The intranuclear location of simian virus 40 polypeptides VP2 and VP3 depends on a specific amino acid sequence.

(1)

The Intranuclear

Location of Simian Virus 40

Polypeptides

VP2

and

VP3

Depends

on a

Specific

Amino Acid

Sequence

CZESLAW WYCHOWSKI,* DANIELE BENICHOU, ANDMARC GIRARD

Unite de Virologie Moleculaire (CNRS UA 545), Institut Pasteur, 75724 Paris Cedex15, France Received 1 June 1987/Accepted 27August 1987

A cDNAfragment codingforpolioviruscapsid polypeptide VP1wasinserted intoasimian virus 40(SV40)

genomeintheplaceof theSV40VP1geneand fused inphasetothe3' end of the VP2-VP3genes. Simiancells

wereinfected withtheresultinghybridvirus in thepresenceofanearly SV40mutant usedas ahelper.Indirect immunofluorescence analysisof the infected cells usinganti-poliovirus VP1 immuneserumrevealed that the SV40/poliovirusfusionproteinwaslocated inside the cell nucleus.Deletions of variouslengthsweregenerated

intheSV40 VP2-VP3 portionof thehybridgeneusingBAL31 nuclease. Theresultingvirusgenomesexpressed splicedfusionproteinswhose intracellular locationwaseitherintranuclearorintracytoplasmic, dependingon the presence or absence of VP2 amino acid residues 317 to 323 (Pro-Asn-Lys-Lys-Lys-Arg-Lys). This was confirmed bysite-directed mutagenesisof theLysresidue atposition 320. Modification ofLys-320intoeither ThrorAsnabolished the nuclear accumulation of the fusionprotein.It isconcluded that at leastpartofthe

sequenceof VP2 aminoacids 317to323 allows VP2 and VP3toremainstablylocated insidethe cellnucleus. The proteinsaremostprobably transportedfrom the cellcytoplasmtothecell nucleusbyinteraction,withVP1 acting as acarrier.

Simianvirus40(SV40) virionsaremadebytheassembly ofthree structuralproteins, VP1, VP2,andVP3,withMrsof

46,000, 39,000,and27,000,

respectively

(9,23, 26).The three

corresponding

viralgenes are

organized

ina

partially

over-lappingmanner on the SV40genome, of whichthey

consti-tutemostof thelate genesportion (31). Capsid proteinsVP2

andVP3areencoded by nucleotides545 to 1601 and899to 1601,respectively, and bothareread inthesameframe.VP3

isthereforeasubsetof VP2,correspondingtotheC-terminal two-thirds of the molecule. Capsid protein VP1 is encoded by nucleotide residues1488to2574.The endoftheVP2-VP3

openreading frame therefore overlaps by113nucleotidesthe

beginning of theVP1 openreading frame, whichis read inan alternative frame (31).

The threecapsid polypeptidesare synthesized duringthe latephase ofthevirusreplicationcycle and aretransported

rapidly after their synthesis into the nucleus of theinfected

cell (30), in whichthey assemble into SV40particles (22). The molecular mechanisms underlying the transport of

proteins from the cytoplasm to the nucleus in mammalian cells are poorly understood. One approach used to

deter-mine the presence and location of possible migration se-quences in nuclear proteins has involved the construction andexpression ofchimeiicgenescontaining variousregions

ofanuclearprotein fusedtoacytosolic protein. Thus,the 13 NH2-terminal amino acids of the Saccharomyces cerevisiae mata2 protein were found to suffice to transport a matot2 ,-galactosidase fusion protein to the nucleus (11). The

amino-terminal portions of the yeast Gal4 (28) and L3 (19)

proteins were sufficient to target

P-galactosidase

fusion

proteins to the cell nucleus. Similarly, fusing the chicken pyruvate kinase molecule to the highly basic stretch of

amino acidsfound in the SV40 T antigen

(Pro-Lys-Lys-Lys-Arg-Lys-Val) resulted in efficient transport of the protein to the cell nucleus (13). Moreover, proteins coupled to a

synthetic peptide with the sequence of these seven amino

acidswerefoundto accumulate in the cell nucleus (16).

*Correspondingauthor.

Wehaverecently inserted intoaSV40 genome the cDNA sequenceof the polioviruscapsid polypeptide VP1,a cyto-solic protein, in such a manner as to generate fusion pro-teins, with poliovirus VP1 at the COOH terminus and various amounts of SV40 VP1 at the NH2 terminus. We observed that all the fusion proteins could be recovered in the cell nucleus as long as they contained the first eight NH2-terminal amino acids of SV40 VP1. This led to the conclusion that a migration signal sufficient to target the fusion protein to the cell nucleus was present within that stretchof amino acids in theSV40 VP1 protein (35).

Whilescreeningthehybrid SV40/poliovirusesthus gener-ated, we noticed that many expressed proteins that were

immunoprecipitated by anti-poliovirus VP1 immune serum showedonsodium dodecylsulfate-polyacrylamide gel

elec-trophoresis a different Mr from thatexpected from a SV40

VP1/poliovirusVP1fusion protein. Aswillbe shown, these

hybrid viruses correspond to SV40 recombinants in which

thepoliovirusVP1 sequencehas beeninserted in phasewith, and downstreamfrom,theSV40VP2andVP3genes.These SV40 VP2-VP3/poliovirus VP1 fusion proteins accumulate

inside the nucleus ofthe infected cell, provided that they contain the domainofVP2covering amino acid residues317 *to 323 and are expressed in the presence ofSV40 polypep-tide VP1. A model is presented to accountfor these data.

MATERIALS ANDMETHODS

Enzymes and reagents. Restriction enzymes were pur-chasedfromBoehringerMannheimBiochemicals,New En-gland BioLabs, Inc., or Amersham Corp.; T4 DNA ligase, Klenow DNApolymerase, and polynucleotide kinase were

purchased from Boehringer Mannheim; BAL 31 nuclease waspurchased from New England BioLabs; andM13mpl8 was purchased from P-L Biochemicals, Inc. All enzymes wereusedaccording to the instructions of the manufacturer.

