The minute virus of mice capsid specifically recognizes the 3' hairpin structure of the viral replicative-form DNA: mapping of the binding site by hydroxyl radical footprinting.

0022-538X/91/094629-07$02.00/0

CopyrightC) 1991,American SocietyforMicrobiology

The

Minute Virus

of

Mice

Capsid

Specifically

Recognizes

the 3'

Hairpin

Structure of

the

Viral

Replicative-Form

DNA:

Mapping

of the Binding Site by

Hydroxyl

Radical Footprinting

KURTWILLWANDAND BERNHARD HIRT*

Swiss InstituteforExperimental Cancer Research, CH-1066 Epalinges, Switzerland Received 29January1991/Accepted24May 1991

Theterminal hairpin structures of the DNA of minute virus of mice(MVM) are essential for viral replication. Here weshow that the hairpin 3' terminus ofMVM replicative-form DNA bindsspecifically toemptyMVM capsids. Binding of the same terminalDNA sequence in itslineardouble-stranded(extended)conformationwas notobserved. After heat denaturation and quick cooling of 3'-terminal extended-form fragments,notonly the virionstrand but also thecomplementary strand was found to bind to the capsid, presumably becauseeach strandre-formedasimilar hairpinstructure.Nobinding affinityfor thecapsidwasfoundtobeassociatedwith hairpin or extended 5' termini or with any other region of the viral DNA. Hydroxyl radical footprinting analyses revealed three protected nucleotide stretches forminga bindingsite at thebranch point of thetwo 3'-terminal hairpin arms looping out from the DNA stem (T structure). Single basechangeswithin this site did notaffect the binding.Inband shiftexperiments, specific bindingto theT structure wasdemonstrated forVP1 butnotforVP2.

The minute virus ofmice (MVM) belongs to the genus Parvovirus of the family Parvoviridae (20). These are non-envelopedicosahedral viruses which replicate autonomously in mammalian cells. The MVM genome is linear single-stranded DNA, 5,149 bases in length (2). At both termini, short self-complementary sequences are folded into stable hairpin structures (3, 18). The 3'-terminal hairpin serves as template andprimer forcomplementary-strand synthesis in vivo to produce a double-stranded replicative-form (RF) DNA (4).The ends of the RF DNA molecules occur either in thehairpin conformationor in the so-called extended form, in which the terminal DNA stretch is base paired to a complementarystrand forming a regular, linear terminus (3). Replication models which account for these conformational differences have been developed (6, 25).

The RFDNAsof the autonomous parvoviruses Aleutian mink disease virus (ADV) and bovine parvovirus have been shown to interact with viral capsid proteins via their 3' termini. The binding was observed only for 3' ends in the hairpin conformation but not for extended forms (15, 29). The ADV DNAhad been shown to interact with only one of thetwo ADVcapsid proteins, VP1 (28, 29). Here we show that the 3' terminus of the MVM RF DNA in its hairpin conformationalso binds to empty viral capsids and purified VP1. Using the hydroxyl radical footprinting technique, we havemapped the binding site to the central branch point of the T-shaped hairpin. We have tested other sequences forming similar T-shaped structures. These included the left-hand end of the MVM complementary strand, a 3'-terminal canine parvovirus (CPV) hairpin DNA fragment, and anoligodeoxynucleotide whichdiffered fromthe MVM genome inpositions 69 and 70. All theseDNAs werefound tobind to the MVMcapsidaswell. In contrast, we could not detectbindingof thecapsidtotheconformationally different 5'-terminal MVM DNA hairpin. Linear double-stranded DNAfragments, whetheroriginatingfromthe 3' end,the5'

*Correspondingauthor.

end, orotherregions of the MVM RF DNA, also were not foundtobind.

MATERIALSANDMETHODS

Materials. Restriction enzymes and DNA-modifying en-zymes werepurchased from Boehringer.Radiolabeled prod-ucts were from Du Pont. Products for radical footprinting werepurchased from Sigma.

Cellsand viruses. The cultivation ofmouse A9 fibroblast cells and thepropagation of MVMp, the prototype strain of MVM,wereperformedasdescribed elsewhere (1). CPVwas grown in feline NLFK cells.

