Short, duplicated sequence indicative of the recombinogenicity of the junction between a unique and an inverted repeat sequence in the S component of the herpes simplex virus type 1 genome.

(1)

0022-538X/89/051877-07$02.00/0

Short, Duplicated Sequence Indicative of

the

Recombinogenicity of

the Junction between

a

Unique

and

an

Inverted

Repeat

Sequence

in

the S

Component of the

Herpes

Simplex Virus

Type 1

Genome

KENICHI UMENE

Department of Virology, Faculty of Medicine, Kyushu University 60, Fukuoka 812, Japan Received16September 1988/Accepted29December1988

A herpessimplex virustype1(HSV-1) strain, B3,wasfoundtohaveashortduplicationonthe left junction

between theuniquesequence (Us)and the inverted repeatsequence

(Rs)

in theS componentof thegenome

DNA. A shortregion of

Rs

contiguoustotheleft

Us-Rs

junctionwasduplicatedinB3. Basedonthe nucleotide sequencesin and around the

Us-Rs

junctions of B3and otherHSV-1 strains,aconceptofjunctionstretchwas

proposed. Theorganization of junctionstretch is

Rs

side5'-(GorAstretch)AGC-3'

Us

side.Introductionof theconceptofjunctionstretch ledtoadefinition ofthestructurein and around the

Us-Rs

junction,in theform

commontoHSV-1 strains. The rightendof

Us

inthe HSV-1 genome wasthe A of theATG initiationcodon

ofgeneUS12, and thus theATGtriplet may actas a bufferto preventexpansion of

Rs,

asisthecasewith

HSV-2. The duplication inB3wasgeneratedbya crossovereventbetweenapointon

Rs

andthe

Us

sideend

ofthe left junctionstretch.These observationssuggestthat the

Us

side endofthejunctionstretchpossessesthe

property of recombinogenicity, responsible for generation ofthe duplication in strain B3 and also for the formation of the

Us-Rs

junction of HSV.

Three alphaherpesviruses ofherpes simplexvirus types 1 and2(HSV-1 andHSV-2)andvaricella-zoster virus (VZV) arecommonhumanpathogens (7, 8, 19). They have aDNA genomecontaining twouniquesegments(ULand Us), each of which is bounded by its own inverted repeats (TRL and IRL, and

IRS

and

TRs)

to form the long (L) and short (S) components(4, 18)(Fig. 1). HSV-1 and HSV-2aresimilar in both genome structure and gene layout. The other alpha-herpesvirus, VZV, differssignificantlyfrom the herpes sim-plex viruses in genome size and base composition. Exami-nations ofthepolypeptide sequencehomology showed that the S components of HSV-1 and VZV are related to a

significant degree, though they do differ in genelayout (3). Itishypothesizedthatarcombinationbetweentwounique sequencesaligned in an opposite orientationcaused expan-sion of the inverted repeat and contraction of the unique sequence in herpesvirus genomes (7, 32). This proposal seemed to account for three alphaherpesviruses HSV-1, HSV-2, and VZV as having descended from a common ancestor. Based on this same idea, Davison and McGeoch presenteda scheme toshow the derivation of the S compo-nents of HSV-1 and VZV from the S component of an ancestral herpesvirus by expansion and contraction of

Rs

(3). The scheme satisfactorily explained the differences between HSV-1 and VZV in gene layout. HSV variants, which were assumed to be generated by recombination between uniquesequences arranged in anopposite orienta-tion withexpanded repeat sequence and contracted unique sequence, were actually isolated (1, 11, 23). The

Us-Rs

junction corresponds to the crossover point. As a factor involved in the determination of the

Us-Rs

junction, Whitton and Clements noted the location of the first translational initiationcodon (32). Thetranslational initiation codon of the IEmRNA-5gene(corresponds to gene US12) of HSV-2 had the Aof the ATGasthefirst base in

Us,

andthisdefined the right

Us-Rs

junction. They assumed that the ATG of the HSV-2 IEmRNA-5 gene acted as a buffer to prevent the expansionof

TRs.

However, the first ATG of the

IEmRNA-5 geneof HSV-1 strain 17was8 basepairs (bp)into

Us

and didnotdirectlydefine theright

Us-Rs

junction (32).

I have now obtained evidence fora novel HSV-1 strain, termedB3, which hasashortduplicatedregionderived from

Rs

on the left

Us-Rs

junction. The nucleotide sequences

around

Us-Rs

junctionsof B3 and other HSV-1 strainswere compared. A concept ofjunction stretch was introducedto define thestructurearoundthe

Us-Rs

junctionsin the form common toanyHSV-1 strain. The rightend of

Us

of HSV-1 defined in this formwasthe A ofthefirstinitiator ATG ofthe US12 (IEmRNA-5) gene. The ATG of IEmRNA-5 gene seemed to act as abufferto preventthe expansionof

Rs

in the HSV-1 genome, as was the case with HSV-2. The duplicationin B3 was assumed to begenerated by a cross-over eventbetweenapointin

R.

and the

Us

side end of the leftjunction stretch. Thus, the

Us

side end of thejunction stretch apparently possesses the property of recombinoge-nicity, responsible forgeneration oftheduplicationin strain B3 andalsofor the formation of the

Us-Rs

junctionof HSV.