[_y-32P]ATP

at 3,000 Ci/mmol was purchased from Amer-sham. Oligodeoxynucleotides

5'-CCTTTTGTTTTTGTTG-GGG-3' (oligodeoxynucleotide 1), 5'-CCTTTTCGTTTTG-TTGGGG-3' (oligodeoxynucleotide 2), and 5'-CCTTTAC-3862

on November 10, 2019 by guest

http://jvi.asm.org/

(2)

TTCTAGGCCTG-3' (oligodeoxynucleotide 3) were synthe-sized by J. Igolen (Institut Pasteur). Oligodeoxynucleotides 1 and 2 were used for site-directed mutagenesis, and oligodeoxynucleotide 3 was used for plasmid sequencing according to the method of Zagursky et al. (37).

Construction of recombinant plasmids. Escherichia coli 1106 (803

rkmk)

(21) was used for the selection and propagation of recombinant plasmids. Plasmid pCW18 has already been described (36). Transformation of bacteria, extraction, and analysis of plasmids were as previously described (1, 17, 18).

Protruding 5' ends of restricted DNA (20

,ug/ml)

were filled in by treatment with Klenow DNA polymerase (40U/ml) in the presence of all four deoxynucleoside triphosphates (200

puM

each). Ligation reactions were performed for 48 h at4°C

orfor 18 h at

15°C

in 60 mM Tris hydrochloride (pH 7.5)-l

mM EDTA-10 mM MgCl2-10 mM dithiothreitol-1 mM ATP-20

,ug

of bovine serum albumin per ml, by using 1 U of T4 DNA ligase per

,ug

of DNA.

Before digestion with BAL 31 nuclease, pCW18 was linearized at the unique AccI site ofSV40. Digestion with BAL 31 nuclease was performed for different periods at30°C

in 120 mM Tris hydrochloride (pH

8.0)-i

mMEDTA-0.6M

NaCl-12mM CaCl2-12 mM

MgC92,

by using an enzyme-to-DNA ratio of 0.08

U/,ug

(35). The reaction was stopped with EDTA, and the trimmed DNA was extracted with phenol and chloroform and precipitated with 2 volumes of ethanol before ligation. In the case ofpCW18A202, the plasmid DNA was linearized at theAccI site ofSV40, digested withBAL 31 nuclease as described above, ethanol precipitated, and cleaved at the uniqueBglIIsite. The large fragment wasthen ligated with the small pCW18A0 BalI-BgII fragment.

Site-directed mutagenesis. Synthetic oligodeoxynucle-otides 1 and 2 (see above) were synthesized on anautomated synthesizer and purified by high-performance liquid chroma-tography and electrophoresis on a 20% polyacrylamide gel containing 7 M urea. Site-directed mutagenesis was per-formed directly on the DNA in the plasmids bythefollowing protocol (20). The supercoiled plasmid DNA was purifiedby cesium chloride gradient centrifugation. Part of it was di-gested withBglIIto linearize the plasmid, and anotherpart was digested with two restriction endonucleases cleavingthe DNA on either side of the region to be mutated (forexample,

BalI and XbaI in the case of pCW18AO). The resulting molecules (0.03 pmol) were mixed together and added to 12.5 pmol of the synthetic oligodeoxynucleotide with the appropriate sequence that had beenphosphorylated atits 5' end by using polynucleotide kinase. The DNA mixture (in 8.6

RI

of

0.lx

TE buffer) was mixed with 2

RI

of lOx concentrated polymerase-ligase buffer (1 M NaCl; 65 mM Tris hydrochloride [pH 7.5], 80 mM MgCl2, 10 mM ,B-mercaptoethanol), dipped intoaboiling-water bath for 3min

to denature the DNA molecule, and thengraduallycooledby incubation for 30 min at 30°C. The tube was further

incu-bated for 30 min at4°C and then in an ice bathfor 10 min. The mixture was then mixed with 4

RI

ofa solution of the four deoxyribonucleoside triphosphates(2.5mMeach): 2 pu1

of 10 mM ATP-3,ul of T4 DNA ligase (3 U)-0.4 pI ofthe Klenow fragment of DNA polymerase (2 U). The final mixture (20

pI)

was incubated at 12.5°C overnight. The ligation mixture was then used to transform competent HB101 bacteria. Ampicillin-resistant colonies were selected and transferred to Schleicher & Schuell membrane filters,

and those harboring a plasmid with the desired mutation weredetected by colonyhybridization with the

[y-32P]ATP

5' end-labeled oligonucleotide as a hybridization probe.

Hybridizationwasdone as describedby Chesebroet al. (5) for 18 h at10°C below thecalculated

Tm,

using 2 x 106cpm perfilter.Thetemperaturefor the washing of the filters was determined experimentallyby step-by-step increase.

DNA sequence analysis. The DNA of the plasmids to be sequenced wastreatedwithStuI (SV40 nucleotide1446) and BglII. The resulting small fragment corresponding to the fused SV40/poliovirus VP1 gene was recovered from low-melting-temperatureagarose gels (34) anddigestedby XbaI (poliovirus nucleotide 2546). The resulting fragments were inserted into M13mpl8 replicative-form DNA molecules cleaved by HincII and XbaI. Sequence determination was performed on the M13 single-stranded DNA by using the four-dideoxynucleotide chaintermination method (27). The

mutation borne by plasmid pCW18A70 or by plasmid pCW18A71 wassequencedby themethodofZagurskyetal. (37).