Purification of MVMp particles. MVMp was purified on CsCl gradients according to the method described in refer-ence 28. Fractions were collected from the bottom of the tube, assayed for their density, dialyzed against 10 mM HEPES

(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic

acid) (pH8.0)-50mMNaClandanalyzed by sodiumdodecyl sulfate

(SDS)-polyacrylamide

gel electrophoresis (PAGE) and Coomassie blue staining. The fraction containing the maximalamountofviralproteins, usually correspondingtoa densityof 1.35

g/cm3,

wasconsideredtocontainpure empty viralparticles and was used in all experiments. The protein concentration was determined by fluorescence

spectropho-tometry.

Purification ofviral proteins. Viral proteins were

purified

in SDS-PAGE gels by using 25 mM Tris-190 mM

glycine-0.1%SDSasrunning buffer. Theywerelocalized

by

staining

aparalleltrack withCoomassie blue andwereelectroeluted, usingthesamebuffer,in thepresence of 300

p.g

ofRNaseas carrier. After 3 h ofelectroelution at 25 mA, 1-ml

samples

were dialyzed against 1 mM HEPES (pH 8.0)-5 mM NaCl and lyophilized to a final volume of 100 ,ul. Proteins were

precipitatedwith 5volumes of acetone,dried,anddissolved in binding buffer(10mM HEPES [pH

8.0],

50 mM

NaCl).

IsolationofMVM and CPV RFDNAs. At 24 h

postinfec-tion, whole-cell DNA was isolated by the method of Hirt 4629

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Ti

jsty

I

5'

CD

Le) ) GOD

CD 10 014

v-Ina0O

CM4

_ ~ CM CM

IUL t (7

f)'.. 0)

0I)CI) NtI aC I

le t-oo

Iqr

0 0 ~ 0 0.*1 .. 0 I

3'

VIUVI VI5'

S v~~~~- Ix olM-m_ v-o I4 b s|, IV ...9@

CD

Cl*.

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5'.

1-1000 2000 3000 4000

Ava

II

Xba

I

r0

_-i-It-MboII

][1 5

,

Mbo

I

HI

5000 nucleotides

FIG. 1. Restriction sitesontheMVMpgenome.Thepositionsofthe 3' end labelsareillustratedby points.Thefragmentswhich havenot

beenlabeledareindicatedby hatchedarrows.

(11). Viral monomer RF DNAs were purified on 5to 30%

sucrosegradients (22).

DNAdigestion andendlabeling of fragments. DNA(1 ,ug)

was digested in the appropriate restriction buffer, phenol

extracted, ethanol precipitated, and dried. 3'-End labeling

was performed with Escherichia coli DNA polymerase I

(largeKlenowfragment) and a-32P-labeleddeoxynucleoside triphosphates. Specific fragmentswerepurifiedin

polyacryl-amidegels andrecovered by electroelution.

Gel retardation assay. Band shift experiments were

per-formedundertheconditions described previously (29). Sam-pleswereanalyzed byelectrophoresison5%polyacrylamide

gels ranginginlength from 10to35 cm.

Hydroxyl radical footprinting. The protocol of Zorbas et al. (30)was followedexceptfor differences in the composi-tion ofbinding buffer, the finalreaction volume, and the time ofreaction. The DNA-proteincomplexwas formed in 85 ,ul

of10 mM HEPES (pH 8.0)-50 mM NaCl as described for

band shift experiments. After 1 h of incubation at room

temperature,the solutionwasmade 1 mM in sodium

ascor-bate,20 p.M inEDTA, 10 p.M in(NH4)2Fe(SO4), and 0.3% in H202. Thefinalreaction volume was 100 ,u1. After addition

oftheH202 solution, the reactionwascarriedoutfor 2min andstopped with 10 ,ul of100 mM thiourea-20mM EDTA. When the DNAprotein complex wasto be separated from unbound DNA, 10% glycerol and 0.1% bromophenol blue

were added and the sampleswere immediatelyloaded onto

5% polyacrylamide gels. After electrophoresis, the DNA complex and free DNA were recovered by electroelution.

Both samples were phenol extracted, ethanol precipitated,

dried, and analyzed in 6% polyacrylamide-8 M urea

se-quencing gels.

