Interactions between the termini of adeno-associated virus DNA

J. Mol. Riol. (1989) 206. 91~100

Interactions Between the Termini of Adeno-associated Virus DNA Roy A. Bohenzky1T2t and Kenneth I. Bern&f ‘Department

and Medical Microbiology University of Florida FL 32610. l’.S.A.

of Immunology

of Medicine,

College

Gainesville,

2Department of Microbiology Hearst Microbiology Research Center Cornell University Medical College New York, N Y 10021, I1’X.A. (Received 28 July

1988, and in revised form

1 November 1988)

The adeno-associated virus (AAV) genome is a linear, single polynucleotide chain with inverted terminal repeats of 145 bases. In order to test whether the terminal repeats at opposite ends of the genome have to be able to completely base-pair during DNA replication, we have created chimeric genomes in which an 11 base symmetrical sequence has been deleted from the terminal repeat at one end of the genome and replaced by a different 12 base symmetrical sequence. We have used these chimeric constructs either as a duplex insert in pBR322 or as purified duplex virion DNA to transfect adenovirus-infected HeLa cells. When chimeric duplex virion DNA was used, all of the progeny virions obtained after two cell passages contained DNA with wild-type sequences in both terminal repeats. When plasmid clones were used, the structure of virion DNA depended on the original orientation. If the mutant terminal repeat was originally at the right end of the genome (terminus of genetic map), all progeny terminal repeat sequences were again wild-type. However, if the original construct contained the mutant sequence in the left termmal repeat, the majority of progeny molecules were parental in type (i.e. mutant left and wildtype right terminal repeat). We conclude (1) although the terminal repeats at opposite ends of the genome may interact during DNA replication, it is not necessary that they be perfectly complementary. (2) In direct competition, the wild-type sequence displays an advantage over the mutant allele. (3) In a plasmid clone, the terminal repeat on the left end of the genome is at an advantage in a competitive situation. We note that the left terminal repeat is adjacent t,o a transcriptional promoter.

1. Introduction

1973; Berns & Kelly, 1974) of which the outboard 125 bases form a palindromic sequence (Lusby et al., 1980). The overall palindrome is interrupted from bases 41 to 85 by two smaller palindromes. Thus, when the sequence is folded on itself to form the maximum number of potential base-pairs, a T-shaped structure results. The terminal repeat is thought to serve as the primer for AAV DNA replication (Straus et al., 1976; Hauswirth & Berns, 1977, 1979; and see Fig. 1). A still unresolved question is whether the incoming parental virion DNA, which is single-stranded, can simply fold over its 3’ end to serve as the primer or whether the terminal repeats at opposite ends of the molecule anneal to form a hydrogen bondal.,

The adeno-associated virus (AAQ) 2 genome is a linear, single polynucleotide chain of 4681 bases (Srivastava et al., 1983). The terminal 145 bases constitute an inverted terminal repeat (Kozcot ef t Present, address: Department of Microbiology. College of Physicians and Surgeons, Columbia University, New York, NY 10032, U.S.A. $ Author to whom all correspondence should be addressed. 0 Abbreviations used: AAV, adeno-associated virus; Ad, adenovirus; bp, base-pair(s); kb, lo3 bases or basepairs; HSV, Herpes Simplex virus; SV40, simian virus 40.

91

0 1989 Academic Press Limited

92

R. A. Bohenzky

and K. 1. Berm

I 3’

, I I D’AC B A ~

ASCA’D f

\

III

T

E longote

ol___--__----_--D_:cB_n_3, D’AC’EJ’A’

Nick

I t ------------------------3

+ DdCBA

,

D’AC’B’A’

Elongate ot nick I

D’ A C’ 0’ A’

Elongote and strand dlsploce ----------t

-------

o_pcce-n_.3’

-

D’AC’E’A’

+ A’CBAD’

DA’CBA

Figure 1. Model for AAV DNA replication. The model is shown starting from the original single-stranded virion DNA. The single strands are depicted in 4 theoretical conformations (I to IV), which become hairpinned to prime daughterstrand synthesis. A nick is introduced into the parental strand opposite the point of initiation and the primer is replicated by repair synthesis. Subsequent rounds of replication occur via hairpin-priming and strand displacement resulting in single strands, which are shuttled into the pathway at the initial steps.