Preparationof virus stocks. The DNA from the

recombi-nant plasmids was digested with BamHI and BglII, recir-cularized by ligation, and transfected into CV1 cells bythe

method ofSompayracand Danna (29). Briefly, transfection

was done by using 106 CV1 cells, plated onto 60-mm-diameter petri dishes, and0.4ml ofa1-,ug/ml mixture ofthe

recombinant SV40DNA and ofthehelper pDR404DNA, in

Dulbecco minimal essential medium containing 0.05MTris hydrochloride, pH 7.3 and 500 ,ugofDEAE dextran per ml. The cells were incubatedat37°Cin a CO2 incubator for2h,

after which they were washed and fed with medium

supple-mented with 5% fetal calf serum. After 15 days, the cells

were disrupted by freezing and thawing, cell debris was discardedbycentrifugation, and thesupernatant wastitrated

and used as a virus stock.

Preparation and immunoprecipitation of infected-cell ex-tracts. Labelingofinfected cells,preparation ofcellextracts,

andimmunoprecipitation byusingproteinA

Sepharose

were asdescribed previously (36).

RESULTS

Construction of recombinant viruses with an SV40

VP2-VP3/poliovirus VP1 hybrid gene. We have

previously

re-ported the construction of

plasmid

pCW18

(Fig. 1),

which carries an SV40 genome into whichthe cDNA

coding

for the

poliovirus capsidpolypeptide VP1

(poliovirus

type 1 nucle-otides 2472 to 3417) has been inserted between the

unique

EcoRI and BamHI sites (36). Upon transfection of simian cells withthe DNA of this

plasmid

cleaved at the

BgIII

and BamHIsites and recircularized, a

hybrid

SV40/poliovirus

is

generated, inwhich thesequence

encoding

SV40VP1 amino

acids 95 to 361is replaced by that ofthe 302 amino acidsof poliovirusVP1 flankedbyafewadditionalamino acids from

poliovirusVP3andprotein2A.This

hybrid

virusis defective for capsid protein

synthesis.

It can be grown in a mixed infectious virus stock by cotransfection ofthe simian cells with the DNA of the SV40T-antigenambermutantam404as ahelper (24, 25).

To generatehybrid viruses

expressing

poliovirus

polypep-tide VP1 fused inphasewithSV40

polypeptides

VP2orVP3 (or both), the DNA of

plasmid

pCW18

was cleaved at the uniqueAccI site(SV40 nucleotide

1611),

digested

with BAL 31 nuclease for various

lengths

oftimebetween 3 and21min,

and then treated withEcoRI, filled in with Klenow enzyme, andrecircularized (Fig. 1).

Resulting

deleted

plasmids

were

cloned, and eachclonewasgrown intoan

independent

virus stock witham404as a

helper.

The fusion

protein

expressed

by each of the truncated

SV40/polio

virus

hybrids

was

on November 10, 2019 by guest

http://jvi.asm.org/

(3)

analyzed by SDS-PAGE of [35S]methionine-labeled infected-cellextractsafterimmunoprecipitation withan

anti-poliovi-rus VP1 immune serum (2). When the poliovirus VP1

se-quence was fused in frame with that of the SV40 VP1

polypeptide, the molecular weight (Mr) of the fusion protein

was in the range of 37,000 to 50,000 (35, 36). When fusion occurred between SV40 VP2-VP3 polypeptidesand

poliovi-rus VP1, two bands of material with MrS in the range of 62,000 to 76,000 were detected (Fig. 2; Table 1). Another

band of material with an apparent Mr of 46,000 was also

observed in all the gels. Part of this material could corre-spond to a cellular protein such as actin, as it was also

present after immunoprecipitation of infected-cell extracts with a control non-immune rabbit serum (data not shown). However, anotherpart of this material was probably SV40

VP1, as judged from the similarity of its electrophoretic

migration with that of the material immunoprecipitated with an anti-SV40 immune serum(see below). A few additional minor bands of materialwerealsoobserved, which could be either discretedegradation productsortruncatedtranslation products resulting from the abnormal use of internal

initia-tion sites. Thesequestionswere notaddressed further. The infected-cell extracts were also immunoprecipitated with a

rabbit anti-SV40 immune serum (Fig. 2): two bands were

observed,onewithanapparentMrof46,000, SV40 VP1,and the other with an Mr of 92,000, probably SV40 T antigen. The anti-SV40 immuneserumdidnotimmunoprecipitate the SV40/poliovirus fusion proteins probably because it only poorly recognized SV40 polypeptidesVP2 andVP3.TheMrs of the fusion proteins expressed by some of the defective recombinant viruses selectedare summarized in Table 1.

Identification ofanamino acidsequence responsible forthe nuclear locationofSV40 capsid polypeptidesVP2and VP3.To

determine the intracellular location of the SV40 VP2-VP3/ poliovirus VP1 fusion proteins expressed by these viruses,

OriSV40

pCW18.A55

AccI,Bal31 EcoR Klenow+4dNTP SV40 VP2 VP3

i

T4 ligase

SV4O~~~~~~~.M.I_{P_BIov,}VP2IVP3

FIG. 1. Construction ofSV40genomescontaininganSV40

VP2-VP3/poliovirus VP1 hybridgene. Plasmid pCW18 (36) contains the

poliovirus cDNA fragment (nucleotides 2472to3417)that encodes theCOOHterminus of poliovirus VP3 (_), thewhole of poliovirus

VP1 (M), and the NH2 terminus of poliovirus polypeptide 2A

(VLUI). rzzJ, Theremaining 5' terminal partoftheSV40VP1gene;

a,theSV40 VP2 and VP3genes.The large circlecorrespondsto theSV40genome,and the small circlecorrespondstothat of pML2,

the EcoRI site ofwhich has been destroyed.

