Functional domains within the a sequence involved in the cleavage-packaging of herpes simplex virus DNA.

JOURNAL OFVIROLOGY, Sept. 1986, p. 605-618 Vol.59, No. 3 0022-538X/86/090605-14$02.00/0

Copyright© 1986, AmericanSocietyforMicrobiology

Functional

Domains

within

the

a

Sequence

Involved in the

Cleavage-Packaging of Herpes Simplex Virus DNA

LOUIS P. DEISS, JOANY CHOU, AND NIZA FRENKEL*

DepartmentofMolecular Genetics andCellBiology, The University of Chicago, Chicago, Illinois60637 Received24February 1986/Accepted 16May 1986

Newlyreplicated herpes simplexvirus(HSV)DNAconsists of head-to-tailconcatemerswhicharecleavedto generate unit-length genomes bounded by the terminally reiterated a sequence. Constructed defective HSV vectors(amplicons)containing aviral DNAreplicationoriginand the asequencearesimilarly replicatedinto largeconcatemers whicharecleavedat asequencespunctuatingthejunctionsbetweenadjacent repeat units, concurrentwith thepackaging of viralDNAintonucleocapsids.Inthepresentstudywetested theabilityof seed amplicons containing specific deletions in the a sequence to become cleaved and packaged and hence be propagated in virus stocks. These studies revealed that two separate signals, located within the Ub and Uc elements ofthe a sequence, were essential foramplicon propagation. No derivative defective genomeswere recovered from seedconstructs which lackedtheUc signal.Incontrast,propagation ofseed constructslacking the Ubsignal resultedin theselectionof defective genomeswithnoveljunctions, containing specific insertions of a sequences derived from the helper virus DNA. Comparison of published sequences of concatemeric junctions of several herpesviruses supported a uniform mechanism for the cleavage-packaging process, involving the measurement from two highly conserved blocks of sequences (pac-l and pac-2) which were homologous tothe required Uc and Ub sequences. These resultsform the basis for general models for the mechanism ofcleavage-packaging of herpesvirusDNA.

The 150-kilobase (kb) DNA genomes of standard herpes

simplex viruses1and 2(HSV-1andHSV-2)arecomposed of two components, L and S, each consisting of unique se-quencesbracketedby invertedrepeats,aband b'a' flanking UL, andacand c'a' flankingUs. The L and S components invert relative to each other, yielding four genomic isomers

(reviewed in reference 25). The L-S junctions and the L termini of all four isomers contain a variable number of tandemly reiterated a sequences. In contrast, eachoftheS termini contains only a single copy ofthe a sequence (15,

38). Thestandard virus genome can thus berepresented by anb-UL-b'a'mc'-Us-ca, with n and m varying from 1 to greaterthan 10.

The linear viral DNA molecules circularize soon after

infection (22), and newly replicated viral DNA molecules

consist of large head-to-tail concatemers,arisingmost prob-ably by rolling-circle replication (2, 3, 12). The large concatemers are subsequently cleaved within a sequences situatedatthejunctions between adjacentviral genomes (6,

21, 25). Studies with temperature-sensitive mutants

sug-gestedthat thecleavageprocess maybe linked topackaging

(13, 14, 23; S. Bachenheimer, personal communication). Defective virus genomes present inseriallypassaged virus stocks are typically composed of head-to-tail repeat units,

eachcontainingaDNAreplication originand the a sequence

(reviewed in reference 8). In the presence of helper virus,

multimeric defective genomes can also be generated from seed plasmids (amplicons) containing a DNA replication

origin and the a sequence, which is required for con-catemericcleavageandpackaging(7, 28, 29, 31, 32, 36). Any

pairofa sequencesalong the concatemeric molecule could become cleaved, resulting in the packaging of monomers,

dimers, and higher multimeric forms of defective genomes into nuclear capsids. However, only full-length

(approxi-* Correspondingauthor.

mately 150kb) defective genomes were found to be present in cytoplasmic virions (37). Like the L-Sjunctions in stan-dard viral DNA, thejunctions separatingthe repeatunits of defective genomes contain a variable number oftandemly

reiterated a sequences, which are cleaved to generate a variable numberof a sequences at one terminus and

predom-inantly asingle a sequence atthe other terminus(7). TheasequencesofHSV-1 and HSV-2strainsrangeinsize

from 250 to 500 basepairs (bp)andcontainbothunique(U) anddirectly repeated (DR)elements(6,18, 19, 35). The size differences predominantly reflect variability in the copy number of the DR elements. For example, the 501-bp a sequenceofHSV-1 (F) is represented by

DR1-Ub-(DR2)19-(DR4)3-Uc-DR1, whereas the 264-bp a sequence of the

Justinstrain containsonlytwocopiesof DR2, asinglecopy

of DR4, and two copies of another reiteration designated

DR3.5 (18, 19). In adouble a (baac)junction the tandemly reiterated a sequences share the intervening DR1 (20 bp) element (6, 19, 21). Furthermore, in the F strain, the L

terminus (ba) contained 18bp plusone 3' overhang base of the DR1 sequence, whereas the S terminus (ca) carried the

remaining 1 1/2 bp. These findings led Mocarski and Roiz-man(21) to propose that the double ajunctions formedthe substrate for cleavage. In this regard, the cleavage ofviral DNA concatemers represented a puzzle inasmuch as,

al-though the number of a sequences at the junctions is variable, the predominantjunctions in both standard and defective viral genomes containa single a sequence (7, 15, 38). We haverecently shown that thejunctions containinga singleasequencecanindeedserve asthe targetfor

cleavage-packaging. Furthermore, thecleavage process itself appears to be accompanied by the amplification ofan a sequence, inasmuch as molecules carrying junctions of the structure xayarecleaved togenerate xaand ya termini (7).

The presentinvestigationwasdesigned to further mapthe signals within the a sequence which are involved in the 605

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606 DEISS ET AL. A.

E PE Ba

)oril' I|test

a

helper virus test DNA construct

I

Cotranstect

I

Propagate virus

Passage 2

DNA 4Restriction

enzymes

E1 CM helper

+ ?detective

B.

an L

L. S

lb

3UL ;bamc ca

....~~~~~~~~~~~~~~~~~~...

Construct Ub Uc

DR1 Ub (DR2)22 (DR4)3 Uc DR1

.F---.---

*, *,A-2 + + SA DBA S S S B BSA E

(DR2),

-D_

{I

A

-e_-B -e_-B

.s*

S S

b-S S

A

...XB.

A A

A A

S

Oa

B I K:

A-i + +

propagatior

H-i +A

B-i +

-C-i +

-E-i +

-I-l

A-C- D-1

L

+ [+helper]

S

G-1 - + t-helper]

FIG. 1. Propagationtest and structureoftest constructs. (A) Scheme of the propagation test (28). (B) Structure of the test a sequences. Top line, schematic representation of the standard viral genome and the location of the a sequence in the L-S junction. Second line, arrangement of the directly repeated (DR) and unique (U) elements in the a sequence ofHSV-1 F DNA as described by Mocarski and Roizman (19). Shownarethe sites forBamHI (Ba),SstII (S),ApaI(A),DraI (D),BssHII (B), andEcoRI(E). The test a sequences (4) are contained between theBamHIandtheEcoRI sitesin thegeneralconstructshown inpanelA.Symbols:+or-,presence or absence of intact UborUcelements;A,deletionswithinthe Ub or Ucelements.The resultsofthepropagation tests are described as efficient propagation(+), lack of propagation (-),andpropagationatlowefficiency coupledtoselectionof genomeswith insertedhelpera sequence (+ helper).

cleavage-packaging process. Analyses ofthe products

re-sulting from the propagation ofseed amplicons containing

various deleted versions of the a sequence revealed the presence of two regions within the a sequence that are

essential for efficient propagation. The results also revealed aspects of the mechanism by which single a-containing junctions become cleaved.

MATERIALS AND METHODS

Cells and viruses. HEp-2 cells and Vero cells were

ob-tained from American Type Culture Collection, Rockville, Md., rabbit skin cells and HSV-1 strain F were obtained

fromB. Roizman, University of Chicago,and HSV-1 strain Justin was obtained from A. Sabin, who was then at the

University of

Cincinnati,

Cincinnati, Ohio.

Derivation of plasmids. Cloning was done as previously described (28) with Escherichia coli DH1 (gift of D.