stabilized, single-stranded circle. A possible advantage of such an interaction would be that the conformation at the termini would be essentially duplex and indistinguishable from the duplex molecule formed during the first round of replication. In this form of the model, the initiation of the first round of DNA replication would be from a structure comparable to those used to initiate subsequent rounds. A similar model has been proposed in the case of adenovirus (Ad) DNA replication to allow displaced single strands to be replicated (Daniell, 1976; Lechner & Kelly, 1977). Interestingly, the genomes of both viruses have inverted terminal repeats and replicate by a singlestrand displacement mechanism. That the terminal repeats at opposite ends of the molecule can interact during replication is known. When the duplex form of AAV DNA is inserted into

a bacterial plasmid, an infectious clone results. Transfection of the cloned DNA into cells infected with Ad leads to excision and replication of the AAV genome (Samulski et al., 1982; Laughlin et al., 1983). Clones with deletions of up to 100 bases at one end of the AAV insert are viable, but the terminal repeat progeny have the wild-type sequence at both ends (Samulski et al., 1983; Senapathy et al., 1984). While this observed repair process could be the consequence of recombination, it appears more likely t’hat the deleted end is using the intact end as a template extending from the base-paired termini of the single-stranded circle described above. The circle would be stabilized by hydrogen-bonding between the remaining complementary sequences (potentially 45 base-pairs in the case of a 100 base deletion from one end). In the experiments described here, we have

AA V Terminal

Interactions

approached the question of whether such basepairing interactions between the two ends are necessary. In a more specific sense, we have asked whether there must be perfect. complementarity. We have demonstrated that deletion of an 11 base symmetrical sequence from the terminal repeat under conditions where the DNA cannot self-repair is lethal (Samulski et al., 1983). However, viability can be restored by substitution of alternative eight base or 12 base symmetrical sequences (Lefebvre et al., 1984). Viral genomes containing the mutant sequences are not, demonstrably less efficient at replication than the wild-type genomes (Bohenzky et al., 1988). Here, we report experiments in which we used chimeric genomes with the wild-type sequence at one end and the mutant sequence at the other to address the question of the need for the ends t,o be able to base-pair perfectly. Although unexpected effects of polarity were observed, it does not appear that AAV DNA replication requires perfect base-pairing between the terminal repeats at opposite

93

replication assay as described by Hermonat & Muzyczka (1984). Adenovirus type 2 (Ad-2), needed m helper for AAV, was propagated as described (Rose et al., 1969) with the exception that HeLa cells were used instead of KB cells. The adenovirus stocks were titrated on KB cells using the plaque assay described by Williams (1971). (b) DNA purification

and transfection

DNA was extracted from purified AAV virions by the alkaline lysis/phenol extraction procedure (Bohenzky et al., 1987). Plasmid DNA was extracted from Escherichia coli HBlOl by alkaline lysis (Birnboim & Doly, 1979) as modified by Maniatis et al. (1982) and purified by isopycnic centrifugation in CsCl/ethidium bromide gradients. DNA was transfected into adenovirus-infected KB cells using the DEAE-dextran procedure of McCutchan & Pagan0 (1968) as modified by Samulski et al. (1982). Plasmid DNA was transfected into E. coli HBlOl by a variation of CaCl, transformation (Rawlins & Muzyczka, 1980).

ends of the genome. (c) Restriction enzyme anaZysis Restriction endonucleases were purchased from Bethesda Research Laboratories, New England Biolabs, International Biotechnologies, Inc. or BoehringerMannheim Biochemicals and the reactions carried out according to the suppliers’ instructions. Following digestion, the DNA fragments were end-labelled using the Klenow fragment of E. coli polymeraae I (Bethesda Research Laboratories) and [a-32P]dCTP (New England Nuclear). The mixtures were then electrophoresed through 10% (w/v) polyacrylamide gels in TBE buffer (50 mM-Tris, 50 mM-boric acid, 3 mm-EDTA). The gels were dried and autoradiographed on Kodak XAR-5 film.

