A bovine papillomavirus type 1-encoded modulator function is dispensable for transient viral replication but is required for establishment of the stable plasmid state.

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Vol.60,No. 2 JOURNALOFVIROLOGY, Nov. 1986, P. 729-742

0022-538X/86/110729-14$02.00/0

A

Bovine Papillomavirus

Type

1-Encoded

Modulator Function

Is

Dispensable for

Transient Viral Replication

but

Is

Required

for

Establishment of the Stable

Plasmid State

MONIKA LUSKYt AND MICHAEL R. BOTCHAN*

Department ofMolecular Biology, University of California, Berkeley, California 94720 Received5 June1986/Accepted6 August 1986

Abovinepapillomavirus (BPV) type 1-encodedfunction (M)which isanegativeregulatorof viral plasmid replication has been described elsewhere(Bergetal. Cell, inpress;Roberts and Weintraub, Cell,in press). We reporthere thatexpression ofM, which isa repressorof transient BPV replication and is notrequired as a

positive factor in theseassays,is required for the establishment ofthe viralgenome as astable nuclear plasmid.

This function is encoded inpart by the5' portionof the BPV Elopenreading frame, whereas the3' partof this open reading frame encodes a positive replication function (R). The R function is required for early replicationevents. We usedtransient replicationassaystodefinethe phenotypes ofmutantsin both theRand Mgenesandcomplementation teststoshowthat RandMdefinetwo separategenes.We showed that R- and M- mutants could also complement each other in stableassays. Incotransfection experiments,M- mutants hadalethaleffectonthe growth of G418-resistantcolonies, and inadditiontheirmorphological transformation efficiencieswere reduced. Therarecolonies which didappearcontained themutantDNAintegrated intothe cellular genome. R- mutants transformed with

wild-type

efficiency, and the mutant DNA was also found integrated. Whencotransfected, R- andM- mutantscouldeachbeestablishedasunrearranged plasmids. Most of the viral information involved in latent bovine

papillomavirustype 1 (BPV-1)replicationandmorphological transformation is localizedto a5.4-kilobase (kb)subgenomic

fragment termed the 69% transforming fragment (20) (see

Fig. 1). Regulatorycis-actingsignals and trans-acting

func-tions implicated in viral replication have been identified withinthis region. Forexample, two discrete plasmid main-tenance sequences (PMS-1 and PMS-2) havebeen defined, either of which can support replication of recombinant plasmids in cellsthatprovide viral trans-acting factors (22).

PMS-1 has been localizedto the viral upstream regulatory

region termed the URR (A. Stenlund, G. L. Bream, and M. R. Botchan, submitted for publication) and consists of

two domains (24). Domain 1 is located just 5' to the 69% transforming fragment and has enhancer activity, and do-main 2, mapped by electron microscopy (39)overlapswith theviral

origin

ofreplication. PMS-2 is located withintheEl

open

reading

frame

(ORF)

(23). Acis-acting site

implicated

in

negative

control of

replication

hasrecently been shownto

overlap withdomain2of PMS-1. Asecond

negative

control of

replication

has been

mapped

to sequences just 5' to

PMS-2 (32a).

Severaloverlappingsplicedandunspliced

polyadenylated

transcripts

all transcribed fromone strand have been iden-tified (1, 15, 37, 41). Whereastheysharea common3'

end,

at least three different 5' ends have been

mapped

(37,

41;

Stenlund et al.,

submitted).

The start site for two RNA

species which contain parts of the El

region

have been

linked both in vivo and in vitro to a promoter,

P1,

which overlaps domain2ofPMS-1 withintheURR

(Stenlund

et

al.,

submitted).

Genetic approaches to

identifying

viral gene

products

required

for latentBPV

replication

have been

guided

by

the

* Correspondingauthor.

tPresent address: ZMBH,

University

of

Heidelberg,

D6900 Heidelberg, FederalRepublicofGermany.

location of eight ORFs withinthe69%transforming fragment as deduced from the DNA sequence (5). However, this approach bears two inherent problems. (i) Splicing can

combine ORFs in complicated ways. For instance, an

out-of-framespliceeventjoinspartof theBPVE6ORF withpart

ofthe BPV E7 ORF to create the E6/E7 gene (3, 41). (ii)

Many ofthe ORFs present in theviralgenome overlap(Fig.

1).Complementation analysishelps to resolve thisproblem,

since mutations in different ORFs that complement each

other canbeassignedtodifferentgenes.Therefore,by useof smallmutationsaffecting onlysingle ORFs, several comple-mentationgroups thatplayarole in viralplasmidreplication

havebeen identified(2a, 3, 9, 13, 23, 24, 33). A geneproduct encoded in part by the 3' portion of the El ORF has been showntobeabsolutelyrequired for early replicationevents

(24). Moreover, mutations in this region lead invariably to

integration of the mutant viral DNA into the host cell

genome in stable assays (13, 23, 33). The mutant function

canbe

complemented

in trans

by

a

wild-type (WT)

gene or mutantsin other

complementation

groups(23, 24).

In an attempt to genetically define the BPV El ORF in

more detail, we introduced frameshift mutations by

using

linker insertions throughout this ORF. The mutants were

analyzed intransientand stableassays. Our results showed

that the BPV El region contains coding information for at least two

complementation

groups. The 3' part of the El

ORFencodesa

positive

replication

function

(R).

The 5'part

oftheElORF encodesamodulator function

(M),

described earlier(2a, 32a),whichcan

negatively

regulate

viral

replica-tion. Evidence is

provided

here that shows that the

modu-latorfunction,

although

not

required

for transient

replication

ofBPV DNA, is

required

for its establishment as a latent

nuclear

plasmid.

We showed that R- and M- mutants

complementeach other in stable and transientassays.

Thus,

M- mutants can

provide

Rintransand

accordingly

allow for

transient

replication

of both genomes in C127 cells. R-mutantscan

provide

Mintransand thus

negatively

regulate

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IE6

H -I T HinCtlI

7

6959, PMS1

, I

l

I E8

EZWII

I E7

I

El

I

775 576 EI-Sma d1l0 i2113-2 d1211

711

I .

Sia Bpfit

945

15T'

PMS2

EcoRI

2113 2878 3089Ncol

Hpol P1

URR

FIG. 1. Organizationof theBPV-1genomeand location of mutations within the viral DNA. Thetopline shows thephysicalmapofthe

BPV-1genomeopenedattheuniqueBamHI site withsomerestriction sites indicated aslandmarks. Theplasmidmaintenancesequences PMS-1 and PMS-2 andtwotranscriptionalpromotersareindicated. The ORFs withinthe 69%transforming fragmentdeduced from theDNA

sequence (5)are shown above themap. The positionsof several BPV mutantsare shownbyvertical barsconnectingthe DNA withthe

particularORF affected. Below themapof thefull-lengthgenome,the location of the E9 ORFwithin the viral URRis shown. Thepositions of mutations in thisregionareindicatedbyvertical bars.

M- mutants in transient assays. In stable transformation assays, bothgenes arerequiredforplasmid replication.