A93 A47

A55

QVP1aSV aVP1 aSV uVP1 eSV

92,5-b-69

,m

_* _Pm* _

m* *_* _*

46 __

30U

FIG. 2. SDS-PAGEanalysis of immunoprecipitated SV40 VP2-VP3/poliovirusVP1fusion proteins.Extractsfrom [35S]methionine-labeled cells infected with the indicated virus were immunopre-cipitated with anti-poliovirus VP1 rabbit immune serum or with anti-SV40 immune serum.Theimmuneprecipitateswereanalyzed byelectrophoresis ina10to18%polyacrylamide linear gradient gel (36) in thepresenceof SDS.Arrows onthe left refertotheposition (103) of '4C-labeled markers. The asterisks correspond to the positions expected for SV40 VP2- and SV40 VP3/poliovirus VP1 fusionproteins, calculated from the size ofthedeletions generated into thegenomeof thehybrid viruses.

infected cells were assayed by immunofluorescence using anti-poliovirus VP1 immune serum. Strikingly different re-sults were observed, depending onthe hybrid virus studied

[image:3.612.344.554.73.342.2]

(Fig. 3). The SV40/poliovirus proteins expressed by pCW18A55 and A47 were localized in the cell nucleus (Fig. 3A and B), whereas those expressed by pCW18A93 or A96 werelocated in the perinuclear region and the cytoplasm of theinfected cells (Fig. 3Cand D). The intracellular location TABLE 1. Characterization oftruncated SV40 VP2-VP3 proteins

Deletion ofSV40 Apparent mol wt of

amino acidsa fusionprotein,103"

Plasmid Poliovirus Poliovirus Locationc VP2 VP3 VP1/SV40 VP1/SV40

VP2 VP3

pCW18A55 335-352 217-234 76 65 N

pCW18A47 330-352 212-234 75 64 N

pCW18A93 318-352 200-234 73 62 C

pCW18A96 310-352 192-234 73 62 C

pCW18A232 340-352 222-234 76 65 C

317-323 199-205

aAminoacidnumbering accordingto Tooze (31).

1ApparentMrwasdeterminedbySDS-PAGEanalysis as described in the

legendtoFig.2.

' Intracellularlocation ofthefusion proteinswas determined by

immuno-fluorescencestaining of infectedcells asdescribedin the legends to Fig. 3 and 6.N, Nuclear;C, cytoplasmic.

on November 10, 2019 by guest

http://jvi.asm.org/

[image:3.612.101.289.435.645.2] [image:3.612.324.565.567.678.2]

(4)

C

D

FIG. 3. Intracellular localization of theSV40/poliovirusfusionproteins. CV1 cellson coverslipswereinfected with 2 PFUof thefollowing virus stocks per cell: A,SV40-A&55-am404; B,SV40-A47-am404;C, SV40-A93-am404;D,SV4O-A96-am404.Coverslips were fixed 40 h after infection and processed for immunofluorescence staining by using anti-poliovirus VP1 rabbit immune serum aspreviously described(36). of the fusion proteins determined by immunofluorescence

staining of infected cells is summarized in Table 1.

The nucleotidesequenceatthe

junction

betweentheSV40

VP2 and VP3 genes and the poliovirus cDNA fragment in each ofthehybrid

SV40/poliovirus

genomes wasdetermined (Fig. 4). In the caseofA55 and A47, the BAL 31 deletions extended upstream to SV40 nucleotides 1546 and 1531, respectively. The fusion proteins these viruses express

therefore lack the 18 and 23 COOH-terminal amino acids fromSV40 VP2-VP3capsidpolypeptides, respectively (Fig. 4). On the other hand, in the case of A93 and A96, the

deletions

generated by

BAL 31 were found to extend to

SV40 nucleotides 1495 and 1471, respectively. As aresult, thefusion

proteins

thesetwovirusesexpresslackthe35and 43 COOH-terminal amino acids from SV40 VP2-VP3,

re-spectively.

The presence of COOH-terminal amino acid residues -35 to -23(i.e., SV40VP2amino acids 317 to 329 or VP3 amino acids 199 to 211) therefore seems to be required forthefusionproteinstobetargetedtotheinfected cell nucleus. Itshould be noted that inthe case ofA93, the BAL 31deletionhadalso reached further downstream than

the EcoRI site,removing the first21 nucleotides of

poliovi-ruscDNA.

The SV40VP2-VP3 sequence identified above containsa

stretch of amino acids (Asn-Lys-Lys-Lys-Arg-Lys; NKKKRK),which is reminiscent ofthat (Pro-Lys-Lys-Lys-Arg-Lys-Val) identified by Kalderon et al. (12, 13) in the

SV40Tantigenand shownbythose authors to be involved in the nuclearlocation of the

protein.

Toascertainthat the

ability to target the VP2-VP3 fusion protein to the cell

nucleus was indeed due to this stretch of amino acids, we

deleted their coding sequence from the SV40 VP2-VP3/ poliovirus VP1 hybridgene(Fig. 5).

This was done by starting from plasmid pCW18A202, which contains thepoliovirus VP1 sequence fused in phase behind the sequence ofSV40 VP1 amino acids9 to 94 (35; Fig. 5). The sequence coding for SV40 VP1 amino acid residues 1 to 8, which has been deleted in pCW18A202, is

LEU LYS ALA TYR GLU ASP GLY PRO ASN LYS LYS LYS ARG LYS LEU SER ARG

..CTAAAAGCTTATGAAGATGGCCCCAACAAAAAGAAAAGGAAGTTGTCCAGG

_pCW18.A6I

,9480 1490 SA i50

15A

CW18. 6 _pW18.A93

GLY SER SER GLN LYS THR LYS GLY THR ASN SER VAL

GGCAGCTCCCAAAAAACCAAAGGAACC-AATTCCGTA

1530 14 t PCW18s9%

LaW8.A47 LpCW18..55 pCW18.A471 pCW18A55

ALA GLN GLY LEU GLY GLN MET LEU GLU

GCACAGGGGTTAGGTCAGATGCTTGAA. tPoliovirus VP1

pCW18.&93i

FIG. 4. Locationand extentof theBAL31-generated deletions. PlasmidpCW18wasdigested withBAL31nucleaseatthe AccIsite, cut with EcoRl, treated with Klenow DNA polymerase, and recircularized (see the text). Theextentofsomeof the deletions thus generatedwasdeterminedby nucleotide sequencing(arrows). The beginningof the sequence ofpoliovirusVP1is indicatedby the short arrow.