2. Materials and Methods (a) Cell lines and viruses KB cells were kindly provided by Dr C. S. H. Young and cultured in Dulbecco’s minimal essential medium supplemented with 10% (v/v) fetal bovine serum (Gibco) and 2 mM-L-glut&mine (Gibco). HeLa-S3 cells were kindly provided by Dr J. Hurwitz and were cultured in suspension using Joklik’s minimal essential medium supplemented with 5% (v/v) bovine calf serum (Gibco) and 2 mm-L-glutamine (Gibco). Adeno-associated virus type 2 (AAV-2) was propagated in HeLa-S3 suspension cultures as described (Rose et al., 1969). Virus particles were purified by lysis of infected HeLa cells with sodium desoxycholate and isopycnic centrifugation in CsCl gradients (Rose et al., 1966). Virus stocks were titrated on KB cells by a fluorescent focus assay as described by Carter et al. (1979) or by a DNA

(d) Plasmid conhwtions All plasmids used in this study are illustrated in Fig. 2. The plasmid containing the AAV genome cloned into the P&I site of plasmid pBR322 (pSM620) and the mutants containing large deletions in the left (pSM609) or right

Terminal mutants of AAV

Figure 2. Chimeric unique region of the the presence of the designates a deletion

Plasmid

Genotype

pSM620

wvwt

Left

terminal repeot s s

Internal sequences

Right terminal repeat

L

pBB704

wt/wt (-3) I

pBBBO8

mt/mt (-3) r

pBB901

rt/mt (-31 r

pBB1002

mt/wt k31r

pSM609

del/wt (-3)

pSM802

wt/del

pLRl208

del/mt (-3)

pBB215

mtldel

33 8 5s

9

I IW

Ad,--‘--j I-r 1’4’1

terminal mutants of AAV. The mutants used in this study are depicted. Each terminus flanks the genome. The wild-type (GGGCTTTGCCC) and mutant (CAGATCTG) alleles are distinguished by unique resttiction sites SmuI (S) or BgZII (B). de1 denotes a deletion of approx. 100 bp. (-3) of 3 bp from the right end of the genome.

K. A. Wohenzky and K. I. Rerns

94

(pSM802) terminal repeat have been reported, as was the mutant containing the additional 11 bp deletion in the right terminal repeat (pSM1205: Samulski et nl., 1982,

1983). The reciprocal 11 bp deletion was constructed in pSM802 (pBB312) by digestion with 9mal and recircularization. The plasmid containing the mutant allele (an 8 bp

f3glTI site (CAGATCTG) in place of an 11 bp symmetrical sequence (QGGCTTTGCCC (nucleotides 57 to 67)) in the right terminal repeat and a large deletion in the left terminal repeat (pLR1208) has been reported (Lefebvre rt

aZ.. 1984). The reciprocal plasmid containing the mutant allele on the left side and a large deletion on the right (pBB215) has been reported (Bohenzky rt nl., 1988). The chimeric plasmids were constructed by digesting the wild-type or mutant plasmids with HindIII, which cuts once within t,he AAV genome and once within the

pBR322 sequences. The fragments containing either the left or right termini reciprocal fragment. structed by ligating

were religated to the appropriate The chimera pBB901 was conthe left side-Hind111 fragment of

pSM802. wit,h the right side fragment of pl,R1$08. Thr ol’posite-orientation chimera pBB1002 was constructed hy ligat,ing t,he left side HindIT fragment, of pB B215. with the right’ side fragment of pSM609. The homologous

plasmids with homogeneous trrmini warp caonstrucsted1)) ligating the left side Hind111 fragment of pSM802. with the right side fragment of pSM609 for the wild-type/wil(l-

type plasmid pBB704 and the left side fragment of pBt3215 with t’he right side fragment, of pLR1208 for the

plasmid pBB808.