MATERIALS ANDMETHODS

Recombinant plasmids. The plasmidpMLBPV5 (WT)and mutants thereof, dl2ll

(E2-),

BallS (E2- E5-), i2113-2

(El-),

dl306

(El1-),

have been previously described (23). Since thesteps takentoproduce the Ball5mutanthavenot beencompletely described, wereportthemhere. pMLBPV-5 DNAwas digested and linearized with KpnI. The DNA was then subjected tolimited digestion with Bal 31. XhoI linkerswereaddedtothe ends of thedigested DNA, and the molecules were recloned. Analysis of one clone chosen showed that the 5' border of the deletionextendedto BPV position 2694. This was measured by electrophoresis of small restriction fragments, including HhaI, Sau3A, and SphI. The 3' border of the deletion extended pastthe pML

SalIsiteatpBR position 650. This DNAwaslinearized again with XhoI at the linker site, and the small PstI-BamHI BPV-1fragment which spans the BPV early 3'poly(A) site andenhancerwasblunt end ligatedtothefilled-inXhoI ends. Both XhoI sites were lost upon cloning; however, the BamHI sitewas reformed. For this study, tocompare this

construction with other BPV-1 mutants inserted in pML-1, weexcised the DNA from the plasmid vector with BamHI

and reclonedit into WTpMLDNAattheunique BamHIsite ofthe vector. The 5' boundary of the deletion in mutant Ball5 is then nucleotide 2694 (±5 basepairs [bp]),and the 3' boundaryis definedbythe BPVPstI siteatnucleotide 4173. Mutant d1576 (E6/E7-) has been previously described (23), and mutant 775 (E6-) has a 25-bp deletion between positions 445 and 470, accompanied by insertion ofanXhoI linker (34) and was provided by J. Schiller. Mutants BPV Dll (PWT), BPV D43 (Rsa43), D134, and D125 have a BamHI linker inserted atpositions 7609(Dll), 7620(D43), 7673 (D134), and 7760 (D125). We had initially introduced these mutations intothe BPVsequencesby usingaplasmid carryingthe BPVXbaI-SmaIfragment (24). Full-length BPV genomes were reconstructed by replacement of the WT XbaI-SmaIfragment with each of themutantfragments. We did not change the names of these mutants in this study. Therfore,Dll, D43, D134, and D125 in thispaperrefertoa full-length BPV genome with a single linker insertion, whereas inourpreviousreport(24)thesesame names were given to abacterial plasmid containing only a mutant frag-ment of BPV. Mutant D144 is a full-length BPV genome which has a BamHI linker insertedat position 883 to 884. Similarly, the El Smamutant isidenticaltothe WTexcept fora4-bp insertionatthe SmaI site (2a). A partof the El ORFwas mutagenized by random linker insertion (14). The

BPVSmaI-EcoRI fragment(945to2113)wasinserted intoa

BomHI

I E4 I

1E311E51

40HI

HindIll

A

a

i I I I

I

i

I I

I

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BPV-1-ENCODED MODULATOR FUNCTION 731

1089

E8S

1479 859

II

I

El

I

I|

l

I

l

813 2663

i i i

Smol Hindl

945 1008 1515SgIII 811D11

EicoRl 2113

C127ceIs

Rplication

-D144

-El-S -D112

-D8

-D28

-D9 D29

I i D18

D13

iI~- -- Dill

II D31

_~~~~~~~~~~~~~~~~-D35i

-d130

-D4

D26

-i211l

4 883/884 +

Sma 945 +

+1001-1006 +

A 952-1030 +

1043-1071 + 113-1121 + +1123-1132 +

A12381279

+1-A1280-1287

+1-I A1361 -1416

+1-A1406-1430

-i+1404-1417

-X61515-1811

+1-A1709 +18071830

3-2 2113

-WT

91% 90% 84%

92%

74% 82% 92% 4% 18% 22%

0 0

9%y

0 0 0

[image:3.612.67.560.64.339.2]

100%

FIG. 2. Deletionand linker insertion mutations within the BPV El ORF:mappositions and transient replication properties in C127 cells. Thetopline showspartof the physicalmapofBPV-1 DNA.Thelocations ofORF E7, El, and E8areindicated above. Vertical bars within

theElORF indicatethe positionsofinternal ATG codonsatpositions 849, 1506, 1596, 1632, 1794, 1938, 1980, 2169, and2619.Thepositions ofthe mutations described here areshown below. A slash (/) betweentwonucleotide positions (D144)refers toasimple linkerinsertion

betweenthesetwopositions. Linker insertionsaccompanied by deletionof adjacentsequences areindicated byaA(delta) followed by the

deletedsequences;for example, inD9 thenucleotides 1113to1121,and thus9bp,aredeleted. Duplications ofsequences areindicated by

a + followed by the nucleotides whichare duplicated; for example, linker insertion in D112 occurred with aduplication of6 bp of the nucleotides 1001to1006.The replicationproperties ofallmutantDNAsin transientassays areshownonthefar right.Theautoradiograms

obtainedfromreplicationassays werequantitatedbydensitometry. Replicationofmutantsis expressed relativetoWTpMLBPV, andzero

indicates thatnoDpnI-resistant supercoils could be detected. The results presentedaretheaverageofthreeseparateexperiments, and results

variedby ±5%.Inno case,however,wasreplication detected for themutantsshowntohave0% replication potential.

pML2-derived vector, pp3 (21, 24), between the SmaI and EcoRIsites of thevector,giving risetopp3SR.pp3SRDNA was randomly cleaved with DNase, and the double-stranded,syntheticoctamer5'CGGATCCG3' (New England BioLabs) encoding the BamHI recognition sitewasinserted at the double-stranded breaks. Sequence analysis of the linker mutationswas done by standard procedures (2, 26). The map and nucleotide positions for the inserted BamHI linker in the individual mutants are shown in Fig. 2. Each

mutantfragmentwas then usedtoreplace theWTfragment in thefull-length BPVgenome.

Cellsand DNAtransfections. C127(10), 576, and775 cells were maintained atlow cell densityin DME plus 10%fetal calfserum.576 cells(3, 23)areC127derivatives and contain one to five copies of the BPV 576 (E6/E7) mutant and, similarly,775cells containonetofivecopiesof the BPV 775 (E6)mutant.For stable and transienttransfections,0.5to20 ,ug of plasmid DNAwasappliedto2 x 105to3 x

105

cells

per 60-mm (diameter) dish in the presence of 30 ,ug of Polybrene (18) per ml, followed by a 22.5% dimethyl sulfoxideshock 6 h after transfection. Forfocus assays, the medium was changed once per week. For selection of G418-resistant colonies, the cells from each 60-mm dish were trypsinized 48 h after transfection and split onto two 100-mm (diameter) dishes each. Selection was done in the

presence of 500 ,ug of G418 per ml (6). Foci and

G418-resistant colonies werecounted after 3 weeks unless other-wiseindicated.

Isolation and analysis of cellular DNA. Total DNAs from morphologically transformed and G418-resistant cells were prepared by the standard modifications (25) of the procedure of Thomasetal. (38). The DNAswere analyzed uncleaved or cleaved with the indicated restriction enzymes. For transientreplicationassays,low-molecular-weight DNAwas extracted from Hirt (16) supernatants at 24-h intervals, starting either 24 or 72 h after dimethyl sulfoxide shock. DNAsamples from theHirt supernatantsweredigestedwith DpnI, which cleaves unreplicated (methylated) DNA (31). DNAanalysis by gel electrophoresis and Southern blotting were aspreviously described (22 and referencestherein).

RESULTS

DeletionofBPV3'earlyORFs and transientreplication. A transient assay for BPV replication has been previously describedwhich allows formeasurementofviralreplication ofcellswithout selection (24). We havepreviously reported that coding information 3' to the BPV El ORF was not absolutely required for plasmid maintenance. A mutant (BallS) lacking ORFs E2, E3, E4, and E5 was maintained stablyin C127 cells(22). A mutant

(dl211)

defectiveonly in the E2 ORF could, under certain conditions (G418

selec-U I

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M3 BaI5 r----

---1

40

...