MATERIALS ANDMETHODS

Cellsand viruses. Vero cellsweregrown inEagleminimal essential medium(MEM) supplementedwith 5% calfserum.

SP23, used as the standard HSV-1 in this work, was a

single-plaque isolatefrom HSV-1 strainPatton (27). HSV-1 strain B3, the duplication of which was analyzed in this work, and HSV-1 strains B4 and B9 wereisolatedatKyushu University Hospital (25).

Working stocks of HSV-1 were made on Vero cells in EagleMEMwith2% fetal bovineserum at alowmultiplicity of infection (0.01 PFU per cell). HSV-1 DNAs were pre-pared from viral particles obtained after glycerol gradient centrifugation (23).

Restriction endonuclease digestion, acrylamide gel electro-phoresis, and Southern hybridization. Restriction endonu-cleaseswere purchased from Takara ShuzoCompany (Ky-oto, Japan) and Toyobo Company (Osaka, Japan), and conditions for digestion were those recommended

by

the 1877

on November 10, 2019 by guest

http://jvi.asm.org/

(2)

(1)

(2)

TRL UL IRL IRSUs TRs

a a a

aIR5Bal Ba2 U5 Ba3 4TR5

IE175 USi US12 IE175

Tal sal Sa Ta2

FIG. 1. Maps of HSV-1 genome. (1) Structure of the HSV-1 genome arranged in prototype orientation (18). HSV-1 DNA is a linear, double-stranded molecule of about 155 kb, consisting of two covalentlylinked components, L and S, that constitute 82 and 18% ofthe genome, respectively. Each component consists of unique sequences (UL and Us) bracketed by inverted repeat sequences (TRL, IRL,IRs,andTRS).Ashortsequence, a, is repeated directly atthe terminiofthe HSV-1 genome and is also present in the inverse orientationat the L-Sjunction. (2) Expansion of the S component. The locations and 5' to 3' orientations of mRNA species are indicatedbyhorizontal arrows. Protein-coding regions are shown as open boxes (13, 14, 17).BarmHI sites(Bi toB4)areindicated (26). (3) Expansions of the regions spanning the Us-Rsjunctions. TaqI sites (Tal and Ta2) and Sau3AI (isoschizomer of Mbol) sites (Sal andSa2) are indicated (13, 14).

manufacturers. The DNAs digested with restriction endonu-cleaseswereseparated in a5% acrylamide gel(22).Southern hybridization was carried out on a Biodyne A transfer membrane (Pall Ultrafine Corp.) asdescribed before (22).

DNAsequencing. An appropriaterestriction fragment was subcloned into both M13mplO and M13mpll and sequenced by usingthedideoxynucleotidechain termination procedure (15, 20).

RESULTS

Detection of an HSV-1 strain with a variation around the left

Us-Rs

junction. Restriction fragment length polymorphism of HSV-1 strains wasanalyzed by using restriction endonu-cleasesrecognizing4-bp motifs (25). It became clear that the HSV-1 strain termed B3 differed from other HSV-1 strains with respect to the length ofrestriction fragments deriving from the region spanning the

Us-Rs

junction. The DNAs of strain B3 and strain SP23 (asastandard)weredigested with TaqI andMboI (isoschizomer ofSau3AI), electrophoresed in a 5% acrylamide gel, and transferred to a nylon mem-brane. The DNAs on the membrane werehybridizedwitha

32P-labeled 0.14-kilobase (kb) TaqI-Sau3AI DNA fragment containingtheright

Us-Rs

junction, which corresponded to the Ta2-Sa2fragment inFig. 1(lanes 1 and 2 in Fig. 2). The fragment of about 0.14 kb, corresponding to the region containingtheright

Us-Rs

junction (Ta2-Sa2 in Fig. 1), was detected in thedigested products of both B3 and SP23. The 0.15-kbfragment, correspondingto theregion containing the left

US-Rs

junction [Tal-Sal in Fig. 1 (3)], was detected in the digests of SP23 but was not present in those of B3. A fragmentof 0.20 kbwasdetected in thedigested products of B3. Therefore, a structural variation around the left

Us-Rs

junction is present inthe B3genome.

Structuresof theregions spanning

Us-Rs

junction of HSV-1 genomes. TheEcoRIHfragment of strain B3, which contains

1 2 3 4 M

-0.271 -0.234

- MO -0.194

-0.118

-0.072

FIG. 2. Southernhybridization analysesof DNAfragments con-taining Us-Rs junctions of HSV-1 genomes. Genome DNAs of HSV-1 strains SP23 (lane 1) and B3 (lane 2), 1- to 3-kb BamHI fragmentsof strainB3(containingrightUs-Rsjunction) (lane 3),and 4- to 7-kb BamHI fragments of strain B3 (containing left Us-Rs junction)(lane 4) weredigested withTaqI and MboI.Thedigested DNAswere electrophoresed in an5%acrylamide gel, transferred to a Biodyne A transfer membrane, and hybridized with 32P-labeled 0.14-kb Taq1-Sau3AI DNA fragment containing right Us-Rs junc-tionof HSV-1 strain Patton[Ta2-Sa2 in Fig. 1 (3)]. Lane M, Marker mixture ofHaellI fragments of (X174 DNA. Sizes of the fragments areshown in kilobases.

allof

Us

and a part of

Rs,

wascloned into XgtWES (26).The

recombinant phage was termed XEH3. The two TaqI-Sau3AI fragments of 0.14 kb and 0.20 kb, which were assumed to correspond to the regions containing the right

and left

Us-Rs

junctions, respectively, were prepared from

XEH3 DNAs and cloned into both M13mplO and M13mpll. Thenucleotidesequencesof the fragments weredetermined by usingthedideoxynucleotide chain termination procedure (Fig. 3 and 4). In the 0.20-kb fragment containing the left

Us-Rs

junction of B3, a duplication of about 40 bp was identified. The presence of the duplication was assumed to be the cause ofelongation of the region spanning the left

Us-Rs

junctionin B3(lane 2 in Fig. 2).