3. Results (a) Replication

of AAV DNA chime&c term&i

with

Tt has been reported that plasmids containing forms of the AAV genome in which the termini differed from one another with respect to their terminal repeat alleles produced AAV DNA and virions when transfected into adenovirus-infected KB cells (Bohenzky et al., 1987). Analysis of the DNA extracted from progeny virions revealed two distinct distributions of terminal repeat alleles. Progeny resulting from an input plasmid that contained the wild-type allele in the left terminal repeat and the mutant allele in the right terminal repeat contained mostly the wild-type allele in both termini. Tn contrast, progeny resulting from an input plasmid in which the terminal repeat alleles were in the opposite orientation contained both alleles present in approximately equal amounts. Furthermore, most of the left terminal repeats contained the mutant allele, while most of the right terminal repeats contained the wild-type allele. Thus, most of the progeny contained the terminal repeat alleles in the same orientation as the input plasmid. A small amount of termini containing the alternative allele could be detected but the level was low and the frequency of transfer in both directions was approximately equal. A possible explanation for the two phenotypes lies in the background of the constructs. The original isolate of AAV cloned into pBR322,

pSM620, contained two intact termini (Hamulaki et nl., 1982). Two plasmids, pSM609 and pSM802, were co-isolated with pSM620 and contained approximately 100 bp deleted from the left and right termini, respectively (Samulski et al., 1983). In addition to the large deletion on the left: pSM609 also contained a 3 bp deletion from the right end. Since p8M802 was not sequenced, it, was unknown whether a similar small deletion existed in its left end. The deleted plasmids were used to construcht, the mutant variants pLR1208 and pBB215 (Lefebvre et nl.. 1984; Kohenzky et nl.. 198X). The previously studied chimeras (Bohenzky et nl.. 1987) were constructed by ligating the mutant containing terminal fragment from either pLR1208 or pK B215 to the appropriat,e wild-type fragment from pSM620. The resulting set) of chimeras (pRK620X and pBB501) were not symmetrical. in t,hat the background for the terminal repeat, alleles in thtb left termini were from pBM620 and pSMSO2 for pRB6208 and pBBfiO1. respectively, and the background for the right termini were from pSM609 and pSM620 for pRB6208 and pKBfiO1, rc:spe(aGvely. It was possible. therefore. that t IIf‘ differences in the two phenot’ypes may have been t,he result of differences in the backgrounds of the (shimeras and not in the terminal repeat alleles themselves. In order to control for this possibility, a set of plasmids was constructed in which all the left’ termini came from the pSM802 background and all the right termini came from the pSM609 background. The resulting plasmids contained either wild-type or mutant alleles in bot’h termini (pBB704 and pBB808, respectively) or were chimeras with the wild-type allele in the left terminus and the mutant allele in the right terminus (pBB901) or in the opposite orientation (pRB1002). These plasmids were transfected into adenovirus-infected cells, which were then harvested after 72 hours. Freeze-thaw lysat’es of the transfected cells were used with adenovirus to coinfect suspension cultures of Hel,a cqells. After incubation, the suspension cultures were harvested and AAV virions isolated and purified by successive rounds of isopycnic centrifugation in C&l. DNA was extracted from the virions, annealed to form duplex molecules, and subjected to analysis with restriction endonucleases. The results of such an experiment, as well as a restriction map of AAV-2, are shown in Figure 3. When virion-extracted DNA is digested with Pstl. six bands are seen. The two largest bands of 2.4 kb and 1.7 kb, labelled A and B, represent the internal sequences of AAV. Two smaller bands of 0.495 kb and 0.415 kb, labelled C and D, represent, the left and right, terminal PstI fragments, respectively. Two sets of triplet bands can be seen migrating between the internal and terminal fragments. These represent aberrant,ly base-paired terminal fragments that occur during the reannealing process when viral DNA containing termini in opposite orientations (flip and flop) come together. They are