Sequencingof DNA.Sequencingreactionswerecarriedout by themethod of Maxam and Gilbert(14).

Synthesisofoligodeoxynucleotides. Thesequencebetween nucleotides 39 and 79 of the MVM genome, including the part of the 3'-terminal hairpin which forms the T-shaped structure, was synthesized as a single 41-mer. After

self-annealing, the DNA was 3' end labeled by using Klenow fragment and [32P]dCTP.

RESULTS

3'-Terminal hairpin of MVM RF DNA binds to MYM capsids. We carriedoutband shift experiments with MVM RF DNA enzymatically digested with StyI. The fragments

were3' end labeled by using radioactive dATP,asshown in

Fig. 1.Afterelectrophoresisina5%polyacrylamide gel,the

pattern seen in Fig. 2, lane 1, was obtained. When the digestion products were electrophoresed after incubation with MVM capsids, the band representing the 3'-terminal hairpin fragment disappeared (lane 2, band h), suggesting binding of this fragment to the capsid. In contrast, the mobilityof therespective extended-form fragment (140 bp, bande)remainedunchanged, indicatingthat thepresenceof theterminalsequence inahairpin conformationis essential for binding. The specificity of this binding is demonstrated by theunchangedmobilities of the otherdigestion products comingfrom differentregionsof the genome. However, in theexperiment described above, we could not analyze the

00) 0)

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3'

lm-

1-c"J

*-+I.*-Iv

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3' e

denat. RFxAva II

ri,7^_r

+

~~~

+ T

-+

-+r

-I

0-Ilw

I~~~~~~-

L-l

~ ~

~~~~~~~

Ii

.S'h~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

->3'h ' w

I'I

1 2 3 4 5 6 7 8 9 10 111213

FIG. 2. Gel mobility shift analysis of MVM RF DNA fragments. Restriction fragments were generated by digestion withStyI,XbaI, and MboI orAvaII. The 3'-end-labeled digestion products were separated in a 5% polyacrylamide gel (lanes 1 to6 and 11 to13). The gel-purified 3'-terminal extended-form fragment of the StyI digest was run in lanes 7 and 8, and its denatured form was run in lanes 9 and 10. For band shift analyses, samples wereincubated with0.05 ,ugof empty MVM capsids (C) (lanes 2, 4, 6, 8, 10, 12, and 13). The sample in lane 13 was additionally incubated with a100-fold molar excess of T oligodeoxynucleotide (T). h, hairpinform; e, extended form.

5'-terminal MVMStyI fragments because they were too long (2,000 bp). To produce short 5'-terminal fragments which could be tested in a band shift assay, the right-hand XbaI digestion product of MVM RF DNA was cleaved withMboI (Fig. 1). Figure 2, lane 3, shows the electrophoresis patterns of the resulting 3'-end-labeled fragments (one additional band with a length of about 110 bp is of unclear origin). The addition of MVM capsids had no influence on the position of the bands, including those representing hairpin or extended 5' termini (lane 4). To identify the latter, we have redigested the fragments with

AvaIl,

which cuts at nucleotide 4874 and therefore cleaves the 5'-terminal fragments labeled at nucle-otide 4802 and leads to the disappearance of the correspond-ing bands, which was in fact observed (data not shown). To confirm the result described above, both sets of MVM DNA fragments, i.e., those produced by StyI digestion and those resulting from cleavage with MboI, were loaded in the same lane (lane 5). After the addition of MVM capsids(lane 6), all bands remained unchanged except for that representing the 3'-terminal hairpin. An

AvaII

digest of MVM RF DNA was

loaded without priortreatment (lane 11) or afterincubation with empty capsids (lane 12). The specific disappearance of theband of the 3' hairpin fragment (about 400 bp, lane 12) was prevented when a 100-fold molar excess of a synthetic

oligodeoxynucleotide

that spanned the region between

nu-cleotides 39 and 79 on the MVM genome was added (lane 13). This oligodeoxynucleotide (T oligodeoxynucleotide) is expected to form the T-shaped structure characterized by

two

hairpin

armsloopingoutfromthestemofthe molecule

(compare withFig. 7). FragmentIVoftheAvaIl digest isnot visible in Fig. 2(lanes 11, 12, and 13), although it should be labeled(Fig. 1). Onthe other hand, there are twoadditional fragments of unclear origin (Fig. 2, between fragment V and VII). If we suppose that the restriction site at nucleotide 4874 is not GGACC, as published (2), but GGTCC, the extended- andhairpin-form5'-terminalfragments could have been labeled with dATP and thus they, instead offragment IV, would have given the observed result.