The known nucleotide sequences around the

Us-Rs

junc-tions of strains 17 and Patton werecompared [Fig. 5 (1 and 2)] (13, 14, 16, 31). With respecttothenucleotide sequence on the

Rs

side, strain 17 was similar to strain Patton. However, there weredifferences in the nucleotide sequence onthe

Us

side. The first threeorfour residueson theright end of

Us

differed between the two strains [Fig. 5(1R and 2R)]. The A of initiation codon ATG of gene US12 is the eighthresiduefromtheright

Us-Rs

junctionin strain 17 and theseventh residue in strain Patton[Fig.5(1R and2R)]. The

Us-Rs

junctionin thesestrainswasdeterminedsimplyasthe point over which the homology between the two

Rs

se-quenceswas nolonger maintained. To search forregulation in thestructureof

Us-Rs

junctions,thenucleotide sequences around the

US-Rs

junctions oftwoother HSV-1 strains, B4 and B9, were determined [Fig. 5 (3 and 4)] and compared with those of strains 17and Patton. If the

Us-Rs

junctions

are definedas the pointover which the homology between thetwo

Rs

sequenceswas nolongermaintained,thelocation of thejunction would not be in a form common to HSV-1 strains.Thus,Iintroduced the concept ofjunctionstretchto define theregions spanning the

Us-Rs

junctions in the form commonto anyHSV-1 strain. Construction of thejunction

on November 10, 2019 by guest

http://jvi.asm.org/

[image:2.612.65.303.73.229.2] [image:2.612.379.505.76.241.2]

(3)

(1) (2)

No. GAT C G A T C No.

*ws

;

W10

26 G-- 1

G -11

U

12

C_C..-*

-12

-55 C- _

* _ -~~A-25

-45 G_.

-44

T---_ _A -44

-- -C -45

-25T--~ump* G -5 5

-25

T-_

--

-G

12

_~

-12 G-_ S-C 26

-11 C _ _

-1 G-

-r

1 G--_

Rs

10

G-Rs

us

FIG. 3. Autoradiographs showing nucleotide sequences of the

novelduplicationonthe leftUs-Rsjunction of HSV-1 strain B3. The

0.20-kb TaqI-Sau3AI fragmentofXEH3, containingthe left Us-Rs junction of strain B3, was cloned in M13mplO (panel 1) and

M13mpll(panel 2) andsequenced bythedideoxynucleotide chain termination method. The products were separated in a thin 6%

polyacrylamide-urea gel. The nucleotide numberswerederivedby comparisonwith thesequencereported byMcGeochetal.(13, 14) with the referencepoint atthe left Us-Rsjunction. The regions of

novelduplication and junction stretchwerenumberedwithaminus (-1to -55).The concept ofjunction stretchwasproposedin this

work, and the detailsaredescribed in thetext.

stretch is

Rs

side5'-(GorAstretch)AGC-3'

Us

side[Fig. 5 (5)].Thelengthof the GorAstretch variedfrom 3bp (right

Us-Rs

junctionof strainB9)to 10bp (left

Us-Rs

junctionof strainPatton) in the four HSV-1 strains examined (Fig. 5). Thejunctionstretchwasconsideredapartof

Rs

withregard to derivation. The nucleotides in thejunction stretch were

numbered and extended from the residuecontiguous to

Rs

[numbered -1 inFig. 5(5Land5R)]. Divergenecefrom the

consensus sequence of thejunction stretch was found at residue 1 in the left

Us

side of strain 17[Fig. 5(1L)] andat

residue 4 in the left

Us

side of strain B4 [Fig. 5 (3L)]. The nucleotide sequences encompassing the junction stretch

were conserved among the HSV-1 strains and could be numbered

uniformly.

The

Rs

side sequence

adjoining

the

junction

stretchstarted from G

[numbered

1 in

Fig.

5

(5L

and SR)]. The

Us

side sequence

adjoining

the left

junction

stretch started from C

[numbered

12 in

Fig.

5

(5L)]

and ended atA

[numbered

12972 in

Fig.

5

(5R)]

onthe

opposite

side of

Us

adjoining

the

right junction

stretch. The number-ingsystemfor

Rs

and

Us

wasthe same asthat of McGeoch etal.(13)and McGeochetal.(14),

respectively.

The

regions

which had been regarded as terminal parts of

Us

in the

junction

stretch in this work

(Fig.

5). This alteration

placed

the A of translationalinitiation codon ATG of gene US12at the first basein

Us

from the

right junction

stretch.

