Proc. Nad. Acad. Sci. USA Vol. 86, pp.

4907-4911, July 1989

Biochemistry

cDNA cloning reveals that the major group rhinovirus receptor on HeLa cells is intercellular adhesion molecule 1
(cytokine induction/viral receptor/transfection)

JOANNE E. ToMASSINI, DONALD GRAHAM, CORRILLE M. DEWITT, DONALD W. LINEBERGER, JOHN A. RODKEY, AND RICHARD J. COLONNO*
Department of Virus and Cell Biology, Merck Sharp & Dohme Research Laboratories, West Point, PA 19486

Communicated by Edward M. Scolnick, April 10, 1989

A 90-kDa surface glycoprotein was previABSTRACT ously isolated and shown to be required for infection by the "major" group of human rhinovirus (IRV) serotypes. In the present work, the amino acid sequence of the receptor protein was obtained from CNBr and tryptic peptides. Using degenerate oligonucleotides predicted from the peptide sequences, we identified four cDNA clones that encode a 3-kilobase mRNA. The clones were ligated, subcloned in a simian virus 40 expression vector, and used to transfect receptor-negative Vero (monkey) cells. Results showed that transfected cells expressed receptor molecules capable of binding HRV and a monoclonal antibody which recognizes the mjor group HRV receptor. The cloned receptor cDNA encoded a protein with a sequence nearly identical to that of the intercellular adhesion molecule 1 (ICAM-1), indicating that the two surface proteins are one and the same. Both proteins have identical mass, carbohydrate composition, and tissue distribution. In addition, major group receptors on HeLa cells could be induced with various cytokines in a manner similar to the ICAM-1 ligand. A similar induction of the HRV "minor" group receptor was not observed. Human rhinoviruses (HRVs) are members of the Picornaviridae and are the major causative agent of the common cold in humans (1). Earlier studies have indicated that HRV serotypes can be divided into "major" (78 serotypes) and "minor" (10 serotypes) groups based on receptor specificity (2, 3). An anti-receptor monoclonal antibody (designated antibody 1A6) has been previously isolated which specifically blocks attachment of the major group of HRVs (4). By using this antibody, a 90-kDa glycoprotein was isolated from HeLa cell membranes and shown to be required for attachment of the major group of HRVs to susceptible cells in culture (5). Chimpanzee and human clinical trials testing the efficacy of antibody 1A6 have also demonstrated that this receptor is involved in HRV infection of the nasal cavity in vivo (6, 7). Further biochemical characterization of the purified 90-kDa protein demonstrated that it was an acidic glycoprotein having a pI of 4.2 (8). Carbohydrates accounted for 30% of the molecular mass of the protein and seven N-glycosylation sites were predicted on the basis of partial digestion with N-Glycanase (8). The intercellular adhesion molecule 1 (ICAM-1) is a cell surface ligand for the lymphocyte function-associated antigen 1 (LFA-1) adhesion receptor (9). ICAM-1 is a singlechain 76- to 114-kDa glycoprotein with a polypeptide core of 55 kDa that can be induced by several cytokines (10). The interaction of ICAM-1 and LFA-1 plays an important role in leukocyte adhesion and in the execution of immunological and inflammatory functions (10).
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. 1734 solely to indicate this fact.
4907

The present study describes the cloning and sequencing of the cDNA that encodes the cell surface receptor required for infection by the major group of HRVs.t Sequence and protein comparisons show that ICAM-1 is the surface glycoprotein utilized by HRVs during infection.

MATERIALS AND METHODS

Peptide Sequence. HRV major group receptor protein was isolated from HeLa R19 cells (2) and purified by immunoaffinity chromatography as previously described (5). Purified protein (40 ug) was reduced in 6 M guanidine hydrochloride/0.1 M Tris'HCl, pH 7.1/0.1% EDTA containing 100 mM dithiothreitol at 500C for 2.5 hr, then alkylated with 0.5 ,uM iodoacetic acid in the same buffer at pH 7.8 for 45 min at 230C. After extensive dialysis against H20, the protein was lyoph' ilized and treated with 0.4 mM CNBr in 70% (vol/vol) formic acid at 230C for 20 hr. The sample was chromatographed on a 10%o polyacrylamide gel and transferred onto Polybrenecoated glass fiber filters (11). Strips containing peptides were cut out and the N-terminal sequence was determined as described (12). Alternatively, 80 1Lg of receptor protein was purified by PAGE and transferred directly onto a nitrocellulose filter. Filter strips were blocked and then digested with 1.2 ug of tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin for 18 hr at 370C (13). The resulting peptides were separated by reverse-phase HPLC and sequenced as above. Preparation and Screening of cDNA Libraries. Polysomal mRNA and total RNA were isolated from dividing HeLa R19 cells by using the procedures detailed previously (14, 15). Both RNA preparations were subsequently purified by oligo(dT)-cellulose column chromatography. Double-stranded cDNA was synthesized (16) by priming poly(A)+ RNA with oligo(dT) (Collaborative Research) or degenerate oligonucleotides (21-24 bases in length) predicted from peptide sequences, using cDNA Synthesis Plus reagents (Amersham). Double-stranded cDNA was blunt-end ligated to EcoRI linkers (New England Biolabs) and inserted into A ZAP dephosphorylated EcoRI arms (Stratagene). The ligated DNA was packaged in Stratagene Gigapack Plus extracts according to directions supplied by the supplier and plated at 35,000 plaque-forming units per 150-mm plate. Multiple filter lifts (17) of phage DNA were hybridized to 32P-labeled oligonucleotides in 6x SSC/Sx Denhardt's solution/0.5% SDS/50 mM sodium phosphate buffer, pH 6.8, containing 0.2 mg of salmon sperm DNA per ml overnight at 10C below the calculated Tm of the oligonucleotides used (1 x SSC = 0.15 M NaCl/0.015 M sodium citrate; lx Denhardt's
Abbreviations: HRV, human rhinovirus; ICAM-1, intercellular adhesion molecule 1; LFA-1, lymphocyte function-associated antigen 1. *To whom reprint requests should be addressed. tThe sequence reported in this paper has been deposited in the GenBank data base (accession no. M24283).