Inlane7, thegel-purified extended 3'-terminal fragment is visible. As expected, there was no shift of this fragment produced by added MVM capsids (lane 8). However, on adding capsids to heat-denatured and quick-cooled samples, all thematerialwas movedtothe top of the gel (lanes 9 and 10). Since the fragment possesses a 3' recessed right-hand endwhich has been filled in by Klenow polymerase, the label is within the complementary strand. The unpaired comple-mentary strand can, like the virion strand, form a T-shaped hairpin structure (compare Fig. 7). This structure, which permits binding to the capsid, has never been observed in vivo. The same result as that seen in Fig. 2, lanes 9 and 10, was obtained with DNA labeled in thevirion strand with T4 polynucleotide kinase (datanot shown).

Hydroxyl radical footprinting. The 3'-terminal hairpin frag-ment ofthe Styldigest (compare Fig. 1 and 2) was used for radical footprinting experiments (Fig. 3). The cleavage pat-tern ofthe free DNA is shown in lane 1 and those of the DNA-protein complex are in lanes 2 and 3. Threeregions of DNA, clearly seenin lane 3, were foundto be protected by bound capsid. This experiment was repeated, and the posi-tions of protection (lane 9) were mapped relative to the migrationof sequencingreaction products (lanes 5 to 8). The material analyzedinlanes 3 and 9 had been recovered from a polyacrylamide gel in which theDNA-protein complex was separated from unbound DNA after the radical reaction. In lane 10, the unbound DNA samplerecovered fromthe same gel was loaded. This sample is specificallyenriched in DNA molecules which are nicked in the binding regions (see Discussion). Lane 11 shows free DNA thatwas cleaved in the absence of any protein.

The capsid binds at the branch point of the terminal T structure. The protected area includes 5 nucleotides in the centerof the T (noted as C in Fig. 3) as well as 6 nucleotides in both the 3'- and5'-oriented hairpin arms (depicted in Fig. 4). Additional nucleotides are semiprotected: 3'-CACT-5' between nucleotides 38 and 41, C in position 48, G in position 62 andTinposition 74. In the 5'-oriented armof the T, wefound a sequence slightly different from that published by Astell et al. (3): starting with nucleotide 68, thesequence observed was not 3'-CTG-5' but 3'-CGT-5'.

Binding of VP1 to the Tstructure. The empty MVMcapsid is formedby twodifferent proteins designated VP1 and VP2. We wanted to know whetherbothoronlyoneof them would bind to the viral DNA. The two capsid proteins were separated by SDS-PAGE and gel purified as described above. A gel retardation experiment was carried out using the MVM StyI digest. The result is given in Fig. 5. Lane 1

5XbalRFxf0bol

I I

RFxStyl RFxSty

C - '+- +' ,§

ll

IV'

5' e

t i L

UU0

i~~

N'.,

W

;!d

it

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A

Sty1-3-h O H

+ +

]5'

]C

797

IM. 0

1:.

] 3

B

StyI-3-h

C Py G Pu- -'

++ C

c

__*

-_ _ i~~

MP

sk

_r I

Z CL a- m C: > > >

3'h

1 2 3 4 5 6 7 8 9 10 11

FIG. 3. Hydroxyl radicalfootprintinganalysis oftheinteraction between the MVMcapsid and the MVMRF3'hairpin terminus. (A) Lane 1,hydroxyl radical-inducedcleavage of the 3'-terminal MVM RFStyIhairpinfragment;lanes2and3,cleavage patternsofDNA capsid complexes formed by1 ,ug(lane 2)or2,g(lane 3) of empty MVM capsids. Priorto theiranalysis, thecomplex andfree DNA had beenpurified in polyacrylamide gels. (B) Mappingofprotection sites. Lane 4, pBR322Hinflfragments (154 and75bp); lanes5 to8, Maxam andGilbertsequencing reactions of theStyIfragment; lane 9, protection by bound MVM capsids against hydroxyl radical-mediated cleavage; lane 10, DNA from the same reaction that migrated as free DNA during gel purification; lane 11, direct cleavage of free DNA in the absence of capsid.