Fixation of theduplicated regionontheleft

Us-Rs

junction

in strainB3.Nucleotidesequence

analyses

ofarecombinant phage clone

carrying

the EcoRI H

fragment

of strain B3 indicated the presence ofthe

duplicated

region

on the left

Us-Rs

junction

ofstrain B3

(Fig.

3 and

4).

To confirm the

fixation ofthe

duplication

on the left

Us-Rs

junction,

B3 genome DNAs were

analyzed directly.

DNA of strain B3 was digested with BamHI and electrophoresed in a 0.8% agarose

gel.

Two agarose

gel

blocks, containing

the BamHI

fragments

of1to3kb

[including

the2.0-kbBamHI

fragment

spanning

the

right

Us-Rs

junction (Ba3-Ba4)

in

Fig.

1

(2)]

and the BamHI

fragments

of4to7 kb

[including

the5.5-kb BamHI

fragment spanning

theleft

Us-Rs

junction (Bal-Ba2)

in

Fig.

1 (2)],

respectively,

were

prepared.

DNAsextracted from each agarose

gel

block were

digested

with

TaqI

and

MboI,

electrophoresed

ina5%

acrylamide

gel,

transferredto a nylon

membrane,

and

hybridized

with the 32P-labeled 0.14-kb

TaqI-MboI

fragment

containing

the

right

Us-Rs

junction (lanes

3 and 4 in

Fig. 2).

The 0.14-kb

fragment,

which was assumed to spanthe

right

Us-Rs

junction,

was detected

only

in

digests

ofDNAsfromthe agarose

gel

block

containing

the BamHI

fragments

of 1 to 3 kb

(including

Ba3-Ba4

fragment

with

right

Us-Rs

junction).

The 0.20-kb

fragment having

the

duplicated region

wasdetected

only

in the

digests

ofDNAs from the agarose

gel

block

containing

the BamHI

fragments

of 4 to 7 kb

(including

Bal-Ba2

fragment

withleft

US-Rs

junction).

Itwasconcluded that the

duplicated region

ofstrain B3 was fixed at the left

Us-Rs

junction

andwas nottransferredtothe

right

Us-Rs

junction.

Effect ofthepresenceoftheduplicated

region

on

transcrip-tion and growth rate of strain B3 in culture cells. The

duplicated

region

ofB3existed within the transcribed

region

of gene US1

(IEmRNA-4)

and was outside the

predicted

protein-coding

regions (14, 17).

Toexamine the effectofthe presence of the

duplicated

region

on the

transcription

of gene

US1,

immediate-early

mRNAs

(IEmRNAs)

ofB3 and SP23were

prepared

and

analyzed by

Northern

blotting (Fig.

6)

(12,

21, 30).TheIEmRNA-3

(gene

IE175)

and IEmRNA-4 (gene

US1) produced

by

infection with B3were

comparable

tothose with SP23. The resultsindicated that the presence of the

duplicated region

on gene US1 hadno

significant

effect onthe

transcription

of gene USL.

Toexamine theeffect ofthe presenceofthe

duplication

on the growth of

HSV-1,

one-step

growth

curves of B3 and SP23 onVero cellswere constructed

(2).

The

growth

curve of B3was

comparable

tothatof

SP23; hence,

the presenceof the

duplicated

region probably

had no

significant

effect on

the

growth

of B3 in culture cells. DISCUSSION

HSV-1strain B3wasfoundtohavea

duplicated

region

on

its left

Us-Rs

junction,

and the nucleotide sequence

on November 10, 2019 by guest

http://jvi.asm.org/

[image:3.612.55.292.76.480.2]

(4)

TaqI

(1) (L) TCGAGGCGACCGGCGGCGACCGTTGCGTGGACCGCTTCC T

50 IRs+ Us 11

GC TCG TCGGGGCGGGGG GA AGCC AC TG TG GTC C TC CGGG AC

10 1 1 12 30

TRs , Us

(R) GC TCGTCGGG:GGGGAGC ATGTCGTGGGCCC TGGAAATGGC

10 1(1) (8) (30)

12979 12972 12950

TaqI o.uR-seq

(2) (L) TCGACGCGACCGGCGGCGACCGTTGCGTGCQACCGC T TCCT

50 lRs5 3534R-seq U-seq 20 11

GC TC GTCGGGGGGGGGGGAGCGACCGTTGCG TGG TC CGCT

' _12g

10 1 - 11u-seq-.Us -25 -30

TC C TGCrC

GTCGGT|GGGGGGGAG~CC

AC TG TGGTCC TC CG

-31 -44-45 -5512 26

TRs . Us

(R) GCTCGTCGGG[G13GGGGGAGCATGTCG TGGGC CCTGGAAAT

10 1-1 -10 12953

12972

FIG. 4. (1) Nucleotide sequences of the regions spanning left (L) and right (R)Us-Rsjunctions ofHSV-1strain17,published by McGeoch etal.(13, 14). The locations of U -Rsjunctions and the numbering system with thereferencepoint at the left U -Rsjunction were as described by McGeoch etal. (13, 14). Thenucleotidenumbers ofUsextending from right Us-Rsjunction are given in parentheses. Thetranslational initiationcodonATGof gene US12 is underlined. The Taql sitecorresponds toTal in Fig. 1 (3). (2) Nucleotide sequences of the regions spanningleft(L) andright(R) Us-Rsjunctions ofHSV-1 strain B3. The nucleotide numbers are the same as those used byMcGeoch et al. (13, 14), exceptforthe regions numbered with a minus, as described for Fig. 3. Two homologous sequences of R-seq (residue 33 onRsside toresidue -11) and U-seq(residue -12 toresidue -55) are indicated. The junction stretches are boxed. Theinitiation codon ATG of gene US12 is underlined.

