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Journal of Virology, July 2008, p. 7167-7179, Vol. 82, No. 14
0022-538X/08/$08.00+0     doi:10.1128/JVI.02664-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Characterization of the New World Monkey Homologues of Human Poliovirus Receptor CD155

Shaukat Khan,¹ Xiaozhong Peng,^1, Jiang Yin,^1, Ping Zhang,² and Eckard Wimmer^1*

Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, New York 11794,¹ Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907²

Received 14 December 2007/ Accepted 5 May 2008

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In contrast to Old World monkeys, most New World monkeys (NWMs) are not susceptible to poliovirus (PV), regardless of the route of infection. We have investigated the molecular basis of restricted PV pathogenesis of NWMs with two kidney cell lines of NWMs, TMX (tamarin) and NZP-60 (marmoset), and characterized their PV receptor homologues. TMX cells were susceptible to infection by PV1 (Mahoney) and PV3 (Leon) but not by PV2 (Lansing). Binding studies to TMX cells indicated that the formation of PV/receptor complexes increased when measured first at 4°C and then at 25°C, whereas PV2 did not significantly bind to TMX cells at either temperature. On the other hand, NZP-60 cells were not susceptible to infection by any of the PV serotypes. However, a low amount of PV1 bound to NZP-60 cells at 4°C, but there was no increase of binding at 25°C. In contrast, both NWM cell lines supported genome replication and virion formation when transfected with viral RNAs of either serotype, an observation indicating that infection was blocked in receptor-virus interaction. To overcome the receptor block, we substituted 3 amino acids in the marmoset receptor (nCD155), H80Q, N85S, and P87S, found in the human PV receptor, hCD155. Cells expressing the mutant receptor (L-nCD155mt) were now susceptible to infection with PV1, which correlated with an increase in PV1-bound receptor complexes from 4°C to 25°C. L-nCD155mt cells were, however, still resistant to PV2 and PV3. These data show that an increase in the formation of PV/receptor complexes, when measured at 4°C and at 25°C, correlates with and is an indicator of successful infection at 37°C, suggesting that the complex formed at 25°C may be an intermediate in PV uptake.

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Poliovirus (PV) is characterized by a highly restricted host and tissue tropism. Infecting by the oral-fecal route, only humans are natural hosts of PV. Disease symptoms are predominantly neurological but they are rare, depending upon the serotype (44). The major determinant of virus pathogenicity is the human cell surface receptor CD155 (PV receptor [PVR]), although other factors, such as interferon, also play an important role (22). CD155 has been thoroughly characterized (44), and homologues of this protein are known to exist in primates and nonprimates (23). Nonhuman primates, such as chimpanzees and African green monkeys (AGMs), which are members of Old World monkeys (OWMs), have been shown to be susceptible to PV infection. In the wild, however, PV infections of nonhuman primates are not well documented.

PV is a nonenveloped, plus-stranded RNA virus, a member of the genus Enterovirus in the Picornaviridae family. Its genome is approximately 7,500 nucleotides long, carrying a small viral protein (VPg) covalently linked to the 5' terminus and a poly(A) tail at the 3' terminus. The genome encodes a single large polyprotein, encoding structural proteins (P1) and nonstructural proteins (P2 and P3). Proteolytic processing of P1, P2, and P3 by the virus-encoded proteinases 2A^pro and 3C^pro/3CD^pro generates the functional proteins. Sixty copies of each of the four capsid polypeptides (VP1 to VP4), processed from P1, assemble to form a capsid shaped as an icosahedron with five-, three-, and twofold axes (18, 51). In contrast to VP1, VP2, and VP3, the smallest capsid polypeptide, VP4, is located inside the capsid. The capsid proteins VP1, VP2, and VP3 fold as eight-stranded antiparallel β-barrels whereby the antigenic regions are hydrophilic β-turns within these structures (19, 52). They give rise to three different sets of neutralization antigenic sites and, hence, the virus exists as three serotypes (types 1 to 3) (7, 9). A notable structural feature of the capsid is the "canyon," a depression characteristic for capsids of all entero- and rhinoviruses, which is a site of cellular receptor binding (51).