FIG. 3. Transient

replication

ofWT

pMLBPV5

and themutants

d1211

and Bal15in C127 cells. After DNAtransfection, DNAwas

isolated from Hirt supernatants every 24 h,

starting

72 h after

dimethyl

sulfoxide shock

(e.g.,

72,96,and 120h,leftto

right

in each

panel). Equal

fractions of each

sample

were

analyzed.

Before

gel

electrophoresis,

the DNAs were cleaved with

DpnI

to

digest

the

unreplicated (methylated) input

DNA. After

gel

electrophoresis

through

0.9% agarose and transfer to

nitrocellulose,

hybridization

wasdone with106countsof

32P-labeled,

nick-translated

pMLBPV5

DNA

(specific

activity,

2 x 108to x 108

dpm/,ug

ofDNA).This

probe

wasused for all

experiments.

LanesMland M3 contained 200 pgeach ofWT

pMLBPV5

andBall5

plasmid

DNAs,

respectively.

LaneM2contained 20pgof

d1211

as amarker. Arrowheadstothe left and

right

indicate the

positions

of form I

(supercoiled)

and II

(nicked circle)

WTand BallSDNAs,

respectively. DpnI-sensitive

DNA

(unreplicated)

was at the bottom of the

gel. Hybridization

throughout

the lane ofthe secondtime

point

inthed1211 panelwas dueto

partial DpnI

digestion.

tion),

be maintained as a

plasmid; however,

when selected

for

morphological transformation,

the DNAwasfound

inte-grated

(23).

Tosubstantiateour

probe

the

replication phenotypes

of thesemutants,itwasof interesttocomparethe initial

replication

properties

of both

mutants side

by

side. WT

pMLBPV5,

BallS,

and

d1211

DNAs were transfected into C127 cells

(1

,ug of DNA per 60-mm

dish).

Low-molecular-weight

DNAwas extractedat 24-h intervals and

digested

with

DpnI,

which cleaves

only

nonreplicated

(methylated)

DNA

(31).

Thus,

accumulation

ofsupercoiled DNA over time is a measure of DNA

repli-cation. Three time

points

fromeach transfection

(72, 96,

and 120

h)

are shown in

Fig.

3. The

experiment

showed that

replication

of mutant BallS is

indistinguishable

from that seenwith WT BPV

DNA,

whereasmutant

dl211

replicates

at a much lower rate

(see

also reference

24).

Densitometric

scanning

of the

autoradiogram

showed that accumulation of

BallS DNA was 97% of that seen with WT BPV

DNA,

whereas d1211 DNA accumulated to

only

11% ofthe level seenwith WT DNA. From these

data,

weconclude that this mutation

solely

within the E2 ORF affects therateof

early

replication events, whereasdeletion ofthe entiresequences

E2throughE5 didnot

impair

theinitial

replication

properties

of the BPVgenome.

Only

partof the ElORF is

required

for transient

replica-tion. Atrans-actingfunctionencoded within theEl ORF had

beenimplicatedin stable

plasmid

maintenance

(13,

23,

33).

Furthermore,aBPV mutant,i2113-2

(23)

(Fig. 1),

within this complementation group had been shown tobe defective in transient replication

(24),

indicating

that the function

af-fectedwas

required

for

early

replication

events. To

analyze

the El ORF in more

detail,

we chose to introduce small

frameshift mutationsthroughoutamajorpartof theEl ORF. Synthetic BamHIlinkers were inserted atrandom

positions

(14) within the BPV Smal-EcoRI

fragment (see

Materials

and Methods). The nucleotidepositions of the linker

inser-tion mutants described here are shown in

Fig.

2. Each

mutant fragment was used to replace the appropriate WT fragment in the full-length BPV genome. The

replication

propertiesof thenewly created linker insertionmutantswere assayedinitiallyin transientexperiments. AllmutantDNAs were transfected into C127 cells. Their behavior in the transient assays is summarized in

Fig.

2.

Representative

replication assays for the mutants D9, D29,D13,D18,

D31,

and d1306 are shown in Fig. 4A. Three time

points

(72,

96,

and120 h) for eachtransfection areshown. Toour

surprise,

wefound that mutants D144 through D29replicated in the

transient assay in a way similar to thatof WT BPV. Mutants D13and Dlll werealso able toreplicate; however, therate of DNAaccumulationseenwiththesemutantsseemedtobe

delayed andquantitatively less whencompared with that of

the WT. Both D13 and Dlll are in-frame

deletion-substitution mutants, and thus, partial activity of a trans-actingfactor may beexpected.However, mutant D18,which

does have aframeshift mutation, alsoreplicated at alower

ratethan WT BPV and mutants that scored positive. Since

theframe shift in D18 would lead to deletion of information for this gene encoded 3' to the mutation (see below), we believe that an internal ATG may be used to initiate a fragment of the protein which has partial activity. The sequence and position of the frame shift for mutant D18

predictsatermination codon at BPV nucleotide 1480, and a potential reinitiation codon in frame with the rest of El is

positioned just5' tothis point at nucleotide 1506. Peabody

and Berg (29, 30) have recently presented compelling data

consistent witha reinitiation (as opposed to scanning)

mech-anism for eucaryote translational use of internal AUGs. However, theyhave shown that the position of the termina-torwith respect to the AUG is critical. If the terminator is too far from the AUG, reinitiation is notdetected. In this regard, it is interesting that our mutantsD31and D35, which are completely negative for replication, have frame shifts

which predict termination at nucleotides 1460 and 1426,

respectively. Therefore, a simple resolution to the apparent

quantitativedifferences between these mutants and D18 may be that thetermination codons for mutants D31 and D35 are

Ml WT

m- M2 d1211

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A

B

Ml D9 D29 D13

I I I

B WT D31

I[ I' .

dl1306

MJ2

.

D29 D29 D31

MIMU2

D31 D26 D26

XI Xh . I....

If . ...

..)~ :. ...2SE'..::

,

.8

...,

rs....

..K It

FIG. 4. (A)Transient DNA replication of BPV ElmutantsD9,D29, D13, D18, D31,d1306, and WT pMLBPV5 in C127 cells. Time points ofDNAisolationand analysiswere asdescribed in the legendtoFig.3. Lanes MlandM2contained200pgeachofpMLBPV5andd1306

DNAs, respectively. Forms I andIIareindicated by arrowheads.(B)TransientDNAreplication of BPVElmutantsincotransfectionassays:

D29plus D31, D29 plus D26, and D31 plus D26. The molar ratio ofthecotransfectedDNAswas1:1 (0.5 ,ug ofuncutplasmid DNA plus 0.4 ,ugofcutplasmid DNA). For cotransfection of D29 plus D31, the BPV DNA in D29wasremoved from thepMLvectorplasmid by partial BamHI cleavage; for the othertwo cotransfections, the BPV DNAin D26was removed from the vector sequences in the same way.

Therefore,thecotransfectedDNAswereofdifferent sizes. Lanes Ml and M2 contained 2,ugof cellularID13DNAand 200pgofpMLBPV5 plaSmid DNA, respectively; forms I and II of the marker DNAsareindicatedbyarrowheads.

too far from the AUG at 1506 to allow for significant reinitiation. Clearly, further work,particularlyidentification of the protein encoded by this gene, will be needed to substantiate thishypothesis. Mutants D4 andD26 failed to showanyDpnIresistance and thusreplicatedDNA. Mutant d1306 was able to replicate in this assay, although with slower kineticsandto alowerlevel than did the WT. Using stableassays, wehave earlierreportedthat this mutant, an in-framedeletion, haspartial activity (23).