ningtheduplicated regionwasdetermined [Fig. 4(2L)]. Two homologous sequences were present around thejunction. One isfrom residue33toresidue -11(R-seq),and the other isfrom residue -12 to residue -55 (U-seq). Both sequences were 44 bp in length and differed at two residues. One difference is between residue 20 on R-seq (A) and residue -25 onU (T). The otheris between residue 1 on R-seq(G) andresidue -44 onU-seq (T). Thebasesatresidues 20 and 1 on R-seq are the same as those at the corresponding residues in

TRs

of strain B3 and

Rs

of other HSV-1 strains. Theduplicationwasassumedtobegenerated byaninter-or intramolecular recombination between a point on

Rs

of a hypotheticalancestorofstrain B3 [I in Fig. 7 (1)] and the

Us

side end of the junction stretch ofthe ancestor [I' in Fig. 7 (1)]. Ahomology of two bases of GC, atresidues 35 and 34 in

Rs

[I of Fig. 7 (1)] and at residues -10 and -11injunction stretch [I' of Fig. 7 (1)] was present on the recombined region. After formation of the duplication, the bases of A at residue -25 and G at residue -44 in the U-seq copy were assumedtohave been substitutedbyT[IIin Fig. 7(2)]. The proposed pathway for generation of the molecule with the duplication from a molecule without the duplication is shown in Fig. 7. However, there remains the possibility that strain B3 might be derived from an HSV-1 ,molecule having dupli-cationsonboth sides of

Us

iftheduplication on the right side is removed.

The duplication in strain B3 was fixed on the left

Us-Rs

junctionandwas nottransferredtotheright side of

Us

(Fig. 2). I had already isolated two novel HSV-1 derivatives, SP22-4 and SP26-3, having duplications of DNA sequences containinggeneUS12 and theorigin ofDNAreplication (27). The duplicated sequences were amplified and transferred fromoneside to theother in

Us

asaresultofrecombinations between the region deriving from

Rs

on the duplicated sequencesand thecorresponding homologous region of

Rs.

Two structural features of theduplicatedsequenceof strain B3 are perhaps involved in fixation of the duplication, in comparison with those of SP22-4 and SP26-3. One is the shortnessof theduplicatedsequenceof strain B3,i.e.,44bp. Recombinationbetween thetwosequenceswith suchashort homology would berare.The other is the absence of origin ofDNAreplicationintheduplicated sequenceof strain B3. The regions around the

origin

are assumed to be unstable and recombinogenic. A DNA fragment with the origin, replicating separatelyfrom viral genomes, may beamplified

or transferred, as was interpreated for the VZV genome rearrangements by VlaznyandHyman (29).

The two copies of the inverted repeats enclosing one

unique sequencerecombine with each other in HSV-1 rep-lication (5, 9, 24, 28). Exchanges ofgeneticmaterialsby the recombinations function to maintain homogeneity between the two copies of the inverted repeats (13). Of the two homologous sequences presentonthe

Us-Rs

junctionof B3 (R-seq and U-seq), the nucleotide sequence of the R-seq

on November 10, 2019 by guest

http://jvi.asm.org/

[image:4.612.159.473.70.367.2]

(5)

IRs+ j.s

(i4 (L)

GTCGGGGGGGGGAAGCCAC

I

6 1 1 12

TRS4H

Us

(R) GTC

GGG

GGAGCATG

6 11 8

IRS |US

(2) (L) GTCGGG

WGGGGGGGGGAGC CAC

6 1 1 14

TRS

4-US

(R) GTCGGG

IAAAAG:CATG

6 11 7

IR5

US

(3) (L)

GTCGGGGGGGGGAGACAC

65 11 4 5

TRs+ US

(R)

G TCG

GG|GG

G A

GC|A

T G

II r -I I

11 6 5 1 1 5

IRS

.j

Us

(4) (L) G

TCGGGGGGGGAGCC

AC

6 11 9

TRs.j*

US

(R)

GTCGGGAAAAGCATG

6 1 1 7

IRs- Junction stretch *-U5

(5)

(L)

GTCGGG

(GorA)

AGCk AC

6 1-1 12

TRS

oJunction stretch *-

U5

(R)

GTCGGG

(G

orA)

AGC ATG

I I I

6 1-1 12972

FIG. 5. (1 to 4)Nucleotidesequencesof the regions spanning left (L) andright (R) Us-Rsjunctions of HSV-1 strains 17 (panel 1), Patton(panel2), B4 (panel3), and B9(panel 4). The sequences of strain 17 (panel 1) are from McGeoch et al. (13, 14), and the sequence aroundright Us-Rsjunction of strain Patton was from WatsonandVandeWoude (31). Other sequences were determined in the present work. The Us-Rsjunctions are placed at the point overwhich the homology between the two Rs sequences was no longer maintained. The nucleotidesare numberedextending inboth directions fromtheUs-Rsjunction. Junction stretches are boxed. (5) Consensus structurein andaroundthe Us-Rsjunction. The num-beringsystems arethe same as those used by McGeoch et al. (13, 14),exceptforthe region ofjunction stretch. The nucleotides within thejunction stretch are numbered with a minus symbol from the residue contiguousto R . Inthis definition- the A of initiationcodon ATG ofgeneUS12is located at the right end ofUs.

copywasthesame asthecorrespondingregion in

TR5

[Fig. 4(2L) and7(2)]. However, the U-seq copydjfferedfrom the corresponding region of

TRS

at tworesidues [Fig. 4 (2L) and 7 (2)]. These results suggest that there were no effective recombinational events to maintain homogeneity between U-seq copy and

TRs.