4908

Biochemistry: Tomassin'i et al.

Proc. Natl. Acad. Sci. USA 86 (1989)

Table 1. Peptide sequences of HRV major group receptor Amino acid location Peptide* Sequence T5 TFLTV 78-82 C1 QPVGKXLTLRCQVE 98-115 GGAP T4 TELDLRPQGLELFE 161-183

solution = 0.02% polyvinylpyrrolidone/0.02% Ficoll/0.029 bovine serum albumin). Filters were then washed in 6: SSC/0.5% SDS for 5 min at 230C and then for 10 min at th hybridization temperature. Hybridization-positive phage wer*e picked and plaque purified by dilution plating and rescreeninj Construction of Full-Length cDNA Clone. A full-lengt ,h DNA fragment [3048 base pairs (bp)] was obtained by joininIg the inserts of clones 3-10 and 21 at a common Sac I site aIt nucleotide 2257 and clones 105 and 20C at a common Bgl I site located at nucleotide 657. The 3-10/21 assembled DNA wats then joined to the 105/20C dimer by means of a common SatC I site at nucleotide 1358 to form plasmid pHRVr1. Purifieid plasmid DNA was sequenced by using the Sequenase Kiit (United States Biochemical). The 3'-recessed ends of th e full-length cDNA were filled in with the Klenow fragment c DNA polymerase and blunt-end ligated into the simian viruIs 40 early promoter transfection vector pSVL (18), by insertion into the filled-in Xba I sites of the vector. The construct wais used to transform Escherichia coli DH5a cells (BRL) t ;o generate plasmid pSVL-HRVrl. Transfection of Cells with HRV Receptor cDNA. CsC]Ipurified plasmid DNA (pSVL-HRVrl) at 10 lOg/ml wats transfected into subconfluent monolayers of Vero (monkeye) cells by the calcium phosphate precipitation procedure de scribed elsewhere (19). After a 48-hr incubation in mediur n containing 10%6 serum at 37C, cells were subjected to binding studies using [35S]methionine-labeled HRV-15 and 1251 labeled 1A6 antibody as indicated (2). Induction of HRV Receptors. Confluent monolayers c )f HeLa R19 cells were treated with the indicated concentraItions of cytokines for 5 or 24 hr at 37C. After incubation1, medium was removed and replaced with a fresh McCoy's 5) medium containing [35S]methionine-labeled HRV-2 or HRVr 15. Following incubation with virus for 1 hr at 34C, thie percent of virus bound and unbound was determined as previously described (2). Specificity of binding was con firmed by parallel binding assays in the presence of unlabele d 1A6 antibody.

XTSAPYQLQ
T2 T1 T3

VTLNGVPAQPLGP VLYGPRXDERD DGTFPLPIGESVXV

313-325 365-375 407-421

*T1-5, tryptic peptides; C1, CNBr peptide.

RESULTS

The standard one-letter code is used; X, undetermined.

sf

Preparation and Sequencing of HRV Receptor Peptides. Previous studies have demonstrated that purified major group HRV receptor protein can be obtained by antibody 1A6 immunoaffinity chromatography (5). Sufficient quantities of