From the data desctibed above it is not possible to distinguish whether mutationswhich do notreplicate lie in cis-acting signals oraffect trans-actingfactors. To address thisissue, weperformedcomplementationtests. Ifdifferent trans-acting functions areaffectedbymutationsthatscored positive and those that scored negative, then a positive

mutantshouldrescuethereplication deficiencyofanegative

mttant.The mutantswerepairwise cotransfectedwith each other. The result ofarepresentative experimentis shown in Fig. 4B.The molar ratio of thecotransfectingDNAswas1:1

foreachexperiment.Forcotransfectionof D29plus D31,the

BPV DNA in D29 was removed from the pML vector by partial BamHI cleavage; for the othertwo cotransfections, the BPV DNA in D26 was removed from the vector se-quences in the same way. Thus, the cotransfected DNAs wereof different sizes. Three time points (72, 96, and120h) were taken from each transfection. The experiment shows that cotransfection of D29 plus D31 and D29 plus D26 resulted in replication ofboth transfected DNAs. In con-trast, cotransfection ofD26plus D31 resulted inno

appear-anceofreplicatingDpnI-resistantDNA ofeither mutant. Weconcluded that thereplication deficiency of the repli-cation-negative mutants (D31 through D26) can be comple-mentedintrans. Mutants i2113-2 and i2405-5(23, 24)did not replicatein transientreplicationassays anddid not comple-mentmutantD31for transientreplication (datanotshown); therefore, they are all part of the same complementation group. MutantsD144through D29definea second comple-mentationgroup within theEl ORF (see below). The func-tion encoded bythis complementation groupis dispensable forinitialreplication events.

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5762-cells

775-cells

M1 M2 M3 WT D43 D144 D43 D144 WT

I I I I I I

1I1) ID- ^

d

<III

resident

DNA

s~~~~~~~~~~~~~~~V

...

'

zeS-FIG. 5. Transient replication assay of BPV mutants D43 (E9) and D144 (5' El) and WT pMLBPV5 in 576 and 775 cells. Before transfection, all BPV DNAswereremovedfrom thevectorplasmidby complete(WT)orpartial (D43andD144)BamHI

cleavage.

Therefore, transfectedDNAs wereofadifferent size(8kb)than residentmutantDNAs(10.6kb).AfterDNAisolation

(time

points

were asdescribed in thelegendtoFig.3), eachsamplewasdigestedwithDpnIand PvuI beforegelelectrophoresis,resultingin

digestion

of all

unreplicated

DNA (DpnI)and linearization of theresidentd1576and 775 DNAs(PvuI).ThePvuIsite is located in thepMLvectorsequencesandwastherefore not present in the transfected DNAs. Thus, replication can be scored as accumulation of supercoiled DNA. Gel electrophoresis and hybridizationwere asdescribed inthelegendtoFig.3. LanesMl,M2,andM3contained2,ug of cellularID13DNAand 200pg each ofuncut

(M2) and linear (M3) pMLBPV5DNAasmarkers.FormsI, II, andIIIof the markerDNAsareindicatedbyarrowheadsontheleft,and the arrowheadontheright indicates linear residentmutantBPV DNAs(dl576and775).

5' El mutants and mutations within the URR define a negative modulator function in transient assays. In results

presented elsewherewe have shown thattransient

replica-tion ofsupertransfectedWT BPV DNAisrepressedincells

(576 and 775) carrying a

low-copy-number

mutant of BPV

(2a). Establishment of high-copy-number WT genomes in thesecells isalsoinhibited(2a, 3). Surprisingly,it was found that a frameshift mutant,

El-Sma

(containing a4-bp

inser-tion atthe BPV SmaI site), could replicate in these cells.

Furthermore, sinceWT DNA whencotransfected with this

particular mutant inhibits replication of both genomes, we reasoned that BPV encodes for a negative modulator

func-tion(M) whichcan actin trans. The results presented here extend theseobservations.

Wefound thatmutations(D43 and D134[Fig. 1]) in a small ORF(E9) within the URR also affect this 5' El gene (see

below).Asdescribed elsewhere(2a), aphenotype of mutants

in this 5' El gene is that they replicate transiently in cells

harboring BPV low-copy-number mutants. This assay dis-tinguishes a unique phenotype of this gene in a quick and convenient way and is therefore usedas a primarymethod forscreening mutants which may be members of the same complementation group. A representative experiment is shown inFig.5. As can be seen with WTBPV,noreplicated supercoils couldbe detected in transient assays after trans-fection of either 576 or775 cells (see also2a). In contrast, mutants D43 (E9-) and D144 (5' El-) replicated equally wellin either cell line. Identical results were obtained with mutantsD134, D112, D28, D9, and D29. Avarietyof other mutants, including Dll, D125, d1576, 775, and Bal15, be-haved as did the WT in these cells; i.e., they did not

replicate. As weexpected, all mutants that are R- also did notreplicate in these cells (data not shown). To determine whether the E9 and 5' El mutation affected a trans-acting

function, we contransfected themtogether with 3' El mu-tants (R-) into 576 cells. We reasoned that both types of ..

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5762-

cells

A

_B

MARKERS WT D26

+b 4

1 23 4 WT EI-SfaEl-SmaD26 El-SmaMock

I I I I I

D43 043 MEl-Sma

a_

D26

I I

_ .

11W

a

0 t

+

-v * 4111

d1576

1l)-or '0W.

(III d1576 Iw. 111)-.

1w .

1'

.4

_..-P.

^

--'.. ..

I

FIG. 6. Complementation of BPV mutants in transient replication assays in 576 cells. (A) Complementation of the El-Sma mutant (5' El) by WT pMLBPV and D26 (3'El).Before transfection, allBPV DNAs were removed from the plasmid vector by complete (WT andE1-Sma) orpartial (D26)BamHIcleavage. The molar ratio of thecotransfected DNAs was 1:1. Time points were taken as described in the legend to Fig. 3. Each DNA sample was cleaved either with DpnI plus PvuI (WT and D26lanes;see thelegend to Fig. 5) orDpnIplusSmaI (E1-Sma, WT+ El-Sma,and D26 +E1-Smalanes).SmaIdid notcleaveE1-SmaDNA but linearized theWT, D26, and residentd1576DNAs.Thus, replication could be measured as accumulation of supercoiledE1-SmaDNA and accumulation oflinear WT or D26 DNA in the cotransfection experiments.Gelelectrophoresis and hybridization were as described in the legend to Fig. 3. The lanes marked Mock contained DNA samples of timepoints taken after mock transfection of 576 cells and cleaved withDpnIplusPvuI.The markerlanes contained 2 ,ug ofHindIll-digested cellular ID13 DNA,resulting in linearization of the resident viralBPV DNA(Ml);2 ,ugof uncut celluar ID13 DNA(M2);and 200 pgeach ofuncut(M3) and linearized pMLBPV5 DNA(M4). Forms I, II, and III of viral BPV DNA resident in ID13 cells and forms I andIIof pMLBPV DNAare indicated by arrowheads onthe left; the arrowhead onthe right indicates form III of the resident d1576DNA. (B) Replicationof El-Sma and D43mutantsandcomplementation ofmutantD43bymutantD26in 576 cells.Beforetransfection,allBPVDNAs wereremoved from thevectorsequences (seeabove). After DNAextraction, each samplewascleaved withDpnIplusSmaI(D43 + E1-Sma lanes)orDpnIplus PvuI (D43 + D26lanes). The marker lane contained 200 pg of linearizedpMLBPV5DNA.The arrowheadtotheright indicates linear resident576 DNA.The arrowheadstothe left indicateformsIandII ofreplicatingEl-SmaDNAandformIIIofreplicating D43 DNA.

genome should be inactivatedfor

replication

ifR- mutants

encode

for

the

putative negative modulator function,

M.The

results of these experiments are shownin Fig. 6.

Cotrans-fection of D26 (R -) with the E1-Sma mutant

(Fig.