Thus, the U-seq copy functionally resembled

Us,

though the sequence was derived from

Rs.

A 1 2

_

-B 1 2

__

-28S

-

18S

FIG. 6. Northern blotting analyses of IEmRNAs of HSV-1 strains SP23(lanes 1) and B3(lanes2). IEmRNAswereprepared by infecting Verocell monolayerswith HSV-1 in medium containing cycloheximide,asdescribed before (30). Polyadenylated RNAwas

selected on an oligo(dT)-cellulose column,denatured with glyoxal anddimethylsulfoxide, electrophoresed on an 1%agarosegel,and transferredtoa Biodyne A transfer membrane(12, 21).The mem-brane washybridized witha32P-labeled0.48-kbSacI-Xholfragment fromahybridphage Dec36carryingtheEcoRIHfragment ofHSV-1 (26) (A). The bands detected with the0.48-kb fragment represent mRNAs ofgene US1 (IEmRNA-4). The membrane was then hy-bridized with 32P-labeled 0.35-kb SmaI fragment of hybrid phage XNDE54 carryingEcoRlfragment ofHSV-1class Idefective DNA (22) (B). The bands detected with the 0.35-kb fragment represent mRNAsofgene IE175 (IEmRNA-3).Thepositions of28S and18S calfliver rRNAs(Pharmacia,catalogno.27-2506-01)areindicated.

The

Us-like

character of the U-seq copy is relevant to the

fixation of theduplicated sequence onthe left side ofUs. There-is thehypothesis that

Rs

regionsofHSVandVZV wereformed by expansion of

Rs

of an ancestral herpesvirus through a nonhomologous recombination between two

Us

sequences arranged in an opposite orientation (7, 32). This approach related the structures of the S components of HSV-1 and VZV by a discrete number of steps involving expansion orcontraction of

Rs

(3). The discoveryof HSV variants with expanded repeat sequences and contracted uniquesequences,aspredictedby the hypothesis,supported the presence of a mechanism which mediates the process suggested by the hypothesis (1, 11, 23). In the hypothesis, the

US-Rs

junctions correspond to recombinedpoints. Un-der thedefinition of

Us-Rs

junctioninthiswork,the

Us

side end of the junction stretch forms one side of the

Us-Rs

junction, i.e., therecombinedpoint.The

R.

side ofthenew recombinedpoint in strain B3[residue -11inFig.4(2L)and 7 (2)], which forms the boundary between the R-seq copy andU-seq copy,wasthe

Us

side endofthejunction stretch. These results suggest that the

Us

side end of thejunction stretch possesses the property of recombinogenicity in-volved in theformation of

Us-Rs

junctions of HSV-1 strains and also in generation ofa novel duplication of strain B3. Other HSV variants generated by such a mechanism may also exist. However, except for strain B3 described in this work, structuralalterations indicating the

recombinogenicity

of the

Us

side end ofthejunction stretch have not been reported. In this sense, probably, the recombinationatthe

Us

side end of thejunction stretchis not sofrequent.

In

HSV-2,

the Aof the ATGasthetranslational initiation codonof the IEmRNA-5 gene (geneUS12)wasthefirst base in

Us

and defined the right

Us-Rs

junction. Whitton and Clements assumed that the ATG acts asabufferto prevent

on November 10, 2019 by guest

http://jvi.asm.org/

[image:5.612.374.498.76.228.2] [image:5.612.60.292.77.482.2]

(6)

IRS5

Junction stretch * Us

35 34 20 5 1 12

I -

---CGGCGA---A---TCGGIGGGGGGGGAGCICA---Recombination

I ----TC

GGG

GGGGGGGGAGCjC

A---5 1-1 -11 12

IR5s

Junction stretch * Us

IRs - US

5 1 -11 -12 -25 -44 -55 12

( 2) E ----TCGG|GGGGGG GGAUC GA--

---TCGGGGGGGGGGAGC|CA---R-seq *

46-T

[image:6.612.100.521.75.329.2]

U-seq *W

FIG. 7. Model for thegeneration of the duplicated region in HSV-1 strain B3. (1) Existence ofan ancestral HSV-1 strain withoutthe

duplicated regionwas assumed. Theoccurrence ofaninter- orintramolecularrecombination between the twoRs sequences on the2-bp

homology(residues 35 and 34 inmolecule I and residues -10 and -11 in molecule F') is shown. The numberingsystemisasdescribedfor

Fig. 5 (5). Junction stretchesareboxed. (2) Product of therecombinationalevent (molecule lI). The numberingsystemis the same asthat

forFig. 4(2L). ThetwohomologoussequencesofR-seq and U-seqareindicated.The base substitutionsatresidues-25 and-44onU-seq

wereassumedtohaveoccurredafter generation of the duplication. theexpansion of

TRs

(32). In theirdefinition,the AofATG

astheinitiation codon ofgeneUS12 of HSV-1 strain 17was

at the eighth residue in

Us

from the right

Us-Rs

junction. They supposed thatthe Aof the ATG of HSV-1 geneUS12 would come to be positioned 1 bp from the right

Us-Rs

junction,likethatofHSV-2, if equal though nonhomologous

crossoverweretakeplaceand thefirst ATGwereto actas a

bufferto prevent the expansionof

TRs.