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this protein were obtained by using this procedure to allow chemical and enzymatic cleavage of the protein into peptides for protein sequencing. Initial attempts to sequence the intact protein indicated that the N terminus of the protein was blocked (data not shown). Therefore, fragmentation of the 90-kDa protein was attempted in two ways. First, receptor protein was cleaved with CNBr, the products were separated by PAGE, and then protein fragments were transferred directly onto Polybrene-coated glass fiber filters. Silver staining and Western blot analyses revealed a predominant 80-kDa protein band in addition to uncleaved protein, a minor 73-kDa fragment, and a barely detectable 10-kDa fragment (Fig. 1 Inset). The 80- and 10-kDa fragments were cut out from the corresponding positions on the filter and submitted for Nterminal amino acid sequence analysis. An 18-amino acid stretch (Cl) was obtained from the 80-kDa fragment (Table 1). The finding that no sequence could be obtained from the 10-kDa fragment suggested that this small fragment may represent the blocked N-terminal portion of the protein. A second approach to obtain the amino acid sequence involved partial enzymatic digestion with trypsin. Receptor protein was digested in situ on nitrocellulose after transfer from polyacrylamide gels. Released peptides were separated by reverse-phase HPLC (Fig. 1) and several were submitted for sequencing. Only the five peptides having retention times of 27.6, 31.8, 35.5, 39.8, and 41.6 min, 5-23 amino acids in length, had discernible amino acid sequences (Table 1). Antisera prepared against synthesized C1 and T4 peptides coupled to thyroglobulin were found to react with receptor protein in a Western blot assay (data not shown), confirming their derivation from authentic receptor protein. Cloning and Sequencing of the Receptor cDNA. HeLa cell mRNA was isolated and used to synthesize double-stranded cDNA according to the methods outlined in Materials and

1
0

3Kb

10

20 30 40 Retention time, min

50

60

105

,
5'

3-10

I,

21-1
-AA 3'

FIG. 1. Isolation of receptor peptides. HRV receptor protein was cleaved with CNBr or digested with trypsin. (Inset) Western blot of the CNBr-cleaved (lane 2) or uncleaved (lane 1) receptor after transfer to Polybrene-coated glass fiber filters. The positions of protein mass markers are as indicated and arrows indicate the position of the 80- and 10-kDa peptide bands. Separation of the tryptic peptides was accomplished by reverse-phase HPLC using a C8 column eluted with a gradient of 30-70%o (vol/vol) acetonitrile in 0.1% trifluoroacetic acid. The elution profile is displayed above. Fractions containing the five indicated peptides yielded the amino acid sequences shown in Table 1.

IH Dl I D2 I

D3 I D4 ID5IT

FIG. 2. Cloning of the HRV major group receptor. The map depicts the relative location of each of the four receptor clones identified. Kb, kilobases. The locations of the Sac I (S) and Bgl I (B) restriction sites used to join the clones are indicated by the broken lines. Map locations of the N-terminal signal sequence (H), protein domains (D), and predicted transmembrane region (T) are shown at the bottom.

Biochemistry: Tomassini et al.

Methods. Because the map locations of peptides were unknown at the time, two strategies were employed to clone the HRV receptor gene. The first method used standard oligo(dT) priming of RNA to synthesize cDNA, while the second used degenerate oligonucleotide primers predicted from peptide sequences T1-4 and C1 utilizing preferred codon frequencies (20). Several A ZAP libraries were prepared and multiple lifts were screened individually with peptide-derived oligonucleotides other than the one used in priming (i.e., Ti-primed libraries would be screened with T2-4 and Cl). Of the >5 million phage screened, only 3 hybridization positives were further characterized. Of these, only one clone (20C) was hybridization positive with more than one probe. In fact, clone 20C was positive with probes from peptide sequences

Proc. Natl. Acad. Sci. USA 86 (1989)

4909

T1, T2, and T4. DNA sequencing confirmed that the 788-bp insert of clone 20C encoded the three peptides. Clones 105 (679 bp) and 3-10 (951 bp) were identified by hybridization with peptides C1 and T3, respectively, and cross-hybridized to the 5' (105) and 3' (3-10) ends of clone 20C. Since clone 3-10 did not contain a poly(A) tract, the 3-10 insert was used to rescreen the oligo(dT)-primed library. Clone 21 (1032 bp) was then identified and shown to contain apoly(A) tract indicative of the 3' end of the putative receptor
gene mRNA. The four clones were ligated (Fig. 2) as described in Materials and Methods and the complete sequence was

determined by dideoxy sequencing (Fig. 3). The full-length clone (pHRVrl) had a single large open reading frame en-

5'GCTA TAAGGATCAC GCGCCCCAGT CGACGCTGAG CTCCTCTGCT ACTCAGAGTT GCAACCTCAG CCTCGCT (71)

ATG GCT CCC AGC AGC CCC CGG CCC GCG CTG CCC GCA CTC CTG GTC CTG CTC GGG GCT CTG TTC CCA GGA CCT GGC AAT GCC CAG ACA TCT (161) MET Ala Pro Ser Ser Pro Arg Pro Ala Leu Pro Ala Leu Leu Val Leu Leu Gly Ala Leu Phe Pro Gly Pro Gly Asn Ala Gln Thr Ser ( 3) GTG TCC CCC TCA AAA GTC ATC CTG CCC CGG GGA GGC TCC GTG CTG GTG ACA TGC AGC ACC TCC TGT GAC CAG CCC AAG TTG TTG GGC ATA (251) Val Ser Pro Ser Lys Val Ile Leu Pro Arg Gly Gly Ser Val Leu Val Thr Cys Ser Thr Ser Cys Asp Gln Pro Lys Leu Leu Gly Ile (33) GAG ACC CCG TTG CCT AAA AAG GAG TTG CTC CTG CCT GGG AAC AAC CGG AAG GTG TAT GAA CTG AGC AAT GTG CAA GAA GAT AGC CAA CCA (341) Glu Thr Pro Leu Pro Lys Lys Glu Leu Leu Leu Pro Gly Asn Asn Arg Lys Val Tyr Glu Leu Ser Asn Val Gln Glu Asp Ser Gln Pro (63)

