214, 559–570 (1995)

CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier Canyon- Publisher Connector Rim Residues, Including Antigenic Determinants, Modulate Serotype-Specific Binding of to Mutants of the Receptor

JAMES HARBER,*,1 GU¨ NTER BERNHARDT,*,2 HUI-HUA LU,* JEAN-YVES SGRO,† and ECKARD WIMMER*,3

*Department of Molecular Genetics and Microbiology, State University of New York, Stony Brook, New York 11794-5222; and †Institute for Molecular Virology, Robert M. Bock Laboratories, University of Wisconsin, Madison, Wisconsin 53706-1596

Received June 20, 1995; accepted October 4, 1995

Several mouse cell lines expressing hybrid human poliovirus receptors (hPVRs) bearing mutations in the first immunoglobu- lin-like domain were previously characterized for their defective binding and replication of poliovirus type 1 Mahoney (G. Bernhardt, J. Harber, A. Zibert, M. DeCrombrugghe, and E. Wimmer, Virology, 203, 344–356, 1994). Here we report that these mutant hPVRs were utilized to explore differences in the binding behavior of the three serotypes of poliovirus. Type 3 polioviruses (both Sabin and the neurovirulent Leon strain) clearly bound to the hPVR mutant Q130G/GD, but were incapable of initiating infection. Also, binding at 25Њ of poliovirus types 2 and 3 to cell lines expressing the hPVR mutants P84SYS/HPGA, L99GAE/AAAA, and D117F was greater than type 1 poliovirus. Further study of the serotype-specific interaction with mutant hPVRs was accomplished with antigenic hybrid viruses. Improved binding by antigenic hybrid viruses demonstrated that serotype-specific binding to mutant hPVRs is, in part, determined by the amino acid sequence of neutralization antigenic sites (NAgs) and the probable conformational rearrangement of amino acids adjacent to the NAg sites. Finally, site-directed mutants of poliovirus were utilized to determine the relative contributions, to hPVR interactions, of individual amino acids with solvent accessible side chains in the viral canyon. Of the 18 viable virus mutants produced, 3 (D1226A, I1089A, and VPEK1166HPGA) expressed impaired replication phenotypes on the mutant hPVR cell lines P84SYS/ HYSA and D117F. A location at the rim of the poliovirus canyon was implicated for the interaction of the amino terminal domain of the poliovirus receptor with conserved and serotype-specific viral surface amino acids. The possible involvement of elements of neutralization antigenic sites in receptor binding may explain, in part, why poliovirus exists in only three serotypes. ᭧ 1995 Academic Press, Inc.

INTRODUCTION 1988a,b) or two heterologous neutralization antigenic se- quences (termed trivalent viruses; see Murdin et al., All wildtype (wt) polioviruses occur, surprisingly, in 1992). Antigenic hybrid viruses, however, usually express only three serotypes (discussed in Wimmer et al., 1993). a small plaque phenotype, and they are impaired in repli- They are closely related both genetically (Wimmer et al., cation. When infecting human and primate cells, all 1993) and structurally (Filman et al., 1989; Yeates et al., known polioviruses use only a single receptor entity, the 1991) and have protein sequences that are at least 85% poliovirus receptor (PVR) (Wimmer et al., 1994). homologous (Toyoda et al., 1984). Much of the sequence Polioviruses are members of the genus Enterovirus of variation occurs in short segments of the three major the family Picornaviridae. Their nonenveloped capsids viral capsid proteins (VP1, VP2, and VP3) which define consist of 60 protomers that are composed of three sur- major (NAgIa, NAgIIa, and NAgIIIa) and minor (NAgIb, face proteins, VP1, VP2, and VP3, and the internal protein, NAgIIb, and NAgIIIb) neutralization antigenic sites on the VP4 (Hogle et al., 1985). The capsid surrounds a positive- surface of the virion (Hogle and Filman 1989; Murdin et stranded RNA molecule. The VP1, 2, and 3 capsid pro- al., 1992; Page et al., 1988). Through genetic manipula- teins fold as eight stranded antiparallel b-barrels tion, it is possible to exchange neutralization antigenic whereby the antigenic regions are hydrophilic -turns epitopes between viruses. The resulting antigenic hy- b within these structures (Hogle and Filman, 1989; Ross- brids carry either one (Burke et al., 1988; Evans et al., 1989; Jenkins et al., 1990; Martin et al., 1988; Minor et mann and Johnson, 1989). The surface structure of the al., 1990; Murdin and Wimmer, 1989; Murray et al., virion is convoluted (Fig. 1a). The fivefold axis of symme- try appears as a prominent star-shaped protrusion, while the threefold axis is located on a broad plateau. Sur- 1 Current address: Division of 156-29, California Institute of rounding each fivefold axis is a deep cleft, termed the Technology, Pasadena, CA 91125. canyon (Rossmann et al., 1985), that has been proposed 2 Current address: Max-Delbru¨ck-Centrum fu¨r Molekulare Medizin, to be the receptor binding site (Rossmann and Palmen- Robert-Rossle-Strasse 10, 13122 , . 3 To whom correspondence and reprint requests should be ad- berg 1988; Rossmann, 1989). dressed. The human poliovirus receptor (hPVR) is a member of

0042-6822/95 $12.00 559 Copyright ᭧ 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

/ m4655$7603 11-21-95 02:04:48 vira AP-Virology 560 HARBER ET AL.

FIG. 1. Structure of the poliovirion and a model of the V-domain of the hPVR. (a) A complete capsid structure of PV1 (M) illustrated as a water- accessible molecular surface. One of the 12 pentameric subunits of the capsid and its five constituent triangular pseudoprotomeric subunits are illustrated. The 51 and 31 labels indicate the locations of the fivefold and the threefold axes of this pentamer. The twofold axes occur at the intersection of the three adjacent pentamers. The central pseudoprotomer illustrates the subunit geometry of VP1, VP2, and VP3(ii). The biologically relevant protomer (to viral assembly) is pear-shaped and consists of VP1, VP2, and VP3(i). The internal VP4 protein is not visible from the surface. The canyon’s north wall (A), south wall (C), and bottom (B) are indicated. The major poliovirus antigenic sites are labeled Ia, Ib, II, and III on an adjacent pseudoprotomer. (b) A three-dimensional model of the immunoglobulin-like V-domain of hPVR and the locations of mutations which compromise receptor function (Bernhardt et al., 1994b). The mutations are abbreviated in the texts as follows: 84, P84SYS/HYSA; 99, L99GAE/AAAA; D117F; and 130, Q130G/GD. The orientation of the hPVR is such that the G؅ strand leads to the two C-domains and transmembrane region ,117 not illustrated). The antibody-like variable loops are at the upper part of the molecule (CDR1, B–C loop; CDR2, C؅ –CЉ loop; CDR3, F–G loop). Viral) attachment has been determined to occur at the right side of the molecule, involving an area that includes the C؅–CЉ loop (not illustrated, see Bernhardt et al., 1994b). the immunoglobulin superfamily with the domain struc- were introduced into the V-domain of hPVR (Aoki et al., ture V–C–C (Mendelsohn et al., 1989; Koike et al., 1990) 1994; Bernhardt et al., 1994b; Morrison et al., 1994). Muta- and a molecular weight of greater than 80 kDa (Bernhardt tions influencing on PV1(M) binding were mapped to the ,et al., 1994a). Analysis of the alternate splicing products putative C؅–CЉ–D region, the D–E loop, the E–F loop of the hPVR gene revealed that two membrane bound and the G؅ strand (see Fig. 1b). Interestingly, mutations (hPVRa and hPVRd) and two secreted (hPVRb and in the largest amino terminal loop between b-strands B hPVRg) isoforms are potentially expressed by the cell and C did not influence virus binding and replication. It (Koike et al., 1990). Expression of the two membrane was concluded that the PV binding region localizes to forms of the hPVR is known to vary in tissue culture cells one side of the V-domain model (Bernhardt et al., 1994b). (Bernhardt et al., 1994a). The function of these hPVRs These studies also suggested that once virus binding remains obscure (Freistadt, 1994; Freistadt et al., 1993). has occurred, the virus is internalized and, hence, viral Several studies have demonstrated that the amino-termi- replication ensues (reviewed by Wimmer et al., 1994). nal V-domain of the hPVR mediates binding of the virus Thus far, mutations mapping to the V-domain of hPVR to the cell (Koike et al., 1991; Selinka et al., 1991, 1992; were studied primarily with the virulent poliovirus type 1, Morrison and Racaniello, 1992). Amino acid substitutions Mahoney [PV1(M)] (Aoki et al., 1994; Bernhardt et al.,