In thedefinition in thepresentwork,the Aof the ATGastheinitiator codonof

gene US12 ofHSV-1 is the first base in

Us,

as is that of HSV-2 [Fig. 5 (5R)]. Presumably, the

Rs

of HSV-1 had alreadyexpandedtothepointat which the initiator ATG of

geneUS12 actedas abuffertopreventtheexpansionof

TRs.

It isprobable that the presence ofgeneUS12may function

advantageously forreplicationormaintenanceof HSV in the human body, although gene US12 is not required forvirus growth in cultured cells (1, 10, 23). With respect to the degree ofexpansion of

Rs,

it is assumed that HSV-1 and HSV-2 are located at the same step in the hypothetical descentfromanancestral herpesvirus (6).

The conceptofjunctionstretchwas introduced todrawa

constant line ofdemarcation between

Us

and

Rs

(Fig. 5). Two aspects in nucleotide sequences around the

Us-Rs

junction support the definition of

Us

side boundary of the junction stretch. Onewas the presence ofthree conserved

nucleotides, 5'-AGC-3', on the

Us

side boundary of the

junction stretch, and theotherwasthepresence of theATG initiationcodononright-side boundaryof

Us.

However,the

Rs

sideboundaryof thejunction stretch wasdefined solely

so as to seta common

Rs

sequence adjoining thejunction

stretch, by comparing the nucleotide sequences of four

HSV-1 strains. Thus, the

Rs

side boundary of thejunction stretch is not so certain as the

Us

side boundary. The

conceptofjunctionstretchmadefeasible the definition of the regions spanningthe

Us-Rs

junctioninthe formcommonto

anyHSV-1 strain [Fig. 5(5)]. For example,thediscrepancy

between strains 17 and Patton in the previously determined distancefrom the

Us-Rs

junction tothe translational initia-tion codonofgeneUS12could be eliminatedby makinguse

of the concept ofjunction stretch [Fig. 5 (1Rand 2R)] (16, 31). Thelengthand basecompositionof thejunctionstretch

are variable among HSV-1 strains and also between two

Us-Rs

junctionsofanHSV-1strain(Fig. 5).Thepresenceof

anadditional44-bpsequenceonthejunctionstretch of strain B3had no serious effects onviral replication. Thejunction stretchesseemtobetolerant of such variations. The loss of uniformitybetweenapairofjunctionstretches derivedfrom

one strain isregarded as a

Us-like

property of thejunction

stretch, althoughthejunction stretches wereassumedtobe derived from

Rs.

Thepolymorphisminnucleotidesequences

ofjunction stretches may serve as physical markers for genetic analyses ofthe HSV-1 genome.

ACKNOWLEDGMENTS

I thank M. Ohara forhelpful comments.

A partofthis workwassupported bygrants from theMinistryof

Education, Science andCulture ofJapan. LITERATURE CITED

1. Brown, M., and J. Harland. 1987. Three mutants of herpes simplex virus type 2: one lacking the genes US10, US11 and

US1andtwoinwhichRshasbeenextendedby6kbto0.91map unit with loss ofUs sequences between0.94 and the Us/TRs junction.J. Gen. Virol. 68:1-18.

2. Dargan, D. J., and J. H. Subak-Sharpe. 1985. The effect of

triterpenoid compoundson uninfected and herpes simplex

vi-(1 )

on November 10, 2019 by guest

http://jvi.asm.org/

(7)

rus-infected cells in culture. 1. Effect on cell growth, virus particles and virus replication. J. Gen. Virol. 66:1771-1784. 3. Davison, A. J., and D. J. McGeoch. 1986. Evolutionary

compar-isons of the Ssegments in the genomes of herpes simplex virus type 1 andvaricella-zoster virus. J. Gen. Virol. 67:597-611. 4. Davison, A. J., and J. E. Scott. 1986. The complete DNA

sequenceof varicella-zoster virus. J. Gen. Virol. 67:1759-1816. 5. Davison, A. J., and N. M. Wilkie. 1983. Inversion of the two segments of the herpes simplex virus genome in intertypic recombinants. J. Gen. Virol. 64:1-18.

6. Gentry, G. A., M. Lowe, G. Alford, and R. Nevins. 1988. Sequence analyses of herpesviral enzymes suggest an ancient origin for human sexual behavior. Proc. Natl. Acad. Sci. USA 85:2658-2661.

7. Honess, R. W. 1984. Herpessimplex and ''the herpescomplex": diverse observations and aunifying hypothesis. J.Gen. Virol. 65:2077-2107.