CD155, the only known cellular receptor mediating uptake of PV into cells, is a highly glycosylated single-span membrane protein (about 80 kDa) belonging to the immunoglobulin superfamily (30, 37, 50). CD155 can be broadly divided into five domains: three extracellular immunoglobulin (Ig)-like domains (one variable [V] and two constant [C] domains), a transmembrane domain, and a cytoplasmic tail (Fig. 1). The human CD155 gene is expressed in cells in four variants (, β, , and ) through alternate splicing of the CD155 transcript RNA. Two of the variants (β and ) occur in a soluble form, while the other two variants ( and ) are membrane bound and serve as the receptor for PV. Human CD155 (hCD155), and , differ only in the length and sequence of their cytoplasmic (C-terminal) tails. Genetic and biochemical evidence has identified the V-domain of CD155 as the virus binding domain (1, 3, 5, 14-16, 40, 54). Interestingly, these genetic modifications have indicated that the three PV serotypes interact with the V-domain in a slightly different manner (14), a phenomenon that we have observed also in our studies of the interaction between the hCD155 homologues of NWMs reported here.

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FIG. 1. The predicted structure of CD155 homologues. Schematic diagram of the CD155 structures of human (hCD155), AGM CD155 (AGM1), tamarin (tCD155), and marmoset (nCD155). Three extracellular immunoglobulin-like domains (circles) are formed by disulfide bonds. The transmembrane domain and the C terminus are shown with binding domains for Tctex-1 (empty box) and µ1B subunit (shaded box). The predicted N-glycosylation sites are shown on immunoglobulin domains (squares), as well as the number of amino acids that compose each domain. hCD155 is produced as four different splice variants (, β, , and ) that differ in the presence of the transmembrane domain and the length of the C-terminal domain. AGM CD155 occurs in two splice variants, AGM1 and AGM1, and AGM2 is encoded by a second gene.

CD155 is the founding member of a family of immunoglobulin molecules whose function has generally been characterized as adhesion proteins (45). Specifically, CD155 has been shown to mediate cell-to-matrix contacts by specifically binding to vitronectin (32). Moreover, it has been shown that CD155 recruits the adhesion molecule nectin-3 to cell-cell junctions through a trans-heterophilic interaction (43). CD155 also acts as a ligand for DNAX accessory molecule 1 (DNAM-1), and this interaction results in DNAM-1-dependent enhancement of NK-mediated lysis of target cells (8). The C-terminal domains of both membrane-bound forms of hCD155, and , interact with Tctex-1, a small protein of the dynein motor complex, and are involved in retrograde axonal transport of the virus-receptor complex (42, 46). Another element found only in the C-terminal domain of hCD155 is the tyrosine-containing binding motif of the µ1B subunit of the clathrin adaptor complex, which directs CD155 transport to the basolateral surface in polarized epithelial cells (47).

Shortly after the discovery of hCD155, putative homologues were described, most of which were related to, but not homologous to, hCD155 (11, 41, 49). The most thoroughly studied human and rodent proteins related to CD155 are called nectins (45), while the rodent orthologue of CD155 has now been named Tage4 (2, 49). Just like the nectins, however, Tage4 has no affinity to PV (49; S. Khan, S. Mueller, and E. Wimmer, unpublished data). The only CD155-related proteins with an affinity to PV are found in chimpanzees, OWMs, and NWMs. OWMs, exemplified by AGMs, have been thoroughly studied because of their susceptibility to PV by oral infection, which is highly inefficient compared to humans (31, 53). AGMs possess two genes coding for three membrane-bound forms of CD155 (31). These are two splice variants, AGM1 and AGM1, as well as AGM2, which is encoded by a second locus in the genome (31). Both AGM1 and AGM2 serve as functional PV receptors and have amino acid similarities to hCD155 of 90.2% and 86.4%, respectively (31).

