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Molecular cloning and expression of a murine homolog of the human poliovirus receptor gene.

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JOURNALOFVIROLOGY, May1992, p. 2807-2813 0022-538X/92/052807-07$02.00/0

Copyright © 1992, American Society for Microbiology

Molecular Cloning

and

Expression

of

a

Murine

Homolog

of the Human Poliovirus

Receptor

Gene

MARY E. MORRISONANDVINCENT R. RACANIELLO* Department of Microbiology, Columbia University College of

Physicians

&

Surgeons,

New

York

New York 10032 Received 23September1991/Accepted9February 1992

The poliovirusreceptor(Pvr) is amemberoftheimmunoglobulin superfamilyofproteins,but itsfunction in the cell is not known. Southern blot hybridization analysis indicated that the murine genome contains a sequencehomologofpvr.Asafirst step towardusingthe murinepvrhomolog(mph) tostudythe function of Pvr, murinegenomicand cDNA clonesencoding mphwereisolated.mphencodesapolypeptidewith extensive

sequence similarity to the extracellular domains of the human PVR. mph mRNAs of 2.0 and 3.0 kb are

transcribed in theadultmousebrain, the spinal cord,thespleen,thekidney,theheart,and the liver.TheMph proteindoesnotfunctionas areceptorforpoliovirus. However,substitution of domain 1 of the Mph protein with thecorrespondingsequencefrompvrproducedachimeric receptor that could bindpoliovirusand leadto productiveinfection. By constructing pvr-mph chimeras, it will bepossible to identify thecontact points of polioviruswithin domain 1 of Pvr.Identificationof theligandand the cellular function of theMph proteinmay

helpusunderstand the roleofPvr in the cell.

Recently, several cell proteins have been identified as

receptorsfor different viruses. Insome cases,these proteins

have known cell functions; for example, the human immu-nodeficiency virus type 1 (HIV-1) receptor, CD4, and the Epstein-Barr virusreceptor, CR2, participate incell

recog-nition, while the majorgrouprhinovirus receptor, ICAM-1, isan adhesionmolecule (5, 12, 15, 28, 29). However, while

the cell receptorfor poliovirus has been identified (14), its naturalfunction remains unknown. Thepoliovirus receptor (Pvr) isanintegral membrane protein that isamemberof the

immunoglobulin superfamily of proteins (14). The Pvr

pro-teincontains oneV-like andtwoC-like extracellular immu-noglobulin domains and thereforemayactas anadhesionor

cell surface recognition molecule.

Informationonthefunction of Pvrmight be obtained from thestudyofamurine Pvrhomolog (Mph). For example, the

distribution of putative Mph ligands would be more easily

determined with mice. Homologous recombination

experi-ments to create an mph null allelle in mice might enable

elucidation of its function. Finally, the ligand- and virus-bindingsites of the human Pvr couldbe explored by creating recombinants betweenpvrandmph.

To facilitate researchinto thenatural function of the Pvr,

we have isolated genomic and cDNA clones for a murine

sequence homolog. mphhasahigh degreeof aminoacid and nucleotide similarity topvraswell as a conserved splicing

pattern. mph transcripts are detected in all adult mouse

tissues tested. Mph is not a receptor for any'of the three serotypes ofpoliovirus; however, substituting the first do-main of the Mphwith that of the Pvr enables the recombi-nant protein to serve as a Pvr. The amino acid residues crucial to interaction with poliovirus therefore reside in domain 1.

MATERIALS ANDMETHODS

Cells and viruses. HeLaS3 cellsweregrowninsuspension cultures in Joklik minimal essential medium containing 5%

* Corresponding author.

horse serum and 10 p,g of gentamicin per ml. For plaque assays, HeLa cells were plated in Dulbecco's modified Eagle's medium (DMEM) containing 10% horse serumand

gentamicin. Mouse L cells were maintained in DMEM containing10% calfserum,100 U ofpenicillinperml,100p,g

of streptomycin per ml, and 20 ,ug of gentamicin per ml. Transient DNA transformants were grown in DMEM with 10% fetal bovineserum,100 U ofpenicillinperml, 100,ugof

streptomycinperml,and 20 ,ugofgentamicinperml. Stable DNA transformants were grown in the same medium with

100 mMhypoxanthine-0.4 mMaminopterin-16 mM thymi-dine(HAT). Poliovirus strainsP1/MahoneyandP2/Lansing

were derivedfromthe infectious cDNA clones (17, 18).

DNA transformation. Itk- aprt- cells were seeded in

plasticcell cultureplates1 day beforeuse(2 x 106cells per

6-cm-diameterplate for transient assay; 7.5 x 105 cells per

10-cm-diameter plate for isolation of stable transformants). The medium was changed 4 to 6 h before transformation. Plates were treated with the following DNA-calcium

phos-phate coprecipitates: for transientassay,0.5ml ofamixture

of 10,ugofplasmidDNA and 10,ugofherringspermDNA;

for stable transformation, 1.0 ml of a mixture of 10 ,ug of

plasmidDNA and 3 ,ugofa plasmid containingthe

herpes-virus thymidine kinase gene. After 18 h of incubation at 37°C, the mediumwas replaced and incubationwas contin-ued for 24 h. For transient assay, cells were infected with

poliovirus 48 h after transformation. For isolation of stable transformants,cellsweregrownin HAT medium for 2weeks and HAT-resistant colonieswere subcultured.

Virusinfection. Monolayers of L-cell transformantswere

infected withPl/MahoneyorP2/Lansingatamultiplicityof infection of 5, as indicated in the figure legends. After a

45-min adsorption period at 37°C, plateswerewashed with phosphate-buffered saline four timesto removeunadsorbed

virus, and the medium was replaced. Aliquots of

superna-tants were removed at different times after infection, and virus titerswere determined by plaque assayon HeLa cell monolayers (10).