50 AR G I

'C

G ,G

CG 40 30 20

GIC'C 3I G

GA3

I I *3

TCPWACGTCACTTACGT

ACATG..

.CCAR

60 CG 1IIIIIIIIIIIIIII 11111

AGTGTGCAGTGARTGCAA

GTGTAC...

GGTTC

I A

A 80 140

AG 90

FIG. 4. Schematic representation of the interaction between the MVMcapsid and the 3'-terminalhairpinMVM RFStyIfragment. Large black boxes represent areas of complete protection, cross-hatched boxes indicatesemiprotected regions, and asterisks indicate positions ofthe 3' end labels.

1 2 3 4 5

FIG. 5. Mobility shiftanalysisof MVMRFStyIfragments.The StyIdigestwas runin lane1. Lane 2shows thepattern of thesame digest in the presence of 15jigof RNase. MVMcapsid proteinsVP1 orVP2 wereadded in lanes3 and4,respectively.BothVP1andVP2 wereaddedin lane5.e, extendedform; h,hairpin.

shows the electrophoresis pattern of the digest in the ab-senceofprotein. Lane 2 isacontrol which confirms thatthe mobilities of the fragments remain unchanged in the pres-enceof 15 ,ug of RNase. RNasewasalso addedtotheviral protein samplesas acarrier. The80-bp3' hairpin fragment partially disappearedin the presence of VP1 (lane 3), sug-gesting thatit bound to theprotein. In contrast, no loss of material was visible for the extended terminal fragment, indicating the involvement of the 3' hairpin structure in bindingtoVP1. VP2 hadnosimilareffect when addedtothe DNA(lane 4),althoughit hadbeenappliedinapproximately a fivefold excess overVP1, corresponding to the stoichio-metric ratio of the viral proteins within the capsid (23). Interestingly, when VP1 and VP2 in the amountsapplied in lane 3 or4 were mixed together, incubated for 30 min in binding buffer,and thenadded to the DNA,even moreof the 3' hairpin fragment disappeared(lane 5).

Theexperimentwasrepeatedwith thesyntheticT oligode-oxynucleotide (usedascompetitorinFig. 2, lane 13). Itwas shiftedbyVP1,confirmingthat theviralprotein binds within the hairpinT structure. Noshift of the T oligodeoxynucle-otidewasproducedbyVP2(datanotshown).

Sequence requirements for binding. Asshown above(Fig. 2),after heatdenaturation andquickcooling, bothstrandsof

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5.-!F.7 86

i'

'179

3'e-l

N1-46_

VI a

-o

-101

3h-F w__

GNP

1 234

FIG. 6. Gel mobility shift analysis ofrestriction fragments of MVMandCPVRF DNAs.Restrictionfragmentswereproduced by digestion ofMVM RF DNAwith StyI or of CPV RF DNA with AccI.The3'-end-labeled digestion products were separated in a5% polyacrylamide gel (lanes 1 and 3). For mobility shift analysis, sampleswereincubatedwith0.05 ,ugofempty MVMcapsids(lanes 2and4). e,extendedform; h,hairpin.

MVM RF DNA were found to bind to the viral capsid, indicating that theDNAsecondary structurerather thanits nucleotidesequenceis important forbinding. Furthermore, bindingwasdemonstrated foranoligodeoxynucleotide com-prisingasequence published elsewhere (3) which was sub-sequently found to be slightly different from the MVMp sequence we observed. To see whetherfurther changes in thenucleotide sequence would have any influence on bind-ing,weusedanAccIdigest of CPVRF DNAinaband shift experimentwith empty MVM capsids. As shownin Fig. 6, theMVMcapsid bindstothe3'-terminal hairpin fragment of AccI-digested CPVRF DNA(lane 4, band h) with the same specificityasit bindstoitshomologous 3'-terminalhairpinin StyI-digestedMVM RF DNA(lane 2).Lanes 1and 3contain therespective free-DNA samples.InFig.7, the sequencesof thefourDNAswhichhavebeenfoundto bindtothe MVM capsid are depicted. Each sequence can form a T-shaped secondary structure which is essential forthe formation of the DNA-protein complex. Differences from the MVM se-quence within the T

oligodeoxynucleotide

and the CPV DNAarenoted.