/ m4655$7603 11-21-95 02:04:48 vira AP-Virology SEROTYPE-SPECIFIC POLIOVIRUS BINDING TO RECEPTOR MUTANTS 561

1994b; Morrison et al., 1994). We therefore investigated DE. Single-stranded DNA was generated from this con- binding and replication of all three serotypes of poliovirus struct (denoted PC-DE) for oligonucleotide-directed mu- with the mutant hPVR cell lines established by Bernhardt tagenesis. The oligonucleotides for mutagenesis (Table et al. (1994b) and were able to observe striking serotype- 1) were designed with the map/silent option of the GCG specific differences. Of particular interest is a receptor Wisconsin DNA program (Devereux et al., 1984), and they with a mutation located outside of the predicted binding were used to create both the capsid amino acid muta- site for PV1(M) (Q130G/GD). It was found to efficiently tions of interest and a diagnostic silent restriction en- bind type 3 poliovirus, and not the other serotypes. The zyme site. Site-directed mutagenesis reactions were car- binding of type 3 viruses, however, did not progress to ried out as previous described (Kunkel et al., 1987; Zoller replication. Also, poliovirus types 2 and 3 displayed in- and Smith 1984). creased affinities [relative to PV1(M)] for three other RNA was transcribed from the T7 promoter of the hPVRs mutated in the predicted virus binding region cDNA clone with T7 RNA polymerase, and transfected (P84SYS/HYSA, L99GAE/AAAA, and D117F). Thus, differ- into mouse L-cells (van der Werf et al., 1986). When CPE ence in the interaction of the three serotypes of poliovirus was observed, the cells were frozen and thawed three with hPVR can be distinguished by a simple panel of times to release the virus. Virus was then characterized mutant hPVR cell lines. We were able to address the by plaque assays. Absence of progeny virus at this stage question of serotype specificity further by studying differ- was retested by transfection of six 10-fold dilutions of ences in binding of antigenic hybrid viruses with the the RNA from the transcription cocktail onto HeLa cell mutant receptors. One result of this approach was that monolayers. The HeLa monolayers were overlaid with antigenic exchanges to PV1(M)-modulated binding to DME containing 2% calf serum and 1% noble agar and hPVRs. This suggests that antigenicity and receptor bind- observed for the formation of plaques. that again ing may be linked to a greater extent than previously failed to yield progeny virus were further analyzed for considered for poliovirus. A possible relationship of this integrity of the open reading frame by in vitro translation phenomenon to evolution of poliovirus serotypes will be (Molla et al., 1991). discussed. Viable viruses from L cell lysates were amplified once In order to investigate the contribution of viral capsid on a single 10-cm plate of HeLa cells; infected cells were residues to hPVR binding, a panel of 21 mutations in frozen and thawed three times, and the lysates were then PV1(M) surface-accessible amino acids was con- spun at 5000 g to remove aggregates. All viruses with structed. Viable viruses were tested for binding and repli- point mutations were passaged no more than three times cation on mutant hPVRs in a manner consistent with in HeLa cells. Viral RNA sequencing using a standard assays of the serotype variants and hybrid polioviruses. protocol (Boehringer Mannheim Biochemicals) con- Three of the mutants displayed a phenotype of defective firmed the presence of the mutations in third-passage binding to wildtype hPVR binding and defective replica- virus. tion on hPVR receptors P84SYS/HPGA and D117F. The location of these mutations and their influence on recep- Mutant receptor cell lines tor binding will be discussed. Mouse cell lines expressing mutant poliovirus recep- MATERIALS AND METHODS tors were established as previously described (Bernhardt et al., 1994b). To simplify the nomenclature of cell lines Polioviruses bearing the mutated hPVR, the following designations are The wt polioviruses, PV1(M), PV2(MEF), and PV3(Leon) made: 82/92, Q82A/L92P; 84, P84SYS/HYSA; 99, L99GAE/ were obtained from the ATCC collection. The antigenic AAAA; 117, D117F; and 130, Q130G/GD. Three cell lines (84, 99, and 117) have reduced binding and replicative hybrids were constructed previously and are designated capacities for PV1(M). The binding and replication of as follows: Site I/type 2 hybrid poliovirus (abbreviated PV1(M) in cell line 99 was consistently marginal relative I/2); site I/type 3 hybrid poliovirus (I/3) (Murray et al., to cell lines 84 and 117. The cell line 130 does not repli- 1988b); site II/type 2 hybrid (II/2) (Murdin and Wimmer, cate PV1(M). Expressions levels of the receptor were 1989); site II/type 3 hybrid (II/3) [Lu et al., manuscript in monitored during each third passage of the cell lines preparation]; sites I/3 / II/2 trivalent poliovirus and sites with a fluorescence-activated cell sorter (Becton Dickin- I/2 / II/3 trivalent poliovirus (Murdin et al., 1992). son and Coulter Instruments). The anti-hPVR monoclonal Construction of poliovirus capsid mutants antibodies p242, p216, and p437 used to monitor recep- tor expression were generous gifts of A. Nomoto. All A fragment (nt 496 to 3625) of the poliovirus cDNA mutant hPVR cell lines retained an uncompromised level containing the capsid-encoding region was excised from of D171 binding relative to the wt hPVR cell line. The a full-length cDNA by PflMI digestion, and it was inserted absence of the p216 antibody epitope and diminished into plasmid pGEM9Zf(0) (Promega) and denoted PC- p242 antibody binding is diagnostic for cell line 117,

/ m4655$7603 11-21-95 02:04:48 vira AP-Virology 562 HARBER ET AL.