RNA and DNA isolation. Total RNA was isolated from

cultured cells and tissues by homogenization in 4 M guani-2807

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dine isothiocyanate followed by centrifugation on a cushion of CsCl (2). Poly(A)+ RNA was purified by chromatography onoligo(dT)-cellulose columns according to the manufactur-er's instructions (Collaborative Research). Genomic DNA was prepared from cultured cells as described previously (13). Mouse genomic DNA was prepared from tail samples as described previously (6).

Northern (RNA) and Southern hybridization. RNA was fractionated on 1% agarose-formaldehyde gels and trans-ferred toZeta-Probe membranes (Bio-Rad)according tothe manufacturer'sinstructions. High-stringency blots were pre-hybridized for 4 h at 42°C in a solution containing 50% formamide, 5x SSCPE(1x SSCPE is 0.15MNaCl, 0.015M sodium citrate, 13mMKH2PO4, and 1 mMEDTA[pH 7.2]),

lOx Denhardt'ssolution, 1% sodiumdodecyl sulfate(SDS),

and 500 ,g ofboiled, shearedherringspermDNAperml and

hybridized for 18 h at 42°C in a solution containing 50% formamide, Sx SSCPE, 2x Denhardt's solution, 1% SDS,

100 ,ug ofherring sperm DNA perml,and 5 ngofDNAprobe

perml.High-stringencyblotswerewashed three times for 5 mineach at room temperature and then once for 30 minat 650C in lx SSC (lx SSC is 0.15 M NaCI plus 0.015 M sodium citrate)-1% SDS. Low-stringency Northern blots were hybridized in the same solution butwere incubated at

370C. Low-stringencyblotswerewashed three timesat room temperature and once at 550C in 2x SSC-1% SDS. For Southern analysis, genomic DNAwas

digested

with4 Uof restriction endonucleases permicrogramovernightand frac-tionated on 0.8% agarose gels. DNA was transferred to nitrocellulose filters as described previously

(27).

High-stringency hybridization conditions were as described pre-viously (Schleicher & Schuell). Low-stringency Southern blots were prehybridized for 4 h at 42°C in a solution

containing 1 M NaCl, 50 mM Tris-HCl

(pH

7.4),

35% formamide,0.5% SDS, Sx Denhardt's

solution,

and1 mgof

herringspermDNAperml and

hybridized

for 18hat

42°C

in asolution containing 1 M

NaCl,

50 mM Tris-HCl

(pH 7.4),

35% formamide, 0.5% SDS, lx Denhardt's

solution,

10% dextransulfate, 1mg ofherringspermDNAper

ml,

and 5 ng of DNA probe per ml. Low-stringency blots were washed three timesat roomtemperature andonce for30 minat

55°C

in2x SSC-0.1%SDS.

32P-labelled

probeswere

prepared by

oligonucleotide-primed synthesis

(Pharmacia).

cDNA, genomic, and cosmidlibraries. cDNAwas

synthe-sized as described previously

(14)

from

twice-oligo(dT)-selected mRNAs from the brains of Swiss Webstermice or

mouseLcells. Double-stranded,

EcoRI-adapted

cDNAwas ligatedtoEcoRI-digestedXZAParms

(Stratagene)

and

pack-aged by using Gigapack Gold extracts

(Stratagene).

A

C57/BL genomic DNA library in EMBL3 was

kindly

pro-vided by Glenn Radice.

Unamplified

cDNA or

amplified

genomic librariesof106 recombinants were

plated

on Esch-enchia coli LE392 cells. Duplicate filters were

hybridized

with DNA probes under the conditions described above for Southern blots. XZAP autoexcision of

insert-containing

pBluescript was carried out according to Stratagene's in-structions. A genomic cosmid library from C57 mice was

obtained from the laboratory of Fred Alt, and duplicate

filterswere screened as describedpreviously (20).

DNA sequencing and sequence comparisons. Subcloned restrictionfragmentsfrom cDNAclones weresequenced by

the dideoxy method (23) either manually or by automated sequencing (ABI,Inc.). Sequenceswerecompiledand ana-lyzed withtheUniversity of Wisconsin GeneticsComputer Group sequence analysis package (4). Alignments of the human PVR sequence and the murine homolog sequence

weremadeby usingthe Genetics

Computer

Groupprogram

BESTFIT,with default

settings.

5' end reconstruction.

Polymerase

chain reaction

(PCR)

was used to reconstruct the 5' end of the

longest

brain-derived cDNA clone.

Briefly, genomic

sequencing

of cosmid 2DNA

provided

thecorrect5' 26-nucleotide sequence. A 5' PCR

primer, bATG2,

containing

anEcoRI

site,

the

missing

26-nucleotides, and a

25-bp

overlap

with braincDNAclone 2 and a3' PCR

primer, bTGA,

overlapping

thetermination codon and also

containing

an EcoRI site were used to

amplify

the

coding

region

ofbrain cDNA clone 2:

bATG2,

5'CTGGAATTCCCCATGGCCCGGGCCGCAGTCCTCC

CGCCGTCCAGATTGTCACCGACGCTG3';

bTGA,

3'CG

TCAAATGCACACTGGGATGCTTAAGGTC5'.

PCR was

carried outfor 30

cycles

at

94°C

for 1min,

55°C

for 2min,

and

72°C

for 3 min. The

product

wascloned into M13vectors for sequenceconfirmation and into othervectorsfor expres-sionstudiesasdescribed below. The reconstructed cDNA in M13 is

designated

pMEM.brmpl8.

Expression

of cDNA clones. The PCR-reconstructed

mu-rine brain cDNA was cleaved with EcoRI, the ends were

made blunt with Klenow enzyme

(BMB),

and the

resulting

fragment

wascloned intoan

SmaI-digested

expression

vec-tor,

pSVL

(Pharmacia).