Hanahan, Cold Spring Harbor Laboratories, Cold Spring

Harbor,

N.Y.). Restriction enzymes and T4 DNA ligase

were purchased from New England BioLabs and Bethesda Research Laboratories. Calf alkaline phosphatase was pur-chasedfromBoehringerMannheimBiochemicals,and

poly-nucleotide kinase was purchased from P-L Biochemicals,

Inc. All enzymes wereusedasrecommendedbythe suppli-ers. The test constructs contained the sets of deleted a sequenceswhichwereconstructedby Chou and Roizman (4) as described in detail by them, starting from a 537-bp a sequencefromHSV-1 strain F. Briefly, theconstructs were made inpUC9 and contained3 bpof b sequences and 12bp

ofc sequences in addition to the a sequences. A series of deleted a subclones were made (Fig. 1B). In the present

study,a 1.90-kb EcoRIfragment, containingtheHSV DNA

replicationorigin

ori-i'

(oris;

20,31,36)wasinserted between theEcoRI sites of the deleted a clones derived by Chou and

Roizman (4), yieldingthegeneral structure in Fig. 1A. The inserted EcoRIfragment contained a0.26-kb EcoRI-to-PvuII segmentofpKC7(24)and the1.64-kbori-' PvuII-to-EcoRI segment, derived from map coordinates 0.856 to 0.867 of HSV-1 F DNA. This ori-i' segment was derived originally frompRB373(20) andwastransferred topKC7 as described in our previous study (7). The construct A-2 contained the intact a sequence(537 bp) andwasderived by the insertion of ori-i' into pRB3119; clone A-1 was picked as a colony during thepreparation of the A-2construct.H-1wasderived frompRB3613 which containedaBssHIIdeletioninUc; B-1 wasderived from pRB3384 whichcontained a SstII deletion fusingthe second DR4 elementto theDR1element; C-1was derived from pRB3385 which contained a SstII deletion fusing the first DR4 element to DR1; E-1was derived from pRB3387 which contained an ApaI deletion fusing the Ub element to DR1; I-1 was derived from pRB3614 which contained a BssHII deletion in Uc as well as an Apal deletion in Ub; D-1 was derived from pRB3386 which contained an ApaI deletion in Ub; G-1 was derived from pRB3389 which contained a SstII deletion fusing the DR1 element to the third DR4 element. Plasmids pRB3386, pRB3613, and pRB3614 underwent intermediate recloning

intopUC9 before theori-i' segmentwas inserted,to obtain a uniform orientation of the deleted a segments in the plasmids. Asisterset ofplasmids was constructed with the EcoRI origin fragment in the opposite orientation. The constructs G-2, G-3, and G-4 were rescued from

[image:2.612.135.489.73.291.2]

G-1-propagated defective genomes, as described in the text. Constructs G-5 and G-6wereconstructed (to eliminate Dral restriction sites) by insertion of a PvuII fragment which contained ori-i' and the G-deleted a sequence into a DraI-cleaved pBR322 plasmid. The PvuII fragment was taken from the sister clone ofG-1, which contained the EcoRI ori-i' segment in reverse orientation to that in G-1. The

...

l

*----.---

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HERPES SIMPLEX VIRUS CLEAVAGE-PACKAGING 607

- A-I B-I C-1 Etl H-l I-I D-1 G-1 .A-iG-l

(Jusi.aaa ba ba ba ba a b a b a a

i v l * - § helper j

~ ~ ~ v

a -

-ti

0-0323

0297

orX90.

1 2

8.a

0-.112C*a -_aOta303 a 04&o 278 *a.

-***or;90 *t

5 8 11 I5

17 18

FIG. 2. Analyses of DNA from transfection-derived virus

stocks.EcoRI digests of32P-labeled DNAfrom cells infected with passage2ofatransfection receivinghelperHSV-1 Justin alone (lane

1)orwithpassage2of duplicatecotransfections(aand b) receiving helpervirus along withthe test constructs shown(lanes 2 through

16). Thegroupof fragments visible in lane 1 correspond tohelper

virusDNAfragments. Shownare bandscontaining the replication

origin (ori), the intact single and double ajunctions (a and a-a,

respectively), thetestasequences (a*), and the chimeric junctions

(a*-a).Fragments arisingfrom digestion ofthegenerateddefective genome are marked by their sizes in kilobases. Lanes 17 and 18

show a shorter autoradiographic exposure of lanes 2 and 15,

respectively.

PvuII-DraI fusion resulted in the loss of both restriction sites. G-5 and G-6 differed in the orientation ofthe PvuII insert.

Cotransfectionandpropagationof virus stocks. Cotransfec-tion of rabbit skin cells with helper virus DNA and test

constructs was done as previously described (28) and as outlined in Fig. 1A.

Analysis of infectedcell DNA. Vero cell cultures (25 cm2)

were infected withpassage 2 virus and labeled with

32Pi

as

previously described(28). At 20 h postinfection, DNA was

extracted from theinfected cells by lysis with amixture of sodium dodecyl sulfate and proteinase K which was fol-lowed byphenol-chloroform extraction and ethanol precip-itationaspreviously described(28). Following digestionwith the appropriate restriction enzyme, the DNA was

electro-phoresed in 0.5% agarose gels; 1/50 of the DNA produced

froman infected 25-cm2culture wasused perlane.

Nucleotidesequenceanalysis. For thenucleotide sequence

analysestheconstructsG-2, G-3, and G-4wereeach cleaved

with either EcoRI-PstI or BamHI-SstI. The appropriate

fragments containing theasequencewereelectroeluted, and theEcoRI orBamHI sites wereend labeled with polynucle-otide kinase. The nucleotide sequence was determined by theMaxam-Gilbert technique (17).

RESULTS

Propagation of amplicons containing deletions in the a

sequence.As reviewedabove, thea sequenceofHSV DNA

wasfoundto be essential forthe cleavage and packaging of viral DNA and therefore for the propagation of defective

genomes in serially passaged virus stocks. To map the

cleavage-packaging signal(s)within theasequencewetested

the ability of test constructs containing specific deletions in the a sequence to becomepropagated invirusstocks. Figure

1Asummarizes the general structure of these test constructs and the features of the propagation assay. All of the test constructs contained the replication origin

ori-i'.

In addi-tion, they contained either an intact a sequence derived from HSV-1 F or various deleted a segments derived from it (Fig.

1B). The intact a as well as the deleted a segments were derived from plasmids constructed previously by Chou and Roizman (4), starting from the cloned a sequence ofHSV-1

F DNA. All constructs used in the present study were designated according to the a sequence which they con-tained; the lettering system of the original study of Chou and Roizman (4) was used. The constructA-1, which was used as a control for this set of propagation tests, contained an a sequence of 285 bp, i.e., close to the average size of the deleted test a constructs (range 86 to 497 bp). The 285-bp variant a sequence contained only a single DR2 element and in that respect resembled the shorter type a sequences found in some HSV-1 strains (6, 18, 35).

In the propagation tests, duplicate cell cultures (a and b) were cotransfected with helper HSV-1 Justin DNA and the control or test constructs. Virus stocks derived from these transfections were serially passaged, and

32P-labeled

DNAs

from

cells infected with passage 2 of the resultant series were analyzed by digestion with EcoRI (Fig. 2) and additional enzymes (not shown) to test for the presence of defective genomes generated from the seed constructs. As seen in the maps shown in Fig. 3 (e.g., for the D-1 andG-1

constructs),

two fragments were expected to arise byEcoRI digestion of concatemers generated by replication of the seed constructs: (i) a fragment of 1.90 kb, which contains the replication origin and was common to all constructs, and (ii) a variable-sized fragment containing the respective test a sequences.

The analyses revealed the following. (i) As expected, the control construct A-1 gave rise to abundant defective genomes composed of head-to-tail repeats of identical size to that of the input seed construct (represented by the 1.90- and 2.97-kb fragments; Fig. 2, lane 2). A minor proportion of the repeat units in the generated defective genomes contained tandemly reiterated a sequences, apparent as the "ladder bands" with increments of ca. 260 bp, extending from the

2.97-kb EcoRI band. (ii) Virus stocks derived from transfections receiving the constructs B-1, C-1, E-1, H-1, and I-1 did not contain authentically propagated defective genomes (Fig. 2, lanes 3 to 12). All of these constructs

containeddeletions affecting the Uc element, with the small-est deletion present in the construct H-1 (40-bp deletion

boundedbythe BssHII sites in Uc). (iii) Virus stocks derived fromcotransfections receiving D-1 and G-1 contained defec-tive genomes with repeat units identical to the input seed

constructs (yielding the 1.90-kb origin fragment and the fragments designated a* in Fig. 2, lanes 13 through 16).

However,the abundance of generated defective genomes in the population was relatively low compared with their

coun-terparts generated from the control construct A-1 (compare the relative intensities of defective and helper virus frag-ments in the short autoradiographicexposures showninFig. 2, lanes 17 and 18). In addition, the defective genomes contained significantproportions of repeats that were ca. 240 bplarger than the input seed plasmids (the 3.37-kb fragment in lanes 13 and 14 and the 3.03-kb fragment in lanes 15 and 16). As shown below, the increase in size resulted from the

insertion of an intact a sequence, derived from the helper virus DNA, within the repeat unit junctions. Consequently, these bands have been designated a*-a in Fig. 2.