6A) abolished replication of El-Sma DNA.

Similarly,

cotrans-fection ofD26 with D43 (E9-)

(Fig.

6B) led to shutoff of replication of D43DNAin576 cells. Thesameresultswere

obtained with the 5' El mutants

D144, D28,

and D29

(data

not shown). In contrast, cotransfection of D43 DNA with

El-Sma DNA resulted in

replication

of both mutant genomestogetherin thesecells.

Thus,

this assayestablished

thatE9and5'El mutations affectthesamegenes.

Further-more,sinceD43and El-Smamutantsboth could

replicate

in C127 cells

(Fig.

7),

they

mustbeR+.

We concludedfromthese

experiments

that BPVencodes anegative

trans-acting

factor. 3' El mutants which cannot

replicatecan provide this factorin trans and are therefore

R- yet M+. Pairwise cotransfections with 3' El mutants

defined E9 mutants and mutants

D144,

El-Sma, D28,

and D29asM-.Transfectionof E9 and 5' Elmutantsinto 576or C127 cells ledto

replication; thus, they

areR+ yetM-.

M- mutants aredefectivefortransformation andlethalto cellgrowth.The

experiments

described abovedemonstrated

oneaspectoftheM-

phenotype-the

ability

of M- mutants VOL.60, 1986

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FIG. 7. TransientreplicationofmutantsD43(E9),D144(5' El), El-Sma (5' El), and WT pMLBPV5 in C127 cells. After DNA transfection, low-molecular-weight DNAwas isolated every 24 h starting 24 h aftertransfection (e.g., 24, 48, 72, and 96 h). DNA analysiswas asdescribedinthelegendtoFig.3. Lane Mcontained 200pgof WTpMLBPV5 plasmidDNA. FormsI,II,andIII(linear

DNA)areindicatedbyarrowheads onthe left.

to replicate transiently in an environment where WT BPV does not. It was of interest to determine whether these mutants could establish themselves stably in transformed cells. When stable transformation assays were performed,

TABLE 1. Focusformation of URR and Elmutants

inC127 cells

No.of foci/jig ofDNA

Plasmid Mutation 1 lig 10,ug 20,ug

Expt ia Expt 2a Expt 2 Expt 1 Expt 2

pMLBPV5 None 136 143 TMTCb TMTC TMTC

(WT)

D1l (qWT) URR 145 141 TMTC TMTC TMTC

D43 URR-E9 0 0 1 1 2

D134 URR-E9 0 0 2 2 3

D125 URR 123 117

D144 El 0 0 0 2 1

El-Sma El 0 0 2 3 1

i2113-2 El 105 TMTC TMTC

aExpts 1 and 2 referto two separateexperiments performed ondifferent

days. Each number is theaverageof the colonies scoredontwoplates. Foci

werecounted20 days aftertransfection.

bTMTC, Toomanyto count.

wefoundtooursurprisethattheabilities ofM- mutantsto

induce foci were reduced at least 100-fold

compared

with that ofthe WT

(Tables

1 and 2). From

these results,

two

interpretations

were

possible.

Either the E9 and 5' El mutations

impaired

a function directly required for

onco-genic

transformation

orthey definedan

important

regulatory

genewhoseabsence would render

establishment

of

the BPV

genome

impossible.

For

instance,

in the absence of the negative modulator

function,

detected

by

the transient as-says(seeabove), BPV infection could

be

lethal. The results

of

cotransfection experiments

with M- mutants with an

integrating marker, the neor gene, are consistent with this latter view. In these

experiments

M- mutants D43,

D134,

D144, E1-Sma, D8, D28, and D29 were

cotransfected

with theneormarker, and the number of

G418-resistant colonies

wasscored. Parallel

cotransfections

of the neor marker with

WTBPV anda mutantDll

(PWT

[Fig. 1])were

performed.

Representative results are shown in Fig. 8. In all of the experiments, the absoluteamountof neor marker DNAwas

kept constant (0.5 p,g of DNA per 3 x

105

cells), and increasingamountsofmutant orWTDNAwere

added

tothe transfection mixtureatthe

indicated

molarratios.Figure 8A showsthat, with increasing

concentration

ofWT BPV

ard

TWT, the number of G418-resistant colonies actually in-creased

slightly.

In contrast,

increasing

the dosage of M-mutant DNAs

clearly

interfered with the growth of G418-resistant colonies. Atratiosof 20:1 (BPVmutant

DNA-neor

DNA), we

observed

atleast a 200-fold difference between the number of colonies

obtained

with WT versus mutant DNA.Theincompletenatureof interference atlowerdoses iscertainlythe sumof several differenteffects,including the frequency ofcotransformation (which is

approximately

80%,

this sets a fivefold upper limit on the effect at 1:1

ratios).

Furthermore, this

lethality

isclearlyaslowkinetic

phenom-enon

(Fig.

8B). At 12

days

afterselection, the difference in the number of

appearing

G418-resistant colonies between the WT and mutants was not nearly as dramatic as that observed 24 days after selection. Thus, although G418-resistant colonies initially appeared, they died before 3

weeks. This could mean that a few cell doublings were

allowedtooccurbefore

cell

deathbecame manifest, which in

turn implies that the lethality may not be absolute. There-fore, atthelevelsofBPV DNAused in the1:1mixture with

TABLE 2. Focusformation ofEl

mutant§

inC127 cells

Plasmid No. of foci/

5 jigof DNA' WTb.*---'---'--- 157C

D12... 1

D8... 0

D28... 3

D9... 1

D29

...0

D18... 4

D13... $6

Dlll... 71

D31... 95

D35... 98

dl306... 165d D4... 142

D26... 131

i2113-2... 129

aFociwerecounted20daysaftertransfection.

bThe

WT

carriesnomutation.

cNumber of foci per1,ugofDNA.

dNumber offoci per0.1 ,ugofDNA.

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BPV-1-ENCODED MODULATOR FUNCTION 737 the neormarker, the absolute amount ofBPV DNA maynot

begreat enough to elicit complete lethality.

To examine the state of the mutant BPV DNAs in the surviving G418-resistant cells, we picked colonies three

weeks after selection and expanded them into cell lines. Attemptsto isolate colonies and expand them into cultures before this time inevitably failed because of cell death. Figure 9 shows the results of Southern blots obtained with mutants D43 (E9-, lanes A to D) andD144(5' El-, lanesE to H). Thecellular DNAs were analyzed uncut. In no case

could supercoiled plasmid DNA be detected, but rather all

hybridizing BPV DNA migrated with the

high-molecular-weight cellular DNA. The same results were obtained with mutantD134 (E9-) and the 5' El mutant El-Sma (data not

shown). Furthermore, the integrated state of these mutant BPVDNAs in stable assays was independent of the molar ratio of neor DNA to BPV DNA used for transfection. These

A

200

z

0

0 0

100

0L t

Ci t

--b- WT

-D1I IVWTI

0-D144 _- EI-Sma

O-043

1:1 1:2 1:5 110

D43+

neo

D144+neo

M

A B

C

D

E

F

G H

I

1II

[image:9.612.317.559.65.326.2]

I

FIG. 9. Southern blot analysis of total cellular DNAs from G418-resistant C127 cells derived by cotransfection ofpNEO5' DNAplus the mutants D43 (E9) and D144 (5'El). Cotransfection was done at a 1:10 molar ratio of marker DNA to mutant BPV plasmidDNA with0.5,ugofpNEO5'DNA.Total cellular DNA was isolated from individual G418-resistant colonies which had been picked and expanded into cell lines 26 days after transfection. Gel electrophoresis and blot and hybridization analyses were as de-scribed in thelegendtoFig.3.LanesAthrough H eachcontained10

jig

ofundigested cellularDNA. Lane Mcontained 100 pg (5 copy equivalent) ofpMLBPV5plasmid DNA. The arrowheads on the left indicate thepositionsofDNAforms IandII.