TABLE 1

Oligonucleotides Used in Single Strand Mutagenesis Reactions to Generate Mutant Poliovirus DNA Clones

Mutation(s) Site //0 Oligo sequence 5؅ to 3؅

I1089A SalI / GCTGGGTTGTCGACGGTCATAGCGGTCACGCATGC T1091K SalI / GCTGGGTTGTCGACCTTCATAATGGTCACGCATGC K1109R SnaBI / GGACAGTATCTTTATACGTAATCCTCCACACTGC D1114E XmnI / CCGTAACTGAACAGTTTCTTTATAAGTGATC D1114T/AA PvuII / CCGTAACTGGACAGCTGCTTTATAAGTGATCTTCC P1162G Sma / GGAGCGCCCCCGGGTACGTAC P1162Q Bsu36I / GGAGCGCCCTGAGGTACGTAC T1143ET/AAA PvuII / GCATGGCCATTGTTAGCAGCTGCGAAATTTGCAG V1166PEK/HPGA SmaI / GTAGTCGTCCCAGGCTCCCGGGTGTGGAGCGCC DD1171/2.TS PmII / GATGAGGTTTGCCACGTGTAGGAGGTCCATTTTTCGGGCAC K1214R NaeI / CTGGTCCTTCAGTGGGACCCTGGAAAAACCGTCG D1226E AvaI / CCATAAAGGGACTCCCCGAGTGCTGCCGAC D1226A AvaI / GCACCATAAAGGGAGGCCCCGAGTGCTGCCGAC D1236H XmnI / CCGAAGTGATTCAGAGATGC D1247E BclI 0 GAACACAACCCGACCAAG K1256T BstEII 0 CACTCTGATTGTGGATGTGACCTTGG KD1287AA PvuII / GGTGTAAGCGTACCAGCTGCGTAATCCACTCC R2172A PvuII / CCGGGCAGAACCTGGCAGCTGGTGATGTCTGG ID 3180.MV SalI / GCCTTCGGTGAAACTGTCGACCATGGTTTGCCGATACG T3229V AccI / GCTCTATATGTACGGTATCTCGCAGTAGACGCACGCTGAAGTC while cell line 84 has partially lost the p242 epitope. Cell temperature. Without washing, the cells were overlaid line 99 retained all antibody epitopes. Cell lines 82/92 with 4 ml of warm (37Њ) DME, and placed in an incubator and 130, which did not bind PV1(M), can be distinguished for 3.25 hr, and subsequently washed twice with warm in that 82/92 has lost the p242 epitope, whereas 130 DME lacking methionine. Each plate was then overlaid displayed mildly diminished p437 binding. with 3.5 ml methionine-free DME containing 5 mg/ml acti- nomycin D (Calbiochem) and 200 mCi of 35S translabel Virus binding and replication assays (ICN). The radioactive medium was gently removed from the plates, and the cells were harvested at 6.5 hr postin- Cells expressing the appropriate mutant hPVR were 6 fection and were lysed by freezing–thawing. The lysate grown to 30–50% confluence in 60-mm dishes (1 1 10 was suspended in 10 ml of DME and centrifuged at 12Њ cells). Cell density was monitored to provide a minimal for 30 min at 10,000 rpm in a Sorvall centrifuge. The variance between samples. A typical assay included 107 5 35 supernatant was then diluted to 30 ml and spun at 28,000 PFU of unlabeled, or 10 cpm of S-labeled virus sample rpm in a Ty50.2 rotor for 4 hr. The radioactive supernatant per 60-mm dish of cells. Virus was incubated at 4 or 25Њ was immediately removed, and the pellet was resus- for 1 hr with the monolayers. For the determination of pended in 200 ml of DME at 4Њ overnight. The solution growth parameters, the plates were carefully washed was clarified in a 1.5-ml tube by centrifugation at 10,000 four times at the incubation temperature with medium rpm for 5 min. The supernatant was removed and stored. (DME at 4 or 25 ), and overlaid with 2 ml of DME (without Њ This protocol allowed for the convenient parallel prepara- serum). ‘‘Zero time points’’ corresponded to cells that tion of viruses. In order to obtain enough radiolabeled were frozen immediately following washing; for subse- viruses I1089A and VPEG1166HPGA (both highly defec- quent time points, the cells were placed in a 5% CO2 tive), a large-scale protocol was used as described incubator. All cells were prepared for titering by freeze– (Harber et al., 1991). The specific activity of virions was thawing three times. Viral titers were determined by determined and ranged from 15 to 45 PFU/cpm. Binding plaque assay on HeLa monolayers in 6-well dishes. For of the radiolabeled virus to receptor-negative MOP cells binding assays involving radiolabeled virions, the cell was typically less than 3% of that of receptor-expressing monolayers were solubilized with a solution of 1% SDS MOP cells. and 0.2 N NaOH, and radioactivity was determined by standard procedures. Assays with site-directed poliovirus mutants

Purification of radiolabeled poliovirus Binding assays (as described above, but with modifica- tions) were used to identify poliovirus mutants defective Virus at an m.o.i. of 10 was applied to a 10-cm HeLa in binding to wildtype hPVR. Radiolabeled viruses were cell monolayer (typically 5 1 107 PFU) for 1 hr at room added at 50,000 cpm per sample. After 1.5 hr of incuba-

/ m4655$7603 11-21-95 02:04:48 vira AP-Virology SEROTYPE-SPECIFIC POLIOVIRUS BINDING TO RECEPTOR MUTANTS 563 tion with cell monolayers at 4Њ, the sample dishes were coating is defective for type 3 virions on cell line 130 placed on ice and washed five times with ice-cold DME. (see below). The samples were then lysed and counted. Growth prop- The binding and replication phenotypes of the three erties were determined as described above. Heat-lability serotypes to other mutant hPVR cell lines was intermedi- was measured by incubating virus (1 1 105 PFU) for 1 ate to that of wt or 130. However, distinct differences hr at 37 or 45Њ. The reduction in titer was determined as in binding between the three serotypes were apparent. the ratio of titer at 45Њ/37Њ. Whereas at room temperature PV1(Sabin) bound to cell lines 84, 99, and 117 at a level corresponding to no more Molecular graphics than 15% of that bound to wt hPVR, Sabin 2 and Sabin 3 viruses bound at high levels to the mutant receptors, Figures 1a and 5a–5c were generated from atomic particularly to cell line 99 (Fig. 2a). In general, binding coordinates obtained from the Protein Data Bank at to receptor cell lines increased when the assays were Brookhaven (Bernstein, 1977) for PV1(M) [entry number performed at 25Њ instead of 4Њ (Bernhardt et al., 1994b; 2PLV (Hogle et al., 1985)], and for the domains 1 and 2 Fig. 2b). One notable exception is the binding of of the CD4 molecule [entry number 1CDH (Ryu et al., PV3(Leon) to cell line 130 (Fig. 2b). The reason for the 1994)]. All molecular surfaces were calculated with the apparent decrease is not known. program GRASP (Nicholls et al., 1991). Domains 1 and 2 The binding data shown here demonstrate that sero- of the CD4 molecule served as a model for the corre- type-specific interactions occur with the hPVR. However, sponding hPVR domains, and the model was docked into they are apparent only with hPVR mutants. A possible the canyon on a Silicon Graphics Crimson computer with explanation of this phenomenon is that subsets of inter- the program MIDAS-PLUS (Ferrin 1988). acting amino acids in the virus/receptor complex are not The ribbon diagram of the molecular model of the the same for the three serotypes of the virus. Therefore, hPVR (Fig. 1b) was generated on a Silicon Graphics IRIS we investigated the possibility that neutralization anti- 4D with the programs molscript (Kraulis, 1991) and In- genic determinants of virions of the different serotypes sight II (Biosym Technologies, San Diego, CA) as pre- influenced the interaction with the receptor. viously described (Bernhardt et al., 1994b).