8. Honess, R. W., and D. H. Watson. 1977. Unityanddiversity in the herpesviruses. J.Gen. Virol. 37:15-37.

9. Knipe, D. M., W. T. Ruyechan, B. Roizman, and I. W. Halli-burton. 1978. Moleculargenetics of herpes simplex virus: dem-onstration of regions of obligatory and nonobligatory identity within diploid regions of the genome by sequence replacement and insertion. Proc. NatI. Acad. Sci. USA 75:3896-3900. 10. Longnecker, R., and B. Roizman. 1986.Generation of an

invert-ing herpes simplex virus 1 mutant lackingthe L-S junction a

sequences, an origin of DNA synthesis, and several genes includingthose specifying glycoprotein E and the a47 gene. J. Virol. 58:583-591.

11. Maclean, A. R., and S. M. Brown. 1987. Deletionand duplica-tion variants around the long repeats ofherpes simplex virus type 1 strain 17. J. Gen. Virol. 68:3019-3031.

12. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: alaboratory manual, p. 200-201. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.

13. McGeoch, D. J., A. Dolan, S. Donald, and D. H. K. Brauer. 1986. CompleteDNAsequenceof the short repeat region in the genome of herpes simplex virus type 1. Nucleic Acids Res. 14:1727-1745.

14. McGeoch, D. J., A. Dolan, S. Donald, and F. J. Rixon. 1985. Sequencedetermination andgeneticcontentofthe short unique region in the genome of herpes simplex virus type 1. J. Mol. Biol. 181:1-13.

15. Messing, J. 1983. New M13 vectors for cloning. Methods Enzymol. 101:20-78.

16. Murchie, M.-J., and D. J. McGeoch. 1982. DNA sequence analysisofanimmediate-earlygeneregionof theherpes simplex virus type 1 genome (map coordinates 0.950 to 0.978). J. Gen. Virol.62:1-15.

17. Rixon, F. J., and D. J. McGeoch. 1985. Detailed analysis of the mRNAs mappingin the short unique region of herpes simplex virus type 1. Nucleic Acids Res. 13:953-973.

18. Roizman, B. 1979. The structure and isomerization of herpes

simplex virus genomes. Cell 16:481-494.

19. Roizman, B., L. E. Carmichael, F. Deinhart, G. de-The, A. J. Nahmias, W. Plowright, F. Rapp, P. Sheldrick, M. Takahashi, and K. Wolf. 1981. Herpesviridae: definition, provisional

no-menclature, and taxonomy. lntervirology 16:201-217.

20. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequenc-ing with chain-terminatsequenc-ing inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467.

21. Umemura, T., K. Umene, J. Nishimura, Y. Fukumaki, Y.

Sakaki, and H. Ibayashi. 1986. Expression of c-myc oncogene during differentiation of human burst-forming unit, erythroid (BFU-E). Biochem. Biophys. Res. Commun. 135:521-526. 22. Umene, K. 1985. Variabilityof theregionof theherpessimplex

virus type 1 genome yielding defective DNA: Sinml fragment polymorphism. lntervirology23:131-139.

23. Umene, K. 1986. Conversion ofa fraction ofthe unique

se-quence to partofthe inverted repeats in the S component of the herpes simplex virus type 1 genome. J. Gen. Virol. 67:1035-1048.

24. Umene, K. 1987.Transition from a heterozygous to a homozy-gous stateof apairof loci in the inverted repeat sequences of the L component of the herpes simplex virus type 1 genome. J. Virol. 61:1187-1192.

25. Umene, K. 1987. Restriction endonucleases recognizing DNA sequencesof four base pairsfacilitate differentiationof herpes simplex virus type 1 strains.Arch. Virol. 97:197-214.

26. Umene, K., and L. W. Enquist. 1981. A deletion analysis of lambda hybrid phagecarrying the Usregion of herpessimplex virus type 1 (Patton). I. Isolation of deletion derivatives and identification ofc/i-like sequences. Gene 13:251-268.

27. Umene, K., andL. W.Enquist. 1985. Isolation of novelherpes simplex virus type 1 derivatives with tandem duplications of DNA sequences encoding immediate-early mRNA-5 and an

originofreplication.J. Virol. 53:607-615.

28. Varmuza,S.L.,andJ. R. Smiley. 1984. Unstableheterozygosity in a diploid region of herpes simplex virus DNA. J. Virol. 49:356-362.

29. Vlazny, D.A., and R.W.Hyman. 1985. Errantprocessingand structural alterations of genomes present in a varicella-zoster virus vaccine. J. Virol. 56:92-101.

30. Watson, R. J., M. Sullivan, and G. F. Vande Woude. 1981. Structures oftwo spliced herpes simplex virus type 1 immedi-ate-early mRNAs which map at thejunctions oftheunique and reiterated regions of the virus DNA S component. J. Virol. 37:431-444.

31. Watson,R.J.,andG. F.Vande Woude. 1982. DNAsequenceof

animmediate-early gene (IEmRNA-5) of herpes simplex virus type 1. Nucleic Acids Res. 10:979-991.

32. Whitton, J. L.,andJ. B. Clements. 1984. Thejunctionsbetween the repetitive and the short unique sequences of the herpes simplexvirusgenomearedeterminedby thepolypeptide-coding regions oftwospliced immediate-earlymRNAs. J.Gen. Virol. 65:451-466.