ATG TGC TAT TCA AAC TGC CCT GAT GGG CAG TCA ACA GCT AAA ACC TTC CTC ACC GTG TAC TGG ACT CCA GAA CGG GTG GAA CTG GCA CCC (431) Met Cys Tyr Ser Asn Cys Pro Asp Gly Gln Ser Thr Ala Lys Thr Phe Leu Thr Val Tyr Trp Thr Pro Glu Arg Val Glu Leu Ala Pro (93)
CTC CCC TCT TGG CAG CCA GTG GGC AAG AAC CTT ACC CTA CGC TGC CAG GTG GAG GGT GGG GCA CCC CGG GCC AAC CTC ACC GTG GTG CTG (521) Leu Pro Ser Trp Gln Pro Val Gly Lys Asn Leu Thr Leu Arg Cys Gln Val Glu Gly Gly Ala Pro Arg Ala Asn Leu Thr Val Val Leu (123)

CTC CGT GGG GAG AAG GAG CTG AAA CGG GAG CCA GCT GTG GGG GAG CCC GCT GAG GTC ACG ACC ACG GTG CTG GTG AGG AGA GAT CAC CAT (611) Leu Arg Gly Glu Lys Glu Leu Lys Arg Glu Pro Ala Val Gly Glu Pro Ala Glu Val Thr Thr Thr Val Leu Val Arg Arg Asp His His (153)
GGA GCC AAT TTC TCG TGC CGC ACT GAA CTG GAC CTG CGG CCC CAA GGG CTG GAG CTG TTT GAG AAC ACC TCG GCC CCC TAC CAG CTC CAG (701) Gly Ala Asn Phe Ser Cys Arg Thr Glu Leu Asp Leu Arg Pro Gln Gly Leu Glu Leu Phe Glu Asn Thr Ser Ala Pro Tyr Gln Leu Gln (183)

ACC TTT GTC CTG CCA GCG ACT CCC CCA CAA CTT GTC AGC CCC CGG GTC CTA GAG GTG GAC ACG CAG GGG ACC GTG GTC TGT TCC CTG GAC (791) Thr Phe Val Leu Pro Ala Thr Pro Pro Gln Leu Val Ser Pro Arg Val Leu Glu Val Asp Thr Gln Gly Thr Val Val Cys Ser Leu Asp (213)

GGG CTG TTC CCA GTC TCG GAG GCC CAG GTC CAC CTG GCA CTG GGG GAC CAG AGG TTG AAC CCC ACA GTC ACC TAT GGC AAC GAC TCC TTC (881) Gly Leu Phe Pro Val Ser Glu Ala Gln Val His Leu Ala Leu Gly Asp Gln Arg Leu Asn Pro Thr Val Thr Tyr Gly Asn Asp Ser Phe (243)

TCG GCC AAG GCC TCA GTC AGT GTG ACC GCA GAG GAC GAG GGC ACC CAG CGG CTG ACG TGT GCA GTA ATA CTG GGG AAC CAG AGC CAG GAG (971) Ser Ala Lys Ala Ser Val Ser Val Thr Ala Glu Asp Glu Gly Thr Gln Arg Leu Thr Cys Ala Val Ile Leu Gly Asn Gln Ser Gln Glu (273)
ACA CTG CAG ACA GTG ACC ATC TAC AGC TTT CCG GCG CCC AAC GTG ATT CTG ACG AAG CCA GAG GTC TCA GAA GGG ACC GAG GTG ACA GTG (1061) Thr Leu Gln Thr Val Thr Ile Tyr Ser Phe Pro Ala Pro Asn Val Ile Leu Thr Lys Pro Glu Val Ser Glu Gly Thr Glu Val Thr Val (303)

AAG TGT GAG GCC CAC CCT AGA GCC AAG GTG ACG CTG AAT GGG GTT CCA GCC CAG CCA CTG GGC CCG AGG GCC CAG CTC CTG CTG AAG GCC (1151) Lys Cys Glu Ala His Pro Arg Ala Lys Val Thr Leu Asn Gly Val Pro Ala Gln Pro Leu Gly Pro Arg Ala Gln Leu Leu Leu Lys Ala (333)
ACC CCA GAG GAC AAC GGG CGC AGC TTC TCC TGC TCT GCA ACC CTG GAG GTG GCC GGC CAG CTT ATA CAC AAG AAC CAG ACC CGG GAG CTT (1241) Thr Pro Glu Asp Asn Gly Arg Ser Phe Ser Cys Ser Ala Thr Leu Glu Val Ala Gly Gln Leu Ile His Lys Asn Gln Thr Arg Glu Leu (363)
CGT GTC CTG TAT GGC COO CGA CTG GAC GAG AGG GAT TGT CCG GGA AAC TGG ACG TGG CCA GAA AAT TCC CAG CAG ACT CCA ATG TGC CAG (1331) Arg Val Leu Tyr Gly Pro Arg Leu Asp Glu Arg Asp Cys Pro Gly Asn Trp Thr Trp Pro Glu Asn Ser Gln Gln Thr Pro Met Cys Gln (393)