Neutralization antigenic (NAg) sequence determinants RESULTS modulate binding of hybrid PV1(M) viruses to mutant Binding of poliovirus serotypes to mutant hPVR hPVR cell lines 84, 99, and 117 cell lines Sequences specifying NAgIa and NAgIIa of PV1(M) We have previously tested several cell lines express- have been exchanged with the corresponding PV2(Lan- ing mutants of hPVR for their ability to bind PV1(M) (Bern- sing) and PV3(Leon) sequences in a series of six anti- hardt et al., 1994b). We have now extended these studies genic hybrid viruses designated as follows: PV1(M) site to the other viral serotypes whose surface properties I/type 2 virus (I/2) (Martin et al., 1988; Murray et al., differ somewhat from PV1(M), particularly with respect 1988a); PV1(M) site I/type 3 virus (I/3) (Burke et al., 1988; to the neutralization antigenic sites. Whereas the binding Murray et al., 1988b); PV1(M) site II/type 2 virus (II/2) to wt hPVR is similar for all serotypes (Bibb et al., 1994; (Murdin et al., 1992); and PV1(M) site II/type 3 (II/3) (Lu and Figs. 2a and 2b), an unexpected variation of binding et al., manuscript in preparation). Two trivalent antigenic was observed when different hPVR mutant cell lines were hybrid viruses were utilized as well. These consisted of tested. This is particularly striking in the case of cell combinations of site Ia and IIa exchanges: the PV1(M) line 130 (hPVR cell line Q130G/GD; see Materials and I/2 / II/3 trivalent hybrid and the PV1(M) I/3 / II/2 trivalent Methods for nomenclature) which fails to bind type 1 and hybrid (Murdin et al., 1992). In binding assays, all anti- type 2 viruses, but binds efficiently type 3 poliovirus (Figs. genic hybrids retained the character of the PV1(M) virus 2a and 2b; column 130). Attachment, however, did not from which they are derived in that at 4Њ binding to any lead to observable replication at 37Њ. Figure 2c shows of the mutant hPVR cell lines showed the PV1(M) profile the results of an assay in which virus was bound at room (data not shown). However, when the binding assay was temperature, and incubated further at 37Њ for 24 hr to performed at 25Њ, half of the hybrid viruses were capable allow replication. The wt hPVR cell line displayed a typi- of significant binding to the mutant hPVR cell lines well cal 0 to 24 hr replication profile for all viruses tested, above the binding levels of PV1(M) (Fig. 3), resulting in including the neurovirulent PV2(MEF) and PV3(Leon) viral replication (data not shown). The increased binding strains (data not shown). In contrast, the type 3 Sabin of the mouse neurovirulent hybrid I/2 to the mutant recep- (Fig. 2c) and Leon (data not shown) virions attached to tor-expressing cell lines suggests that a component of cell line 130, but the virus titer did not increase. Because the receptor binding site of type 2 virus which mediates the titer did not change significantly during the 24-hr 25Њ binding with the mutant receptors 84, 99, and 117 incubation, we consider it likely that the process of un- resides in the structural region of NAgI of type 2 viruses.

/ m4655$7603 11-21-95 02:04:48 vira AP-Virology 564 HARBER ET AL.

FIG. 2. Binding of the three poliovirus serotypes to different hPVR constructs. (a) Serotype-specific binding patterns of Sabin strains 1, 2, 3, and PV1(M) at 25Њ. Mutant hPVR cell lines are 84, 99, 117, and 130. MOP, a hPVR-negative mouse cell line; wt, a MOP cell line expressing the hPVR/ ICAM 1315 chimera containing the wt hPVR (Bernhardt et al., 1994b). (b) Comparison of radiolabeled PV1(M) and PV3(Leon) binding to wt and mutant hPVRs at 4 and 25Њ. The binding of PV1(M) and PV3(Leon) to mutant hPVRs 84, 99, and 117 is inducible by increasing temperature, whereas PV(3) is decreased for binding to cell line 130 at 25Њ vs 4Њ. No temperature effect could be detected with mutant receptor 82/92. The binding profiles for PV3(Leon) and PV3(Sabin) have been found to be identical (data not shown). (c) Binding and replication assays of Sabin polioviruses types 1, 2, and 3 on hPVR cell lines. PV3(Sabin) was the only virus able to bind to cell line 130.

The site II/3 virus also displayed elevated levels of cell three serotypes (A. Palmenberg, personal communica- bound virus at 25Њ on cell lines 84 and 99, but none tion), 21 virus mutants with changes in amino acid sites of the chimera carrying type 3 antigenic determinants in the region of the viral canyon and rim were constructed resembled the type 3 pattern seen in Fig. 2. Instead, they (Table 2). Site-directed mutagenesis yielded 18 viable retained two characteristics of PV1(M): a level of binding derivatives of PV1(M). Three viral RNA constructs to cell line 117 and the absence of measurable binding (DD1171TS, KD1287AA, and R2172A) were incapable of over background to cell line 130. A trivalent hybrid virus producing infectious virions by transfection, despite con- containing both exchanges (site I/type 2 / site II/type 3) firmation of the integrity of the poliovirus polyprotein and had a 25Њ binding level which was intermediate of its proteolytic processing by in vitro translation using a HeLa constituent hybrid virus counterparts. The binding of the cell-free extract (Molla et al., 1991; data not shown). reciprocal hybrid viruses (site I/type 3; site II/type 2 and Three viruses (I1089A, VPEK1166HPGA, and D1226A) the trivalent containing both) showed no influence of tem- were greatly diminished in their ability to bind to a wild- perature (Fig. 3). In fact, the I/3 virus and the trivalent type hPVR cell line at 4Њ, and they failed to replicate on hybrid carrying this exchange both showed reduced mutant hPVR cell lines 84 and 117 (Table 2) (cell line 99 binding to the mutant cell lines 84, 99, and 117. was not included in the replication screen because of very low levels of PV1(M) replication). The one-step PV1(M) canyon mutants with reduced binding to wt growth patterns of these viruses on HeLa cells is shown hPVR are replication-defective in hPVRs mutant cell in Fig. 4. The D1226A mutation did not display a delay lines 84 and 117 in eclipse and was not heat sensitive, an observation indicating that its defect may be correlated with binding The binding and replicative properties of poliovirus to the hPVR. In contrast, the I1089A virus was markedly were further investigated with a panel of PV1(M) variants delayed in eclipse and both the I1089A and VPEK1166H- generated by site-directed mutagenesis. Using maps of PGA viruses were slower in producing newly synthesized the surface of the virus (A. Palmenberg and J.-Y. Sgro, infectious particles. In addition, P1162G, a small plaque unpublished results), and amino acid alignments of the virus, was also found to display a delay in eclipse (data