AA V Terminal Interactions

wt /wt P

PS

PB

wt/mt

mt /mt P

PS PB

P

PS PB

95

mt/wt P

PS PB

A 0

Ca Da

C D

b

b

I

B A ti SC Figure 3. Structure of AAV progeny virus DNA from chimeric plasmid mutants. DNAs from virions isolated from infections of cells with virus stocks of various chimeric mutants were digested with restriction endonucleases, radioactively labelled at their 3’ termini, and electrophoresed on a 10% polyacrylamide gel. The alleles are designated wt for wild-type and mt for mutant, and the chimeras are designated by their terminal alleles in a left terminus/right terminus format. The restriction digests performed are designated thus: P = P&I, S = SWMZIand B = BgZII. A restriction map of t#heAAV type 2 genome is shown below and the fragment labels A to D refer to the labelled migration distances. Because of the inversion of the terminal 125 bases during DNA replication, the 2 SmaI sites in the wild-type allele may be either 47 and 57 or 68 and 78 bases from the end. Because of the inversion, when the complementary strands of virion DNA anneal, the duplex structure at the termini may be aberrant if the terminal repeats are in opposite orientations and so the P&I fragments migrate aberrantly slowly (C, and D,). Several putative aberrant structures are possible and are reflected by the heterogeneity of C, and D,. This heterogeneity is exacerbated after digestion with both PstI and either SmaT or BgZII and many new bands are seen. In the PstI digests, the additional rapidly moving band is a hairpinned terminal fragment. Other faint bands possibly represent the defective variant genomes found in virion preparations. wt/wt, pRB704; mt/mt, pBB808; wt/mt, pBB901; mt/wt, pBB1002.

labelled C, and D, for aberrantly base-paired C and D fragments, respectively. When the AAV DNA is digested with a second enzyme specific for a particular terminal repeat allele (Smal for wild-type and BgZII for mutant), both the normal and base-paired aberrantly terminal fragments disappear and a variety of lower molecular weight digestion products are seen. As is seen in Figure 3, the progeny of pBB704 and pBB808 exclusively contain either the wildtype or mutant alleles, respectively. The progeny of the chimera pBB901 also contain the wild-type allele exclusively. Therefore, the DNA exhibits the same gene conversion phenotype seen with the pRB6208 chimera previously reported (Bohenzky et al., 1987). The progeny of chimera pBB1002 contain

both the wild-type

and mutant

alleles and,

from analysis of their digestion products, show the same low-level reciprocal transfer distribution as was seen with pBB501. The dependence of the genotype of the progeny terminal repeats on the orientation of the parental terminal repeat alleles is consistent.

(b) Replication of chimeric forms of virion-extracted AA V DNA The chimeric AAV DNAs transfected were all in plasmid form. It was therefore not possible to distinguish between terminal interaction during the rescue process or during the process of DNA replication. Thus, it was decided to construct chimeric forms of AAV DNA that could replicate without the burden of rescue. The inability to

K. A. Hohenzky and K. 1. Berns

I%

propagate a pure stock of chimeric AAV from plasmids without at least some level of transfer between termini necessitated the construction of chimeras from virion-extracted DNA by direct ligation. Duplex virion AAV DNAs from pure wildtype and mutant stocks were digested with the restriction endonuclease BamHI, which cuts AAV once at map position 22. The two fragments from each digestion were isolated and purified by two successive rounds of agarose gel electrophoresis followed by electroelution. The left-side fragments were mixed with right-side fragments with either their own or alternative alleles, and the ligation mixture was transfected directly into adenovirusinfected KR cells. The cells were harvested at 72 hours and freeze-thaw lysates used to infect suspension cultures as described above. Virions were prepared and the DNA extracted and subjected to restriction endonuclease analysis. The results of this experiment are shown in Figure 4. The ligation mixtures of fragments with homologous terminal repeat alleles produced progeny with the appropriate markers propagated.

wt/wt P

PS PB

mt/mt P

PS PB P

In contrast, the ligation mixtures of fragments with heterologous terminal repeat alleles produced virus with only the wild-type allele. This was true of either allelic orientation of input DNA. Thus, in contrast to what was seen with plasmid input, where the polarity of input alleles determines the phenotype, in the case of virion DNA input,, only the wild-type allele persisted in progeny. (c) Orientation effects on phenotype with intermolecular competition In addition to serving as tools for studying the interactions between termini, the AAV chimeras represent a form of competition between the wildtype and mutant alleles. This is an intramolecular competition in which either wild-type has an advantage or both wild-type and mutant alleles are equivalent. The results are dependent on the form of the DNA introduced and the orientation of the alleles within the AAV genome. An attempt was made to assay the orientation effects on the phenotype of intermolecular competition also.