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[image:3.612.65.284.72.275.2]

C

I

E

c

oR

I

a*

Oral

Dra

I

a-a*

E

co

RI

EB

Bp c

D- D

G1 D

E

L 312 1.90 3.12 1.90

2.78 1.90 2.78 1.90

E B E E B E EB

DDD D DDC D DOD

_

69 4A31 69 4.31

I

_.69

iI~

_{3.98 .69}I _3.98

69 2.97 i 162 69 2.97 i162

E0B 69i 297 }125 0691 297 1255

E

DB.69

E.

E©B E E®B

D D D E DD

ED

D DD

_a

a t + t"a+ a-

a+--a--33 11.90 3.36 1.90

3 0 3 1.90 3.03 1.90

D-1 G-1

D-1 G-1

D-1 G-1

0-1 G-1

FIG. 3. Structureof chimeric junctions. (A)Dral-digested32P-labeledDNAs from cells infected with passages 2 of transfections receiving HSV-1 (Justin)helper DNA alone (J) or the constructs G-1and D-1. The DNAs correspond to those shown in Fig. 2, lanes 1, 16, and 14, respectively.(B) structure ofthe two testconstructs. Shownare locations of theori-l'fragment (stippled area), the test asegment(a*) and the sites forEcoRI(E),BamHI (B),DraI (D),ClaI (C), andPvuII (P). (C) predicted structures of generated defective genomes with repeat unitscontainingthe original deleted a sequence (a*) or the chimeric repeats (a-a*) with inserted helper virus a sequence. Sizes of generated EcoRIandDraI fragmentsaregiven inkilobases forthe D-1-generated (above the line) and G-1-generated (below the line) concatemers. The DraI sitepresent in the Ub element of theinsertedhelper virus a sequence is circled.

Two conclusions can be reached on the basis of these

observations. First, the failure of the constructs lacking the Ucelement (Uc-) to be propagatedin virus stocks revealed that the sequences bounded by the BssHII sites within the Uc element were essential for cleavage-packaging. These

boundaries are based on the 40-bp deletion in the H-1 construct. Second, the low-efficiency propagation of D-1

(containing a 64-bp deletion in Ub) suggested that the sequences bounded by the ApaI sites in Ub affected the efficiency of amplicon seed propagation. In addition to

testingthe constructs above, we tested (in duplicate sets) the

corresponding a deletions in plasmids which contained the EcoRI 1.90-kb fragment (carrying the replication origin) in

the opposite orientation to that in Fig. 1A. The results of

these studies(data not shown)yieldedthe sameconclusions asabove.

Structure of the larger repeat units derived fromthe

Ub--Uc+ constructs. The experiments described in this and the next sections revealed that the larger repeats found in the D-1- and G-1-derived defective genomes (Fig. 2, lanes 13 through 16; band a-a*) contained an insertion of an a sequence derived from the helpervirus DNA. Specifically,

because the increased size of the repeat units reflected

changesin thefragment carrying theasequence(Fig. 2; data notshown), the larger repeats could be derived by tandem reiterations of the deleted a sequence or, alternatively, by

insertion of an a sequence derived from the helper virus DNA. To test these alternatives we took advantage of the fact that the input test constructs did not contain the Ub sequence and therefore lacked the DraI site (TTTAAA) at position 31 of the a sequence. TheDral enzyme cleavesin three locations within the bacterial plasmid portion of G-1

andD-1,generatingcommonfragmentsof 19 and 692 bp, and the variable (a-containing) fragments of 3.98 and 4.31 kb

(Fig.3). Self-amplification ofthe test a sequences in G-1 and D-1was predicted toresult in increased size of the variable bands. In contrast, insertion of the a sequence from the

Justin helperDNA should result in cleavage ofthese

frag-ments, owingto the presence ofa DraI sitein the inserted Ub sequence. In this case, two new fragments would be

generated, with sizes dependent on the extract site of insertion. The DraI cleavagepatterns(Fig. 3) revealed that the novel repeats indeed containedan additional DraI site,

consistent with an insertion ofan intact (Ub+) a sequence derivedfrom thehelpervirus DNA. In addition,the results showed that thehelpervirusa sequencewasadjacenttothe test (Ub-deleted) a sequence on the side proximal to Uc, creatingachimericdoubleajunction.Specifically,defective genomesgeneratedfrom theconstructsD-1andG-1 yielded a common0.69-kb Dral fragment generated from the

plas-mid region, the a-containing Dral bands of sizes 4.31 and 3.98 kb representing theinputDNA (see map labeleda* in

Fig. 3), and two novel fragments not present in

the

input seed,of sizes 2.97 and 1.62 kbforD-1,and 2.97 and 1.25 kb for G-1. The sizes of these novelfragmentswereconsistent with the insertions of helper a (Ub+ and hence DraI con-taining) occurring on the side proximal to Uc (see map labeled a-a* in Fig. 3).

Nucleotide sequence of the novel chimeric junction. To

further analyzetheeventswhich ledtotheformation of the chimericjunctions we shuttled the repeat units of the G-1-derived defective genomes back into bacteria and obtained the nucleotide sequence for three of the resultant clones.

Specifically, unlabeled passage 2 DNA from a

G-1-A_

A *- 2.97 DraI

1- -1.62

123

_ _

__-4.31

_0

3.98

00

-2.97

#& -1.62

-t.25

-0.69

X 2 3

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HERPES SIMPLEX VIRUS CLEAVAGE-PACKAGING

Inserted a

-'4i1 J ---'I" ?/ F

DR cDRI1 Ub DR2/3 S/4 Uc DRI

Sathl

CZWAG CCCGGGCtGCg CCGCCGCgc

(RauHI)... CCG

(BRaHI)...CCG

CCGCGGGGGGCCCGGGCTGC cg CCGCCGC

DR11

-(CGCTCCTCCCCc)19

TTMAAGGGCCGCGCGCGACCCCCGGGGGGTGTGTrCGGGGGGGGCCCGTTTT

TTTAAAGGGCCGCGCGCGACCCCcGGGGGGTGTGTTTCGGGGGGGGCCCGTTTT

(c(;CTCCTCCCC

Sstll

GCTCCCGCCCCGCCCCC _ ACGCC)3 CGGCCCCGCCCCC a ACGCC CGGCCCCGCCCCC AACGCC

tggggtctggc (CGCTCCTCCCC )2 (gtctgtgggtgggGCTCCTCCCC)2GCTCCCGCGGCCCCGCCCCC[cACGCC

--DR2-- - DR4

- DR353

CGCCGCGCGCGCGCACGCCGCCCGGACCGCCGCCCGCCTTTrrTGCGCGCCGCCC CGCCGCGCGCGCGCACGCCGCCCGGACCGCCGCCCGCCTTTTTTGCGCGCCGCCC

CGCCGCGCGCGCGCACGCCGCCCGGACCGCCGCCCGCCTTTTTTGCGCGCCGCCC

CGCCGCGCGCGCGCACGCCGCCCGGACCGCCGCCCGCCTTTTTTGCGCGCCGCCC

-a-Deleted al

cgc CCGCGCCGGGCCCGGGCTGC cgc CCGCGGGGGGCCCGGGCTGC ...(REcoRI)

cgc CCGCGGGGGGCCCGGGCTGC g CCGCGGGGGGCCCGGGCTGC

- *~ DR1

I

Inserteda_-.

MgCCGCCGC ic TTAAG CGCCGcCGCGACCCCCGGGGGGTGTGTTTCGGGGGGGGCCCGTTTT

Icg ICCGCCGCcgcTTTAAAGGGCCGCGCGCGACCCCCGGGGGGTGTGTTTCGGGGGGGGCCCGTTTT 1CgCCGCCGCI TrTAAAGGGCCGCGCGCGACCCCCGGGGGGTGTGTTTCGGGGGGGGCCCGTTTT

HSV-1(F) (CGCTCCTCCCCc)lg G-3/4 tggggtctggc (CGCTCCTCCCC )2 HSV-1(J) tggggtctggc (CGCTCCTCCCC )2

_ DR2 _

HSV-i(F) CGCCGCGCGCGCGCACGCCGCCCGGACC4 G-3/4 CGCCGCGCGCGCGCACGCCGCCCGGACC4 HSV-1(J) CGCCGCGCGCGCGCACGCCGCCCGGACC(

C.