This

plasmid

is

designated pMEM.

brSVL7. Some recombinants with the human pvr cDNA wereconstructed asdescribed below. Todetermine the Pvr

activity

of these

cDNAs,

L cells were transformed with

CsCl-purified

plasmids

as described above. Transformants were

assayed

fortheir

sensitivity

to

poliovirus

infection and stable cell lines were isolatedasdescribed above.

Oligonucleotide-directed

mutagenesis.

Oligonucleotide-di-rected

mutagenesis

was

performed

on human pvr cDNAs subcloned in M13 and grown in E.

coli

CJ236as described

previously

(1).

The enzymes usedwere T4

polynucleotide

kinase

(BMB)

and Klenow enzyme

(BMB).

Nucleotide

changes

wereintroduced into humanreceptorcDNA inserts

corresponding

to

plasmids

pSVL-H20A

and

pSVL-H20B

(14)

to create a KpnI site between

immunoglobulinlike

domains 1 and 2 in a

position

analogous

tothis

KpnI

site in themurine brain cDNA sequence. These mutationsresultin a

coding

change

fromserine tothreonineatamino acid 132. The M13 human pvr clones with the new

KpnI

site are

designated

pMEM.20AKpn

and

pMEM.20BKpn.

The

muta-genized

coding

regions

were cloned back into

expression

vector

pSVL

asdescribed

previously

(14).

Theseconstructs are

designated

pMEM.20AKpnSVL

and

pMEM.20BK

pnSVL.

Constructionofrecombinants. Recombinants between the human pvr cDNAclone in

pMEM.20BKpn

and the

recon-structed murine brain cDNA clone in

pMEM.brmpl8

were

constructed.

Briefly, plasmid

pMEM.brmpl8

was

digested

with

SmaI

and

KpnI

andtreated with calf intestine alkaline

phosphatase;

the

373-bp

fragment

containing

murine domain 2andthe

7,872-bp

fragment

containing

the

M13mpl8

vector with

mph

domain 3

through

the

cytoplasmic

tailwere

iso-lated from

low-melting-point

agarose

gels.

Plasmid

pMEM.20BKpn

was

digested

with

SmaI

and

KIpn,

and the

507-bp

fragment

containing

pvr domain 1 and the

373-bp

fragment

containing

pvrdomain 2 were isolated from

low-melting-point

agarose

gels.

The

507-bp

pvrdomain 1

SmaI-KpnI

fragment

was

ligated

into the

7,872-bp

mph

domain

3-cytoplasmic

tail-M13mpl8

fragment,

creating

plasmid

pMEM.1.

In this process, the SmaI and

BamHI

sitesfrom the M13 vector were

deleted,

but the XbaI site was left intact. Thedomain2sequencewasrestored to

pMEM.1

by

linearizing

this

plasmid

with

KpnI

and

ligating

in the

373-bp

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MURINE POLIOVIRUS RECEPTOR HOMOLOG 2809

B B BH B H B H B H B B B H B H B

l- l l I... I II--- li min i ii I

exon 1 2 3 45 6

2kb

5.5kbHindIII

exonsequence

9kbBamlHI

cosmid clone 4

genomic clone 6 cosmid clone 2

FIG. 1. Murine DNA

contains

sequenceshomologoustothose of the human pvr.(A)Genomic DNA from C57BL mouse spleen was digested with the indicated restriction enzymes and analyzed by Southern hybridization, at low stringency, with a 0.97-kb EcoRI human pvr cDNA probeencodingthe 3' half of domain 1 and all of extracellular domains 2 and 3. The positions of molecular weight markers(in kilobases) are indicated on the left. (B) Restriction map and exon distribution of the mph gene. A combination of low-stringency hybridization with human pvr cDNAs and high-strin-gencyhybridization withmph cDNAwas used toisolate and map

the genomic DNA region shown. Solid bars indicate exon se-quences; numbers below these bars correspond to the exon numbers inFig. 2. Open bars indicate therestrictionfragments observed by

low-stringency Southern hybridization with human pvr probes.

Stippledbars indicate the extentof individualgenomicandcosmid clones. B, BamHI; H,HindlIl.

1.1

--domain 2KpnI fragment, creating plasmids pMEM.h5 and pMEM.ml. The chimeric junctions were confirmed by

re-striction endonuclease digestion andDNAsequence

analy-sis. The chimeric inserts were excised by total XbaI and

partialEcoRI digestion, the termini were made blunt with Klenow enzyme, and the inserts were cloned into SmaI-digested pSVLvector. The finalconstructs are as follows:

pMEM.ml-1 contains PVR domain 1 up to the

KpnI

site introducedbymutagenesis,followedbymurinesequences3' of theKpnIsite,andpMEM.h5-1 containspvrdomains 1 and 2uptothenaturally occurringKpnIsite betweendomains 2 and 3, followed by murine sequences for domain 3, the putative transmembrane-spanning region, and the cytoplas-mic tail. pMEM.h5-1 contains the

KpnI

site introduced by mutagenesisofthe humanreceptor cDNA.

Nucleotide sequence accession number. The GenBank

ac-cession number for themph cDNAsequence is M12197. RESULTS

Isolation ofgenomicand cDNA clones encoding Mph. To determine whether themouse genome containsa sequence

homolog of the human Pvr, mouse DNAwas subjected to low-stringency Southern blotanalysiswitha0.97-kb EcoRI DNA fragment probe from humanpvr cDNA (14). This hybridization probe detected one DNAspecies, suggesting the presence of a single homologous gene (Fig. 1A). The

humanpvrcDNA probewas then used to screen a C57BL mouse genomiclibrary, and onetype ofrecombinantphage represented by genomicclone6wasisolated(Fig. 1B).This

genomic clonecontained the5.5-kbHindIII and the 2.1-kb EcoRI restriction fragments detectedbygenomic Southern analysisin Fig. 1A. Genomicclone 6 also containeda 9-kb

BamHI fragment, whichwasdetectedby Southern analysis

ofmouse DNA(16).