wt /mt PS PB

mt/wt P

PS PB

-A 4

-C -D

-78 -aa -57 -47

b II

b I

B *C A d Figure 4. Structure of AAV progeny virus DNA from chimeric virion DNA mutants. DNAs from virions isolated from infections of cells with virus stocks of various chimeric mutants were digested with restriction endonucleases, radioactively labelled at their 3’ termini, and electrophoresed on a 10% polyacrylamide gel. The alleles are designated wt for wild-type and mt for mutant, and the chimeras are designated by their terminal alleles in a left terminus/right

terminus format. The restriction digests performed are designated thus: P = P&I, S = SWKZIand B = &III. A restriction map of the AAV type 2 genome is shown below and the fragment labels A to D refer to the labelled migration distances. See the legend to Fig. 3 for additional details.

AA V Terminal Interactions Deletion mutants were used in which one terminus contained a deletion of approximately 100 bp and the other terminus contained a largely intact terminus with either a wild-type or mutant allele. The wild-type plasmids were cotransfected with mutant plasmids, in either the same or opposite orientation, into adenovirus-infected KB cells, incubated, lysates made and introduced into suspension cultures of HeLa cells. Virions were purified from the suspension cultures and the AAV and subjected to restriction DNA extracted endonuclease analysis. The results of this experiment are shown in Figure 5. Each of the mutants was capable of replicating independently and propagating the appropriate allele (data not shown). In three of the four

cotransfections,

however,

only

the wild-type

allele was present in progeny (Fig. 5). These transfections included both cases in which the two alleles were on tbe same side of the genome,

del /wt X del /mt P

PS PB P

97

left or right, as well as the case in which the wildtype allele was on the left side of the genome and the mutant allele was on the right side. In one case, both alleles were propagated. This was the case in which the mutant allele was introduced on the left side and the wild-type allele was introduced on the right side. Thus, the orientation dependence of the two phenotypes in intramolecular plasmid competition is mimicked by the results of intermolecular plasmid competition experiments.

4. Discussion Our purpose in creating the chimeric constructs was to ask whether

replication.

Our

either

pairing

between

wt /de1

wt /de1 X del/mt

del /wt X mt fdel

X mt /de1

perfect

base-pairing

between

terminal repeats of AAV DNA, as implied by structure III in Figure 1, is an obligatory step in the initiation of the first round of AAV DNA

PS PB P PS PB

conclusion

the

is that

terminal

perfect

repeats

base-

is

not

P PS PB

A B ca Da

b

b

sC Figure 5. Orientation-dependent

B

’

A

0”

intermolecular competition between internal palindromic sequence substitution mutants in plasmid DNA form. DNAs from virions isolated from infections of cells with virus stocks of various competition experiments were digested with restriction endonucleases, radioactively labelled at their 3’ termini, and electrophoresed on a 10% polyacrylamide gel. The alleles are designated wt for wild-type, mt for mutant and de1 for a deletion of approx. 100 bp. The chimeras are designated by their terminal alleles in a left terminus/right terminus format. The restriction digests performed are designated thus: P = P&I, S = SmaI and B = Bg6II. A restriction map of the AAV type 2 genome is shown below and the fragment labels A to D refer to the labelled migration distances. del/wt, pSM609; del/mt, pLR1208; wt/del, pSM802; mt/del, pBB215. See the legend to Fig. 3 for additional details.