HSV-1 (F) HSV-1 (J) HSV-1 (KOS) HISV-1 (17)

HSV-1 (USA-8)

chimera G-3/4

49/50 conserved

.T..TTTTGCGCGC

... TTTTTGCGCGC ... TTTTTGGCCGC

... ITT GOGCGC

... TITTTGCGCGC

... TTT GCGOGC

(cGCTCCTCCCC GCTCCCGCGGCCCCGCCCCC aACGCC)3

gtctgtgggtgggG... GCTCCCGCGGCCCCGCCCCCcACGCC

(gtctgtgggtgggGCTCCTCCCC)2GCTCCCGCGGCCCCGCCCCC cACGCC

__________ DR4

8

DR3.5.-CCGCCCGCCTTTTGCGCCGCCC cgc CCGCGGGGGGCCCGGGCTGC

GCCGCCCGCCrTTTTTGCGCGCCGCCC cgc CCGCGGGGGGCCCGGGCTGC ...(EcoRI)

CCGCCCGCCTTTT]rTGCGCGCCGCCCLg CCGCGGGGGGCCCGGGCTGC

Uc DR1

, 1a DRl-- I'I a -Ub

variable 19/20conserved variable 51/53conserved

cg5cccgc CCGCGGGGGGCCCGGGCTGC gccgccgcgc TTTMA ...

cgcCCg CCGCGGGGGGCCCGGGCTGC cgccgccgc TTTAAA

gcgcgcgc CCGCGGGGGGCCGGGCTGC ccgccgcccgcc TTTAAA ...

gcgcgcgc CCGCGGGGGGCCCGGGCTGC ccgccgccaccgc TTMA ...

cgccccgc CCGCGGGGGGCCCGGGCcGC ccgccgccgcgc TTTAAA ...

[image:5.612.136.483.71.455.2]

cgccccgc CCGCGGGGGGCCCGGGCTGC cgccgccgccgc TTTAA

FIG. 4. Nucleotidesequencesof the chimeric junctions andsequenceconservation in the Ub and Uc elements of different HSV-1strains. (A) Generalstructureof the chimericjunctions in clones G-3 and G-4. The Ub--Uc+asequencefrom theconstruct(denotedasdeleteda)

and the insertedasequence arearranged in tandem. The heavy line denotessequencesoriginating from F; thinner line denotes sequences originating from strain Justin (J). DR1*and DR4* indicatepartial elements thatarefusedattheSstll siteasinthe seed G-1construct. ?,A

region conserved between F and Justin; therefore, the site of transition from Justin to F sequences in Uc is unknown. (B) Nucleotide sequencesof theasegmentsin the authentically sized (G-2), and larger (G-3 and G-4)repeatsandthesequences of doubleajunctions of

HSV-1strains F(19, 21) and Justin (18). Sequences which differ in the Justin and F strainsareboxed. The locations of the EcoRl and BamHl sites inthepolylinkerareshown. Thedotted line in the DR3.5repeatindicatesareaofsequenceuncertainty. (C) Sequence conservation in the Ub and Uc elementsin differentHSV-1 strains and in the G-3 and G-4 chimericrepeats.Theregions proximaltothe DR1arevariable,

whereas thosefartheraway areconserved. Nucleotidesequences arefrom Mocarski andRoizman (19, 21) for strain F, Mocarskietal. (18) forJustin(J), Varmuza and Smiley (35) for KOS, and Davison and Wilkie (6) for strains 17 and USA-8.

propagated virus serieswascleaved with ClaI which cleaves oncewithineachrepeatunit(site denotedby Cinmap [Fig.

3, top]). The cleaved fragments were resolved by gel

elec-trophoresis,andmonomericfragments correspondingtothe authentically sizedrepeatsand the larger (chimeric)repeats

wereelutedfrom the gel, circularized, and usedtotransform bacteria. We then determined the nucleotide sequences of

the a sequences in one of the authentically sized clones

(designated G-2) and in two ofthe larger chimeric clones (designated G-3 and G-4). The results are shown in Fig. 4

along with the corresponding DNA sequences of HSV-1

strain F (21), from which the deleted a construct was

derived, and HSV-1 strain Justin (18), which served as

helper virus. Theasequencein G-2wasfoundtobeidentical tothedeletedasequenceinthe seedHSV-1(F)-derived G-1

clone(4). Thus,itcontained the fused(attheSstIIsite) DR1

and DR4 elementsand the entire Ucelementfollowedby the DR1and linkersequences. Thenucleotide sequencesofthe chimericdouble ajunctionsin the twolarger cloned repeat units G-3 and G-4 were identical and contained several

interesting features. (i) Starting from the DR1-DR4 fusion site (SstII), the sequences were identical to those in the rescued, authentically sized clone (G-2) up to the DR1 junction. Thisincluded the DR4 and Uc elements in which the Justin strain differs from the F strain by 1 and 2 nucleotides, respectively (Fig. 4B, the boxed A and C in DR4 and theboxedCGC and G inUc). Thus,the insertionof the helper virus a sequence evidently occurred within the DR1 sequence. (ii) The inserted a sequence was generally

similar to the a sequence of the Justin helper virus DNA. Thus, the chimeric junctions contained the Justin-specific

sequence TGGGGTCTGGC between the Ub and DR2 ele-VOL. 59, 1986

A.

b

DRl

Dolsted a

- F

609

B.

HSV-1(F) G-2

U-3/4

HSV-1(J)

HSV-1(F)

G-2

G-3/4 HSV-1(J)

HSV-I( F)

G-2

G-3/4

HSV-1(J)

IHSV-1(F) G-3/4 HSV-1(J)

_--

-L

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A...,.EcORI EcoRI

Just in Helper F FHe1 er

a|b | a

|b

a|b Iab ab

520[4hI

240

480

24 1~~480

ON

1 2 3 5 7 9 11

B. EcCORI

FG

5i

G 5 A 2 1A 2 a b a b |

a.. -aa

a..

3 81 *

318

* * ~1.90

3 5

FIG. 5. Propagation of test constructs in the presence of Justin and F helper viruses and in transfection competition tests. (A) EcoRI digests of32P-labeledDNAsfrom duplicate transfections (lanes a and b) receiving the HSV-1 strains Justin (lanes 1 through 6) or F (lanes 8 through 11) helper virus DNA along with the indicated testconstructs. Lane 7 contains theEcoRI-digestedF helper DNA. The increments (in base pairs) between fragments containing single a and double a junctions are indicated. (B) Propagation competition tests. EcoRI digests of32P-labeledpassage 2 DNAs from transfections receiving helper virus DNA alone (lane 1) or with theG-5 construct (lanes 2 and 3), the A-2 construct(lane 6), or an equal mixture of both constructs (lane 4 and 5). Fragmentsderivedfrom thedefective genomes are indicated by size. a anda* denote fragmentscontaining the intact and Ub--Uc+ a sequences present in the seed A-2 and G-5 constructs, respectively. a* + aj denotes the fragment containing the chimeric junction with helper Justin a sequence; a + aFdenotes the double a junction arising by self-amplification of the intactasequencein A-2.

ments, two copies of the DR2 repeat, the Justin-specific DR3.5 repeat, and a single copy of the DR4 repeat (these

elementsareboxed inFig. 4B). (iii) two sequence alterations appearedinbothchimericrepeatclones.First, 10 bp into the

Ubelement,thechimericjunctions in G-3and G-4 contained

the triplet CGC (boxed in Fig. 4B) which was absent from the Justinhelper virusasequence.Second, the distal portion

ofthe Uc elementappearedtobederived fromF(construct) rather thanJustin (helper) DNA (Fig. 4A), inasmuch as at

the Uc-DR1transition, theinserted a sequences resembled thesequanceofthe F rather thanthe Justin strain(Fig. 4B,

boxed CGC and G).

Twopointsarenoteworthyconcerning thisresult. First,it

islikely that the sequence variations reflectfeatures ofthe

recombination events leading to the formation ofthe

chi-meric junctions, rather than sequence variation related to propagation in virus stocks. Thus, the nucleotide sequence

of the a sequence in the G-2 repeat (cloned following propagation) was identical to that in the input a construct

(G-1), whereas both chimeric a clones exhibited the same sequencealterations. Second, the sequence alterationswere ofinterest inthat, in the HSV-1 strains sequencedthusfar,

the sequences immediately adjacent to the DR1 (at the Uc-DR1-Ubtransitions)arequite variable,whereas the bulk

ofthe sequences making up the Uc and Ub elements are highlyconserved (Fig.4C). Itispossiblethat thevariability

in the Uc-DR1 andDR1-Ubtransitions within the chimeric

double a junction reflected inherent variability in these

regions. Apossible explanation for the source of sequence variations is proposed in the Discussion.