GCT TGG GGG AAC CCA TTG CCC GAG CTC AAG TGT CTA AAG GAT GGC ACT TTC CCA CTG CCC ATC GGG GAA TCA GTG ACT GTC ACT CGA GAT (1421) Ala Trp Gly Asn Pro Leu Pro Glu Leu Lys Cys Leu Lys Asp Gly Thr Phe Pro Leu Pro Ile Gly Glu Ser Val Thr Val Thr Arg Asp (423)

CTT GAG GGC ACC TAC CTC TGT CGG GCC AGG AGC ACT CAA GGG GAG GTC ACC CGC AAG GTG ACC GTG AAT GTG CTC TCC CCC CGG TAT GAG (1511) Leu Glu Gly Thr Tyr Leu Cys Arg Ala Arg Ser Thr Gln Gly Glu Val Thr Arg Lys Val Thr Val Asn Val Leu Ser Pro Arg Tyr Glu (453)
ATT GTC ATC ATC ACT GTG GTA GCA GCC GCA GTC ATA ATG GGC ACT GCA GGC CTC AGC ACG TAC CTC TAT AAC CGC CAG CGG AAG ATC AAG (1601) Ile Val Ile Ile Thr Val Val Ala Ala Ala Val Ile Met Gly Thr Ala Gly Leu Ser Thr Tyr Leu Tyr Asn Arg Gln Arg Lys Ile Lys (483)
AAA TAC AGA CTA CAA CAG GCC CAA AAA GGG ACC CCC ATG AAA CCG AAC ACA CAA GCC ACG CCT CCC TGA (1670) Lys Tyr Arg Leu Gln Gln Ala Gln Lys Gly Thr Pro Met Lys Pro Asn Thr Gln Ala Thr Pro Pro . (505)

ACCTATCCCG GGACAGGGCC TCTTCCTCGG CCTTCCCATA TTGGTGGCAG TGGTGCCACA CTGAACAGAG TGGAAGACAT ATGCCATGCA GCTACACCTA CCGGCCCTGG (1780)

GACGCCGGAG GACAGGGCAT TGTCCTCAGT CAGATACAAC AGCATTTGGG GCCATGGTAC CTGCACACCT AAAACACTAG GCCACGCATC TGATCTGTAG TCACATGACT (1890)
AAGCCAAGAG GAAGGAGCAA GACTCAAGAC ATGATTGATG GATGTTAAAG TCTAGCCTGA TGAGAGGGGA AGTGGTGGGG GAGACATAGC CCCACCATGA GGACATACAA (2000)

CTGGGAAATA CTGAAACTTG CTGCCTATTG GGTATGCTGA GGCCCCACAG ACTTACAGAA GAAGTGGCCC TCCATAGACA TGTGTAGCAT CAAAACACAA AGGCCCACAC (2110)
TTCCTGACGG ATGCCAGCTT GGGCACTGCT GTCTACTGAC CCCAACCCTT GATGATATGT ATTTATTCAT TTGTTATTTT ACCAGCTATT TATTGAGTGT CTTTTATGTA (2220)
GGCTAAATGA ACATAGGTCT CTGGCCTCAC GGAGCTCCCA GTCCATGTCA CATTCAAGGT CACCAGGTAC AGTTGTACAG GTTGTACACT GCAGGAGAGT GCCTGGCAAA (2330) AAGATCAAAT GGGGCTGGGA CTTCTCATTG GCCAACCTGC CTTTCCCCAG AAGGAGTGAT TTTTCTATCG GCACAAAAGC ACTATATGGA CTGGTAATGG TTCACAGGTT (2440)

CAGAGATTAC CCAGTGAGGC CTTATTCCTC CCTTCCCCCC AAAACTGACA CCTTTGTTAG CCACCTCCCC ACCCACATAC ATTTCTGCCA GTGTTCACAA TGACACTCAG (2550)

CGGTCATGTC TGGACATGAG TGCCCAGGGA ATATGCCCAA GCTATGCCTT GTCCTCTTGT CCTGTTTGCA TTTCACTGGG AGCTTGCACT ATTGCAGCTC CAGTTTCCTG (2660)
CAGTGATCAG GGTCCTGCAA GCAGTGGGGA AGGGGGCCAA GGTATTGGAG GACTCCCTCC CAGCTTTGGA AGCCTCATCC GCGTGTGTGT GTGTGTGTGT ATGTGTAGAC (2770)
AAGCTCTCGC TOTGTGAOOO AGGOTGGAGT GCAGTGGTGC AATOATGGTT GACTGOAGTO TTGAOOTTTT GGGOTCAAGT GATOOTOOGA OOTGAGOOTO OTGAGTAGOT (2880)