9x

R. A. Bohenxky

required, because of the demonstrated ahilit’y of chimeric molecules to persist through many rounds of replication and two cell passages so that encapsidation has occurred twice (Fig. 3, pBB1002). However, when the wild-type allele was at t,he left end of the chimeric plasmid construct, the progeny virions contained genomes with wild-type terminal repeats at both ends. The same results were obtained when chimeric duplex virion DNA molecules with the wild-type allele at either end were tested. Thus. the ability of the terminal repeat heterogeneity to persist through replication is dependent on the initial structure of the chimeric parental genome. We consider that this variability is a consequence of two factors. The first is that genomes with wild-type terminal repeats have an advantage in replication over those with mutant terminal repeats. The advantage is small, because in parallel infections or transfections, the rates and extent of DNA replication were indistinguishable. In cotransfections of wild-type/wild-type and mut,ant/mutant plasmids or coinfections of wildtype/wild-type and mutant/mutant virions, the same results were L obtained. However, cotransfect,ion of wild-type/wild-type and mutant/mutant duplex virion DNA led to only wild-type/wild-type progeny (Bohenzky et al., 1988). Thus, whenever one allele displayed an advantage, it was always the wild-type allele. Again, in the experiments described here, the wildtype allele displayed an advantage. In the case of the chimeric plasmid constructs, placement of the mutant terminal repeat at the left end neutralized the wild-type advantage. This was not the case for chimeric duplex virion DNA. Thus, the wild-type advantage and/or the neutralization of it appears to depend on the form of the genome (in a virion, plasmid, or as duplex virion DNA) presented to the cell. Because of this dependence on structure, we believe that the wild-type advantage involves an early step in the process during conversion of the artificial plasmid or duplex virion DNA substrate to a physiological replication structure. The reason for the difference in results obtained with plasmid or virion DNA forms is unknown. It may reflect a significant difference in effective substrate concentration with respect to the replication apparatus; i.e. all of the virion DNA taken up hy the cell may be a potential substrate, whereas only a small fraction of the plasmid DNA may eventually assume a conformation essential to be a substrate for replication. Why positioning of the mutant allele in the left terminal repeat of the construct seems to balance out the presumed inherent advantage of the wild-type allele is uncertain. If indeed the plasmid must assume a special conformation for rescue and replication to occur, then the polar effect observed in these experiments reflect an enhanced may well propensity for the region of the left terminal repeat to assume such a conformation. This propensity might well be associated with the leftward-most transcriptional promoter and upstream regulatory

and K. I. Hems

sequences in AAV DNA. which are adjacent to the left terminal repeat (Lusby & Berns, 1982) and seem likely to extend into the terminal repeat’ (A. R. Beaton & K. T. Berns, unpublished results). This transcription unit, represents the AAV equivalent) of an immediate early gene (i.e. the gene product(s) regulate subsequent AAV gene expression: Labow et al., 1986; Trempe & Carter, 1988). The association of viral origins of DNA replication with transcriptional regulatory elements has been well-documented in the cases of polyomaviruses (Wirak et al., 1985; Veldman et al., 1985; DeLucia et al., 1986; Li et al., 1986; Campbell & Villarreal, 1986; Hertz & Mertz, 1986), papillomaviruses (Lusky & Botchan, 1986; Stenlund et al., 1987; Baker & Howley, 1987) and adenoviruses (Adhya et al., 1986; Wides et al., 1987; Rosenfeld et al., 1987; Pruijn et al.. 1987), as has the apparent need for positive transcriptional regulatory protein interaction with the sequences for initiation of DNA synthesis. Furthermore, mitochondrial origins of replication are associated with transcriptional elements (Chang & Clayton, 1985; Chang et al.. 1985). Gottlieb $ Muzyczka (1988) have described an enyzmic activity that cleaves at t’he junctions between the AAV terminal repeat and vector sequences. One of the plasmids they investigated was the parental construct from which the plasmids used in this work were derived. No preference for cleavage at either of the terminal repeat/vector junctions was noted by Gottlieb & Muzyczka (1988) and, therefore, this does not appear to be a likely alternative possibility. We have reported that, in a normal productive AAV infection, half of t,he progeny genomes are chimeric, in that the terminal repeats at the two ends are in opposite orientations (i.e. 1 quarter of the molecules have both terminal repeats in the flip orient,ation, 1 quarter have both terminal repeats in the flop orientation, and half have 1 terminal repeat in the flip and 1 in the flop orientation; Lusby et al., 1981). The current model for AAV DNA replication would predict that this would be the case when a strand was directly displaced from the replicative complex and encapsidated. In this study, we have looked more directly at the requirements for perfect complementarity in the process of replication itself. DNA genomes have specialized All linear sequence organization at the termini. To a large extent, this is a consequence of the requirement of all known DNA-dependent DNA polymerases for a primer to initiate replication. In the case of AAV DNA, the terminal repeat is the primer and the process of hairpin transfer from parental to progeny strand both maintains the integrity of the 5’-terminal sequences and then serves as a template to allow repair of the 3’-terminal sequences of the parental strand (Straus et al., 1976; Hauswirth & Berns, 1979). The terminal repeats at opposite ends can also interact to allow repair of other types of terminal deletions (Samulski et al., 1983). This is potentially important in replication and in rescue of