I

on November 10, 2019 by guest

http://jvi.asm.org/

[image:4.576.44.522.63.379.2] [image:4.576.293.533.518.659.2]

(5)

also that which codes for the PNKKKRK sequence of VP2-VP3 (see Fig. 8). The DNA ofpCW18A202wascleaved

at AccI with BAL 31 nuclease for 1 min and then cleaved with BglII, and theresulting large fragment (7.02 kilobases)

was ligated to the small BalI-BglII fragment (0.95 kilodal-tons) from pCW18A0 (35), which contains the sequence of the 2 COOH-terminal amino acids of poliovirus VP3, fol-lowed by the 302 amino acids of poliovirus VP1 and the 12 NH2-terminal amino acids of poliovirus protein 2A. A li-brary ofhybrid virus genomes was thusgenerated.

After conversion into individual virus stocks through transfection of CV1 cells in the presence of SV40 am404 helper DNA, extractsfrom cells infectedby each of 20 such virus stocks were immunoprecipitated with anti-poliovirus

VP1 antiserum and the SDS-PAGE pattern of the immuno-precipitates was examined. Six of the extracts analyzed showed adouble band of materialmigratingwiththe

appar-ent Mr expected for SV40 VP2-VP3/poliovirus VP1 fusion proteins. The location of these proteins was intracyto-plasmic, asjudged by immunofluorescence analysis of the

infected cells (Fig. 6A).

The nucleotide sequence at thejunction between the 3' part of the SV40 VP2-VP3 sequence and the 5' part of the poliovirus sequence was determined in four ofthe selected hybrid viruses. One ofthem, pCW18A232, showed a polio-virus VP1 cDNA sequencefused in frame to SV40 DNAat position1561, i.e., 13 codonsupstreamfrom thetermination codon of the VP2 and VP3 genes. Thus, the fusion protein expressed by pCW18A232 lacks the sequence PNKKKRK (amino acids 317 to 323 in VP2) together with the 13 last COOH-terminal amino acids from VP2-VP3 (not shown). Thisprotein is located inside the cell cytoplasm. Comparison of the sequenceofthepCW18A232 fusion protein with that ofpCW18A55,whichlacks the last 17 COOH-terminal amino acids of VP2-VP3 but has kept the internal PNKKKRK

sequence, strongly suggests that the latter is indispensable

FIG. 5. Deletion of the sequence PNKKKRK from the SV40 VP2-VP3/poliovirus VP1 hybrid gene. Plasmid pCW18A232 was

constructedasindicated (see thetextfor details). Thesequence at

thejunction of SV40 VP2-VP3 and ofthe poliovirus cDNA was

determined and is indicatedatthe bottom of the figure. Thesame

symbolsareusedas inFig. 1. kb, Kilobase.

Ball,Bglll isolate0.95kbfragment

FIG. 6. Intracellular localization of the SV40/poliovirus fusion protein. CV1cellson coverslipswereinfected with: (A)

SV40-A232-am404at3PFU percell;(B)SV4O-A55-am404; and (C) SV40-A47-am404at10-4PFUpercell.Cellswerefixedat40hafter infection

and processed for immunofluorescence staining by using anti-poliovirus VP1rabbit immuneserum.

for the location ofSV40 VP2-VP3capsid polypeptides in the cell nucleus. No information is available, however, on the

possible role of the last 13 amino acids of SV40 VP2-VP3. The size of the deletions of SV40 VP2-VP3 in the fusion proteins is summarized in Table 1.

Mutation of SV40 VP2 Lys-320 alters the intracellular location of the polypeptide. Asanadditional proofof the role

played by the PNKKKRK sequence in the migration of

SV40 VP2-VP3 polypeptides to thecell nucleus, we

under-took the site-directed mutagenesis of the SV40 VP-2/poliovirus VP1 gene in pCW18zA55 through the use of