DISCUSSION

Wehaveanalyzedthebinding ofempty MVMcapsids to the 3'terminus of MVM RF DNA. Thecapsid recognizesthe specific structure of the 3'-terminal hairpin and does not interactwith the 3'endinitsextended form. Thebindingis duetoone ofthe twocapsidproteins, VP1. Like the entire capsid,gel-purified VP1 wasfoundtodiscriminate between

A,CG

Gc' TCATCAC...3'

A

AGT GTG...

"cc AG

3 TERMINALTSTRUMRE OFMVMCp)DNA

A,Gc

CG/oGCG

Gc' TCATCAC.. .3'

C \ AGT GTG... A G

STRUCTMRE OFT-OLIGODEOXYNUCLEOTIDE

T C0

Co TCA CAC...3'

III III

GC\ AGTAGTG...

GC\suGC

cc

\'C

\\GC

T C

T-STRUCTURE OF THECOMPLEMENTARYSTANDOFTET-TERMINUSOFMV (p)DNA

R G

G TCATCAC...3'

-~ III III

AGT GTG... A G

3'- TERMINALTSTRUMREOFCPUDNA

FIG. 7. Schematicrepresentation ofthe secondary structuresof fourDNAsbindingto the MVMcapsid.The left-handpartsofthe virionandcomplementarystrands of MVM RF DNA are shown at thetop. Thepositions ofsingle base changes between the sequence of the MVM virion strand and two other DNA species, the T oligodeoxynucleotide and the 3'-terminal sequence of the CPV virion strand,areshown.

the extended andhairpin forms ofthe 3' terminus, binding onlytothe latter. The aminoacidsequencesof VP1 and VP2 areidentical exceptforaregionattheamino terminus which isuniquefor VP1 (24). This suggests that theamino-terminal region is involved in binding to the hairpin. In band shift experiments (Fig. 5, lane 3), the VP1-DNAcomplexes did not enter the gel. Complexes with the T oligodeoxynucle-otide were visible as clear bandsjust at the topofthe gel (data not shown). This suggests that VP1 formed large aggregates. The existence ofparvovirus capsid protein ag-gregateshasbeen observedinvivo(17). Although nospecific DNA-bindingactivity wasfound forVP2alone, its addition toVP1markedlyenhanced thebindingtothe 3' hairpin (Fig. 5, lane 5). The incubation of the viral proteins before the addition ofDNAmayhave resulted in thereconstitution of a capsidlike structure with higher affinity for the hairpin region.

Thespecific binding ofthe MVMcapsidtothe MVM DNA 3' terminus isanalogous to previous findings obtained with ADV and bovine parvovirus (15, 29), suggesting that this interaction plays a general role during the recognition of parvovirus DNA by the parvovirus capsid. In many other systems, the specificrecognitionofaviralgenomeby viral capsid proteins serves to ensure the

encapsidation

of the viral genome and preventspackaging ofextraneous nucleic acid (13,21,27;forareview,seereference5).The

synthesis

ofsingle-stranded

parvovirus

progeny DNAandits

packag-ing are simultaneous processes whichrequire the presence

of

preformed

emptyviral

capsids

(9, 16, 19). The

binding

of

the parvovirus RF DNA 3' end to the

parvovirus

capsid

mightthus serve for both the synthesis of progeny strands and their concomitant

encapsidation.

A model for these processes hasbeenproposed elsewhere(29).

It hasbeenassumed thata

T-shaped

structure, character-izedbytwohairpinarmsloopingoutfrom the DNA stem, is formed by intramolecular base

pairing

within the MVM DNA 3' terminus (3).

Figure

4 showsa schematic represen-tation of this structure,

including

the

capsid-binding site,

as

mapped by radical

footprinting.