A0.6-kb BgIII-EcoRI fragmentwasisolatedfromgenomic

clone6and usedtoscreenseveralcDNA libraries. Themost complete cDNA clone was isolated from a mouse brain

library and is designated pMEM.b2. The nucleotide

se-quence of this clone is discussed below. The entire 1.4-kb coding region of pMEM.b2 was used to screen a cosmid libraryofC57BLmouseDNA.Twoclasses of cosmid clones

wereisolated;thebarsbelow thegenomicDNAmapin Fig.

1B indicate the location of DNAs from cosmid clones 2 and 4.Theseclonesbothhybridizedwithprobesfromthe5'end of brain cDNAclone 2, but only cosmid 2 also hybridized with a 3' end probe (16). Both classes of cosmid clones

contain DNA fragments found ingenomic clone 6, as

indi-cated byrestriction mapping.

Nucleotidesequenceanalysisofmph cDNA clones. Nucleo-tide sequence analysis ofmph cDNA and genomic clones revealed the intron-exon structure and the coding regions

shown in Fig. 2. pMEM.b2contains anopenreadingframe of 1,377 bp which encodes a 459-amino-acid polypeptide

composedofa24-residueamino-terminalhydrophobic signal

sequence, a314-amino-acid extracellular domain, a 28-ami-no-acid transmembrane domain, and a 93-amino-acid

cyto-plasmictail. Amino acidsequencealignmentwiththe human Pvrindicates conservation of the three extracellular immu-noglobulinlike domains across the species. The three do-mains(defined byintron-exonboundaries) share52, 57, and 64%identityand64, 68,and80%similarityattheamino acid

level (Fig. 3). The positions of the splice sites in the

extracellular domainsareconserved betweenthehumanand murinegenes.Nonconsensusbases within thesplicesites lie in the same positions, further emphasizing the homology between thepvrand themph genes. Thepossible sites for N-linked glycosylation, indicated in Fig. 3, differ between

A

+sC\\

0GCb

9>i 9>4

.S si,

le

13--

8.7--4.3

-.

3.3

--2.5

--VOL.66, 1992

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[image:3.612.104.235.77.438.2]
(4)

exon 1.... -181.. GGATCCTGAGAGCCAGGCGAGGGAAAGCTGGGCCGAACGAACTGATCCGGGGAGCCGTGAGCGGCGGAAGGGCTCTGGAGCC

GACACTTCAGACCCCTGACTGCCCTCCCAGCCGATCGGTACACGAAGAGTGGTCCCTAGGCACCCCCTGCCCGGGCCCAGTCCCTCCCCGGGCCCCCCG ATGGCCCGGGCCGCAGTCCTCCCGCCGTCCAGATTGTCACCGACGCTGCCGTTGTTGCCGCTGCTACTGCTCCTGCTTCAGGAAACAGgtgaaaacgcg

MetAlaArgAlatAlayalLeuProProSrArUTe-uSrProThrTLePuProLeuLeuP-ILeuL

uuLeunlnGluThr*

[image:4.612.70.557.70.576.2]

...-exon 2 cggatgcct... cctcttttctctctccccagGAGCCCAAGAT

... lyAlaGlnAsp

-100 -1

88

30 99 33

GTGCGGGTACGAGTGCTTCCCGAGGTCCGGGGCCGCTTGGGAGGCACCGTGGAGTTACCGTGCCACCTGCTCCCACCCACGACGGAGCGCGTCTCTCAG 198

ValArgValArgValLeuProGluValArgGlyArgLeuGlyGlyThrValGluLeuProCvsHisLeuLeuProProThrThrGluArgValSerGln 66

GTGACCTGGCAGCGCCTGGATGGCACAGTTGTGGCTGCTTTCCACCCATCCTTCGGAGTGGATTTCCCCAACTCTCAGTTCAGCAAGGACCGTCTGTCC 297

ValThrTrpGlnArgLeuAspGlyThrValValAlaAlaPheHi3ProSerPheGlyValAspPheProAsnSerGlnPheSerLysAspArgLeuSer 99

TTTGTCAGAGCGAGACCAGAAACAAAsCGAGACCTGCGGGATGCCACaCGCACTGheAGGGTyIAVa1G1uAGGAsAGuGGCAATTACACCTGCGAG PheValArgAlaArgProGluThrAsnAlaAspLeuArgAspAlaThrLeuAlaPheArgGlyLeuArgValGluAspGluGlyAsnTyrThrCy.aGlu 396132

TTTGCCACGTTTCCCAACGGTACCCGCAGGGGGGTGACCTGGCTCAGAGTCATAGgtgagtggagggaaccctgg... 451

PheAlaThrPheProAsnGlyThrArgArgGlyValThrTrpLeuArgValIleA... 151

exon 3 .ttctttggtcatctccctagCCCAGCCTGAGAACCACGCTGAAGCCCAGGAGGTCACAATTGGC

...laGlnProGluAsnHisAlaGluAlaGlnGluValThrIleGly

495

165

CCCCAGTCGGTGGCTGTAGCCCGCTGTGTCTCCACTGGGGGCCGCCCCCCTGCCCGAATCACCTGGATCTCATCTCTGGGTGGAGAGGCCAAAGATACT 594

ProGlnSerValAlaValAlaArgCy&ValSerThrGlyGlyArgProProAlaArgIleThrTrpIleSerSerLeuGlyGlyGluAlaLysAspThr 198