GGGACCATAG GCTCACAACA CCACACCTGG CAAATTTGAT TTTTTTTTTT TTTTTCAGAG ACGGGGTCTC GCAACATTGC CCAGACTTCC TTTGTGTTAG TTAATAAAGC (2990) TTTCTCAACT GCCAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAA 3'(3048)

FIG. 3. Complete amino acid and nucleotide sequences of the HRV major group receptor. The N-terminal signal and transmembrane sequences are underlined. Amino acids that correspond to sequences derived from isolated peptides are shown by bold underlines.

4910

Biochemistry: Tomassini et al.

60

Proc. Natl. Acad. Sci. USA 86

A
50
5 hr

(1989)

coding 532 amino acids if the first ATG was utilized. This would indicate a 71-nucleotide 5' noncoding region and a 1333-nucleotide 3' noncoding region, excluding the poly(A) tail. No additional long open reading frames could be found in the 3' noncoding region, suggesting that the stop codon at position 1668 is the site for termination of protein synthesis. This was verified by in vitro translation of RNA derived from pHRVrl, which resulted in a single polypeptide of 55 kDa (data not shown) that is in close agreement to the 54- to 60-kDa size found for deglycosylated receptor protein (8). The location of each of the peptide sequences is shown in Fig. 3. Placement of the C1 peptide within the sequence indicated that a methionine does not exist in close proximity to the cleavage site and that cleavage apparently occurred at a tryptophan residue as a result of oxidation during isolation or chemical cleavage procedures. This type of cleavage has been reported previously (21). Transfection with HRV Receptor Gene cDNA. To demonstrate that the cloned HRV receptor sequence did indeed encode the receptor protein of major group HRVs, a DNA insert representing nucleotides 48-2988 was subcloned in a simian virus 40 transfection vector and used to transfect a HRV receptor-minus cell line (Vero). After a 48-hr incubation, specific binding of radiolabeled antibody 1A6 and HRV-15 was observed in pSVL-HRVrl-transfected Vero cells (Table 2). No specific binding could be demonstrated with nontransfected cells. Homology to ICAM-1 Sequence. The sequence predicted for the 90-kDa HRV cell receptor from HeLa cells displayed striking homology to the sequence obtained for the ICAM-1 gene obtained from cytokine-induced HL-60 cells (22, 23). Direct comparison of the sequences revealed only six nucleotide differences. The HRV receptor gene had a G-to-A substitution at nucleotide 1476 that resulted in a Glu-to-Lys amino acid change at residue 442, a GG-to-CC substitution at nucleotides 2733-2734, and insertion of a C at nucleotide 2043 and a GT at nucleotide 2743. Review of the protein characteristics of the 90-kDa HRV receptor and the ICAM-1 ligand molecule shows that both are surface proteins of similar molecular weight, are ubiquitous among human-derived cells, and have an equivalent number of glycosylation sites (8, 22, 23). Induction of HRV Major Group Receptor. Previous studies (10) have demonstrated that ICAM-1 receptors can be induced after treatment with different cytokines. This induction was dependent on the cells used and cytokine selected. To confirm that the HRV major group receptor is indeed the ICAM-1 ligand molecule, HeLa R19 cells were treated with several cytokines and assayed for virus attachment. Results (Fig. 4A) clearly demonstrate that treatment with at least two of the cytokines, y interferon (IFN-y) and tumor necrosis factor a (TNF), resulted in an enhancement of HRV-15 binding from 17% to 35-38% after a 24-hr induction. An additive effect could be demonstrated when IFN--y and TNF were used together. Under these conditions, HRV-15 binding increased 3.3-fold to 57%. The induction obtained in HeLa
Table 2. Transfection of Vero cells with HRV receptor gene Binding,* % HRV-15 Ab 1A6 Trans- Ab Cell line - Ab + Ab + Ab fection 1.5 HeLa 25.8 1.0 42.9 Vero 1.7 1.6 0.8 1.1 + Vero 5.2 1.7 9.0 0.8 *Cell binding assays in six-well plates used [35S]methionine-labeled HRV-15 and 125I-labeled antibody 1A6 in the presence (+ Ab) or absence (- Ab) of saturating amounts of unlabeled 1A6 antibody as previously described (2).