/ m4655$7603 11-21-95 02:04:48 vira AP-Virology SEROTYPE-SPECIFIC POLIOVIRUS BINDING TO RECEPTOR MUTANTS 565

cally independent of immune selection (Domingo et al., 1993). Our approach of testing the binding and replication of wt poliovirus, or of engineered viral variants, with cells expressing mutated hPVR molecules provides a new strategy for defining the relationship between the three poliovirus serotypes and their interaction with their re- ceptor. By conventional binding assays with intact cells, we have been unable to observe serotype-specific differ- ences in the interaction between polioviruses and the wt hPVR (Bibb et al., 1994; this study). This suggests that the binding energy between the virus and wt receptor appears to be roughly the same within the contact do- main amongst the three serotypes. In this study, however, striking differences in serotype-specific receptor interac- tions were revealed when receptor molecules with alter- ations in the V-domain were used in binding experiments. We interpret this to mean that both conserved and variant amino acids in the region of the viral binding site (canyon and elements of the antigenic sites) are responsible for serotype-specific binding to hPVR. This phenomenon may also serve to explain preferred inhibition of PV2 docking to HeLa cells by mcAbs to CD44. CD44 may form a complex with hPVR or may alter the conformation of hPVR through the cytoskeleton or signal transduction pathways (Shepley, 1988; Shepley and Racaniello, 1994). FIG. 3. Binding of antigenic hybrids to mutant hPVRs. The three The phenomenon of serotype-specific polymorphism hybrid viruses I/2, II/3, and I/2 / II/3 were found to bind to cell lines in V-domain binding is most pronounced for mutant re- 84, 99, and 117. The I/3 divalent and I/3 / II/2 trivalent viruses exhibited ceptor 130 which carries two amino acid replacements little or no binding to the mutant cell lines. The II/2 divalent virus (Q130G and G131D) in the region located between the ,.exhibited the poor binding of PV1(M). proposed G and G؅ b-strands (Fig. 1b; Bernhardt et al 1994b). This region is located adjacent to the C2 domain not shown) but exhibited no binding deficiencies on the of the receptor. At 25Њ, the complete inhibition of binding wt hPVR cell line or replication deficiencies (in 0/24 hr of poliovirus serotypes 1 and 2 to this receptor derivative assays) on wt, 84, and 117 hPVR cell lines. A further was surprising, given the efficient binding of type 3 - characterization of the mutant viruses revealed that two virus to the 130 cells. At present, we have no explanation of the viruses (I1089A and VPEK1166HPGA) were tem- for the phenomenon of serotype-specific polymorphism perature sensitive. Analyses of other viruses showed that in V-domain binding. The mutant receptor domain ex- the DT1114AA variant was also temperature sensitive pressed by 130 cells may be unable to convert the type (Table 2). 3 virions to A-particles, a process thought to be the first step in poliovirus uncoating (Wimmer et al., 1994). Cell line 130-bound type 3 viruses (Sabin or Leon), when incu- DISCUSSION bated for 24 hr at 37Њ, did not go through a phase of In view of the genetic plasticity of viral RNA genomes, typical eclipse but showed a slight loss of input titer. the restriction of poliovirus to only three serotypes is a Indeed, numerous attempts have failed to detect replica- perplexing phenomenon (Wimmer et al., 1993). If neutral- tion of PV3 in 130 cells even if the virus was administered -ization antigenic regions surrounding the canyon had no at high m.o.i. (unpublished observations). The G–G؅ re other function than to assist in hiding the receptor bind- gion of the V-domain may therefore be involved in mediat- ing site, we would expect to find far more than three ing conformational changes that unlock the virion struc- poliovirus serotypes because new antigenic determi- ture and lead to the release of VP4. The fact that the nants would evolve as the virus attempted to evade the binding of PV3 to 130 cells does not progress to produc- immune system. If, however, neutralization antigenic tive infection appears to be a unique case in which bind- sites contribute to the docking process of virions to the ing and uncoating are separated. This property makes hPVR, then the serotype restriction may be in part ex- the complex PV3/130 receptor a desirable candidate for plained. Indeed, evidence has been presented sug- structural analysis by crystallography since destabilizing gesting that some RNA viruses have diverged antigeni- changes which accompany the formation of a picornavi-

/ m4655$7603 11-21-95 02:04:48 vira AP-Virology 566 HARBER ET AL.

TABLE 2

Characterization of Mutant Polioviruses Constructed by Site-Directed Mutagenesis

Replication Three Temperature Defective impeded PV1 (M) surface serotypes Viable Plaque Receptor sensitivity of binding on wt on mutant mutation(s)a conservedb virusc sized footprinte the virusesf PVR cell lineg PVRsh Structural locationi

I1089A Yes / SNo / Yes Yes VP1 B-Strand T1091K No / MNo 0 No No VP1 B-Strand V1107M No / L Yes 0 No No VP1 C-Strand K1109R Yes / M Yes 0 No No VP1 C-Strand D1114E Yes / M Yes 0 No No VP1 C-D loop/helix DT1114AA Yes / S Yes / No No VP1 C-D loop/helix TET1143AAA No / LNo 0 No No VP1 D-E loop P1162G Yes / SNo 0 No No VP1 E-F loop VPEK1166HPGA No / M Yes / Yes Yes VP1 E-F loop DD1171TS No 0 — No N/A N/A N/A VP1 E-F loop K1214R Yes / L Yes 0 No No VP1 G-H loop D1226E Yes / L Yes 0 No No VP1 G-H loop D1226A Yes / M Yes 0 Yes Yes VP1 G-H loop D1236H Yes / M Yes 0 No No VP1 G-H loop D1247E Yes / MNo 0 No No VP1 H-I loop K1256T Yes / MNo 0 No No VP1 I-Strand KD1287AA Yes 0 — No N/A N/A N/A VP1 C-Terminus DNNQ2164AAAA No / MNo 0 No No VP2 E-F loop R2172A Yes 0 — Yes N/A N/A N/A VP2 E-F loop ID 3180.MV No / L Yes 0 No No VP3 G-H loop T3229V Yes / LNo 0 No No VP3 Carboxyl Terminus

a Nomenclature of the poliovirus type 1 Mahoney coordinates deposited to the Protein Data Bank (Hogle et al., 1985). b Derived from alignments (courtesy of Dr. Ann Palmenberg). c Production of virus was monitored by RNA transfection (see Materials and Methods). d Following 2 days incubation on HeLa cells, S, õ1 mm; M, 1–2 mm; L, ú2 mm. e The hypothetical footprint of the hPVR on the poliovirus capsid assumes that the interaction resembles the structure of HRV16 complexed with a two domain fragment of its receptor, ICAM-1 (Chapman and Rossmann, 1993). f A score of / indicates that reduction of titer of ú1 log over the PV1(M) value of infective virus occurred in the heat activation assay (see Materials and Methods). g Viruses displaying 10% or less of the PV1(M) value in the 4Њ binding assay (see Materials and Methods). h The replication of a designated virus on the mutant cell lines (84 and 117) in a single-step growth curve displaying less than a 5% increase in plaques of the wt value. i The structural location of mutations using the strand assignments of Hogle et al. (1985). rus–receptor complex are mitigated. Analyses of the ki- large temperature-dependent differences of binding have netic parameters of PV3/130 cell binding, A-particle for- not been observed with the 130 cell line (Fig. 3C), and mation in the presence of 130 cells, and attempts by PV1(Sabin) has been found to bind to the 130 line at 37Њ blind passage to find PV3 mutants able to replicate on at 87% of the level of binding to the wt receptor (data not 130 cells are in progress. shown). The crystal structure of poliovirus (Hogle et al., The hPVR mutant 82/92 (carrying the double mutation 1985) provides a fixed view of the capsid whereby spe- Q82A/L92P) that completely suppressed PV1 binding and cific segments of the amino acid chains of VP1 and all replication (Bernhardt et al., 1994b) did not show any of VP4 are clearly an internal feature. In solution, at 25Њ variation in serotype-specific binding. In contrast, we or physiological temperatures, however, such ‘‘internal’’ have observed pronounced binding differences of the segments may be exposed to the outside solvent since poliovirus serotypes with cell lines 84 (P84SYS/HYSA), they become accessible, in part, to antibody binding (Li 99 (L99GAE/AAAA), and 117 (D117F). Cell line 99 stands et al., 1994). This strongly suggests that certain regions out in that at 25Њ it bound both serotypes 2 and 3 to wt of the poliovirion are subject to spontaneous structural levels. (reversible) rearrangements when incubated in appro- Binding of PV1 to wt hPVR or its derivatives is higher priate buffers at physiological temperature. Whether at 25Њ than at 4Њ (Bernhardt et al., 1994b). This is also structural alterations or kinetic parameters, or both, ac- the case for PV3 (Fig. 2b) and PV2 (data not shown). count for the serotype-specific difference in receptor Surprisingly, whereas PV3(Leon) binds at wt levels to cell binding remains to be determined. line 99 at 25Њ, its binding is greatly impaired at 4Њ. Such It is highly likely that the viral canyon is a major contact