Essential role of Ub in cleavage-packaging. Insertions of

helper virusDNA sequencesinto defective genomes had not been detected inourprevious studies thatinvolvedthe use of amplicons with heterologous helper viruses (F strain-derived amplicons in combination with Justin helper, and

viceversa; see, e.g.,reference7). Apossible explanation for

theobserved insertions into the D-1- and G-1-derived defec-tive genomes was that the Ub deletion in these constructs affected the cleavage-packaging signal and therefore

chi-mericjunctionswhichrestored the deletedUbsequencesin an appropriate arrangement were selected forpropagation. Threepredictions of thishypothesis weretested below and

found to be correct. The first was that helper virus DNA insertions would not be detected in defective genomes

propagatedfromconstructscontaininganintactasequence. The secondwasthat incompetitiontests,theUb--Uc+ test plasmids willbe propagated with lowerefficiency than the

intacta constructs. Thethirdwasthat theUb--Uc+ repeat units will be propagated from passage to passage within heteroconcatemers, i.e., mixed concatemers containing (i)

repeats with the Ub- a sequence and (ii) repeats with the chimeric (helper a-containing) junctions. The cleavage-packaging functions of these heteroconcatemers could be

provided, at least in part,by thechimericjunctions. Transfer of the helper asequence into different seed con-structs.Totestthe firstpredictionweanalyzedthe patternof aamplificationin defective genomesderivedbypropagation

of the intactaconstructA-2and theUb-constructsD-1 and G-1 (Fig. 1B). Each of these constructs was used to cotransfect

duplicate

cultures in the presence ofHSV-1

(F)

orHSV-1(Justin) helperDNAs(possessinga sequences of 501 and 264 bp, respectively) to determine whether the

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HERPES SIMPLEX VIRUS CLEAVAGE-PACKAGING 611

A.

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2.

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5.r ? 49

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B D E E BC3 E E BE *E B DE

2.06 *. 3 --- or ' o

EcoRI 206 256 206 281

1 5 6 10 Oral _ 005 ,1657

FIG. 6. Structure of concatemers arising by propagation of the G-5 and G-6 test constructs. (A) EcoRI andDraI digests of 32P-labeled passage2DNAsfrom transfections receiving helper Justin DNA alone or with the G-5 or G-6 test constructs. Sizes are indicated in kilobases. The underlined sizes correspond to fragments which arise from the large inserts of helper virus DNA. The corresponding maps for the G-5-derived genomes areshown in panel C3 and accountfor the novel 8.88-kbEcoRI band and the 10.95 and 16.57-kb Dral bands. The constructG-6 also produces largerecombinant repeat units with expected 6.07-kb novelEcoRIfragment and 10.95- and 16.57-kb novel DraI fragments (maps not shown). (B) Structure of the constructs G-5 and G-6. The EcoRI-to-PvuIl segment contains ori-l'; a* denotes the Ub--Uc+asegment. The locationsof theEcoRI (E), BamHI (B), andthefusedDraI-PvuII (D/P) sites are shown. (C) Concatemeric structures for the G-5 construct.Cl,Homoconcatemerscontaining authentically sized (input) repeat units. i,DraI-PvuII fused site at the border of the ori-1' fragment. C2, Structure of heteroconcatemers containing interspersed chimeric and authentic junctions. Black rectangles denote the Ub--Uc+asequence (a*), and open rectangles denote the inserted helper virus a sequence. Cleavage of the heteroconcatemers with Dral isexpectedtoproducebands with increments ofauthentically sized repeats. The firsttwobands (5.87 and 11.49 kb) are shown both here and inpanel A, lanes 7 through 10. C3,Heteroconcatemers containing repeat units with an inserted helper DNA segment spanning the region from the a sequence to the replication origin ori-1'. The predicted sizes of fragments generated from EcoRI and Dral cleavages of the heteroconcatemers are shown for the construct G-5. The corresponding first two bands in the predicted ladder generated byDral cleavage (10.95and16.57kb) areunderlined in panel A, lanes7through 10, as is the novel 8.88-kbEcoRIfragment.

amplified a sequences were derived from the helper virus

DNA or from the seed test plasmid. Restriction enzyme

analyses ofpassage 2 DNAs from thesetransfections (Fig.

5A for EcoRI; additional data not shown) revealed the

following. (i)Theincrementbetween thebands representing

the double a and single ajunctions in defective genomes

propagated from A-2(intacta) in the presence of the Justin helper virus was ca. 520 bp (fig. SA, lanes 1, 2). Thus, the

double a junction was derived by self-amplification ofthe

537-bp a sequence ofthe seed construct(minus the shared

DR1 element) rather thanby insertion of the 264-bp helper

virus a sequence. (ii) In contrast, the increments between the double a and single ajunctions in defective genomes

propagated fromthe D-1 and G-1 constructs varied with the

helpervirus.Specifically, inthepresence of the Justin helper

(possessing a 264-bp a sequence) the increment

corre-spondedto ca. 240bp (Fig.5, lanes 3 through 6), whereas in thepresence ofthe Fhelper (501-bp a sequence) it was ca.

480 bp (lanes 8 through 11). These values were consistent

with insertions of the helper a sequences into the chimeric

junctions. Furthermore, bands arising from self-amplifica-tion of the Ub- a sequences (100 bp in G-1 and 470 bp in D-1) were notdetected.

The data support three conclusions. (i) The intact a sequence contains a complete set of cis-acting signals needed for the cleavage-packaging of viral DNA, as well as for the self-amplification of a sequences. (ii) Fordefective genomes containing intact asequence, the trans insertion of

a sequences from helper virus DNA is less efficient than

self-amplification of the a sequences. (iii) the Ub deletion affectsfunction(s) requiredfor aamplification, as well as for

defective genome propagation. Hence, defective genomes derivedfromthese constructsrequirethe presenceofat least some chimericjunctions (in the same concatemer, see

be-low) for their propagation.

Competition propagation tests. Studies described below

revealed that constructs containing the Ub--Uc+ a se-quences werepropagated invirusstocks less efficientlythan

theircounterparts containingthe intact a sequence. Specif-ically, duplicate cultures were cotransfected with helper

Justinvirus DNA and (i)the A-2intact a construct, (ii)the construct G-5, which contained a Ub--Uc+ a sequence

identical to that in G-1 (map in Fig. 6), or (iii) an equal mixture ofbothplasmids.Therelative propagationefficiency

ofA-2 and G-5 could be determined fromthe ratios ofthe

distinct restriction enzyme fragments which they were

ex-pectedto yield.

Figure SB shows the EcoRI digests of passage 2 DNAs from these transfections. Defective genomes with repeat units derived from the intact a construct were abundant in stocks derivedfromthe single(A-2; lane 6) and mixed (A-2 +G-5,lanes4and5) transfections(see bands 1.9 and 3.18 kb in these lanes). Defectivegenomes derived from the Ub- a construct were propagated in stocks derived from the transfectionsreceivingthe G-5plasmid singly(lanes 2 and 3; bands 2.06 and 3.56 kb). However, in stocks derived from VOL.59, 1986

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[image:7.612.91.530.78.301.2]

themixed transfections, they were present only in very low abundance (lane 4) or were not detected at all (lane 5). (In lane 4, compare the intensities of the 1.90 and 3.18 kb bands

representingthe intact a construct with those of the 2.06 and 3.56 kbbands representing the Ub--Uc+ construct.)

These results demonstrated that the G-5 clone which

contained the Ub--Uc+ a sequence was propagated

ineffi-ciently, consistent with a defect in the cleavage-packaging

signal(s). In addition, the increments observed between

fragments containing the double a and single a junctions in the A-2- and G-5-derived repeats were consistent with the

results discussed earlier. Thus, A-2 repeats contained a

self-amplifieda sequence (a + aF; Fig. SB, lane 6), whereas

G-5-derivedrepeats containedaninserted Justin a sequence

(a* +aj; lanes 2 and 3).

Structure of concatemeric DNA molecules. As discussed

above, test constructs lacking the Ub element appeared to contain a nonfunctional cleavage-packaging signal. At the same time, authentically sized repeat units carrying the

Ub--Uc+junctions were recovered in propagated defective genomes (along with chimeric junctions containing helper a

sequence).Infact,they represented the major repeat units in

the serially passaged virus populations. The studies

de-scribed below provide an explanation for this puzzling

observationin thattheyrevealed that repeat units containing the

Ub--Uc'

junctions were propagated as constituents of

heteroconcatemers containing interspersed chimeric

junc-tions. Specifically, in these studiestheUb--Uc+ asequence was first introduced into plasmids lacking Dral sites. Since the DraI site in the Ub element could serve as the hallmark

for the inserted helper virus a sequences, the degree of

interspersion of chimeric and authentic repeat units within the same defective genome molecules could be determined

by analyzing the distribution ofDral sites along defective genome concatemers.

For the construction of the test clones lacking DraI, a PvuII fragment containing the replication origin and the

Ub--Uc+ a sequence was introduced into a Dral-cleaved

pBR322, resulting in the inactivation of both sites and

yieldingtheplasmidsG-5and G-6,differingin the orientation

ofthe PvuII insert(Fig. 6B, top). ThepredictedEcoRI and

Dral cleavages of homoconcatemers and (the simplest)

heteroconcatemers are shown forthe construct G-5 in Fig.