CAGGAGCCAGGGATACAGGCTGGCACCGTCACTATCATCAGCCGATACTCCTTGGTGCCCGTGGGCCGAGCGGATGGCGTCAAGGTCACGTGTAGAGTG

GlnGluProGlyIleGlnAlaGlyThrValThrIleIleSerArgTyrSerLeuValProValGlyArgAlaAspGlyValLysvalThrcvEArgval 693231

GAACACGAGAGCTTCGAAGAGCCGATCCTGCTGCCAGTGACCCTCTCTGTGCGCTgtgagtgtgccgggaggtgg... 748

GluHisGluSerPheGluGluProlleLeuLeuProValThrLeuSerValArgT... 250

exon 4.... cagtcatctttttctcccagACCCTCCAGAAGTATCCATCTCCGGCTATGATGACAACTGGTAC 792

...yrProProGluValSerIleSerGlyTyrAspAspAsnTrpTyr 264

CTTGGCCGCAGTGAGGCCATACTGACCTGTGATGTACGAAGCAACCCAGAGCCCACAGACTATGACTGGAGCACgtgagtgatgtgaacctaga... 866

LeuGlyArgSerGluAlaIleLeuThrcv.aAspValArgSerAsnProGluProThrAspTyrAspTrpSerTh... 289

exon 5... acctcacccacacccttagGACCTCGGGCGTCTTCCCAGCCTCT 891

...rThrSerGlyValPheProAlaSer 297

GCAGTGGCCCAGGGCTCTCAGCTGCTTGTCCACTCTGTGGATCGAATGGTCAACACTACCTTCATCTGTACAGCCACCAACGCTGTGGGGACAGGCCGT

AlaValAlaGlnGlySerGlnLeuLeuValHisSerValAspArgMetValAsnThrThrPheIleCyvThrAlaThrAsnAlaValGlyThrG1VArg

990330 GCTGAGCAGGTCATCCTGGTGCGAGgtaaggaagccttgtggtgt...1015

AlaGluGlnValIleLeuValArgA... 339

exon 6.... tctccttcctttctcctcagACACCCCCCAGGCCTCCCGAGATTT(TC ACA MCTArT 1089

...spThrProGlnAlaSerArgAs 363

CTGGCTGGEsC4GGG Tr-r-T CTTGATCTGCTAnGGGGGAGGAGGAGGCGGAAGAGCCCTGGAGGAGGAGGAAATGATCGCGACAGAGGATCCTACGAT 1188

uAlaGlyGlyPh

eT.P-iiTt-i3krgGlyArgArgArgArgLysSerProGlyGlyGlyGlyAsnAspGlyAspArgGlySerTyrAsp

396

CCAAAGACTCAGGTGTTTGGGAACGGGGGTCCTGTCTTCTGGAGGTCAGCATCCCCTGAGCCCATGAGGCCAGATGGCAGGGAGGAAGATGAGGAGGAG 1287

ProLysThrGlnValPheGlyAsnGlyGlyProValPheTrpArgSerAlaSerProGluProMetArgProAspGlyArgGluGluAspGluGluGlu 429

1386 462

CGGGCAGTTTACGTGTGACCCTACGATATAGACACTGGACACATGGAAACACCAAGTTCCACCCTCACTGCCAACCACACCAATGCCAGCCAGCAACGA +84

ArgAlaValTyrVal***... 467

TGGTAGGGACCGGTTGGACTGGTTCTTCTGGGGCACACTGGAGTTGGAAGGCACCGCCCCTGCTTCAGGATAGAGGACAAGTGGAACCACACAGACTCC +183

TATCTTTAGGGCCTCATGGAGTAGGGGACCCCAGGAGCGCCATGGTGCACACTCAGGACTCCTCAGAGCTTGCTTTCGCCCCAGCCTAGCCCTGGCCCC +282 GAAACACTCAGGAGCTAATAAATGCCTTGTCGGAAAACCTTGGGGTGTTTTGT... +335

FIG. 2. Intron-exon structure of themph2.0-kbtranscript.Capitallettersrepresentexonnucleotidesequences;lowercase lettersindicate

intron sequences. Nucleotide positions relative to the coding region are shown at the right. Amino acid sequencesare givenbelow the

nucleotide sequences, and amino acid positions are shown at the right. The putative signal peptide is underlined, and the putative

transmembrane domain is boxed.Cysteinesimportanttoimmunoglobulin familydomainstructure arealsounderlined,andthepoly(A) signal is indicated byaline above thenucleotide sequence AATAAA.

the two cDNAs: the Pvr protein contains eight potential sites,whereasMph contains only three.Thetransmembrane and cytoplasmic domains share little or nosignificant simi-larity.

AlthoughpMEM.b2 containedanextensivecodingregion,

it lacked an AUG translation initiation codon. Nucleotide sequencing of subcloned cosmid DNAs indicated that the

missing AUG lay 23 bp upstream from the 5'-terminal nucleotideof brainclone2,onthe same exon as the65bp of signal sequence includedin pMEM.b2 cDNA.

Invivo expression ofmph mRNAs. Northernblot

hybrid-ization of RNA from adult mouse tissues was used to determine the distribution of mph transcripts. A murine cDNA probe containing sequences encoding extracellular domains2 and 3, the transmembrane region,and the

NH2-terminal half of the cytoplasmic tail hybridizedwith 2-and 3-kb mRNAs in all tissues tested, includingthe brain, the

spinal cord, theheart, theliver, thekidney, and the spleen

(Fig. 4). Northern analysis using 3'-noncoding-region

hy-bridization probes indicated that all the cDNA clones iso-GAGGAAGAAATGAAGGCAGAGGAAGGTCTCATGCTACCTCCACACGAGTCACCTAAGGACGACATGGAGTCCCATCTGGATGGCTCCCTCATCTCTCGG

GluGluGluMetLysAlaGluGluGlyLeuMetLeuProProHi3GluSerproLYSASpASpMetGluSerHiSLeuA3PGlYSerLeuIleSerArg

_-"..^,.1I..0- .I

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MURINE POLIOVIRUS RECEPTOR HOMOLOG 2811

1 MARA. .MAAAW ...PLLLVALLVLSWPiGTGTFV%VC1APrTQVPGFLGDSVP 45 Pvr

M1AAVLPPSRLSPTLPLLP,' . GTV 50 Mph

48 ?RQ9.P..r.