/ m4655$7603 11-21-95 02:04:48 vira AP-Virology SEROTYPE-SPECIFIC POLIOVIRUS BINDING TO RECEPTOR MUTANTS 567

FIG. 4. Single-step growth curves of three virus mutants and PV1(M). HeLa cell monolayers were infected at similar m.o.i. with PV1(M) and the mutants and incubated for the indicated times (given in hours). The titer of each virus was determined by plaque assay. area with hPVR (see below), as was predicted by Chap- types. The three virus variants that showed clear pheno- man and Rossmann (1993). However, the binding differ- types with respect to binding to wt receptor carry muta- ences discussed above could be the result of interac- tions mapping either to (1) the ‘‘north wall’’ of the canyon tions involving not only the canyon but also neutralization (I1089A; located near the fivefold axis in a border region antigenic sites lining the canyon. These sites, NAgI (VP1 between VP1 subunits), (2) a region adjacent to NAgI B–C loop) and NAgII (VP2 E–F loop), are highlighted (V1166PEK/HPGA), or (3) the south rim of the canyon (magenta shading) in Fig. 5. Indeed, results with anti- (D1226A). It is possible that all these regions are involved genic hybrid viruses reported here suggest that NAgI in the various stages of receptor binding. Interestingly, may influence binding to the receptor variants as the all three mutant viruses were impaired in replication on binding phenotype to cell lines 84 and 99 clearly covaries cell lines 84 and 117. The srr mutants selected by incuba- with the presence or absence of the type 2 NAgI loop. tion of virus with soluble receptor (Kaplan et al., 1990) A contribution of the type 3 NAgII loop to binding of have yielded amino acid changes in regions identical to PV1 was also observed, but none of the hybrid viruses those described here (Colston and Racaniello, 1994). acquired the phenotype of binding to the 130 cell line. Most recently, these authors have reported differences As noted above, the involvement of neutralization anti- of growth properties of poliovirus serotypes in cell lines genic determinants in receptor binding could be, in part, expressing hPVR mutants (Colston and Racaniello, 1995). responsible for the observation that poliovirus occurs in Although no binding studies were carried out, this obser- only three serotypes (Wimmer et al., 1993). Divergence vation may support our hypothesis of serotype-specific of the neutralizing determinants may shift receptor speci- interaction of polioviruses with hPVR variants. ficity of the virion which could lead to the emergence The D1226A mutation is located in the G–H loop of of a new ‘‘species’’ of enteroviruses with altered tissue VP1 (canyon rim) where the wt aspartic acid is part of tropism. For example, a phylogenetic tree derived from an amino acid triplet (G–D–S) that is conserved in all amino acid similarities in capsid proteins of picornavi- serotypes of poliovirus. This sequence aligns with the ruses suggests that the coxsackie viruses A 21 and A conserved R–G–D triplet of the VP1 G–H loop of foot- 24 (CAV21 and CAV24) are enteroviruses that are very and-mouth disease viruses (FMDV) that has been shown closely related to polioviruses (L. Kinnunen, T. Po¨yry, and to participate in FMDV binding to host cells (Fox et al., T. Hovi, personal communication). Whereas polioviruses 1989; Mason et al., 1994; Berinstein et al., 1995). Residue can cause poliomyelitis, CAV21 and CAV24 cause respi- D1226 of the poliovirus GDS triplet has been suggested ratory disease. Perhaps, CAV21 and CAV24 were at one to form a salt bridge to R2172 of the E–F loop of VP2 time polioviruses until divergence of the antigenic sites (Hogle et al., 1985). Whereas the mutation D1226A did drove them apart by allowing them to accept distinct not abrogate viral replication, an R2172A change was cellular receptor moieties. found to be lethal (Table 2). The significance of this ob- Making use of surface maps of poliovirus (A. Palmen- servation is not apparent. Our hypothesis that the poliovi- berg and J.-Y. Sgro, unpublished results), we have enter- rus G–D–S adhesion motif plays a role in receptor inter- tained the possibility of uncovering contact points be- action may be supported by the observation that several tween the V-domain and the virion by generating viral of the srr mutants selected by Colston and Racaniello mutants and analyzing their receptor binding pheno- (1994) were mutated at D1226. The implication of the

/ m4655$7603 11-21-95 02:04:48 vira AP-Virology 568 HARBER ET AL.

FIG. 5. Locations of hPVR binding mutations on the poliovirus capsid and a virus-receptor model. (a) Stereo view showing details of the fivefold depression, referred to as the canyon. The axes of icosahedral symmetry are labeled around a single representative of the 60 triangular pseudoprotomeric facets. The view is seen along the icosahedral twofold axes of symmetry looking down upon the canyon area. Residues exchanged in the antigenic hybrids (NAgI and NAgII) are represented in magenta color, while the amino acid substitutions resulting from site-directed mutagenesis are colored in cyan. The sphingosine molecule occupying the hydrophobic pocket of the VP1 protein is shown in yellow. (b) Stereo view of the same area as that shown in (a) except that the view is perpendicular to the axes of the icosahedral five- and twofold symmetry. (c) A poliovirus receptor modeled after the CD4 molecule is docked into the canyon. Orientation is the same as that in (b). Domain 1, an immunoglobulin V-like domain (gold) enters the canyon. The smaller domain 2, an immunoglobulin C-like domain, is colored green and sits above the surface of the virion. It is possible that domain 1 contacts residues of the north wall (nearest NAgI) and south wall (the NAgII face) simultaneously based on spatial considerations alone. Also, binding of the receptor to the canyon rim regions does not necessarily involve contacts of the receptor to the bottom of the canyon. involvement of the G–H loop in receptor interaction may residues in positions analogous to those previously re- lead to the elucidation of possible evolutionary mecha- ported for defective rhinoviruses (Colonno et al., 1988), nisms that alter picornavirus receptor specificity and tis- the canyon floor mutations yielded only viruses with ts sue tropism. and plaque size variants in our assays. Of interest was We had expected to find mutations of the canyon floor canyon floor mutation DT1114AA of the VP1 C strand that which greatly impaired replication on the mutant receptor lies adjacent to the hydrophobic pocket and confers a ts cell lines, but this was not the case. Although we mutated phenotype. It will be interesting to see whether this vari-