EJF- 24h r

4030 20-

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0

r
B

40-

*30.C
20-

10
0

Control

IL- 1

IL-2

LPS

IFN-a IFN-y

TNF

TNF +

IFN-y

Cytokine treatment of cells

FIG. 4. Cytokine induction of HRV receptors. Confluent HeLa cell monolayers in duplicate 48-well cluster plates were untreated (control) or treated with interleukin 1 (IL-1) at 50 units/ml, interleukin 2 (IL-2) at 200 units/ml, Escherichia coli lipopolysaccharide B (LPS) at 25 ,ug/ml, a interferon (IFN-a) at 2000 units/ml, y interferon (IFN-y) at 2000 units/ml, tumor necrosis factor a (TNF) at 10 ng/ml, or TNF at 10 ng/ml + IFN-y at 2000 units/ml (TNF + IFN-y) in a total volume of 0.2 ml for 5 (solid bars) or 24 (open bars) hr. After incubation, cells were washed with phosphate-buffered saline (GIBCO) and used in binding studies with [35S]methioninelabeled HRV-15 (A) or HRV-2 (B) as previously described (2). The percentage of total labeled HRV specifically bound to cells is indicated.

cells was low compared to that observed in hematopoietic cells but clearly indicates that the HRV receptor is inducible by cytokines. It has been previously postulated that the receptor molecules used by the major and minor groups of HRVs, polioviruses, and coxsackie B viruses may share some structural homology (24). As a preliminary experiment to ascertain whether the minor group HRV receptor is another adhesion molecule that can also be induced by cytokines, HeLa cells were induced as described above and assayed for their ability to bind the minor group virus HRV-2. As shown in Fig. 4B, no comparable induction of receptors could be observed. Nearly identical results were obtained in experiments involving poliovirus and coxsackie B-1 virus (unpublished data).

DISCUSSION
Previous studies have clearly demonstrated that the vast majority of HRVs require the presence of a 90-kDa surface receptor molecule to gain entry into cells (4, 5). Antibody attachment to this surface protein establishes a "receptor blockade" which effectively protects susceptible cells against infection by HRVs. The molecular characterization reported here indicates that the well-characterized HRV receptor protein on HeLa cells is synonymous with the ICAM-1 ligand. In addition to the virtually identical sequences, both the HRV receptor and ICAM-1 proteins have

Biochemistry: Tomassini et al.

equivalent mass, tissue distribution, and carbohydrate moieties (5, 8, 10, 23). Amino acids -27 to -1 and 454 to 477 represent putative signal and transmembrane sequences of ICAM-1, respectively (Fig. 3; ref. 23). Several nucleotide differences were found between the two sequences in the 3' noncoding region and one change occurred in the coding region, replacing a Glu residue by a Lys residue. An additional 14 bases were identified at the 5' end of the HRV receptor cDNA that were not reported in the ICAM-1 sequence (22, 23). Our clone may not represent the entire 5' end of the mRNA, since Northern blot analysis indicated that the ICAM-1 mRNA is 3.2-3.3 kb in length (22, 23). ICAM-1 is a cell surface ligand for LFA-1, and the interaction between these molecules plays an important role in several immunological and inflammatory functions mediated by leukocyte adhesion (10). This cellular adhesion pathway is one of at least three mechanisms utilized (10). The ICAM-1 ligand is a member of the immunoglobulin gene superfamily and is predicted to have five homologous immunoglobulinlike domains defined by amino acids 1-88, 89-185, 186-284, 285-385, and 386-453 (Fig. 2) (23). In addition, ICAM-1 is closely related to two adhesion proteins of the adult nervous system, namely neural cell adhesion molecule (NCAM) and myelin-associated glycoprotein (MAG) (22, 23). Both the ICAM-1 and HRV receptor molecules have been shown to be ubiquitous on human-derived cells (4, 10) and clearly cannot account for the observed restriction of HRVs to the nasal cavity during a common cold. The significance of HRVs utilizing receptors having immunological and inflammatory functions is unclear. It has been reported that HRV infection involves a very limited number of cells in the nasal epithelium and that the clinical symptomatology that we associate with a cold may instead result from the generation of inflammatory mediators such as kinins (25). It is tempting to speculate that virus interaction with ICAM-1 somehow plays an important role in the production of kinins, other mediators, or both. Recent mutagenic studies using an infectious DNA clone of HRV-14 have demonstrated that the deepest regions of the virion canyon structure are involved in receptor interaction (24). The finding that this region of the canyon is highly conserved among a number of picornaviruses belonging to different receptor families led to the supposition that the receptor molecules that interact with these different viruses have a structural binding domain in common (24). It was further suggested that the specificity of the receptor protein utilized for attachment was determined by the rim of the canyon, since this region of the capsid was highly variable and thus would define the structural requirements of receptor interaction with the virus. Support for this hypothesis may be found in the recent cloning and sequencing of the poliovirus receptor gene (26). The poliovirus receptor molecule also appears to be a member of the immunoglobulin gene superfamily, and has three homologous immunoglobulin-like domains and some sequence homology to neural cell adhesion molecule. It is very likely that receptor genes for the HRV minor group and coxsackie B viruses also will be members of the immunoglobulin gene superfamily. It is interesting to note that, unlike the major group receptors, none of the other picornavirus receptors tested could be induced in HeLa cells by cytokine treatment and that the basal level ofthe minor group receptor on HeLa cells is almost equal to the cytokine-induced level of the major group (Fig. 4 and unpublished data). Formerly, viral receptor studies were restricted to biochemical and biological characterization. The availability of