The

capsid

binds to

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quences which are held close together within the branch

point region

ofthe Tandprotectseachof the threejunctions.

This

finding

is in strong support of the T-type structure

predicted

by the sequence analysis. An extended,

non-branched arrangement of the same binding sequences

changes

their positions in space and may thus prevent

interaction with thecapsid.The branchpoint regionof the T structure is not in aB-type helix. The DNA will be bent at

the

junctions

like DNA within cruciform structures (7).

These bends may represent signals which contribute to the

recognition

ofthe

capsid

asit has been observed for other

systems

(reviewed

in reference 26).

Hydroxyl

radicals attack the DNA within the minor

groove (10). Hence, the efficient protection of the DNA within the capsid-binding region suggests that the capsid binds totheminor groove. In contrast, we couldnotdetect

protection

of the

binding

region against dimethyl

sulfate-induced DNA cleavage: one single G at position 47 was foundtobe

protected (data

not

shown).

SinceG residuesare

methylated

atthe N-7

position

in the

major

groove(14), the

effect of

minor-groove-binding proteins

on Gmethylation is limited.

Samples

treated with

hydroxyl

radicals generally

con-tained both the

DNA-capsid

complex and unbound DNA. Afterthe

hydroxyl

radical reaction, thecomplexwas sepa-rated from free DNA

by

PAGEbefore it wasanalyzed ina

sequencing gel.

When the free DNA was also recovered

from the polyacrylamide gel and analyzed in parallel, a

peculiar

patternwasobserved(Fig. 3,lane10).Itlookedlike

a

negative footprint,

showing

signals

only in those regions

which were

protected

in the complex (Fig. 3, lane 9). To

explain

this

pattern,

we assumethat there is an

equilibrium

between theunbound-DNA

pool

and DNA inthe

complex.

Ifafree-DNA molecule which is nickedin the

binding region

has alower

affinity

forthe

capsid,

thismoleculewill remain

longer

in the free-DNA

pool

than will molecules with an

intact

binding region.

Its contribution to the free-DNA

digestion

patternwillresultina

higher signal intensity

within

regions

correspondingto the capsid-binding site. Thus, the

information

implied

in the patterninlane 10isequivalentto data obtainedfromaninterference

experiment:

the strongest bands

correspond

to those sequences which are

indispens-ablefor

binding.

The MVM

capsid

was found to bind to four different

T-shaped species containing

different nucleotide sequences

(Fig. 7), indicating

thatbindingcantolerate certain changes

in the

primary

sequence.Thequestionsarise:is any

T-form-ing

sequence acceptable, or are there more constraints to

binding?

There are reasons to favor the first possibility. Parvovirusesareverywell conserved with regard totheir3'

hairpin

structures but much less so with regard to the

primary

sequencewithinthesestructures (8). Furthermore,

the

replication

of adeno-associated virus DNA clearly

de-pends

onthe presenceof the Tstructure withinthe inverted

terminal repetition but not on the sequence within the

repetition

(12). Binding experiments with modified

se-quences will

provide

information on the importance of the

primary

structure.

ACKNOWLEDGMENTS

We thankP. Beard and J. Benhattarforsuggestions and discus-sions. We are grateful to H. Zorbas, Institute for Biochemistry,

UniversityofMunich, Munich, Germany, for invaluablehelp with the radical footprintingtechnique. G. Siegl kindly provided CPV. We thank B. Bentele for technical assistance and A. Emery-Goodman foroligodeoxynucleotide synthesis.

This workwassupported inpartbyagrantfromtheCommission oftheEuropeanCommunitytoK.Willwandandbyagrantfrom the Fonds National Suisse delaRechercheScientifiquetoP.Beard and B. Hirt.

REFERENCES

1. Antonietti,J.-P.,R. Sahli,P.Beard,and B. Hirt.1988. Charac-terization ofthecelltype-specific determinant in thegenomeof minute virus of mice.J. Virol. 62:552-557.

2. Astell, C. R., E. M. Gardiner, and P. Tattersall. 1986. DNA sequence of the lymphotropic variant of minute virus ofmice, MVM(i), andcomparison withtheDNA sequenceof the fibro-tropicprototype strain. J. Virol. 57:656-669.

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