1 91.lPCHLI.PPTTE. RVSc'vTiN'rr'4T--*"'PNSi--SKDR 97 Msph

92 LEFVAAR..PLGAELRNAST.hD'GL8R' SRSV:OWl. 139 P.r

98 l,SF'VRAR.PETNADIF,P ATLAil' v, VELDI-!2jU- 147 I.r.

140 RVLAKPQNTAEVQKVCQl,'TG:'-0PVFMtARCVS-.GGRPPAQl,)In.TWIHL.ISG- ,-M-P.1>H9 148 RVIAQPENHAEAQ--)V-TVCPQ'"VAVARCVSTGGRPPARTWTIS-SLO,AKT 197 'ph

190 SQVPGFLSG'TV7'VTSLWILVpISSQV'DGKNVTCKVTEHES;?'S'--P pQ:'::vT 3' >'

198 TQEPGIQAGTVTIISRYSLVPVGRADGVKVTCRVEHESFP1EPILIP'.T', 24'. Mrh 240 VYYPPEVSISGYDNNWYLGQNEATLTCDARSI;PEPTpYpWSTTMGPLiD' 289 Prr 248 VRYPPEVSISGYDDNWYLGRSEAILTCDVRSNPEPT'DYDWSTTSG\'FPA' ,'J,' !.lph

290 AVAQGAQLLIRPVDKPIUTLICNJNALGARQAELTVQVKE;_PPSEH. 337 298 AVAQGSQLLVHSVDRMVNFICTATNAVGTGRAEQVlLVRDTPQASRDV 347

338 SGISRNAIIFLVLGILVFLILL... 359

.... .. . .. l,II?

348 GPLVWGAVGGTLLVLLLAGGFLALILILRGRRRRKSPGGGGNDGDRGSYDP 397 360 ... GIGIYFYWSKCSREVLWHCHLC0PSSEHHQSCRI' 392...

IK .:1...1.1.::.

...:..

398 KTQVFGNGGPVFWRSASPEPMRPDGREEDEEEEE.EMK}A 435...

Pvr

Mph

Pvr

Mph

Pvr

[image:5.612.57.293.82.307.2]

Mph

FIG. 3. Amino acidsequencecomparison ofMphand Pvr. The UniversityofWisconsin GeneticsComputer Groupprogram BEST-FIT was used to align the Mph and Pvr amino acid sequences.

Identical and conserved amino acidsare indicated byvertical lines and dots, respectively. The Pvr sequence is above and the Mph

sequence isbelow theidentityindicators. Potential N-linked glyco-sylation sitesare underlined.

lated in these experiments are derived from the 2.0-kb

mRNA (16). The 3.0-kb mRNA hybridized with pMEM.b2 probes from domain 1, domain 2, or domain 3 but did not hybridize with cytoplasmic tail or 3' noncoding sequences

fromthesamecDNA clone. These results indicate that the

2-and 3-kb mRNAs differ inthe 3' end. Bothmph transcripts

arepresentin Lcells,amurinefibroblastline thatisresistant to infection with allserotypesofpoliovirus (Fig. 4).

Pvractivity of theMph. Thepresenceofmph transcriptsin

mouse L cells, which do not express poliovirus-binding

sites, suggested thatthe Mph protein doesnotserve as aPvr.

Toconfirm thisassumption, mph cDNAwasassayed for Pvr

activity. Because brain cDNA clone pMEM.b2 lacked the

first23 nucleotides of thepolypeptide, thecorrect5' end for the coding region was reconstructed by using PCR, as

described in Materials and Methods. The reconstructed

coding region wassequenced inits entiretyandcloned into the eukaryotic expression vector pSVL. This construct,

designatedpMEM.brSVL7,wastransformed into L cells. At

48 h after transformation, the cells were infected with

poliovirus P2/Lansing, a mouse-adapted strain, and 48 h

laterthe supernatantswereassayedforinfectiouspoliovirus by plaque assay on HeLa cells. As shown in Table 1,

expression of mph in L cells did not permit poliovirus infection of thesecells, whereas control plasmids containing

pvr cDNA conferred susceptibility to infection.

Further-more,virus-binding assays withstable cell lines expressing mph indicate thatP2/Lansingdoesnotbind tothe Mph (16). pvr-mph recombinants. It was of interest to construct pvr-mph chimeric molecules to identify the regions of the

Mph that prevent its function as a Pvr. To facilitate the construction of these recombinants, pvrcDNA H20B was

mutagenizedto create aKpnI site atthe samelocation as a

KpnI site in mph cDNA between domains 1 and 2. This

18S

--FIG. 4. Northern blothybridization analysis of mph transcripts. Poly(A)' mRNAsfrom Swiss Webstermouseorganswere electro-phoresed in formaldehyde-agarose gels and subjectedto Northern hybridization analysis with an mph probe encoding extracellular

domains 2 and3,the transmembrane region, and theamino-terminal half of the cytoplasmic tail (bp 463 to 1182 on the nucleotide

sequenceinFig. 2).Thepositionsof rRNAsareindicatedattheleft.

alteration results in a change from serine to threonine at

human Pvr amino acid 132. Thiscoding change hasnoeffect

on the mutagenized cDNA's ability to serve as a Pvr, as

shown in Table1; transformation ofplasmids20BpSVL and pMEM.20BKpnSVL into L cells lead to susceptibility to poliovirus infection. In this assay system, twofold

differ-encesare not significant.