Proc. Natl. Acad. Sci. USA 86 (1989)

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receptor gene clones should greatly facilitate our studies of virus-receptor interactions by providing a means to map binding domains via mutagenesis.
Note. Studies identifying the major HRV receptor as ICAM-1 have also been recently reported by two other independent laboratories (27, 28).
We thank Alan Rosenthal for discussions relating our cloned sequences with ICAM-1 and to Doug Testa for providing the cytokines used in these experiments. In addition, we acknowledge Bob LaFemina for performing DNA transfections; Mo Sardana, Mark Riemen, and Rich Dixon for helpful discussions concerning peptide sequences and receptor gene cloning; Pete Kniskern and Carl Bennett for oligonucleotide synthesis; and Abner Schlabach for peptide synthesis.

1. Gwaltney, J. M., Jr. (1982) in Viral Infection ofMan: Epidemiology and Control, ed. Evans, E. A. (Plenum, New York), 2nd Ed., pp. 491-517. 2. Abraham, G. & Colonno, R. J. (1984) J. Virol. 51, 340-345. 3. Colonno, R. J. (1987) BioEssays 5, 270-274. 4. Colonno, R. J., Callahan, P. L. & Long, W. J. (1986) J. Virol. 57, 7-12. 5. Tomassini, J. E. & Colonno, R. J. (1986) J. Virol. 58, 290-295. 6. Colonno, R. J., Tomassini, J. E. & Callahan, P. L. (1987) in Positive Strand RNA Viruses, eds. Brinton, M. A. & Rueckert, R. R. (Liss, New York), pp. 93-102. 7. Hayden, F. G., Gwaltney, J. M., Jr., & Colonno, R. J. (1988) Antiviral Res. 9, 233-247. 8. Tomassini, J. E., Maxson, T. R. & Colonno, R. J. (1989) J. Biol. Chem. 264, 1656-1662. 9. Makgoba, M. W., Sanders, M. E., Ginther Luce, G. E., Gugel, E. A., Dustin, M. L., Springer, T. A. & Shaw, S. (1988) Eur. J. Immunol. 18, 637-640. 10. Dustin, M. L., Staunton, D. E. & Springer, T. A. (1988) Immunol. Today 9, 213-215. 11. Vandekerckhove, J., Bauw, G., Puype, M., Van Damme, J. & Van Montagu, M. (1985) Eur. J. Biochem. 152, 9-19. 12. Hewick, R. M., Hunkapiller, M. W., Hood, L. E. & Dreyer, W. J. (1981) J. Biol. Chem. 256, 7790-7797. 13. Aebersold, R. H., Leavitt, J., Saavedra, R. A. & Hood, L. E. (1987) Proc. NatI. Acad. Sci. USA 84, 6970-6974. 14. Colonno, R. J. & Pang, R. H. L. (1982) J. Biol. Chem. 257, 9234-9237. 15. Han, J. H., Stratowa, C. & Rutter, W. J. (1987) Biochemistry 26, 1617-1625. 16. Gubler, U. & Hoffman, B. J. (1983) Gene 25, 263-269. 17. Benton, W. D. & Davis, R. W. (1977) Science 196, 180-182. 18. Strader, C. D., Sigal, I. S., Register, R. B., Candelore, M. R., Rands, E. & Dixon, R. A. F. (1987) Proc. Natl. Acad. Sci. USA 84, 4384-4388. 19. O'Hare, P. & Hayward, G. S. (1984) J. Virol. 52, 522-531. 20. Lathe, R. (1985) J. Mol. Biol. 183, 1-12. 21. Blumenthal, K. M., Moon, K. & Smith, E. L. (1975) J. Biol. Chem. 250, 3644-3654. 22. Simmons, D., Makgoba, M. W. & Seed, B. (1988) Nature (London) 331, 624-627. 23. Staunton, D. E., Marlin, S. D., Stratowa, C., Dustin, M. L. & Springer, T. A. (1988) Cell 52, 925-933. 24. Colonno, R. J., Condra, J. H., Mizutani, S., Callahan, P. L., Davies, M.-E. & Murcko, M. A. (1988) Proc. Natl. Acad. Sci. USA 85, 5449-5453. 25. Naclerio, R. M., Proud, D., Lichtenstein, L. M., Kagey-Sobotka, A., Hendley, J. O., Sorrentino, J. & Gwaltney, J. M., Jr. (1988) J. Infect. Dis. 157, 133-142. 26. Mendelsohn, C. L., Wimmer, E. & Racaniello, V. (1988) Cell 56, 855-865. 27. Greve, J. M., Davis, G., Meyer, A. M., Forte, C. P., Yost, S. C., Marlor, C. W., Kamarck, M. E. & McClelland, A. (1989) Cell 56, 839-847. 28. Staunton, D. E., Merluzzi, V. J., Rothlein, R., Barton, R., Marlin, S. D. & Springer, T. A. (1989) Cell 56, 849-853.