/ m4655$7603 11-21-95 02:04:48 vira AP-Virology SEROTYPE-SPECIFIC POLIOVIRUS BINDING TO RECEPTOR MUTANTS 569 ant displays a difference in thermal denaturation patterns Colston, E., and Racaniello, V. R. (1994). Soluble receptor-resistant po- in the presence or absence of drugs such as the WIN liovirus mutants identify surface and internal capsid residues that control interaction with the cell receptor. EMBO J. 13, 5855–5862. compounds (Rombaut et al., 1991). Colston, E., and Racaniello, V. R. (1995). Poliovirus variants selected A model of the binding of the V and C2 domains of on mutant receptor-expressing cells identify capsid residues that the hPVR to the poliovirion is shown in Fig. 5. Although expand receptor recognition. J. Virol. 69, 4823–4829. many aspects of this model are hypothetical, the illustra- Devereux, J., Haeberli, P., and Smithies, O. (1984). A comprehensive tion serves to depict the size relationship and a possible set of sequence analysis programs for the VAX. Nucleic Acids Res. 12, 387–395. orientation of the receptor toward the canyon. This model Domingo, E., Diez, J., Martinez, M. A., Hernandez, J., Holguin, A., Bor- is based on the assumption that the C؅–CЉ region of the rego, B., and Mateu, M. G. (1993). New observations on antigenic V-domain and the canyon (rim G–H loop) are likely to diversification of RNA viruses: Antigenic variation is not dependent interact in the docking event. The orientation of the virus- on immune selection. J. Gen. Virol. 74, 2039–2045. bound hPVR illustrated here accounts for our observation Evans, D. J., McKeating, J., Meredith, J. M., Burke, K. L., Katrak, K., John, A., Ferguson, M., Minor, P. D., Weiss, R. A., and Almond, J. W. (1989). that the antibody-like loops CDR2 and CDR3 may interact An engineered poliovirus chimera elicits broadly reactive HIV-1 neu- with the PV1(M) virion during docking, but the prominent tralizing antibodies. Nature 339, 385–388. CDR1 region does not. Ferrin, T. E., Huang, C. C., Jarvis, L. E., and Langridge, R. (1988). The MIDAS display system. J. Mol. Graphics 6, 13–21. ACKNOWLEDGMENTS Filman, D. J., Syed, R., Chow, M., Macadam, A. J., Minor, P. D., and Hogle, J. M. (1989). Structural factors that control conformational The authors are indebted to Akio Nomoto for providing hPVR-specific transitions and serotype specificity in type 3 poliovirus. EMBO J. 8, monoclonal antibodies, and to Tapani Hovi and Ann Palmenberg for 1567–1579. communicating unpublished data to us. We thank Michael Shepley for Fox, G., Parry, N. R., Barnett, P. V., McGinn, B., Rowlands, D. J., and editing the manuscript and Jim Simone (Stony Brook) and Pat Burfeind Brown, F. (1989). The cell attachment site on foot-and-mouth disease (Cold Spring Harbor) for assistance with analytical flow cytometry and virus includes the amino acid sequence RGD (arginine –glycine– cell sorting. The assistance of Rebecca Rowehl, Andrea Pevney, and aspartic acid). J. Gen. Virol. 70, 625–637. Susha Zachariah of the Stony Brook tissue culture facility is greatly Freistadt, M. S. (1994). Distribution of the poliovirus receptor in human appreciated. Susanne Meyer is acknowledged for her assistance in tissue. In ‘‘Cellular Receptors for Animal Virus’’ (E. Wimmer, Ed.), pp. screening virus-receptor combinations. J.H. was a member of the inter- 445–461. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, institutional genetics Ph.D. program of Stony Brook, Cold Spring Harbor NY. Laboratories and the Brookhaven National Laboratory. G.B. is a recipi- Freistadt, M., Fleit, H. B., and Wimmer, E. (1993). Poliovirus receptor ent of a postdoctoral fellowship from the Stipendienprogramm Infek- on human blood cells: A possible extraneural site of poliovirus repli- tionsforschung, Heidelberg Germany. This work was supported by Na- cation. Virology 195, 798–803. tional Cancer Institute Grant CA28146, and NIH Grants NIHAI15122 Fricks, C. E., and Hogle, J. M. (1990). Cell-induced conformational and NIH1RO1AI32100. change in poliovirus: Externalization of the amino terminus of VP1 is responsible for liposome binding. J. Virol. 64, 1934–1945. Harber, J. J., Bradley, J., Anderson, C. W., and Wimmer, E. (1991). Cataly- REFERENCES sis of poliovirus VP0 maturation cleavage is not mediated by serine Aoki, J., Koike, S., Ise, I., Sato-Yoshida, Y., and Nomoto, A. (1994). Amino 10 of VP2. J. Virol. 65, 326–334. acid residues on human poliovirus receptor involved in interaction Hogle, J. M., Chow, M., and Filman, D. J. (1985). The three dimensional with poliovirus. J. Biol. Chem. 269, 8431–8438. structure of poliovirus at 2.9 A resolution. Science 229, 1358 –1365. Bernhardt, G., Bibb, J. A., Bradley, J., and Wimmer, E. (1994a). Molecular Hogle, J. M., and Filman, D. J. (1989). The antigenic structure of poliovi- characterization of the cellular receptor for poliovirus. Virology 199, rus. Philos. Trans. R. Soc. London Ser. B 323, 467–478. 105–113. Jenkins, O., Cason, J., Burke, K. L., Lunney, D., Gillen, A., Patel, D., Bernhardt, G., Harber, J., Zibert, A., DeCrombrugghe, M., and Wimmer, McCance, D. J., and Almond, J. W. (1990). An antigen chimera of E. (1994b). The poliovirus receptor: Identification of domains and poliovirus induces antibodies against human papillomavirus type 16. amino acid residues critical for virus binding. Virology 203, 344–356. J. Virol. 64, 1201–1206. Berinstein, A., Roivainen, M., Hovi, T., Mason, P. W., and Baxt, B. (1995). Kaplan, G., Freistadt, M. S., and Racaniello, V. R. (1990). Neutralization Antibodies to the vitronectin receptor (integrin avb3) inhibit binding of poliovirus by cell receptors expressed in insect cells. J. Virol. 64, and infection of foot-and-mouth disease virus to cultured cells. J. 4697–4702. Virol. 69, 2664–2666. Koike, S., Horie, H., Ise, I., Okitsu, A., Yoshida, M., Iizuka, N., Takeuchi, Bernstein, F. C. (1977). The protein data bank: A computer archive. J. K., Takegami, T., and Nomoto, A. (1990). The poliovirus receptor Mol. Biol. 112, 535–542. protein is produced both as membrane-bound and secreted forms. Bibb, J. A., Witherell, G., Bernhardt, G., and Wimmer, E. (1994). Interac- EMBO J. 9, 3217–3224. tion of poliovirus with its cell surface binding site. Virology 201, 107– Koike, S., Ise, I., and Nomoto, A. (1991). Functional domains of the 115. poliovirus receptor. Proc. Natl. Acad. Sci. USA 88, 4104 –4108. Burke, K. L., Dunn, G., Ferguson, M., Minor, P. D., and Almond, J. W. Kraulis, Per J. (1991). Molscript: A program to produce both detailed (1988). Antigenic chimeras of poliovirus as potential new vaccines. and schematic plots of protein structures. J. Appl. Crystallogr. 24, Nature 332, 81–82. 946–950. Chapman, M. S., and Rossmann, M. G. (1993). Comparison of surface Kunkel, T., Roberts, J. D., and Zakour, R. A. (1987). Rapid and efficient properties of : Strategies for hiding the receptor site site-specific mutagenesis without phenotypic selection. Methods En- from immune surveillance. Virology 195, 745–756. zym. 154, 367–382. Colonno, R. J., Condra, J. H., Mizutani, S., Callahan, P. L., Davies, Li, Q., Gomez-Yafal, A., Lee, Y., Hogle, J., and Chow, M. (1994). Poliovirus M. E., and Murcko, M. A. (1988). Evidence for the direct involvement neutralization by antibodies to internal epitopes of VP4 and VP1 of the rhinovirus canyon in receptor binding. Proc. Natl. Acad. Sci. results from reversible exposure of these sequences at physiological USA 85, 5449–5453. temperatures. J. Virol. 68, 3965–3970.