The mph-pvr recombinants that were constructed are

shown schematically in Table 1. Plasmid pMEM.ml-1

en-codesamolecule with human Pvr domain 1 in place ofthe corresponding Mph domain, and plasmid pMEM.h5-1

en-codes amoleculewith human Pvr domains1 and 2 in place

of the Mph domains. As shown in Table 1, both of these plasmids confersensitivity topoliovirus infectiononL cells. Therefore, the amino acid differences in domain1of theMph and the Pvr prevent the Mph from functioning as a Pvr.

Since the Pvr and the Mph share extensive homology in domain 1, it should be possible, by substitution of murine MphforPvr residuesinthe individualloopsandcstrandsof the first immunoglobulinlike domain, to identify the Pvr residues critical forpoliovirus binding and infection.

DISCUSSION

Murine genomic and cDNA clones which encode a

poly-peptide, the Mph, with structural similarities to the human

Pvrwereisolated. Boththe Pvr and theMphincludeasignal peptide, three extracellular immunoglobulinlike domains, a

transmembranedomain, andacytoplasmictail. High amino

acid and nucleotide sequence conservation in the extracel-lular domains (64 to 80%) indicate a closc relationship

betweenpvrandmph; this level of conservation is

compa-rableto thatbetween human and murine ICAM-1(7, 26) or

human and murine CD4 (11), receptors for major group

human rhinoviruses and HIV, respectively, and also

mem-bers of the immunoglobulin superfamily ofproteins.

Chro-mosomemapping indicates thatmph islocated inaregionof

28S

--VOL. 66,1992

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11

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C940,

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1.0

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TABLE 1. Pvractivity test of mph andmph-pvrchimerasa

DNAtransformed PFU/mlat(h): Receptorstructure"

0 48

Herring sperm 46 5.2 x 104

pSVL 212 4.6 x 104

20BpSVL 128 2.9 x 107

pMEM.20BKpnSVL 210 6.0 x 107

pMEM.brSVL7 20 4.1 x 104

pMEM.ml-1

142 6.0 x 107

p

pMEM.h5-1 150 3.5 x 107

aTheplasmids indicatedatthe left weretransiently transfected intoLcells as described in Materials and Methods. Twodaysposttransformation,thecellswere infectedwithP2/Lansing virusat amultiplicityof infectionof5.The inoculum was washedoff,supernatantaliquotswereremoved at0and 48h,andvirustiters were determined by plaque assay on HeLa cellmonolayers.

bTheexpectedproteinstructures, withstippleddomainsrepresentingPvrsequencesand open domainsrepresentingMphsequences.

mousechromosome 7 (8) which is known to be syntenic with human chromosome 19, to which the human vvr has been mapped(9, 24, 25). One significant difference between mph and pvr is thelack ofalternative splicingof the 2.0-kbmph transcript. Examination ofmph mRNAs by PCR showed that there is no form of the 2.0-kb message lacking a transmembrane domain(16), asis found for the Pvr(9).

The natural functions of both the Mph and the Pvr are unknown. It ispossible that this new member of the immu-noglobulin genefamily is involved in cell adhesionorsurface recognition, as are many otherimmunoglobulinlike proteins, including the rhinovirus receptor, ICAM-1 (5, 28, 29); the HIVreceptor, CD4 (12); and the Epstein-Barr virus recep-tor, CR2(15). Adhesion assays arebeing conducted to test this hypothesis. We have alsobegun homologous recombi-nation experiments to explore the effectsoL anullallelle of themph gene duringmouse development. These studies of themphgene mayprovideclues about its function. Whether or not the Mph protein is a functional homolog of the Pvr alsoremainsto be determined.

Theubiquitous expression of mph inmouseorgans, which isalso observed with pvr in humantissues, does not ruleout the possibility of highly regulated cell-specific expression within those tissues. Examination of pvr expression in

transgenic mice by in situ hybridization indicates that the transgeneisexpressedonlyinspecificcelltypes, including, for example, neurons within the central and peripheral nervoussystems,glomeruli and tubularepithelial cells inthe kidney, endocrine cells in the cortex of the adrenal gland, T lymphocytes in the cortex of the thymus, and alveolar macrophages (21). Whether or not a similar pattern of expression ofpvr occurs in humans remains to be deter-mined. Also of great interest is whether the pattern of expression of mph in mice is similar to those of pvr in transgenic mice and humans.Insituhybridization using mph probes is planned to define the patterns of mph expression withinadult mouse tissues and during prenatal development. Moststrains ofpoliovirus cannot infect mice. This species restriction is due to the lack of receptors in mice, as

demonstratedpreviously bythe observation thatexpression ofpvrin transgenicmiceleadstosusceptibilitytopoliovirus infection(20). However, some strains ofpoliovirus, suchas the P2/Lansing strain, have been adapted to grow in non-transgenic mice. Itis believed that these strains recognizea receptorinmicethatcannotberecognized bystrainsthatdo not infect mice. A logical candidate for the mouse P2/ Lansing receptor ismph. However, despite the close rela-tionship betweenpvr and mph and the fact that a closer sequencehomologofpvr does not seem to exist inmice,our results indicate that mph cDNA encoding the 2-kb brain mRNA does not act asa Pvrwhen expressedin Lcells. One possible explanation for this finding is that mph cDNA transformed into L cells does not result in theexpressionof a cell surface protein. We cannot rule out this possibility, because antibodies that recognize the Mph are not yet available. However, we believe it is unlikely that the Mph fails to reach the surfaces of transformed cells, because a proteininwhich domain1of theMph is substitutedwith the corresponding sequencefromthePvrisclearly expressedon thecell surface. It is possible thatLcells lackthe modifica-tion machinery necessary for Mph receptor activity. An-other possibility is that the 3-kb mph mRNA encodes a receptor for P2/Lansing. Molecular clones of this mRNA have notyetbeenobtained, and thereforedirect tests of this hypothesis are not possible. However, because the 3-kb mRNAhybridizes with all extracellularportions of the2-kb cDNAand,inaddition,theextracellular domain1of the Pvr is sufficient to confersusceptibility to poliovirus infection, we believe it is unlikely that the 3-kb mRNA encodes a Lansing receptor.

