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,
NewYork
New York 10032 Received 23September1991/Accepted9February 1992The 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'ssolution,
and1 mgofherringspermDNAperml and
hybridized
for 18hat42°C
in asolution containing 1 MNaCl,
50 mM Tris-HCl(pH 7.4),
35% formamide, 0.5% SDS, lx Denhardt's
solution,
10% dextransulfate, 1mg ofherringspermDNAperml,
and 5 ng of DNA probe per ml. Low-stringency blots were washed three timesat roomtemperature andonce for30 minat55°C
in2x SSC-0.1%SDS.32P-labelled
probeswereprepared by
oligonucleotide-primed synthesis
(Pharmacia).
cDNA, genomic, and cosmidlibraries. cDNAwas
synthe-sized as described previously(14)
fromtwice-oligo(dT)-selected mRNAs from the brains of Swiss Webstermice or
mouseLcells. Double-stranded,
EcoRI-adapted
cDNAwas ligatedtoEcoRI-digestedXZAParms(Stratagene)
andpack-aged by using Gigapack Gold extracts
(Stratagene).
AC57/BL genomic DNA library in EMBL3 was
kindly
pro-vided by Glenn Radice.Unamplified
cDNA oramplified
genomic librariesof106 recombinants were
plated
on Esch-enchia coli LE392 cells. Duplicate filters werehybridized
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
GroupprogramBESTFIT,with default
settings.
5' end reconstruction.
Polymerase
chain reaction(PCR)
was used to reconstruct the 5' end of thelongest
brain-derived cDNA clone.Briefly, genomic
sequencing
of cosmid 2DNAprovided
thecorrect5' 26-nucleotide sequence. A 5' PCRprimer, bATG2,
containing
anEcoRIsite,
themissing
26-nucleotides, and a
25-bp
overlap
with braincDNAclone 2 and a3' PCRprimer, bTGA,
overlapping
thetermination codon and alsocontaining
an EcoRI site were used toamplify
thecoding
region
ofbrain cDNA clone 2:bATG2,
5'CTGGAATTCCCCATGGCCCGGGCCGCAGTCCTCCCGCCGTCCAGATTGTCACCGACGCTG3';
bTGA,
3'CGTCAAATGCACACTGGGATGCTTAAGGTC5'.
PCR wascarried outfor 30
cycles
at94°C
for 1min,55°C
for 2min,and
72°C
for 3 min. Theproduct
wascloned into M13vectors for sequenceconfirmation and into othervectorsfor expres-sionstudiesasdescribed below. The reconstructed cDNA in M13 isdesignated
pMEM.brmpl8.
Expression
of cDNA clones. The PCR-reconstructedmu-rine brain cDNA was cleaved with EcoRI, the ends were
made blunt with Klenow enzyme
(BMB),
and theresulting
fragment
wascloned intoanSmaI-digested
expression
vec-tor,
pSVL
(Pharmacia).
Thisplasmid
isdesignated pMEM.
brSVL7. Some recombinants with the human pvr cDNA wereconstructed asdescribed below. Todetermine the Pvractivity
of thesecDNAs,
L cells were transformed withCsCl-purified
plasmids
as described above. Transformants wereassayed
fortheirsensitivity
topoliovirus
infection and stable cell lines were isolatedasdescribed above.Oligonucleotide-directed
mutagenesis.
Oligonucleotide-di-rected
mutagenesis
wasperformed
on human pvr cDNAs subcloned in M13 and grown in E.coli
CJ236as describedpreviously
(1).
The enzymes usedwere T4polynucleotide
kinase(BMB)
and Klenow enzyme(BMB).
Nucleotidechanges
wereintroduced into humanreceptorcDNA insertscorresponding
toplasmids
pSVL-H20A
andpSVL-H20B
(14)
to create a KpnI site betweenimmunoglobulinlike
domains 1 and 2 in aposition
analogous
tothisKpnI
site in themurine brain cDNA sequence. These mutationsresultin acoding
change
fromserine tothreonineatamino acid 132. The M13 human pvr clones with the newKpnI
site aredesignated
pMEM.20AKpn
andpMEM.20BKpn.
Themuta-genized
coding
regions
were cloned back intoexpression
vector
pSVL
asdescribedpreviously
(14).
Theseconstructs aredesignated
pMEM.20AKpnSVL
andpMEM.20BK
pnSVL.
Constructionofrecombinants. Recombinants between the human pvr cDNAclone in
pMEM.20BKpn
and therecon-structed murine brain cDNA clone in
pMEM.brmpl8
wereconstructed.
Briefly, plasmid
pMEM.brmpl8
wasdigested
with
SmaI
andKpnI
andtreated with calf intestine alkalinephosphatase;
the373-bp
fragment
containing
murine domain 2andthe7,872-bp
fragment
containing
theM13mpl8
vector withmph
domain 3through
thecytoplasmic
tailwereiso-lated from
low-melting-point
agarosegels.
PlasmidpMEM.20BKpn
wasdigested
withSmaI
andKIpn,
and the507-bp
fragment
containing
pvr domain 1 and the373-bp
fragment
containing
pvrdomain 2 were isolated fromlow-melting-point
agarosegels.
The507-bp
pvrdomain 1SmaI-KpnI
fragment
wasligated
into the7,872-bp
mph
domain3-cytoplasmic
tail-M13mpl8
fragment,
creating
plasmid
pMEM.1.
In this process, the SmaI andBamHI
sitesfrom the M13 vector weredeleted,
but the XbaI site was left intact. Thedomain2sequencewasrestored topMEM.1
by
linearizing
thisplasmid
withKpnI
andligating
in the373-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 mapthe 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 theKpnI
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, 1992on November 9, 2019 by guest
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[image:3.612.104.235.77.438.2]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...1015AlaGluGlnValIleLeuValArgA... 339
exon 6.... tctccttcctttctcctcagACACCCCCCAGGCCTCCCGAGATTT(TC ACA MCTArT 1089
...spThrProGlnAlaSerArgAs 363
CTGGCTGGEsC4GGG Tr-r-T CTTGATCTGCTAnGGGGGAGGAGGAGGCGGAAGAGCCCTGGAGGAGGAGGAAATGATCGCGACAGAGGATCCTACGAT 1188
uAlaGlyGlyPh
eT.P-iiTt-i3krgGlyArgArgArgArgLysSerProGlyGlyGlyGlyAsnAspGlyAspArgGlySerTyrAsp
396CCAAAGACTCAGGTGTTTGGGAACGGGGGTCCTGTCTTCTGGAGGTCAGCATCCCCTGAGCCCATGAGGCCAGATGGCAGGGAGGAAGATGAGGAGGAG 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
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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
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[image:5.612.320.541.82.277.2]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 107p
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 conformationaltransi-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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