MOLAR RATIO pNEOS'NA BP DNA

B

WT

pMLIPV

043

12days iBdeas 24days

FIG. 8. Cotransfection of BPV E9 and 5' El mutants with the neomarkergeneon pNEO5'. (A)Effect ofincreasing BPV DNA

dosageonthenumber of G418-resistantcolonies. TheDNAswere cotransfectedattheindicatedmolar ratios with 0.5 ,ugofpNEO5' DNA. The values of each curve represent the average of two independent experiments for each cotransfection. Colonies were counted 20 days after transfection. (B) Representative plates of

G418-resistant C127 cellsstained12,18,or24daysaftertransfection

with the indicated DNA. The cellswere fixed withglutaraldehyde and stained with 1%methyleneblue. Cotransfectionwasdoneata

pNEO5'DNA-BPVplasmidDNA molar ratio of 1:10.

results showed that the M- mutants could not be stably maintainedas

plasmids.

Weconcludedthat the Mfunction is

required

for establishment of latentBPV

plasmid replication.

Todeterminewhether theseparticular phenotypes of M-mutants

(lethality

and

integration)

wereindeed duetoloss of function ofa

trans-acting factor,

we

performed

complemen-tationassays. Theresultsareshownin Tables 3 and4.

First,

variousM- mutants were

cotransfected

withmutantsin the E6/E7 and E6

complementation

groups

(d1576

and

775,

respectively),

and

ability

to induce foci was measured.

Mutants D43,

D134, D144,

and E1-Sma could all

comple-mentthemutantsd1576 and775fortransformation

(Table 3).

Thetransformation

efficiency

wasatWT

levels,

demonstrat-ing that the M- mutants were not dominant lethal. In contrast,cotransfection ofM-mutantswith each other

only

induced few fociat

high

DNAconcentrations.Todetermine whetherthelethal

phenotype

ofM- mutants could also be

rescued by an R- mutant we

performed

the

following

transfection

experiments.

The mutants

D43, D134,

D144,

and E1-Sma

(M-)

were each cotransfected with mutant

i2113-2

(R-) andthe

integrating

neormarkergene.Withthe

appropriate

transfection

mixes,

WTlevels ofG418-resistant colonieswere detected

(Table 4). Futhermore,

observation

ofthe

appearing

coloniesovertime didnotshowanylossin

number or cell

death,

and the colonies were

readily

ex-panded

into cell lines. Not

unexpectedly,

crosses between twoM- mutantsdidnotleadto

complementation (Table 4).

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TABLE 3. Complementationof BPV mutants in C127 cells-focusformation

No. offoci/,ugof DNAb Plasmida Mutation

Expt1 Expt2

pMLBPV5 (WT) None 156 123

d1576 E6/E7 10 12

775 E6 12 9

D43 + dl576 E9 x E6/E7 101 95

D43 + 775 E9 xE6 124 112

D134 + d1576 E9 x E6/E7 98 100

D134 + 775 E9 xE6 105 111

D144 + d1576 5'El x E6/E7 125 113

D144 + 775 5'El x E6 112 110

E1-Sma + d1576 5'El x E6/E7 125

ElSma + 775 5' Elx E6 128

D43 + D134c E9 x E9 2 3

D43 + D144c E9 x 5' El 1 0

D144 + El-Smac 5'El x 5'El 2 2

aSee Table1for the transformationefficiencyof each individual E9orEl

mutant.

bThe molarratio ofcotransfecting DNAswas1:1. Fociwerecounted 20 days after transfection.

cNumberof foci per 10

pLg

ofDNA.

For example, cotransfection of D43 with El-Sma and

pNEO5'

ledto atleasta20-fold reductionin G418-resistant colonies. Thesameresultwasobtainedwhen D43 DNAwas

linked to the neor gene (D43-neo) and cotransfected with El-Sma (Table 4). Lack of

complementation

between M-mutants wasalsomanifestwhen BPV DNAwas

analyzed

in

the rarely appearing G418-resistant colonies.

Figure

10B shows the DNA analysis of G418-resistant cells from the

cotransfection experiment with D43-neo plus El-Sma. The

Southern blot showed that bothmutant DNAs appeared to

existasintegrated copies in genomicDNA. In contrastand consistentwith ourresultsdescribed in transientassays,Fig.

10Ashowsthat R-

MI

x R+ M- crosses(e.g., i2113-2 x D43 and

i2113-2

x D144)yielded cell lines whichcarryboth

mutantDNAs asunrearranged plasmids.

Thus, in stable assays M- mutants D43, D144, and El-Sma all integrated as did R- mutants. Together, they complemented each other and established themselves as

latentreplicating plasmids.

DISCUSSION

The results presented abovedescribe a new BPV gene (M)

required forestablishment of stable plasmids in transformed cells. We showed that this gene is encoded in part by sequenceswhichmap to the 5' end of theElORF.

Hereto-fore,

onehadassumed that theentire

El

ORF was involved inencodingasinglefunction, since its size and location are

intact and conserved among all of the papillomavirus genomes sequenced which have replication potential (7).

This new gene is clearly distinct from the positive factor

previously defined which is required in trans for transient BPV DNA replication (24). The positive factor (R) is en-coded in part by the 3' sequences of this sameORF. We are

confidentthat the two genes, R and M, must define separate

polypeptides, since a series of frameshift insertions at the 5' end ofEl do not destroy R activity. We used a series of assays to demonstrate the uniqueness of eachfactor and to

probe theirphenotypes. These assays are summarized be-low.

(i) WT BPV DNA or mutants in other ORFs do not

replicate in transformed cells which carrylow-copy-number

mutants (2a,

3).

Mutations in the 5' part of

El, however,

allowed for

replication

after

supertransfection

into these cells.

Furthermore,

these mutants

replicated

in uninfected C127 cells.

(ii)

Stable

morphological

transformation was

severely

re-duced with mutants that

interrupt

the

coding

sequences in

the 5'

portion

oftheEl

ORF,

and thesemutantsinterfered in

cotransformation

experiments

with the

outgrowth

of G418-resistantcolonies. Incolonies that could be established with suchmutants, the DNAwas

integrated

in cellular DNA.

(iii)

Mutations in El ORF sequences 3' to this gene, in

contrast, did not

replicate

in transient or stable assays and didnotaffecttransformation efficiencies.

The

phenotypes

described in i and ii define the

properties

ofM-mutants,whereas those in iii describe the

phenotypes

ofR- mutants. We used the

following

complementation

tests toshow thatR and Mdefine two separate genes.

R-mutantsdidnot

replicate

transiently

ineither

C127

cells or

low-copy-number

cells

but,

cotransfected

together

with M-mutants, both genomes

replicated

in

C127

since the latter classofmutant

provided

the

positive

replication

function.In

low-copy-number cells,

neither genome could

replicate

when cotransfected because R- mutants

provided

the

neg-ative

regulator.

In stable transformation assays, neither

genome was

carried

as a free

plasmid

when transfected

separately,

but

together they

complemented

each other.

The outlines of a model for BPV

replication

control,

as

described

by Berg

et al.

(2a),

provide

the basis for

under-standing

theassays used here todefine these twogenes. In

particular,

thismodel

provides

a

hypothesis

useful in

design-ing

future

experiments

aimed at

probing

the role ofM in stable transformation. The

experiments

described here

cer-tainly

suggest that this new gene

plays

some role in the

establishment

(and

perhaps

maintenance)

of the

plasmid

state and transformation.