Bal31

Bgl II

isolate 702kbfragment

T4DNALigase^

~~~~~~~

VP2 VP3

GCAAGIGCCCCAGCACAG

SV.O VP21VP3.L.. Pot...,w.*cDNA

on November 10, 2019 by guest

http://jvi.asm.org/

[image:5.612.332.558.73.532.2] [image:5.612.70.304.447.667.2]

(6)

B

FIG. 7. Intracellular localization of the SV40-poliovirus fusion protein from two hybrid viruses mutated at VP2 Lys-320 in the accumulationsequence.CV1 cellswereinfected (A) with

SV40-A70-am404or(B)withSV4O-A71-am404, andprocessed for

immunoflu-orescencestainingasdescribed inthe legendtoFig. 6.

custom-made oligodeoxynucleotides (see Materials and Methods). The sequence of the oligonucleotides (see Mate-rials and Methods) was such as to generate a mutation of

VP2 amino acid Lys-320 to Thr or Asn. Among several clones ofputative mutantsobtained, two wereselected and sequenced:pCW18A70,in whichthe AAGcodon ofLys-320 was replaced by ACG (Thr), and pCW18A71, in which the sameAAG codon was replaced by AAC(Asn). Both plas-mids weregrown independentlyinto mixed virus stocksby transfection of CV1 cells in the presenceofpDR404 DNA, afterwhich cellswereinfected with theresultingvirusstocks and processed for immunofluorescence by using an anti-poliovirus VP1 immune serum. In both cases, the SV40 VP2-VP3/poliovirus VP1 fusion proteins were found to be located in the cytoplasm and the perinuclear region of the infectedcells (Fig. 7). Therefore,modification ofLys-320to Thr or Asn abolished the nuclear accumulation of SV40 VP2-VP3. The role of the adjacentamino acids ispresently being determined by additional site-directed mutagenesis experiments.

The intranuclear accumulation of the fusion proteins de-pendsonthepresence ofSV40VP1.Successful infection and virus production in the system described above depend on

the am404 helper virus supplying the three SV40 capsid

polypeptides required forpackaging of both its genome and that of the hybridSV40/poliovirus. The question was raised of whether theseproteins could play a role in thetargeting of the SV40/poliovirusfusion proteins into the nucleus.

To examine the possibility that the fusion proteins were transported to the nucleus as a complex in association with the am404 helper virus capsid proteins, cells were infected

with dilutions of the A47 and A55 virus stocks, i.e., at an approximate multiplicity of infection of

10-4

PFU per cell. Under these conditions, the probability for a cell to be simultaneously infected by both a recombinant virus and a helperviruscould be considerednegligible. In each instance, theSV40/poliovirus fusionprotein expressed by the diluted recombinant virus stock was detected inside thecytoplasm

of the infectedcell, as judgedby immunofluorescence

stain-ing usstain-ingananti-poliovirus VP1antiserum(Fig. 6B andC).

These results indicate that the accumulation of the SV40 VP2-VP3/poliovirusVP1protein in the cell nucleusdepends

ontheparticipation of a factor provided by the helperSV40

virus, probably SV40 VP1 (14). The PNKKKRK sequence present at the COOH terminus of SV40 VP2-VP3 seems therefore to be necessary, but not sufficient, to ensure the effective transport and accumulation of the fusion proteins inside the cell nucleus.

DISCUSSION

Translocation ofproteins across cellular membranes is a

keystep in theintracellularsortingandsecretion ofproteins. Forproteins targetedforimportintomitochondria or export

throughtheendoplasmic reticulum,thetargetingisgivenby theleader aminoacidsequence atthe amino acid terminus of the polypeptide. Thus, mitochondrial precursor polypep-tides have a leader peptide that is generally highly basic, contains no acidic residues, and has a high percentage of

leucine, serine,andarginine (32),whereas excreted proteins carryattheir NH2terminusasignal peptide that is madeup ofastretch ofhydrophobic residues,often flankedbybasic

residues (33). At present, more than 200 procaryotic and

eucaryotic signal peptides have been sequenced(33). Both leader peptides and signal peptides are cleaved from the precursorpolypeptide at thetime oftranslocationby mem-brane-associatedprocessing proteases (signalases).

Itisstill, however, totallyunclear what targets aproteinto the cell nucleus. The entry of proteins into the nucleus cannotbe entirely explained by diffusion through the pore

complexesbecause someof these proteins areofvery high

Mr(7)andmany

proteins

withlowMrenterthe nucleus with

muchfaster kineticsthanexpected from moleculardiffusion

only

(3, 4). Nuclearproteinsdonotappeartobesynthesized

in the form of precursor proteins, with added sequences

playingthe partofaleaderpeptideor asignal peptide.It has

been

suggested

that the

specificity

ofthe transport to the nucleus can be determined by an amino acid migration

sequence located within the sequence ofthe protein itself (7). This sequence could be involvedin theinteractionwith a nuclear membrane receptor or nuclear membrane

trans-location

machinery.

Nuclear

migration

sequences have

in-deed beenidentified in the SV40 Tantigen (12, 13), in the

SV40

capsid polypeptide

VP1 (35),in the estradiolreceptor

(10), and in the yeast mata2 protein (11) and ribosomal

protein

L3(19).Severalofthese sequences arecharacterized

by

the fact that

they

containa

proline

residuefollowedby a stretchofseveralbasic amino acid residues.Another typeof sequence has been identified in the estradiol receptor (10)

on November 10, 2019 by guest

http://jvi.asm.org/

[image:6.576.44.263.68.415.2]

(7)

and in the yeast mato2 protein (11). There is some dispute over the actual significance of the yeast mato2 protein migration sequence, because several migration sequences appear toactually exist in the protein.

In the case of the SV40 capsid polypeptides, our data show that the intranuclear location of SV40 VP2 and VP3 dependson thepresenceof VP2 amino acids 317 to 323(VP3 amino acids 199 to 205). Fusion proteins containing this stretchof seven amino acids with thesequence PNKKKRK readily migrated into the nucleus of the infected cell, whereas those in which this sequence had been deleted remained inside thecytoplasmof the infected cell. Different authors have shown that the modification ofLys-128to Thr (6, 12)orAsn(15)abolishesnuclear accumulation ofSV40T