Themostlikely explanation forthelackof Pvrfunction of the Mph is that mouse-adapted strains ofpoliovirus use a different molecule fortheir receptor. Multiple-receptor us-agebya single virus has been observed withother viruses. For example, the coxsackievirus B3 variant CB3-RD was selected forgrowth in RDcells butmaintained itsabilityto growin HeLacells (19).The findingthat CB3saturation of HeLa cell receptors failed to block CB3-RD binding

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MURINE POLIOVIRUS RECEPTOR HOMOLOG 2813

cated a second receptor present on HeLa cells which the parent virus failed to recognize (3). Unfortunately, the molecularidentityof these receptors isnotyetknown. Thus, it is possible that P2/Lansing uses a mouse receptor that is not the murine sequencehomolog of the human pvr.

The close sequence homology of Mph and Pvr should enable identification of Pvr residues important for virus

binding bysubstitution ofMph aminoacids into the Pvr and vice versa. The results presented here demonstrate that domain1of thePvrissufficienttoconfer full Pvractivityon the Mph protein. It is now a matter of mutagenesis and

cloningto define the individual loops and fi strands of Pvr domain 1 thatare important forvirus binding, aswas done for HIV-1 interaction with CD4(22). It willbe of interest to determine whether other parts of Pvr are required for

subsequent steps in the poliovirus life cycle, such as the conformational transitions of the virion that are associated with virus entry into the cell, including the loss of the internal virionprotein VP4, externalization of the VP1 NH2

terminus, and extrusion of RNA from the capsid. CD4 sequences that are important for binding of HIV-1 are different from sequences that permit gpl20-mediated mem-brane fusion (22). By analogy, different parts of Pvr may

participate

in binding and triggering conformational

transi-tions, and Pvr mutants that separate those functions may

provide a dynamic picture of virus-receptor contact and communication atthe molecular level.

ACKNOWLEDGMENTS

We thank CathyMendelsohn for stimulating discussions during the earlypartof this work and Roy Bohenzky for useful technical

suggestions.

Thisworkwassupported bygrants ACSMV-324 from the Amer-ican Cancer Society and GM38125 fromthe National Institute of General Medical Sciences. M.E.M. is the recipient of a National ScienceFoundationpredoctoral fellowship.

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14. Mendelsohn, C., E. Wimmer, and V. R. Racaniello. 1989. Cellular receptorfor poliovirus: molecular cloning, nucleotide sequenceandexpression ofanewmember of the

immunoglob-ulinsuperfamily. Cell 56:855-865.

15. Moore, M. D., N. R. Cooper, B. F. Tack, and G. R. Nemerow. 1987. Molecularcloningof the cDNAencoding theEpstein-Barr

virus/C3d receptor (complement receptor type 2) of human B lymphocytes. Proc.Natl. Acad. Sci. USA 84:9194-9198. 16. Morrison, M. E., and V. R. Racaniello. Unpublished results. 17. Racaniello, V. R. 1984.Poliovirus type II produced from cloned

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18. Racaniello, V. R., and D. Baltimore. 1981. Cloned poliovirus complementary DNA is infectious in mammalian cells. Science 214:916-919.

19. Reagan, K. J., B. Goldberg, and R. L. Crowell. 1984. Altered receptor specificity of coxsackievirus B3 after growth in rhab-domyosarcoma cells. J. Virol. 49:635-640.

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22. Ryu, S., P. D. Kwong, A. Truneh, T. G. Porter, J. Arthos, M. Rosenberg, X. Dai, N. Xuong, R. Axel, R. W. Sweet, and W. A. Hendrickson. 1990. Crystal structureofanHIV-binding

recom-binantfragment of human CD4. Nature (London) 348:419-426. 23. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequenc-ing with chain-terminatsequenc-ing inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467.

24. Schonk, D., P. van Dik, P. Riegmann, J. Trapman, C. Holm, T. C.Willcocks, P. Sillekens, W. van Venrooij, E. Wimmer, A. Geurts van Kessel, H.-H. Ropers, and B. Weiringa. 1990. As-signment of sevengenes to distinct intervals on the midportion of humanchromosome 19q surrounding the myotonic dystrophy gene region. Cytogenet. Cell Genet. 54:15-19.

25. Siddique, T., R. McKinney, W. Y. Hung, R. J. Bartlett, G. Bruns, T. K. Mohandas, H. H. Ropers, C. Wilfert, and A. D. Roses. 1988. The poliovirus sensitivity (PVS) gene is on

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28. Staunton, D. E., V. J. Merluzzi, R. Rothlein, R. Barton, S. D. Marlin, and T. A. Springer. 1989. A cell adhesion molecule, ICAM-1, is the major surface receptor for rhinoviruses. Cell 56:849-853.

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VOL.66, 1992

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Figure

FIG.1.gencyquences;markersextracellularthedigestedclones.stringencythehumanSouthernandlow-stringencyinStippled Fig
FIG. 2.transmembranenucleotideisintron indicated Intron-exon structure of the mph 2.0-kb transcript
FIG. 4.phoreseddomainssequencePoly(A)'hybridizationhalf Northern blot hybridization analysis of mph transcripts

References

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