. Of course, it is

pos-sible that only one arrangement is expressed

from any one genome. Furthermore, if

expres-sion is from nuclearHSVDNA,whichexists as

concatemers orcircles, then mostrepetitive

se-quencesbounding UL-may atthetimeof

expres-sion beinternal.

ACKNOWLEDGMENTS

We thank R.Cortini formakingavailable restriction en-donuclease maps before publication and S. M. Brown for providingseed stocks of HSV-1 and forhelpfuladvice.

V.G.P. wassupported by a CommonwealthScholarship. LITERATURE CITED

1. Aloni, Y.,R.Dhar, 0. Laub,M. Horowitz, and G. Khoury. 1977. Novel mechanism for RNA maturation: the leader sequences of simian virus40mRNAare not transcribed adjacentto codingsequences. Proc.Natl. Acad. Sci. U.S.A. 74:3686-3690.

2. Berget,S.M., C.Moore,and P. A.Sharp.1977.Spliced

segmentsatthe 5' terminusof adenovirus 2 late mRNA. Proc. Natl. Acad. Sci. U.S.A. 74:3171-3175.

3. Brown, S.M.,D. A.Ritchie,and J. H.Subak-Sharpe.

1973.Geneticstudies withherpes simplexvirus type1: the isolation oftemperature-sensitive mutants, their arrangement intocomplementationgroups and recom-binationanalysis leadingto alinkagemap. J.Gen. Virol. 18:329-346.

4. Clements,J.B., R.Cortini, and N. M.Wilkie.1976. Analysis of herpesvirus DNA substructurebymeansof restriction endonucleases. J. Gen. Virol. 30:243-256. 5.Clements,J.B.,R.J.Watson,and N. M.Wilkie.1977.

Temporal regulationof HSV type 1transcription: lo-cation of transcripts on the viral genome. Cell 12:275-286.

5a.Cortini, R.,and N. M. Wilkie.1978.Physicalmaps for HSVtype2DNA withfive restrictionendonucleases. J.Gen. Virol. 39:259-280.

6. Delius, H.,and J. B. Clements.1976. Apartial denatur-ationmap ofherpes simplexvirustype1DNA:evidence for inversions of the unique regions. J. Gen. Virol. 33:125-133.

7. Esparza, J., M.Benyesh-Melnick,and P. A. Schaffer. 1976. Intertypic complementation and recombination betweentemperature-sensitivemutantsofherpes sim-plexvirustypes1and2.Virology70:372-384. 8. Grafstrom,R.H.,J. C.Alwine,W.L.Steinhart,and

W. C.Hill.1974.Terminal repetitionsinherpessimplex virus type 1 DNA. ColdSpringHarborSymp. Quant. Biol. 39:679-681.

9. Grodzicker, T.,C.Anderson,J.Sambrook,and M. B. Mathews. 1977. Thephysicallocations of structural genesinadenovirusDNA.Virology80:111-126. 10. Halliburton,I.W.,R. E.Randall,R. A.Killington,

http://jvi.asm.org/

(19)

VOL. 28,1978

and D. H. Watson. 1977. Someproperties of recom-binants between type 1 and type 2 herpes simplex viruses. J.Gen. Virol. 36:471-484.

11. Hayward, G.S., R. J. Jacob, S. C. Watsworth, andB. Roizman. 1975. Anatomy of herpes simplex virus DNA: evidence for fourpopulations that differ in the relative orientationsof their long and short components. Proc. Natl. Acad.Sci. U.S.A.72:4243-4247.

12.Honess, R.W., and B. Roizman. 1973. Proteins specified by herpessimplex virus. XI. Identification and relative molar rates of synthesis of structural and nonstructural herpes viruspolypeptides in the infected cell. J. Virol. 12:1347-1365.

13. Honess,R.W., and B. Roizman. 1974. Regulation of herpesvirus macromolecular synthesis. I. Cascade regulation of the synthesis of three groups of viral proteins. J.Virol.14:8-19.

14. Jones,P.C., G. S. Hayward, andB. Roizman. 1977. Anatomyofherpes simplex virus DNA. VII. a RNA is homologoustononcontiguoussites in both the Land S components ofviral DNA. J. Virol. 21:268-276. 15. Kieff, E. D., S.L. Bachenheimer, and B.Roizman.

1971. Size,composition, and structure of the deoxyri-bonucleic acid ofherpes simplexvirussubtypes 1 and 2. J. Virol. 8:125-132.