antigen. Our results show that when VP2 Lys-320 was changed to Thror Asn (by analogy with Lys-128 of the T

antigen), the nuclear accumulation of SV40 VP2 and VP3

polypeptides wasabolished.

However, thissituationwasobservedonlyincellsdoubly

infected with both the hybrid SV40/poliovirusand a helper SV40. When the multiplicity of infection was greatly re-duced, to ensure that cells were infected with the hybrid

virus (or the helper virus) alone, all the SV40

VP2-VP3/poliovirus VP1 fusion protein detected was seen to remain intracytoplasmic. It has been suggested that SV40

VP1aloneisactively translocated into the cellnucleus, VP2 and VP3 being passively transported through interaction with VP1 (14). Thisconclusion was basedontheobservation

that mutations within thecodingsequenceofVP1could alter the transport of VP2 and VP3 to the nucleus. The results reported in this reportstrengthen the hypothesisthat SV40

VP2 and VP3 migrate into the cell nucleus onlyin

associa-tion with SV40 VP1. A possible explanation is that SV40 polypeptide VP1, which possesses an endogenous nuclear

migrationsequencewithin the sequence of its firsteightNH2 terminal amino acids (35), is responsible for the active

transportof polypeptidesVP2-VP3 into the cell nucleus. The PNKKKRK sequenceidentified here couldplayaroleeither in theinteraction ofVP2-VP3 with VP1 orin thebindingof thepolypeptidesto areceptoror amatrix in thecell nucleus.

Alternatively, VP2 amino acids 317to 323 could be simply required for the proper folding of the protein in order to correctlyinteract with VP1 beforetransporttothe nucleus. Thefactthat aband of 46kilodaltons, mostprobably SV40 VP1 could be immunoprecipitated from all infected cell extractswith ananti-poliovirus VP1immune serum (Fig. 2) suggeststheexistence inside the infected cell ofapoliovirus

VP1/SV40 VP2-VP3-SV40 VP1protein complex. This com-plex wasobservedindependent of the extent of the deletion

affectingtheSV40VP2-VP3/poliovirusVP1fusionproteins, thus suggesting that the presence orabsence of VP2 amino

acids 317 to 323 was irrelevant to the formation of the complex. This implies that the PNKKKRK sequence in VP2-VP3 is not involved in the interaction with VP1 but ratherplays the roleof a nuclear accumulation signal.

Paradoxically, the sequence PNKKKRK shows a striking

homologywith the nuclear migration sequence identified by Kalderon etal. (12, 13)in the T antigen of SV40. It is rather puzzling that such similar sequences would be involved in twoquitedifferent mechanisms: direct translocation into the cell nucleus in thecaseofTantigen, and accumulation in the nucleus or specific interaction with SV40 VP1 (or both) in the case of VP2-VP3. Indeed, if the nuclear migration sequenceofTantigenwassufficient to target T antigen to the cell nucleus, there should be no reason why a very similar sequence should not be sufficient also to target VP2-VP3.

pCW18.,&202

1 5 10

ALA PRO THR LYS ARG LYS GLY SER CYS PRO GLY SV40VP1

GATGkC_rAU AAKn A-AA- 'T f -AGC

ASPGLY PRO ASN LYS LYS LYS ARG LYS LEU SER ARGGLY SV40VP2/VP3

315 320 325

pCW18.A232

FIG. 8. Thesameregion ofthe SV40genomeencodesthe VP1 nuclearmigrationsequence andthe VP2-VP3nuclear accumulation sequence.pCW18A202expressesaSV40VP1-poliovirusVP1 fusion protein in whichthefirsteightVP1 NH2-terminal amino acidsare

deleted(arrows). pCW18A232expressesSV40VP2-VP3/poliovirus VP1fusionproteins deleted for VP2 amino acids 317to323(arrows) and the 13 VP2 COOH-terminal amino acids (340 to 352). The deleted fusion proteins expressed by both plasmids remained lo-cated inside the cell cytoplasm. The reading frames used for the synthesis of SV40VP1 andofSV40VP2-VP3 areindicatedby the bars aboveorbeloweachcodon,respectively.Amino acid number-ingisafterTooze (31).

Perhaps

tertiary

and quaternary structures ofthe

proteins

are

responsible

forthis difference.

In agreement with our

conclusions,

it was reported re-cently that in the absence of its 35 COOH-terminal amino

acids, SV40 VP3 remains limited to a

cytoplasmic

and

perinuclear location

(8).

The authors of that report con-cluded that the 35 COOH-terminal amino acids shared

by

VP2and VP3arerequired forthe nuclearlocalization ofboth

proteins

and are

likely

to contain the

karyophilic signal

needed for their nuclear

targeting. They

also showed that nuclear localization of VP3 could be observed in the total

absence of VP2 or the SV40

agnoprotein. Interestingly,

quasi-normal transport ofVP3 tothe nucleus(and

presum-ably of VP2also) wasobserved when

only

one-third ofthe amino-terminal sequence of VP1 was present. This VP1

fragment contains the nuclear migration

signal

(35). The intracellular localization of the truncated VP1

protein

was unfortunately notreported by those authors.

The sequence

responsible

forthe

migration

ofSV40 VP1 andthatresponsible fortheaccumulationofSV40VP2-VP3

intothe cell nucleusareencodedbythesame

portion

ofthe

SV40 genome

(nucleotides

1492 to 1513), which is read in two different frames

(Fig.

8). This leads us to

predict

that mutations in this region of the genome should profoundly

affect the

viability

of the

virus,

because

they

would prevent the nuclear location of all three SV40 capsid

polypeptides

VP1, VP2, andVP3.

ACKNOWLEDGMENTS

Wethank J. Feunteunforthe gift of pDR404 andB. Blondel for thegiftofpolyclonal antipoliovirus antibodies.

Thisworkwassupported inpartby CNRSgrant UA 545 andby ATPgrants 155198 and 955339.

LITERATURE CITED

1. Birnboim,H.C.,andJ. Doly. 1979. Arapid alkalineextraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513-1523.