6C,

panels 1and 2. Usingthe G-5 and G-6testconstructsas

input

seed clones, we expectedthat homoconcatemers

con-taining solely the Ub- a sequences would be resistant to DraI(Fig. 6C, panel 1). In contrast, randominterspersionof the chimericjunctions (each containing a Dral site in Ub) within heteroconcatemers containing the authentic repeat units(devoid oftheDral site)wouldcreate aladderof Dral

fragments

with increments of single, authentically sized repeats. Because the authentically sized repeats were 5.62 kb and because the repeats withchimericdouble a were 5.87

kb,we predictthatDralcleavage of the heteroconcatemers will generate a ladder described by 5.87 kb + n (5.62 kb), wheren isan integer, i.e., 5.87, 11.49, 17.11 kb, etc. (Fig.

6C;

panel 2).

TheEcoRI andDral digestionpatternsof passage2 DNAs fromduplicatetransfectionsreceiving HSV-1 (Justin) helper

alongwith the G-5 and G-6 constructsareshown inFig. 6A. The DraI cleavage pattern of the G-5 and G-6 defective genomes contained bands of sizes predicted from random

interspersionof chimeric junctions within the concatemeric defective genomes (Fig. 6A). Interestingly, the DraI and EcoRI patterns revealed the presence of an additional type of chimeric repeat unit within the same defective genome

concatemers. Thefragments resulting fromcleavageofthese interspersed novel repeats are underlined in Fig. 6A and their structure is depicted in Fig. 6C, panel 3, for the construct G-5. In these novel repeats, the construct se-quences between the deleted a and the replication origin

havebeenreplaced by helper virus DNA sequences extend-ing from the a sequence to the replication origin in the S component. The presenceofthese repeatunits in the serially

passaged virus stocks was surprising in that, as already

noted above, we did not encounter chimeric repeat units containing inserted helper virus DNA sequences during propagation ofamplicons containing afunctional replication origin and anintact a sequence. Mostlikely, thesechimeric

repeats were selected for propagation and might in fact represent intermediate structures in the formation of the

chimeric junctions serving to propagate the

Ub--Uc+-containing seeds. Models for the evolution of these large repeats are considered in the Discussion.

DISCUSSION

Distinct roles of Uc and Ub elements in the cleavage-packaging process. We used amplicon propagation tests (28) to mapthe signals required for the cleavage and packagingof

HSV DNA. The constructs tested in these studies fell into three distinct categories. Constructs in the first category (A-1 and A-2) contained an intact a sequence and yielded defective genomes which did not contain additional se-quences derived from the helper virus genome. Constructsin the second category (D-1, G-1, G-5, and G-6) contained deletions in the Ub element but retained the Uc element. Defective genomes with authentically sized repeats were recovered from these constructs. However, propagation resulted in the selection of chimeric repeat units contain-ing intact a sequences derived from helper virus DNA. Moreover, in transfection competition tests the Ub--Uc+ defective genomes did not compete efficiently with their counterparts containing an intact a sequence. Finally, test constructs in the third category (B-1, C-1, E-1, and H-1) contained deletions in the Uc element and could not be propagated into defective genomes. Taken together, these data revealed that the Ub and Uc elements played critical yet distinct roles in the cleavage-packaging process. The in-volvement of the DR2 and DR4 elements in the cleavage-packaging process is at present unknown.

Directional packaging modelfor cleavage-packaging of viral DNA. In this and the next section we propose two alternative models for the cleavage-packaging of viral DNA. Both models are designed to explain the data summarized in this paper and to account for previous observations related to the cleavage-packaging process. The first model (the directional cleavage model) modifies the "theft" model proposed re-cently by Varmuza and Smiley (35) to include polarity inthe cleavage-packaging process. The model is schematically diagrammed in Fig. 7A for standard virus DNA concatemers and includes the following steps. (i) A packaging complex encounters the HSV DNA anywhere in the L or S compo-nent and traverses along the concatemeric DNA in either direction (random walk; Fig. 7A) until a junction containing a Uc signal is found. (ii) Cleavage (producing 3' single-base overhangs) occurs at the DR1 element proximal to the first Uc signal encountered (cleavage 1; Fig. 7). If the complex traverses into a

ba,c

type junction starting from the L component (ca. 80% of the cases, based on the relative sizes of L and S, and assuming random initial binding) this first cleavage will produce ba and

ca,

-1termini (Fig. 7B andC).

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HERPES SIMPLEX VIRUS CLEAVAGE-PACKAGING 613

A.