A A V Terminal Interactions the genome from the integrated state that occurs during latent infection (Hoggan et al., 1972; Berns et al., 1975; Handa et al., 1977; Cheung et al., 1980; Laughlin et al., 1986). Repair of mutations in inverted terminal repeats by the other end has been seen in adenoviruses (Stow, 1982; Hay & McDougall, 1986; Haj-Ahmad & Graham, 1986), retroviruses (Colicelli & Goff, 1985) and yeast (Matsuzaki et al., 1988). In all cases, the repair resulted in wild-type progeny. The only previously reported case of mutant propagation was with a deletion mutation in the Herpes Simplex virus (HSV) inverted repeat (Poffenberger & Roizman, 1985); et al., 1983; Poffenberger however, the propagated mutant was incapable of undergoing the inversion characteristic of HSV. The gene conversion process observed in many of the experiments reported here might be the result of a repair process in which one terminal repeat is used as a templete for repair of that at the opposite end of the genome. This was the primary model suggested by Samulski et al. (1983). However, a conversion process that involves recombination might be possible. The results in Figure 5 strongly suggest that repair is more important than x deletion/wild-type recombination. In the mutant/deletion cotransfection, both wild-type and mutant alleles were present in progeny genomes. Roth mutant and wild-type sequences were present at both termini. These are the results predicted by intramolecular repair and not the results predicted by intermolecular recombination (such recombination would lead to mutant sequences in the left’ terminal repeat and wild-type sequences in the right terminal repeat). Intramolecular homologous recombination would have to take place within the terminal repeat sequences by definition. We have not ruled out this possibility, nor are we certain of the extent to which recombination within the terminal repeats would differ at the molecular level from the repair process suggested by Samulski et al. (1983).

Previous studies have shown t’hat the terminal repeat plays a significant role in integration. The junctions between viral and cellular DNA usually involve the terminal repeat, and the insert is most often present as a head-to-tail tandem repeat et al., 1986; (Cheung et al., 1980; Laughlin McLaughlin et al., 1988). In studies of recombination between AA\’ and simian virus (SV40) in monkey cells, the structure of the rec>ombinant reflected the initial method of delivery of the DNAs (Grossman et al., 1984, 1985). When transfection was used with an AAV plasmid or duplex virion DNA, the recombinant had a simple deletion-m substitution motif and the substitut’ed AAV sequences represented all parts of the genome equally. When virion coinfection was used, 80% of the recombinants contained the AAV terminal repeat and all of these that were cloned represented tandem repeats of five to six copies of both the SV40 regulatory region and one end of the AAV genome. Thus, t’he immediate ability of the end of

99

the AAV genome to fold over on itself after uncoating apparently made a significant difference in the products obtained and may be a clue to the variable results obtained in the present study, which were also dependent on the original construct used for transfection. In summary, we conclude that the ends of AAV DNA can interact during replication but that replication does not require that the ends be able t,o base-pair perfectly. We conclude also that the wildtype sequence has a slight advantage in replication over the mutant allele and that, in the case of a chimeric plasmid transfection, this advantage can be counterbalanced by placing the mut,ant terminal repeat on the left end of the genome. We thank P. Burfeind, H. Homar and M. Bremmer for able technical assistance throughout this study. We acknowledge M. Labow, A. Beaton, R. Kotin, C. Leonard. M. O’Donnell, R. Samulski and E. Muzyczka for helpful discussions and for critical reading of the manuscript. This research was funded by grant AI-22251 from the National Institutes of Health. One author (R.A.B.) was partially funded by grant AI-071 IO from t#he I’S Public Healt,h Service.

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K. L., Tabares, E. t Roizman,

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by N. Sternberg

5, 2924-2935.