Of the monkey species, the least-studied with respect to PV pathogenesis are NWMs, whose habitats are limited to the tropical forests of Central and South America. NWMs are composed of four families; Cebidae, Aotidae, Pitheciidae, and Atelidae. Unlike OWMs, early studies have indicated that NWMs cannot be infected with PV by the oral route, and any susceptibility by injection may depend not only on the animal species but also on the PV serotype (21). Similarly, early tissue culture studies showed that only capuchin monkey cells displayed susceptibility to PV1, but with no cytopathic effect (27-29). Recently, Koike and his colleagues analyzed the gene specifying the hCD155 homologue of brown capuchin monkeys (subfamily Cabinae, genus Cebus). They showed that the V-domain of capuchin monkey cDNA, when spliced onto the C-domain of hCD155, could promote infection of mouse L-cells that were transformed with the chimeric receptor (23). Infection, however, was restricted to only one serotype (PV1), and no analyses of the mechanism of restriction have been reported (23).

The aim of our studies was to examine whether the susceptibility of NWMs to infection by different PV serotypes is due to the virus-receptor interaction or to translation and replication. We have selected two kidney cell lines from tamarins (TMX) and marmosets (NZP-60) of the genera Saguinus and Callithrix, respectively, belonging to the subfamily Callitrichinae of NWMs. Marmoset cells were found to be resistant to PV infection with all PV serotypes, while tamarin cells showed sensitivity only to PV1 and PV3. These phenotypes have been observed in spite of high expression of the CD155 homologues by both NWM cells and by normal translation and replication of PV RNAs or of the PV luciferase (PV-Luc) replicon once transfected into the cells. However, we have observed significant differences in the binding of PV serotypes to NWM cell lines. Only PV1 is able to bind (but not infect) NZP-60 cells, whereas PV2 and PV3 display no virus/receptor-bound complexes on these cells. Moreover, we detected virus/receptor-bound complexes with PV1 and PV3 but not PV2 on TMX cells. Generally, if the formation of virus-receptor complexes was higher at 25°C than at 4°C, the binding would lead to cellular infection at 37°C, an observation suggesting that the complex formed at 25°C may be an intermediate in the early steps of infection. When mutations were introduced into the V-domain of the marmoset CD155, the mutant receptor was able to bind PV1 and also catalyze uncoating and infection. Our results indicate that the restriction of PV infection of NWM cell lines is related to their receptors' abilities to form initial complexes with the virus serotype in question.

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Cloning of NWM CD155 cDNAs. Based on the conserved cDNA sequences of human and AGM CD155, primers were designed corresponding to hCD155 cDNA sequences 122-136 (5'-TGGGCGACTCCGTGA-3') and 942-977 (5'-CACCAATGCCCTAGGAGCTCGCCAGGCAGAACTGAC-3'). Reverse transcription-PCR was performed on total RNA of the NWM cell lines, and the 800-bp fragment was amplified and sequenced. Sequence-specific primers from partial NWM CD155 cDNAs were designed, and the 5' and 3' ends were amplified using the 5'/3' rapid amplification of cDNA ends kit (Roche). The full-length CD155 cDNA was amplified using primers TMX 5' (5'-GGCGATATCAGAGCTAGGCCGCCGCGTG-3'), TMX 3' (5'-GTTCTAGAATCCTAGGAAGAGTTGGCCACAG-3'), NZP 5' (5'-GGCGATATCAGAGCCATGGCCGCCGCGTG-3'), and NZP 3' (5'-ACTCTAGACTGTCACCTTGTGCCCTTTGTCTG-3'). The TMX and NZP CD155 cDNAs were cloned into mammalian expression vector pcDNA 3.1(+) (Invitrogen) using restriction sites EcoRV and XbaI. The N-terminal signal peptide and transmembrane helix were identified in the deduced protein sequence using SignalP and TMHMM on the CBS prediction server (http://www.cbs.dtu.dk/services) (4, 26).