Briefly,

the model suggests

that,

afterinfectionortransfection of naive

cells,

BPV

undergoes

slow but transient

amplification;

this

amplification

is fol-lowed

by

amaintenance mode of

replication

which is

char-acterized

by

coordinate cell

cycle

replication

of viral

plas-mids.The M

protein

either

directly

or

indirectly

assuresthis modeof

replication

evenin thepresenceofanexcessofR.

Wedonotknow how theM

protein

worksbut

hypothesized

that it is a direct

DNA-binding protein

(see below). Some

TABLE 4. Complementationof BPVmutantsinC127 cells

No. of G418-resistant

Plasmid Mutation coloniesa

Expt1 Expt2

pNE05' None 126 144

pNE05' + pMLBPV5 (WT) None 148 156

pNE05' + i2113-2b 3'El 113 122

pNE05' + D43 + i2113-2 E9 x 3'El 155 163 pNE05' + D134 + i2113-2 E9 x 3'El 123 136 pNE05' + D144 + i2113-2 5' El x 3'El 143 152 pNE05' + E1-Sma + i2113-2 5' El x 3' El 135

pNE05' + D43 + E1-Sma E9 x 5' El 10 12

pNE05' + D144 + E1-Sma 5' El x 5' El 4 8

D43-neo + El-Sma E9 x 5' El 13 11

D144-neo + El-Sma 5' El x 5' El 6 9

aThe molar ratio of

pNEO5'

DNA to mutant DNA was1:10;theratio ofthe

mutantDNAstoeachotherwas1:1(e.g.,pNEO [0.5 ,ug] + mutant 1[5jig]

+ mutant 2 [5 ,ug]). G418-resistant colonies were counted 20 days after

transfection.

bi1223-2DNAwasremovedfromthe vectorsequencesby cleavage with

BamHIin allexperiments.

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uncut D43

BamHI

I

D144 D43 12113-2

B D43-neo+EI-Sma

uncut B&mHI

m

I

m-

I

D144 i2113-2

A B C M1M2 D E F

m

m.-s

F G H I K

- 8kb

44.8kb 44.3kb 3.5kb 3.1 kb

42.6kb

FIG. 10. Southern blot analysis of cellularDNAsfrom G418-resistant C127 cellsderivedupon cotransfection ofpNEO5' DNA with

pairwisecombinations of E9, 5'El,and 3'ElmutantBPV DNAs. (A)Cotransfectionsof D43 (E9) plus i2113-2(3' El) andD144(5' El)plus

i2113-2.The molarratio of the cotransfectedmutantBPVplasmid DNAswas1:1. Theratio of pNEO5' DNAtototalBPVplasmidDNAwas

1:10. We cleaved i2113-2 DNA withBamHI before transfection to remove the BPV sequences from the vector DNA, creatinga size

differentialbetween the cotransfecting DNAs. Individual G418-resistant colonieswerepicked 20 days after transfectionand expanded into celllines, and total cellular DNAwasisolated. Gelelectrophoresis and blot analysisweredoneasdescribed in thelegendtoFig. 3. Lanes Athrough E each contained 10 ,ug of undigested cellular DNA.Lanes FthroughK eachcontained10,ugof thesamecellular DNAs cleaved

with BamHI.Forexample,lanesAandForBand G analyzed thesamecellular DNA. Witht2113-2DNA(removedfrom the plasmidvector),

BamHIcleavage resulted inafragment of 8 kb (full-lengthBPV).Cleavage ofmutantDNAsD43 and D144,both linkedtothe pMLvector, gaverisetofragments of 4.8 and 3.1 kb (D43) and 4.3 and 3.5 kb (D144), respectively, andacommon2.6-kbfragmentwhich isthe pMLvector. Lanes Mland M2contained 100pgof WT pMLBPV5 DNA and 2,ug of cellular DNA isolated from ID13 cells(18)whichcontain viral BPV DNA.Thepositions of formsI and IIofpMLBPV5DNAand viralBPV DNAresidentinID13 cellsareindicated by arrowheadsonthe left. Thearrowheadsontheright indicate the sizes of DNAfragments obtained after cleavage of themutantDNAswithBamHI.(B) Cotransfection of BPVmutantD43 (E9) linkedtopNEO5'(D43-neo) plus E1-Sma (5' El). Cotransfection wasdoneatamolar ratioof 1:1. Establishment ofcelllines,isolationofDNA,and DNAanalysisweredoneasdescribedin thelegendtoFig.6A. LanesA, B,and Ceachcontained 10 iLg

ofundigested cellular DNA isolated fromthree individual clones. LanesD, E, and F contained the same DNAsdigestedwith BamHI.

Arrowheadstotheright indicate the positions of the BamHI fragmentsof thetwomutantDNAs.Digestionof themutantDNAs with BamHI gaverisetotwofragments of 8 and2.6 kb fortheE1-Sma mutant,whereasD43-neoDNAwascleavedinto threefragmentsof 5.1(pNEO

DNA), 4.8, and 3.4 kb.

system marks resident plasmids toassure such replication which is coordinate with the chromosome. This is shown clearly in Fig. 5 and 6; notice that, even though the input DNAsreplicated and amplified, the resident plasmids didnot join this replication pool.Thetransientassaysprovedthat M isanegative regulator. Asanaturalextension of thesedata, we speculated that M plays some role in the marking process.

Thus, according tothis model, when WT DNA enters a low-copy-number cell lineit does notfindan excesspoolof R(seeBergetal. [2a] forfurtherdiscussion)provided bythe resident genome. In these cells, the WT DNA can only expressR and Matlevels coordinate with cell cycle

repli-cation. However, an R+ M- mutant enters such cells, producesonly R, and thus canamplify.Akey featureofthe modelis that theresidentplasmidsinlow-copy-numbercell lines provide only enough factors to allow for stable repli-cation ofthemselves and thatM,once bound, is noteasily released. The differences in the dynamics of replication betweenanaive andatransformed cellmustthen bedictated by thestate of the cell itself and the levels of Rprotein.

Thetransientassays showed that the M factor is capable ofnegativeregulation,andthe stableassaysshowed thatthis factor is requiredfor plasmid establishment. A strong pre-diction of the model is that, upon removal of M from the system,the BPV genome should notbe able toregulateits

A

i2113-2 i2113-2 M1M2 A B C D E

I I

:..''

I1.

1I)

_

i,

8 kb

'5.1kb c4.8kb

-'3.1kb

4c2.6kb VOL.60, 1986

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replication, andrunaway replication would ensue. Lethality

maythenbe the resultofan excess ofBPV DNA or agene product whose expression is normally limited by the WT

replication system. Here an analogy to other small but

unrelated DNA tumorvirusesmay be relevant. With simian

virus 40 or polyomavirus, transformation in permissive

systems can onlybe achieved with large-T-antigen mutants

(see, for example, reference 11). In special cases in which

thereplication origin is specifically mutated, large-T antigen plus permissive lines can be obtained (11). In contrast, however, in cellsystemsin which only limited replicationis

detected, transformation frequencies have been reported to

rise with temperature-sensitive large-T-antigen mutants

when thetransformation wasassayed atpermissive

temper-atures (8). In these latter cases, presumably, the effects of limitedreplicationwere notdeletions for establishment, and the increased persistence ofthe viral DNA afforded by the

limited replication increased the chance for stabilization.

Therefore,

different effects of any particular M- or R-mutant on transformation might be expected for BPV,

de-pending on the cell state. In other words, uncontrolled replication may be deleterious, but limited replication may

be advantageous. In this contextit is interesting thatd1306,

an in-frame R deletion mutant, transformed C127 cells at

frequencies

aleast 10-fold greater than did theWT(Table2)

(23).