2. Blondel, B., R. Crainic, and F. Horodniceanu. 1982. Le polypeptide structural VP1 du poliovirus type 1 induit des anticorpsneutralisants. C.R.Acad. Sci. 294:91-93.

3. Bonner, W. M. 1975. Protein migration into nuclei. I. Frog oocytenuclei accumulatemicroinjected histones,allowentry to small proteins, and exclude large proteins. J. Cell. Biol. 64:421-430.

4. Bonner, W. M. 1975. Protein migration into nuclei. II. Frog

on November 10, 2019 by guest

http://jvi.asm.org/

[image:7.612.330.556.72.141.2]

(8)

oocyte nuclei accumulate aclass ofmicroinjected oocyte nu-clear proteins and exclude aclass ofoocyte cytoplasmic pro-teins. J. Cell. Biol.64:431-437.

5. Chesebro, B., R. Race, K. Wehrly, J. Nishio, M. Bloom, D. Lechner, S.Bergstrom, K. Robbins, L. Mayer, J. M. Keith, C. Garon, and H. Haase. 1985. Identification of scrapie prion protein-specificmRNAinscrapie-infectedanduninfectedbrain. Nature(London) 315:331-333.

6. CoUedge, W. H., W. D. Richardson, M. D. Edge, and A. E. Smith. 1986. Extensive mutagenesis of the nuclear location signal of simian virus 40 large-T antigen. Mol. Cell. Biol. 6: 4136-4139.

7. Dingwall, C., S. V. Sharnick, and R. A. Laskey. 1982. A polypeptide domain that specifies migration of nucleoplasmin into the nucleus. Cell30:449-458.

8. Gharakhanian, E., J.Takahashi, and H. Kasamatsu. 1987. The carboxy 35 amino acids ofSV40 VP3 areessentialfor its nuclear accumulation. Virology 157:440-448.

9. Girard, M., L.Marty, and F. Suarez. 1970.Capsid proteins of simian virus 40.Biochim. Biophys. Res. Commun.40:97-102. 10. Green, S., P. Walter, V. Kumar, A. Krust, J. M. Borneert, P.

Argos, and P. Chambon. 1986. Human oestrogen receptor cDNA:sequence,expression and homologytov.erb.A.Nature (London)320:134-139.

11. Hall, M. N., L. Hereford,andI.Herskowitz. 1984.Targeting of E. coli

0-galactosidase

to the nucleus in yeast. Cell 36:1057-1065.

12. Kalderon, D., W. D. Richardson, A. F. Markham, and A. E. Smith. 1984. Sequence requirements for nuclear location of simian virus40largeTantigen. Nature (London) 311:499-509. 13. Kalderon,D., B. L. Roberts, W. D. Richardson, and A. E. Smith. 1984. A short amino acid sequence able to specify nuclear location. Cell 39:499-509.

14. Kasamatsu, H.,and A.Nehorayan. 1979. VP1affects intracellu-lar localization of VP3 polypeptide during simian virus 40 infection. Proc. Natl. Acad.Sci. USA76:2808-2812.

15. Lanford,R.E.,andJ.S. Butel.1984. Construction and charac-terization ofanSV40mutantdefectivein nuclear transportofT antigen. Cell 37:801-813.

16. Lanford, R.E., P. Kanda, and R. C. Kennedy. 1986.Induction of nucleartransportwithasynthetic peptide homologoustothe SV40Tantigen transportsignal. Cell 46:575-582.

17. Mandel, M., and A. Higa. 1970. Calcium dependent bacterio-phage DNA infection. J. Mol. Biol. 53:159-162.

18. Maniatis, T., E. F.Fritsch,andJ. Sambrook. 1982. Molecular cloning: alaboratory manual. ColdSpring HarborLaboratory, ColdSpring Harbor,N.Y.

19. Moreland,R.B.,H.G.Nam,L. M.Hereford,and H. M. Fried. 1985. Identification ofa nuclear localization signal ofayeast ribosomalprotein.Proc. Natl. Acad. Sci. USA82:6561-6565. 20. Morinaga, Y.,T.Franceschini,B.Inouye,and M.Inouye. 1984.

Improvement ofoligonucleotide directed sitespecific mutagen-esis using double-stranded plasmid DNA. Biotechnology 2:636-639.

21. Murray,N. E., W. H. Brammer, and K. Murray. 1976.Lamboid phages thatsimplifythe recoveryof in vitro recombinants. Mol. Gen. Genet. 150:53-61.

22. Pages, J., S. Manteuil, D. Stehelin, M. Fiszman, M. Marx,and M. Girard. 1973. Relationship between replication of simian virus 40 DNA and specific events of the host cell cycle. J. Virol. 12:99-107.

23. Prives, C. L., H. Aviv, B. M. Paterson, B. E. Roberts, S. Rozenblatt, M.Revel,and E. Winocour. 1974. Cell-free transla-tion of messenger RNA ofsimian virus 40: synthesis of the major capsid protein. Proc. Natl. Acad. Sci.USA 71:302-306. 24. Rawlins, D. R., P.Collis, and N. Muzyczka. 1983.

Characteriza-tion of am404, an ambermutation inthe simian40 Tantigen gene. J. Virol. 47:202-216.

25. Rawlins, D. R., and N. Muzyczka. 1980. Construction of a specific ambercodon in the simian virus 40T-antigengeneby site-directedmutagenesis. J. Virol.36:611-616.

26. Rozenblatt, S., R. C. Mulligan, M. Gorecki, B. E. Roberts, and A.Rich. 1976.Directbiochemical mapping of eukaryotic viral DNA by meansofa linked transcription translation cell free system. Proc.Natl. Acad. Sci. USA73:2747-2751.

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

28. Silver, P. A., L. P. Keegan, and M. Ptashne. 1984. Amino terminus of theyeastGal4geneproduct issufficient for nuclear localization. Proc.Natl. Acad. Sci. USA 81:5951-5955. 29. Sompayrac,L.M., and K. J. Danna.1981.Efficient infection of

monkey cells withDNAof simian virus40. Proc. Natl.Acad. Sci. USA 78:7575-7578.

30. Tegtmeyer, P., J. A. Robb, C. Widmer, and H. L. Ozer. 1974. Alteredproteinmetabolismin infectionby the latetsBll mutant of simian virus40. J. Virol. 14:997-1007.

31. Tooze, J. 1980. Molecular biology oftumor viruses, 2nded., part2,p. 799-831. ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.

32. Von Heijne, G. 1986. Mitochondrial targeting sequences may formamphiphilic helices. EMBOJ. 5:1335-1342.

33. Watson, M. E. E. 1984. Compilation of published signal se-quences.Nucleic AcidsRes. 12:5145-5164.

34. Wieslander, L. 1979. A simple method to recover intact high molecularweight RNAandDNAafterelectrophoretic separa-tion in low gelling temperature agarose gels. Anal. Biochem. 98:305-309.

35. Wychowski, C., D. Benichou, and M. Girard.1986.Adomainof SV40capsidpolypeptide VP1 that specifies migration into the cell nucleus. EMBO J. 5:2569-2576.

36. Wychowski, C., S.vanderWerf,and M.Girard. 1985.Nuclear localization ofpoliovirus capsidpolypeptideVP1expressedas a

fusionprotein with SV40-VP1. Gene37:63-71.

37. Zagursky,R.J., K. Baumeister,N.Lomax,and M. L. Berman. 1985.Rapid andeasysequencing of large linear double-stranded DNAandsupercoiledplasmidDNA.Gene Anal.Tech.2:89-94.