Cells. HeLa R19, mouse fibroblast Ltk^– cells, and stably transfected Ltk^– cells with PV receptor variants were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Primary kidney cells from a black-tailed marmoset (Callithrix argentata) were obtained from ATCC (CRL-1924; designation, NZP-60). A tamarin (Saguinus mystax) primary kidney cell line, designated TMX, was generously donated by S. U. Emerson and R. H. Purcell of the National Institutes of Health. TMX cells were maintained in DMEM containing 5% FBS, 5% bovine calf serum (BCS), and 1% penicillin-streptomycin. NZP-60 cells were maintained in 50% DMEM and 50% F-12 nutrient medium containing 10% FBS and 1% penicillin-streptomycin.

Generation of stable cell lines. Ltk^– mouse fibroblasts were maintained in DMEM containing 10% FBS. Cells were plated 24 h before transfection on a 35-mm plate. The cells were transfected with pcDNA 3.1(+) plasmid containing NWM CD155 variants with Lipofectamine 2000 transfection reagent (Invitrogen). After 24 h the cells were selected in the presence of Geneticin (Invitrogen). After selection, cell pools were labeled with mouse monoclonal antibody (MAb) p286 (donated by A. Nomoto) at a concentration of 20 µg/ml. Each sample was washed with phosphate-buffered saline solution (PBS) and then stained with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (BD Bioscience). Labeled cells were sorted by fluorescent-activated cell sorting with a Vantage apparatus (Beckton Dickinson). Sorted cell lines were maintained in DMEM containing 10% FBS and 1% penicillin-streptomycin.

Preparation of purified PV serotypes and viral RNA. Polioviruses used in this study were the most commonly studied representatives of the three serotypes (Mahoney, Lansing, and Leon). It could be argued that experiments with different strains of a given serotype would show different results. We cannot exclude this possibility, but we believe strain variations would not change the basic conclusions drawn from the experiments described here. This is because structurally, all three serotypes have a similar architecture (12, 18, 33), the serotypes largely being defined by numerous surface changes in exposed protrusions of the virion (38). Changes to switch serotypes would be dramatic, while changes to yield different strains would be subtle. We therefore assume here that our experiments of receptor/virus interactions with, say, serotype 1 PV(M) likely reflect experiments with different strains of the same serotype. PV1 (Mahoney), PV2 (Lansing), and PV3 (Leon) were grown in HeLa cells at 37°C, and the cells were harvested 8 h postinfection. The plates were subjected to three consecutive freeze-thaw cycles, and the virus titer was determined by plaque assay on HeLa cell monolayers, as previously described (48).

To obtain viral RNA genome, the PV serotypes were additionally purified by CsCl gradient centrifugation. Viral RNA was isolated from the purified virus stocks with a 1:1 mixture of phenol and chloroform. The purified viral RNA was precipitated by the addition of 2 volumes of ethanol and resuspended in RNase-free water.

One-step growth curve of PV serotypes. Cell monolayers (1 x 10⁶ cells) were incubated with a multiplicity of infection of virus of 10 for 30 min, at room temperature, on a rocker platform. After 30 min, cells were washed three times with PBS and incubated at 37°C in DMEM containing 2% BCS. The cells were harvested at 0, 2, 4, 6, 8, 12, 24, and 48 h postinfection. The plates were subjected to three consecutive freeze-thaw cycles, and the viral titers of the supernatants were determined by plaque assay on HeLa cell monolayers, as previously described (48).

Transfection of viral RNAs into cells. The purified viral RNA was transfected into monolayers with the TransMessenger transfection reagent (Qiagen). Transfected cells were incubated in DMEM supplemented with 2% BCS at 37°C either until complete cytopathic effect was observed or for at least 24 h posttransfection. After three freeze-thaw cycles, the lysate was clarified of cell debris by low-speed centrifugation. Virus titers were determined by plaque assay (48).