The lethal phenotype ofthe M mutants will probably be

themost difficultaspect of the phenomenon described here tostudy further. In ourtransfection experiments, only

10-4

to

10'

cells would eventually becometransformedalthough 1 to 10% of transfected cells transiently expressed and

replicated

BPV (24). Thus, events proceeding toward stable

replication

andtransformation may happen in only a small

fraction of transfected cells. The cell lethality reported, of course, only measuredevents in this subpopulation.

There-fore,

relevant virus-cell interactions mediated by the M

protein

in the transition from transient amplification to the

establishedstatemaybedifficulttofollow becausetransition occurs in this minor fraction of cells.

We reliedoncomplementationtests todocumentmany of

the

points

made inthis study. Our results are notconsistent with recombination playing a significant role. Two distant mutants, e.g., D43 (E9-) and D29 (5' El-), did not

comple-menteach other, whereas two closely spaced mutants such asD29 andD31did. Furthermore,in stable assays no sign of rearrangement was detected by Southern blots ofthe two

complementation

genomes. This is in contrast to a recent report (13) in which a high frequency of recombination betweentwoBPV mutants was readily detected. It is known that transfecting DNA presented to cells in large calcium

phosphate

precipitates is built into large oligomeric struc-tures, forming "peckalosomes" (32). With methods that do not form such precipitates, recombination frequencies are

reportedly low. Forexample, in mixedtransfections oftwo

different simianvirus 40 circles withDEAE-dextran, almost allofthedimers detectedin vivo werehomopolymeric (12),

and high frequencies ofrecombination are only detected if two circles are first linked in vitro (40). The Polybrene methodusedin our studies does not depend on a precipita-tion step fortransfection.

The genetic analysis provided here for R and Mprobably does not define the structure of the entire genes. For example, mutations which interfere with geneexpression,as well as protein structure,can be scored as M- orR-. The productswork in trans, and any BPVgenome containinga

wild-type gene complements a defective gene whether the

defect affects a cis function

required

for

expression

or one

required forprotein information. A summary ofthe

genetic

definitions

provided

in this paperis shown in

Fig.

11.The E9

ORF which is part of theMgene is small

(only

108

bp

[Fig.

1]),but this

region

is encodedinto stableRNA

(A.

Stenlund,

J. Choe,and M.Botchan,

unpublished data)

and islocated3'

toapromoter,

P1, expressed

in transformed cells

(Stenlund

et al., submitted). Furthermore, frameshift mutantsDll and D125, which map

just

5' and 3' tothis

ORF,

donothave the M- phenotype

(Table

1;

Fig.

8A).

Thus,

whereas the E9 region may be a cis-acting regulatory site needed for M

expression, it seems possible that it is part of the

protein

encoding information. Interestingly, anRNA species with a

splice donor at BPV nucleotide position 1234 has been reported in an earlier study of Stenlund et al. (37). Recent

unpublished studies from our laboratory detected at least two RNAspecies (1.4 and1.8kb) which could encodeforthe

M protein and, indeed, analysis of cDNA clones showedthat

a splice between 1234 and 3224 is present in a

relatively

abundant RNA. It is intriguing that the genetic data

summa-rized in Fig. 11 indicate an endpoint of one exon ofthe M

gene around position 1300 in the BPV genome.

The putative M protein may work by itself or require cellular machiriery to manifest negative regulation. The available genetic data eliminate the need for any of the encoding information 3' to the El ORF as required for modulation in transient and stable assays. For example, Ball5 is established as a stable nuclear plasmid, and in an

independent assay system Roberts and Weintraub (32a)have

also shown that these ORFs are dispensable for negative regulation. From their data one can also rule out any role for M action that requires the R protein simultane-ously.

Whereas R and M play pivotal roles in the regulation of BPV replication, their function interacts in unknown but important ways with other viral genes. For example, the E6 and E6/E7 gene products (2a, 3, 23) are required to maintain high levels of viral DNA, perhaps by keeping the pool of R protein high (2a). A role for the 3' ORFs (E2, E3, E4, and E5) is also indicated by several results; however, much of these data are at present confusing. For example, mutant d1211 has a significantly reduced replication rate in transient assays (Fig. 2), implying a role for E2. Consistent with this observation, others have found that E2 mutants integrate in stable assays (9, 13; P. Howley, personal communication). However, BallS, which deletes all of the 3' ORFs including E2, displays WT activity with respect to transient replication (Fig. 2) and stable plasmid maintenance (22). Furthermore,

Grof

and Lancaster (13) have reported that small E5 mu-tants affect stable assays, whereas others have not detected such effects (D. DiMaio and P. Howley, personal communi-cation). Several explanations present themselves, including

the following. (i) The presence of E5 (or any downstream

ORF) makesthe functions of other ORFs, for example, E2, obligatory (13). (ii) The mutations affect the structure or expressionofothermRNAs, and the particular mutant may

ormay not affect the activity of a variety of other genes by alterations of a class of mRNAs. (iii) The mutants all manifest their phenotypes because of an indirect effect on replication, and results are highly influenced by

experimen-tal protocols. Forside-by-side differences between mutants BallSandd1211, we believe that some aspects of explanation

i or iimay be relevant. However, because different groups

have obtained conflicting results with the identical deletion

mutant (9, 13; P. Howley, personal communication), it

seems likely that differences in cell conditions or

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http://jvi.asm.org/

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BPV-1-ENCODED MODULATOR FUNCTION 741 P1

Hindlil

I

P2

HpaI

II1

1E91

Smal EcoRI

I

El

I

11

-M

D43x D134 x

D144

R*M-

El-Sma

D28 D9 029 D31

R-M

i2113-2

*-2405

M

R

x

(X)

FIG. 11. Summary of genetic data. Mutants D43 and D134 define the most 5' region of the M gene, and mutantsD144through D29 define another part of this same gene. Mutant D31 defines the 5' border of the R gene, whereas mutant i2113-2 is close to the 3' border of this gene. AnotherEl mutant at position 2405 described previously (23) did not maintain itself as a plasmid in stable assay and did not complement mutant i2113-2for plasmidreplication. Mutant i-2405 did not complement mutant D31 for transient or stable replication. Therefore, we concluded that i-2405, i2113-2, and D31 are all part of the same complementation group and are R-. We did not test i-2405 for the M phenotype, hence the parentheses in the figure. The solid bars show our conclusion about the position of the M gene; the open bar denotes the region encoding R.

tion protocolshave influencedsomeofthese results. This is particularlytruefor E2 since thisgeneisapositive transcrip-tional activator (35) and the requirements for such viral genesinothersystemscanbeinfluenced by the multiplicity ofinfectionorthetypeof cellused (27, 28, 35; see also the unpublished observations discussedin reference 17).

Anotherpuzzle thatwemustaddressconcerns conserva-tion of the El region as an intact ORF in all papil-lomaviruses, ifin fact it encodes fortwodiscrete genes. A simple resolutiontothisparadoxicalsituationmaybe solved by understanding the complete structures of RNAs which encode the protein information. By linking donors and acceptors forthe two genes in complex ways, deletions or substitutions which would change reading frames may be impossible. On the other hand, latentreplicationof BPV in C127 cells provides no information regarding vegetative replication. It is, of course, possible that this El ORF encodes anothergenethatuses the ORF inanotherway or intact atthis stageof its replication cycle.

ACKNOWLEDGEMENTS

WethankA.Stenlund forcommentsonthemanuscript. This work was supported by Public Health Service grant CA 30490 from theNational Cancer InstituteandgrantMV-91 from the

American CancerSociety.

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