/ m4655$7603 11-21-95 02:04:48 vira AP-Virology 570 HARBER ET AL.

Martin, A., Wychowski, C., Couderc, T., Crainic, R., Hogle, J., and Girard, 51711 and R 78206 as stabilizers of poliovirus virions and procapsids. M. (1988). Engineering a poliovirus type 2 antigenic site on a type 1 J. Gen. Virol. 72, 2153–2157. capsid results in a chimeric virus which is neurovirulent for mice. Rossmann, M. G. (1989). The canyon hypothesis. Hiding the host cell EMBO J. 7, 2839–2847. receptor attachment site on a viral surface from immune surveillance. Mason, P. W., Rieder, E., and Baxt, B. (1994). RGD sequence of foot- J. Biol. Chem. 264, 14587–14590. and-mouth disease virus is essential for infecting cells via the natural Rossmann, M. G., Arnold, E., Erickson, J. W., Frankenberger, E. A., receptor but can be bypassed by an antibody dependent enhance- Griffith, J. P., Hecht, H. J., Johnson, J. E., Kamer, G., Luo, M., Mosser, ment pathway. Proc. Natl. Acad. Sci. USA 91, 1932–1936. A. G., Rueckert, R. R., Sherry, B., and Vriend, G. (1985). Structure of Mendelsohn, C. L., Wimmer, E., and Racaniello, V. R. (1989). Cellular a human common cold virus and functional relationship to other receptor for poliovirus: Molecular cloning, nucleotide sequence, and picornaviruses. Nature 317, 145–153. expression of a new member of the immunoglobulin superfamily. Rossmann, M. G., and Johnson, J. E. (1989). Icosahedral RNA virus Cell 56, 855–865. structure. Annu. Rev. Biochem. 58, 533–573. Minor, P. D., Ferguson, M., Katrak, K., Wood, D., John, A., Howlett, J., Rossmann, M. G., and Palmenberg, A. C. (1988). Conservation of the putative receptor attachment site in picornaviruses. Virology 164, Dunn, G., Burke, K., and Almond, J. W. (1990). Antigenic chimeras of 373–382. type 1 and type 3 poliovirus involving antigenic site 1. J. Gen. Virol. Ryu, S.-E., Truneh, A., Sweet, R. W., and Hendrickson, W. A. (1994). 71, 2543–2551. Structures of an HIV and MHC binding fragment from human CD4 Molla, A., Paul, A. V., and Wimmer, E. (1991). Cell-free, de novo synthesis as refined in two crystal lattices. Structure 2, 59. of poliovirus. Science 254, 1647–1651. Selinka, H. C., Zibert, A., and Wimmer, E. (1991). Poliovirus can enter Morrison, M. E., and Racaniello, V. R. (1993). Molecular cloning and and infect mammalian cells by way of an intercellular adhesion mole- expression of a murine homolog of the human poliovirus receptor cule 1 pathway. Proc. Natl. Acad. Sci. USA 88, 3598–3602. gene. J. Virol. 66, 2807 –2813. Selinka, H. C., Zibert, A., and Wimmer, E. (1992). A chimeric poliovirus/ Morrison, M. E., He, Y.-J., Wien, M., Hogle, J., and Racaniello, V. R. CD4 receptor confers susceptibility to poliovirus on mouse cells. J. (1994). Homolog-scanning mutagenesis reveals poliovirus receptor Virol. 66, 2523–2526. residues important for virus binding and replication. J. Virol. 68, 2578– Shepley, M., and Racaniello, R. (1994). A monoclonal antibody that 2588. blocks poliovirus attachment recognizes the lymphocyte homing re- Murdin, A. D., Lu, H.-H., Murray, M. G., and Wimmer, E. (1992). Poliovirus ceptor CD44. J. Virol. 68, 1301–1308. antigenic hybrids simultaneously expressing antigenic determinants Shepley, M., Sherry, B., and Weiner, H. L. (1988). Monoclonal antibody from all three serotypes. J. Gen. Virol. 73, 607–611. identification of a 100-kDa membrane protein in HeLa cells and Murdin, A. D., and Wimmer, E. (1989). Construction of a poliovirus type human spinal cord involved in poliovirus attachment. Proc. Natl. 1/type 2 antigenic hybrid by manipulation of neutralization antigenic Acad. Sci. USA 85, 7743–7747. site II. J. Virol. 63, 5251–5257. Toyoda, H., Kohara, M., Kataoka, Y., Suganuma, T., Omata, T., Imura, Murray, M. G., Bradley, J., Yang, X. F., Wimmer, E., Moss, E. G., and N., and Nomoto, A. (1984). Complete nucleotide sequences of all Racaniello, V. R. (1988a). Poliovirus host range is determined by a three poliovirus serotype genomes: Implication for genetic relation- short amino acid sequence in neutralization antigenic site I. Science ship, gene function and antigenic determinants. J. Mol. Biol. 174, 241, 213–215. 561–585. Murray, M. G., Kuhn, R. J., Arita, M., Kawamura, N., Nomoto, A., and van der Werf, S., Bradley, J., Wimmer, E., Studier, F. W., and Dunn, J. J. (1986). Synthesis of infectious poliovirus RNA by purified T7 RNA Wimmer, E. (1988b). Poliovirus type 1/type 3 antigenic hybrid virus polymerase. Proc. Natl. Acad. Sci. USA 83, 2330–2334. constructed in vitro elicits type 1 and type 3 neutralizing antibodies Wimmer, E., Harber, J. J., Bibb, J., Gromeier, M., Lu, H-H., and Bernhardt, in rabbits and monkeys. Proc. Natl. Acad. Sci. USA 85, 3203–3207. G. (1994). Poliovirus receptors. In ‘‘Cellular Receptors for Animal Vi- Nicholls, A., Sharp, K. A., and Honig, B. (1991). Protein folding and ruses’’ (E. Wimmer, Ed.), pp. 101–127. Cold Spring Harbor Laboratory association: Insights from the interfacial and thermodynamic proper- Press, Cold Spring Harbor, NY. ties of hydrocarbons. Proteins: Struct. Funct. Genet. 11, 281–296. Wimmer, E., Hellen, C. U. T., and Cao, X. M. (1993). Genetics of poliovi- Olson, N. H., Kolatar, P. R., Oliveira, M. A., Cheng, R. H., Greve, J. M., rus. Annu. Rev. Gen. 27, 353–437. McClelland, A. M., Baker, T. S., and Rossmann, M. G. (1993). Structure Yeates, T. O., Jacobson, D. H., Martin, A., Wychowski, C., Girard, M., of a human rhinovirus complexed with its receptor molecule. Proc. Filman, D. J., and Hogle, J. M. (1991). Three-dimensional structure of Natl. Acad. Sci. USA 90, 507–511. a mouse-adapted type 2/type 1 poliovirus chimera. EMBO J. 10, Page, G. S., Mosser, A. G., Hogle, J. M., Filman, D. J., Rueckert, 2331–2341. R. R., and Chow, M. (1988). Three-dimensional structure of poliovirus Zoller, M. J., and Smith, M. (1984). Oligonucleotide directed mutagene- serotype 1 neutralizing determinants. J. Virol. 62, 1781–1794. sis: A simple method using two oligonucleotide primers and a single Rombaut, B., Andries, K., and Boeye, A. (1991). A comparison of WIN stranded DNA template. DNA 3, 479–488.

/ m4655$7603 11-21-95 02:04:48 vira AP-Virology