In vitro transcription and transfection of PV-Luc replicon. The PV-luciferase (PV-Luc) replicon (34) was digested with DraI before transcription with T7 RNA polymerase. RNA transcripts were transfected into monolayers of various cell types in 35-mm dishes with the TransMessenger transfection reagent (Qiagen).

Measurement of PV RNA translation and replication using luciferase replicons. After transfection with PV-Luc replicon, various cell lines were incubated at 37°C in DMEM with 2% BCS. The cells were grown with or without 2 mM guanidine hydrochloride (GnHCl). At 12 h posttransfection, the growth medium was removed from the dishes, and the cells were washed gently with 2 ml of PBS. The cells in the 35-mm dishes were lysed with 300 µl of passive lysis buffer (Promega). A 50-µl volume of luciferase assay reagent (Promega) was mixed with 20 µl of lysate, and the firefly luciferase activity was measured with an Optocomp I luminometer (MGM Instruments, Inc.).

Detection of CD155 expression levels on cell surfaces by flow cytometry. A total of 1 x 10⁶ cells in suspension were incubated for 30 min at room temperature with or without MAb p286 at a concentration of 20 µg/ml. The MAb recognizes an epitope of the V-domain of hCD155 (55). Each sample was washed with PBS and then stained with FITC-conjugated goat anti-mouse IgG (BD Bioscience). After washing, 1 x 10³ cells were analyzed with a FACSCalibur (Beckton Dickson).

Binding assay of PV serotypes. PV1, PV2, and PV3 capsid proteins were labeled with [³⁵S]methionine, and the viruses were purified by CsCl gradient centrifugation as previously described (6). A total of 1 x 10⁶ cells were incubated with 10⁸ PFU of labeled virus at 4°C or 25°C for 30 min. After incubation, the virus-cell complex was pelleted by microcentrifugation and the cell pellets were washed three times with PBS. The amount of radioactivity of the cell pellet was quantitated with a liquid scintillation counter (Packard Tri-Carb) in counts per minute. All samples were done in triplicate.

Alteration assays. Alteration assays were performed as described previously (13). Purified [³⁵S]methionine-labeled virions (approximate multiplicity of infection of 10) were added to cells in DMEM at a density of 5 x 10⁶ cells per ml and incubated for 30 min at 25°C. The cells were washed, fresh DMEM with 2% FBS was added, and cells were incubated at 37°C for 45 min. The cells were pelleted and dissolved in 0.5% Triton X-100 in PBSA (PBS containing 0.01% bovine serum albumin). The solution was layered on top of a 15% to 30% sucrose gradient in PBSA. The gradients were centrifuged for 75 min at 40,000 rpm in an SW41 rotor. Fractions were collected from the bottom and counted as described above. The gradient markers were made by heating labeled PV1 at 56°C for 10 min followed by incubation on ice for 20 min. Equal amounts of heated and unheated virus were layered on the gradient.

Site-directed mutagenesis. The following primers were used to make mutations in the marmoset CD155 and tamarin CD155 cDNA in the pcDNA 3.1(+) vector with a site-directed mutagenesis kit (Stratagene): H77Q sense (5'-CGTCTTCCACCAAACTCAGGGCCCC-3'); H77Q antisense (5'-GGGGCCCTGAGTTTGGTGGAAGACG-3'); NZP N82YP/SYS sense (5'-CAGGGCCCCAGCTACTCGGAGTCCGAAC-3'); NZP N82YP/SYS antisense (5'-TCGGACTCCGAGTAGCTGGGGCCCTGAG-3'), TMX N80YL/SYS sense (5'-CAGGGCCCCAGCTACTCGGAGTCCGAACG-3'), and TMX N80YL/SYS Antisense (5'-TTCGGACTCCGAGTAGCTGGGCCCTGAG-3').