16. Macpherson, I., and M.Stoker. 1962.Polyoma trans-formation inhamstercellclones-aninvestigation of genetic factors affecting cell competence. Virology 16:147-151.

17. Marsden, H. S., I. K. Crombie, and J. H. Subak-Sharpe.1976. Controlofprotein synthesisin herpes-virus-infectedcells: analysis of polypeptides induced by wild type and sixteen temperature-sensitive mutants of HSV strain17.J. Gen.Virol.31:347-372.

18. Mautner,V.,J.Williams,J.Sambrook,P. A.Sharp,

and T.Grodzicker. 1975. The location of the gene coding forhexonand fibreproteinsinadenovirus DNA. Cell5:93-99.

19. Morse,L.S.,T.G.Buchman,B.Roizman, andP. A. Schaffer.1977.Anatomyofherpessimplexvirus DNA. IX.Apparentexclusionof someparentalDNA arrange-mentsin thegenerationofintertypic(HSV-1xHSV-2) recombinants. J. Virol. 24:231-248.

20. Powell,K.L.,and R. J.Courtney. 1975.Polypeptides synthesised in herpes simplex virus type 2-infected HEp-2 cells.Virology66:217-228.

21. Powell, K.L.,R.Mirkovic,and R. J.Courtney.1977. Comparativeanalysis of polypeptides induced bytype 1and type 2 strainsofherpessimplexvirus. Intervirol-ogy 8:18-29.

22. Rakusanova,T.,T.Ben-Porat,M.Himeno,and A. S.

Kaplan. 1971. Early function of the genome of herpes-virus. I. Characterization of the RNA synthesised in cycloheximide-treated, infected cells. Virology 46: 877489.

23. Rigby, P. W. J., M.Dieckmann, C. Rhodes, and P. Berg. 1977. Labeling deoxyribonucleic acid to high specific activity in vitro bynick-translation with DNA polymerase I. J. Mol. Biol.113:237-251.

24. Sambrook, J., J. Williams, P. A.Sharp,and T. Grod-zicker. 1975. Physical mappingof temperature-sensi-tive mutations of adenoviruses. J. Mol. Biol. 97:369-390.

25. Sheldrick, P., and N. Berthelot. 1974. Inverted repeti-tions in the chromosome of herpes simplex virus. Cold SpringHarborSymp.Quant. Biol. 39:667-678. 26. Skare, J., and W. C.Summers. 1977. Structure and

functionof herpes virus genomes. II. ECoRl, Xba I and Hind III endonuclease cleavage sites on herpes simplex virus type 1 DNA. Virology76:581-595.

27. Southern, E. M. 1975. Detection ofspecific sequences amongDNAfragmentsseparatedby gel electrophore-sis. J.Mol. Biol.98:503-533.

28. Timbury,M.C.1971.Temperature-sensitivemutantsof herpessimplex virus type 2. J. Gen. Virol. 13:373-376. 29. Timbury,M.C.,and J. H.Subak-Sharpe. 1973. Genetic interactions between temperature-sensitive mutants of types 1 and 2herpes simplexviruses. J. Gen.Virol. 18:347-357.

30. Wilkie,N. M. 1973. The synthesis and substructure of herpesvirus DNA: thedistributionof alkali-labile single strand interruptions in HSV-1 DNA. J. Gen. Virol. 21:453-467.

31. Wilkie,N. M. 1976. Physical maps forherpes simplex virus type 1 DNA for restriction endonucleases Hind III, Hpa-1 and X.bad.J. Virol. 20:222-233.

32. Wilkie,N.M., and R.Cortini.1976.Sequence arrange-ment inherpes simplex virus type 1 DNA: identification of terminal fragments in restriction endonuclease di-gests and evidence for inversions in redundant and unique sequences. J. Virol. 20:211-221.

33. Wilkie,N.M.,N. D.Stow,H.S.Marsden,V.Preston, R. Cortini, M. C. Timbury, and J. H. Subak-Sharpe.1977.Physical mappingofherpes simplexvirus coded functions andpolypeptides bymarker rescue and intertypic recombinants. In G.de The,W.Henle,and F.Rapp (ed.), Proceedingsof the 3rdSymposiumon Herpesviruses and Oncogenesis. I.A.R.C., Lyons, France.

34. Williams,J. F., T.Grodzicker, P. A.Sharp,and J. Sambrook. 1975.Adenovirusrecombination:physical mapping ofcrossover events.Cell 4:113-119.

http://jvi.asm.org/