bind; cleave

random -. atfirst scan I-eS C___cleave

walk Uc while packaging at Ub

[image:9.612.136.466.76.333.2]

~~~''Uc UIL UbU, SU LII L

degraded -| packaged b |-degraded

B. bind- walk 2

OLSS

L

leftI

S

UCm

b

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Cb.b* - * E bac

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U,Lb:S{ Uc S YPU@M b bUb: vUc -6

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(S) (L)

L,right - c.an- - ban.I ab -- ca

L,left - can-I - ban_I ab -- ca S,right - c - ban.I anb -- ca

S,left - c - ban-I anb -- ca

b]n.L

Jucton

FIG. 7. Directional cleavage model.(A)Basicfeatures ofthemodel.Thepackaging complex(open circle)bindsto thestandardviral DNA concatemer at arandomlocation andtraverses the concatemer in a random walk(rightorleftinthefigure).Thefirstcleavage[1] occurs at aconstantdistancefromthefirstUcsignalencountered, leavinga3'single-baseoverhang. Inthe standard viralgenomethiscleavage occurs inthe DR1 element.PackagingbeginsatthegeneratedLterminus(whichcontainsatleast one a sequence),whereas thegenerated S terminus (which is devoidofa sequences) is rapidly degraded. While packaging in the L toS direction, theDNA isscannedfor the nextdirectly repeated junction, skipping the invertedjunction (underlined).Asecondcleavage[2]occurs at aconstantdistancefrom the first Ubsequence encountered in the secondjunction, leaving 3' single-base overhangs and producingapackaged molecule (heavy line) which carries a ca

terminus, and anunpackaged terminus which isdegraded. (B)Diagram of thecleavagewithinsingleajunctions inastandard virus DNA concatemer,featuringthefourpossible permutations of initial binding and random walk. Assuming randombinding, ca. 80%oftheinitial bindingeventsshouldoccurinL(lines1and2) andca. 20% shouldoccurinS(lines3and4). (C) Termini produced in packaged and free moleculesas aconsequenceof thecleavage-packaging processstarting from each ofthefourinitial binding permutations.nrepresentsthe number ofasequencesinthejunctions whicharebeingcleaved.

If the process began within the S component, the first cleavage will generate ban and c termini. In both cases

directionalpackaging will begin fromthegeneratedLtermini (baorban), whereas the generated S termini(ca, - 1orc) will

be rapidly degraded. (iii) Packaging will continue by

scan-ningin theL-to-S direction,untiladirectly repeatedjunction

is encountered. (iv) A second cleavage (also yielding 3' single-base overhangs) will thenoccur proximal to the first Ubsignal encounteredwithin thatjunction.This cleavageis expectedtoproducea caterminusonthepackagedmolecule andafree(ban,)- terminuswhichmightberapidly degraded

orform the substrate for a newround ofpackaging (intoa

separate capsid).

The directional cleavage model can account for several previously unexplained observations. First, the proposed scanning for a second directly repeated junction (step 3)

accountsfortheobserved cleavageatalternate(i.e., directly repeated) junctions within standard virus concatemers. Fur-thermore, the polarity requirement for the two

prospec-tive termini has also been demonstrated in studies with constructed defective genomes, supporting a scanning

mechanism (9; L. P. Deiss and N. Frenkel, manuscript in preparation). Second, the proposed specificities ofjunction cleavages and termini degradation account for the produc-tion of two a-containing termini (with 3' overhangs [20]) from single a-containing junctions (7). Furthermore, the modelaccountsfor thepresence ofa variable numberofa

sequencesat the Lterminus, andasingle a sequenceat the S terminus. Third, the predicted generation of ba termini from

ba,c

junctions in the majority(thoseresulting with first encounterin L[ca.80%]) of first cleavages mightaccountfor theobservedpredominance ofLterminicarrying asingle a sequence(15). Moreover, itmight constitutethemechanism forthediminution

of

a sequencesfrom multiple a-containing junctions arisingas consequenceofgenome circularization, following virus entry into the cells (21, 22). Finally, the

proposed roles for the Ub and Uc elements in cleavage-packaging might account for the distinct propagation pat-terns ofamplicon constructs tested in the present study. Specifically (Fig. 8 a through e), the model predicts that

defective genome concatemers arising by replication of Ub--Uc+ constructs (Fig. 8a) could nevertheless undergo

thefirstcleavageatthe Ucsignal, unlike theirUc- counter-parts. The resultant terminus, which carries the Ub- a sequence, might undergo an aberrant fusion with the S terminus of the helper virus DNA (Fig. 8b), yielding

chi-mericjunctions, afterrounds ofhomologousrecombinations

and replication (Fig. 8 c through e). Finally, the resultant concatemers mightserve as substrates for thenormal cleav-agepackagingprocess, with the first(Ucproximal)cleavage

occurring within a Ub--Uc+

junction,

followedby the sec-ond (Ub proximal) cleavage within a chimeric

junction

present in the sameheteroconcatemer.

While the directional packaging model may account for VOL. 59,1986

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614 DEISS ET AL.

a. _ lori I lUcI lori I lucI Iori I IUcI ,

c

icleave b. Ior' lucIUc Ior I I Ub U

detectivegenome termi,ius e v helper S terminus

f

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a a

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....---

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iori UcI oriI cUcTl or'i iuci

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J. I orII lUclUb Uc' I ori I Uc lUb Uci

cleave 4 cleave

cleave/package

FIG. 8. Generation and cleavage oftheheteroconcatemers from Ub--Uc+constructs. (a)through(e)Chimeric junctionsare gener-ated and cleaved by the directional cleavage mechanism. Concatem-ersarising by replication of the Ub--Uc+ seedconstructs(thin line inpanela) arecleavedattheDRI proximaltotheUcsignal by the firstcleavagestep.The generatedterminus, carryingUc,fuses with the S terminus of standard virus DNA (thick line in panel b). Homologousrecombinationatthe ori-1 region followed by rolling-circle replication of the resulting circular repeat generates heteroconcatemers (panel d). Homologous recombination in theUc elements within a large chimeric repeat and an authentic repeat along the same concatemer results in the formation of chimeric junctions (panele)containing the inserted helperasequence, with the last portions of the Uc sequence derived from the seed con-struct.Cleavage of theheteroconcatemers occursby the directional cleavage mechanism, thefirstcleavageoccurring withinthedeleted ajunction (atthe Ucsignal) and the secondcleavage occurringinthe chimeric junction (at the Ub signal). The model predicts that concatemersderived byreplication of theUc-constructswillnotbe cleaved instep (a). (f) through(j) Generation and cleavage of the chimeric repeats by the double-stranded break-gap repairmodel. Homologous double crossover recombination occurs between helpervirus DNAwith a double a L-Sjunction (heavyline) anda

defective viralgenome (thinline) whichwasgenerated by replica-tion oftheinput Ub--Uc+construct(panel f).Thecrossoverpoints are in the homologous Uc and ori-l' regions, yielding the heteroconcatemer shown (panel g). Achimericjunctionarises by homologous recombination betweenthe Uc sequences in an authen-tic repeat and the Uc segment intheinserted helpersegment. The chimericjunction contains the deleted a andthe intact inserted a carrying aUcelement which isderived in partfromthe construct (panel h). The heteroconcatemer is a substrate for the cleavage-packaging process, occurring by the double-stranded break-gap repairmechanismdepictedinFig.9. In the aamplificationprocess the Ub--Uc+ junctions is used as junction 1 and the chimeric junction isused asjunction2. ThesequencevariabilityinUbarises during the resolution ofthe Holliday (11) structures. This model

many of the previous observations, it suffers from two drawbacks. First, as recognized by Varmuza and Smiley (35), the modelpredicts the formation of termini devoid of a sequences. Such termini have not been observed in total nuclear DNA (7, 20). Therefore, it must be assumed that

they are rapidly degraded. Second, although the model explains some of the structural features of the chimeric

junctions, it fails to explain the observed variations in the

Ub-DR1 portion of the inserted a sequence. It appears unlikely that such variations could result from the fusion of the defective genome Uc terminus with the S terminus of standardvirusDNA.

Double-strand break and gap repair model for a amplifica-tion.The secondmodel proposed for the cleavage-packaging process is based on the double-strand break and gap repair

mechanism proposed by Szostak et al. (33) to explain

recombination events resulting in gene conversion. Specifi-cally, the model involves the interaction of two directly

repeatedjunctions (Fig. 9; junctions 1 and 2), resulting in a

amplification by a gene conversion-like mechanism. The resultant junctions are then cleaved to yield the genomic

termini. Cleavage-packaging thus includes the following steps.(i)Asin thedirectionalcleavage model, the packaging

complex traverses along concatemeric viral DNA, starting

from anypoint in the Sor L components, until the first Uc

signal is encountered injunction1 (Fig. 9A). (ii) Scanning-packaging begins, proceeding in the L-to-S direction, until thedirectly repeatedjunction2, along thesame concatemer,

is encountered. The twojunctions are juxtaposed for the nextseries ofevents(depictedschematically in Fig. 9B). (iii) AUcsignal-directed double-strandedcleavage occurs within the DR1 elementof either junction,e.g., injunction 1,(Fig.

9B, step b). (iv) The resultant 3'-terminated strand invades

the homologous sequence injunction 2 and is extended by

copyingthe a sequenceinthatjunction,whiledisplacingthe

equivalent strand (steps c and d). (v) The displaced strand

fromjunction2 serves asthetemplatefor repair synthesis of

the second strand of junction 1 (step e). (vi) The process

terminates by the resolution ofthe two Holliday structures (11);this is predictedtooccur at or neartheDR1sequences (stepf). (vii)Ifjunction2containedasingleasequence(with Uc+ element), it too will become a recipient for an a sequence fromjunction 1 in asimilarsequence ofevents as

above.Theentireprocess thus results in theamplification of

the a sequence within each of the twojunctions (step g). Each ofthedoubleajunctionsnowcontainsaDR1 sequence flanked by the Uc and Ub signals. (viii) Following the a amplification, both double ajunctions arecleaved

(generat-ing a 3' single-base overhang) between the newly inserted

and the original a sequence (step h). This cleavage may

require a structure in whichthe DR1 element is flanked by

Ub and Uc

signals,

anarrangementwhichis present

only

in the double ajunctions. The model predicts that ca.

80%

of thepackagedgenomes (initialencounter inL)will carry ba and ca termini, regardless ofthe number ofa sequences in the cleaved junctions, whereas the remaining packaged

genomes (initial bindingin S) will be terminated withcaand

bansequences

(Fig.

9C). The termini ofthe

generated

free molecules(predictedtobeofthe typebanand ca, or ca;

Fig.

9C)might eitherbecomedegradedor serveastargetfornew packaging cycles.

predicts that constructs containing the Uc+ repeats will form heteroconcatemers which willnotbecleavedandpackagedowingto

thelackof Uc element injunction1.

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HERPES SIMPLEX VIRUS CLEAVAGE-PACKAGING 615

Junction i Junction 2

bind; start to amplify c

random + scan at + scan L-a.S at fi a cleave walk first Uc while packaging Uc atUb/Uc

Las L aL a

L gUb:cI 'S- W--c IUw 'UbUcI UC: :Ub" ' L

I

I amplification

~~p

L~~~

a ap

L*Ub::Uc S /UC::Ub L

Uc:: Ub -6

--I

Uc::b

S Uc: :Ub Uc: :Ub L

'Ub: :Uc S /Uc: :Ubw L

Icleavage

Uc: :Ub

Ub_Ub::.Uc-

=-Uc:

:UC _{5 Uc: :Ub}

I

U_Jc:

-f

_:UbbI _L

free-I

|. packaged , |_tree

C.

B.

I._u

as

a. Ub Uc

,4 ,_

-~~~~--- 11111 1i<112

_. _ -a s

4 l.dvU%t U*c

4

X

I-

W

.c

rDc_ixz

'.____________4

4. copy

C.ZD

_

-a _

_a

1

a - 6.u7iiillf

a a . jUiticIti

_Ik

I. Ub Uc Ub Uc

1.*a -a

1.-l,== -

e-11. _ _ _

II.

I a

-FIG. 9. Thedouble-stranded break-gap repair model fora amplification. (A) Basic features of the modelare shown fortwo singlea

junctions. Following randombinding and random walk, packaging beginsat the first Uc sequence encountered, proceeding in anLto S

direction, untiladirectly repeated junction is encountered. Thetwojunctions interact bythedouble-strandedbreak-gap repair mechanism (detailed in panel B), resulting inreciprocal copying ofanasequencefromonejunctiontotheother, theainsertionsoccurringatathe first Uc signal encountered in both junctions. Thegenerated doubleajunctionsarecleavedwithintheDR1elements flankedbythe Uc and Ub

signals.Unpackaged moleculesareeitherdegradedorutilized inanewroundofpackaging.(B) Proposedstepsinthe double-strandbreak-gap repair model foraamplification, featured after thegeneconversionmodel of Szostaketal.(33). (a)Twodirectlyrepeatedsingleajunctions

(junction 1, heavy line; junction 2, thin line)arejuxtaposed. (b) A double-strandbreakoccursattheDR1eIenient proximaltothefirst Uc

signal. (c)The resultant 3' terminus ofjunction1invadesjunction 2,displacingthehomologousstrand.(d) The invading3' terminus has been

extended(dotted line) bycopyingthecomplementary junction 2 strand;thedisplacedstrandpairsin theDR1regionwith thesecond cleaved

strand ofjunction 1, forming atemplate for extension of this strand (dotted line). (e) The newly synthesized portions join the original corresponding strands, forming the Hollidaystructures(11). (f)Resolution has occurred within theDR1elementsoradjacentsequences.(g)

Asimilar series ofsteps results in theamplificationof the asequence injunction 2. (h)Cleavage producing3' single-base overhangs has

occurred within theDR1 elements, separatingthenewandoriginalasequencesin eachjunction.Thiscleavagecould be directedbythe Ub andUcsignals which flank the cleavage site. Smallarrowsindicate the 3' end of each strand.(C)Terminiproducedinpackaged and free

moleculeas consequence ofcleavage-packaging starting from each of the four initialbinding permutations. nrepresents the number ofa

sequencesin thejunctions whicharebeing cleaved.

Unlike the directional packaging model, the double-strandedgaprepairmodel doesnotpredictthegenerationof termini devoid of a sequences. Furthermore, multiple-a-containing S termini have been observed in stocks contain-ingveryhigh ratios of defectivetohelpervirusgenomes (7), supporting the hypothesis that they represent intermediary products of the packaging process. Inaddition, the double-strandedbreak-gap repairmodelaccountsfor thesamesetof observations explained by the directional cleavage model, including the cleavage of alternate junctions in standard virus DNAand thegeneration of packaged DNA molecules terminated with a single a sequence at the S end and with predominantly singlea(but also multiple a)sequencesatthe Lends.Finallyitalsoprovidesaschemeforthepropagation

of the Ub--Uc+ construct and explains the structural fea-tures of the resultant chimeric repeats (Fig. 8f through j). Briefly, this scheme involves homologous recombination between helper virus DNA and the replicated

Ub--Uc+-defective genomes (Fig.

8f),

followed by intramolecular recombination between the created recombinant junction and an adjacent deleted junction in the same concatemer (Fig. 8g).This lastrecombinationcreateschimericjunctions with the inserted Justin a sequence carryinga Uc element

derived in part from the F construct (Fig. 8h). The heteroconcatemer is then cleaved-packaged by the double-stranded break-gap repair mechanism (Fig. 8i), with the

sequence variation in Ub arising as a result of slippage duringthe resolutionof the Holliday structures (Fig. 8i and j). In contrast, heteroconcatemers, which are predicted to ariseby similarrecombinational events withUb+-Uc-

con-structs, would notbecome cleaved and packaged owingto

the lack of Uc element in junction 1. Thus, unlike the directional cleavage model, thepresent model requiresthat bothjunctions (which are cleaved to generate the termini) containthe Uc signal.

pac-l and pac-2 homologies. The proposed role of the

A.

1iCti(ltioIl I

2.Invadc

3d( ISI)lIuC/

_3cdislacc

Bind,Walk

Free

Packaged

(S)

h

(L)

L,right

-can

-ban

ab

--

ca

L,

lef

t

-can

-ban

ab

--

ca

S,right

-ca

-ban

anb

-- ca

S,left

-ca

j

-ban

anb

-- ca

I.resolve

u

.n2

VOL.59, 1986

1-ip--S.- s

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a _a

Ub

---

Uc

DR1

Ub

---

Uc

---

pac-2*

v

*pac-1

---B.pac-2

ICGmotif

F

Justin

KOS 17 USA-8

HG52

vzv

EBV HVS 11

BHV-l

.... CGCCG;CG

.... CGCCGCC .... CGCCGCC .... CGCCGCC .... CGCCGCC .... CGCCGC( .... CGCCcC( ....gGCCGCC

. CcCCGaC

....CGCCGC(

HSV-1 (F) HSV-1 (Justin) HSV-1 (KOS) HSV-1 (17) HSV-1 (USA-8) HSV-2 (HG52)

VzV

EBV/Namalwa HVS (11)

D.

S S S

S

S S S

s

BssHII

1--

Bt9HII

T.

--I I ---I

cgcgcgcacgccgcccggaccgccgcccgcc gcgcgccgccc ; cgcgcgcacgccgcccggaccgccgcccgcc =.TTA gcgcgccgccc cgcgcgcgcgccgcccggaccgccgcccgcc gcqcgcgcgcc ; cgcgcgcacgccgcccggaccgccgcccgcc TTTTP gcgcgcgcgcc

cgcgcgcgcgccgcccggaccgccgcccgcc TTTT gcgcgccgcc B ggctgccttccgcggcgcccccgcgcggc =TLT cccgcgcccg ; caaacgcgcggggaggtggggtcgc ¶TTTTTPTT ctctctcgagc B ggggccagccacgccccctccac TTT ccaggaatgcc B ctcgctctagccacgcccaggaca T¶TT ccagctgccci

B cagagaaaa N=1T ctccgcgcccc

--- S terminal region -Cn-ri----II---

I

DraI SmaI

gcgccgccgcgctttaaagggccgcgcgcga CCCCC GGGGGG

gcgqccgccgctttaaagggccgcgcgcga CCCCC GGGGGG

geccgccgcccgcctttaaagggccgcgcgcga CCCCC GGGGGG

jgcccgccgccaccgctttaaagggccgcgcgcoga CCCCC GGGGGG

gEcccccgccgcgctttaaagggccgcccgcga CCCCC GGGGGG

ctcccgccccgcctcttttcccccggggagtcaa CCCCC GGGGGGGG

cctctcccqgggtccgccgggcgcccagaaa CC GGGGGGG ccccccatgccctctccaacaccccccgaag CCCCCC GGGGG

ccccttccccctctttqcctaccaaqttat CCCCC GGGGGG

L Terminal region ---I end SiaI

ccgcccgcggggggcccgggetgc L

cgccgcggggggcccgggct3c L

gcgcccgcggggggcccgggctgc L ;cgcccgcggggggcccgggctgcc L

ccgcccgcggqgggcccggqccgEc L cccccgcgcggcaggacggggactcccgccc L

ggqgccgcgagagggctggcctcc L gcggcggcccatgctccccccatgc

igcqcccactgcttggggcccc

ccccgcgqggggqctgggcc L

IT motifi I

---GnI---Apal

TGTGTTTC! GGGGGGGG cccgt ....

TGTGTTTC GGGGGGGG cccgt ....

TGTGTr rGGGGGGGG cccgt ..

TGTGTTTT GGGGGGGG cccgt ....

TGTGTTTT GGGGGGGG cccgt

TTTTTT GGGGGGGGG cgcga ....

TTATTTTC GGGGGGGGG tccga TCTTTCCT GGGGG ccttt ....

AAAATCAGT GGGGG ctgca

E.

summary:

~~~~~~~~~~~~~DRi

DR1 Ub ,

(DR2)22

(D~R4)3

UcDR

[ III

---IIN

I {1

A A

[1

It- -- -

---

Construct D-1

Construct H-l

pac-1

I 100 bp

pac-2

Homology

FIG. 10. pac homologies in different herpesviruses. (A) Alignment ofthe pac-J and pac-2 sequences flanking the cleavage site. (B)

Conservedpac-2 (Uchomologous) sequences(capital letters) foundatconcatemeric junctions of variousherpesviruses. References for the

nucleotide sequences arelisted in panel D. Cleavage sites reportedinthe literature areunderlinedatthe end of thesequences shown.The Epstein-Barr virussequence shown is that of the integratedgenomeinthe Namalwa cell line (16). Hence nocleavage (underlined) site is

indicated. (C)Conserved pac-J (Ub homologous) sequencesfound at concatemericjunctions of various herpesviruses. (D) Summary of

distances fromthe Tmotifinpac-I (arrowinpanelA)andT,, element in pac-2 (arrowinpanel B)tothe respectivegenomic termini, and the

percentG+Cresidues inthe Sand L terminalregions.TheaverageG+Ccontentof therespectiveviralgenomes aretaken from reference

26.(E)Deletionsdefinedbythe D-1(Ub)andH-1(Uc)constructsoverlapthe conservedpac-J andpac-2sequences.TheApaI (A)andBssHII

(B) sites which define thedeletions areindicated.

616

A.

S terminus/pac-1 L Terminus/pac-2

Virus Reference Distance %GC Ster Distance %GC Lter Ave %GC end + T region T + end region qenome

F Mocarski and Roizian, 1982 41-42 83 33-34 97 67

Justin Mocarski et al., 1985 40-41 83 31-32 97 67

KOS Varmuza and Smiley, 1985 43-44 84 33-34 97 67

17 Davison and Wilkie, 1981 44-46 80 33-35 97 67 USA-8 Davison and Wilkie, 1981 42-44 84 33-35 100 67

HG52 Davison and Wilkie, 1981 44-47 79 30-41 90 69

VZV Davison, 1984 41-42 83 33-34 76 46

EBV Matsuo et al., 1984 ? 79 ? 74 59

HVS 11 Bankier et al., 1985 40-41 66 31-32 78 46

BHV-1 Haamerschaidt etal., 1986 - - 30 93 72

B B

-2 I

ommmmmmmmmmmMME IL

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