Nucleotide sequence accession numbers. The nucleotide sequence data for the cDNAs for marmoset CD155 (nCD155) and tamarin CD155 (tCD155) have been submitted to GenBank under accession numbers EU277851 and EU277852, respectively.

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Identification of CD155 homologues in NWM cell lines. To ascertain whether TMX and NZP-60 cells have a homologue of CD155, we first stained the cells with a panel of monoclonal antibodies against hCD155, of which MAb p286 bound to receptors expressed on both NWM cells with the same intensity as on Ltk^– cells overexpressing hCD155 (data not shown). This result suggested that the NWM cells expressed putative homologues of hCD155 that share structural motifs. To identify this receptor, we first amplified an 800-bp fragment by PCR from a cDNA library of the NWM cell lines, using primers based on highly conserved sequences in human and AGM CD155. This 800-bp fragment was sequenced, and species-specific primers were used for the rapid amplification of the 5' and 3' cDNA ends to obtain the full-length cDNA sequences of tamarin CD155 (tCD155) and marmoset CD155 (nCD155).

On analysis of the cDNA sequences, we found that tCD155 has an open reading frame of 1,167 bp which encodes a 388-amino-acid polypeptide, while the nCD155 has a slightly longer open reading frame of 1,200 bp, encoding a 399-amino-acid polypeptide (Fig. 1). tCD155 and nCD155 share 74% and 75% amino acid identity and 87% and 88% nucleotide identity, respectively, with hCD155 (data not shown). In addition to sequence similarities with hCD155, both tCD155 and nCD155 express the Ig-like structure V-C2-C2, a hallmark of the immunoglobulin superfamily, similar to hCD155 and AGM CD155 (Fig. 1). In comparison to hCD155, which has eight N-glycosylation sites, tCD155 and nCD155 have six, while AGM1 has seven, AGM1 has six, and AGM2 has five (31) (Fig. 1). The cytoplasmic tails of tCD155 and nCD155 differ in length; however, both share a consensus sequence for the binding of Tctex-1 and the tyrosine-containing binding motif for the µ1B subunit with hCD155 (Fig. 1) (39, 47). The close relationship of the extracellular domains and the presence of consensus binding sequences in the cytoplasmic tails in all receptor molecules (Fig. 1) suggest that the NWM CD155 homologues have biological functions similar to that of hCD155. It should be noted that an examination of the NWM receptors did not reveal to us distinct isoforms or receptors expressed from gene duplications.

Susceptibility of NWM cell lines to infection with PV serotypes. Given that the NWM cell lines express a CD155 homologue, we performed one-step growth curve experiments to determine their susceptibility to infection with different PV serotypes (Fig. 2). In TMX cells, PV1 showed delayed early growth compared to that observed in HeLa cells; although by 48 h postinfection the viral titers were comparable (Fig. 2A). PV3, on the other hand, produced lower viral titers after 48 h postinfection compared to HeLa cells (Fig. 2C). PV2 did not show growth on TMX cells; however, there was a decrease in titer at 5 h followed by an increase at 12 h (Fig. 2B). Nevertheless, the titer of the virus at incubation and after 48 h was similar (Fig. 2B). Finally, NZP-60 cells were resistant to infection with all PV serotypes (Fig. 2).

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FIG. 2. One-step growth curves of PV strains on various cell lines. One-step growth curves in mouse (Ltk–), human (HeLa), marmoset (NZP-60), and tamarin (TMX) cells were carried out as described in Materials and Methods. (A) PV1; (B) PV2; (C) PV3. Each point represents the mean virus titer from three experiments.

Resistance of NWMs to PVs is not related to inhibition of translation or RNA replication. There are several possible explanations for the NZP-60 cells' resistance to infection with all PV serotypes and for the TMX cells' resistance to only PV2. First, resistance to infection could be due to a defect in the interaction between virus and receptor. Second, there could be an intracellular block at the stage of translation or replication. To test the latter possibility, both TMX and NZP-60 cells were transfected with PV1, PV2, and PV3 RNA and found to produce virus titers comparable to those obtained on transfected Ltk^– cells, regardless of which cell lines were used (Fig. 3A).

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FIG. 3. Translation and replication of PV RNA and of PV-Luc replicon in different cell lines. (A) Virus titers of PV1 (Mahoney), PV2 (Lansing), and PV3 (Leon) in marmoset (NZP-60), tamarin (TMX), and mouse (Ltk–) cells after transfection of RNA as described in Materials and Methods. (B) Firefly luciferase activity of PV-Luc replicons. Methods for the transfection of PV-Luc replicon RNAs into various cell lines, with or without 2 mM GnHCl, and the measurement of luciferase activity are described in the text.

To study translation in the absence of replication, we used a PV-Luc replicon that contained the firefly luciferase gene instead of the P1 domain of the PV polyprotein (34). After transfection with the replicon, the cells were incubated in the presence of GnHCl, a potent inhibitor of PV RNA replication, and the luciferase activity was measured 12 h posttransfection. The luciferase activity obtained in cell cultures with GnHCl represents translation of the input mRNAs. In mouse Ltk^– cells there was a 10-fold increase in luciferase signal when GnHCl was omitted from the culture, and similar increases were also observed with the TMX and NZP-60 cells (Fig. 3B). We conclude from this observation that intracellular replication of genomic RNA of all three serotypes analyzed is not blocked. These data suggest that the resistance to infection of NWM cell lines with different PV serotypes is related to one or more of the earliest steps in the viral life cycle: receptor binding, uptake, or uncoating.

CD155 expression levels on NWM cell lines. To determine whether susceptibility of the NWM cells to PV infection is related to the surface expression of the CD155 homologues, we employed flow cytometry and MAb p286 isolated against an epitope of the V-domain of hCD155 (55). By this procedure we found the signal to CD155 on TMX cells to be comparable to that on HeLa cells and twofold higher on NZP-60 cells than on HeLa cells (Fig. 4). Considering the possibility that MAb p286 may recognize the different receptors with different affinities, we cannot firmly conclude that more nCD155 is expressed on NZP-60 cells than hCD155 on HeLa cells. However, there can be no doubt that both NWM receptors are expressed on the respective monkey kidney cell lines and, consequently, it is unlikely that the inability of some PV strains to grow on the NWM cell lines is the result of insufficient levels of receptor molecules for interaction with PV.

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FIG. 4. Analysis of expression levels of CD155 on the cell surface by flow cytometry. The CD155 expression levels on the surface of human (HeLa), mouse (Ltk–), tamarin (TMX), and marmoset (NZP-60) cell lines were determined by flow cytometry using MAb p286 and a secondary Ab anti-mouse-FITC conjugate, as described in Materials and Methods. The data are expressed in arbitrary units.

Binding efficiency of PV strains to NWM cell lines. Infection by PV is initiated when the virus docks to CD155 receptor molecules at the cell surface. This interaction subsequently leads to uncoating and internalization of the viral genome (15). Having established that PV can translate and replicate in both TMX and NZP-60 cells, we were interested in determining the binding ability of the PV serotypes to the NWM receptors by a receptor-excess assay. It has been shown previously that PV interacts with CD155 in two distinct steps (36). The first step can be studied at 4°C and is probably electrostatic in nature. The second step occurs at physiological temperatures and leads to irreversible structural changes of the virion, resulting in the formation of an A-particle from which VP4 is absent (17). The first step in the entry pathway is the formation of an initial binding complex, which can be isolated at temperatures below physiological temperatures. Here we have isolated this initial binding complex into two separate steps, at 4°C and at 25°C, using ³⁵S-labeled virus. At temperatures ranging from 0°C to 25°C, the virus bound to cells can be recovered in an active form (20); thus, we can study virus-receptor binding complexes at 25°C without forming A-particles.

On analysis of the data from the binding assays, we found PV1-receptor complexes on TMX cells at 4°C, but the amount of virus-receptor complexes increased fourfold at 25°C (Fig. 5A). In contrast, PV3 showed lower amounts of bound complexes at 4°C compared to PV1, but the amount of bound complexes increased 10-fold at 25°C (Fig. 5C). PV2, on the other hand, showed the same amount of bound complexes as Ltk^– cells at 4°C or at 25°C (Fig. 5B). PV1-receptor complexes were observed on NZP-60 cells, but without an appreciable increase in binding from 4°C to 25°C (Fig. 5A). The levels of PV2- and PV3-bound complexes on NZP-60 cells were similar to background levels on Ltk^– cells at either 4°C or 25°C (Fig. 5B and C).

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FIG. 5. Binding assay of PV strains to various cell lines. The binding of 108 PFU [35S]methionine-labeled PV1 (A), PV2 (B), and PV3 (C) to 1 x 106 cells of human (HeLa), tamarin (TMX), marmoset (NZP-60), and mouse (Ltk–) cells was measured as described in Materials and Methods. The values are averages of three experiments.

The observed increase in binding of PV1 and PV3 to tCD155 at 25°C, in comparison to 4°C, suggested to us that the increase in association of the virus to the receptor led to successful infection. This would also explain why PV1 does not infect NZP-60 cells: binding of this strain to nCD155 is very low and did not significantly increase at 25°C compared to that at 4°C.

Conformationally altered virus particles. As noted above, the increase in the amount of virus-receptor complexes from 4°C to 25°C correlated with successful infection of TMX cells at 37°C (Fig. 2 and 5). Next, we wanted to determine whether the transition from native to subviral particles could be demonstrated directly upon incubation of the NWM cells with PV. As illustrated in Fig. 6, TMX cells produced 135S particles, although the conversion was inefficient under the conditions of the experiment. NZP-60 cells, on the other hand, did not yield a significant amount of subviral particles.

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FIG. 6. Sucrose density gradient fractionation of extracts of [35S]methionine-labeled PV1-infected cells. Purified native virions were attached to the cells at 25°C and incubated at 37°C for 45 min before lysis. The lysates were laid on a sucrose gradient, and the gradients were fractionated from the bottom. Unheated and heated labeled virions were used as controls.

Mutational analysis of the V-domain of nCD155 and tCD155. Unlike TMX cells, which showed an increase in receptor-bound complexes from 4°C to 25°C with PV1, NZP-60 cells did not bind a significant amount of virus, and there was no appreciable change in complex formation from 4°C to 25°C. nCD155, therefore, can bind PV1 weakly, but it cannot catalyze the structural changes in the virion necessary for infection. Since the V-domain of CD155 is the site of virus binding, we analyzed the V-domains of human, AGM, tCD155, and nCD155 by alignment to identify potential amino acids that are involved in binding and uncoating of PV1 (Fig. 7; numbering of amino acids in the V domains of tCD155 and nCD155 is according to the numbering of hCD155). Previously, mutations of hCD155 in the C'C", C"D, DE, and EF loops and in the N-terminal part of the C" β-strand were found to affect both PV1 binding and replication (1, 5, 10, 14, 35, 40).

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FIG. 7. Amino acid alignment of the V-domain of CD155 homologues (modified from that reported in reference 16). The figures shows the amino acid alignments of the V-domains of human (hCD155), African green monkey (AGM1 and AGM2), tamarin (tCD155), and marmoset (nCD155) proteins. Residues identified for binding all three PV strains are shown in bold, residues identified as binding two serotypes are shaded and bold, and residues identified as binding only one serotype are shown in gray. The arrows indicate the β-strands of the V-domain. Residues implicated in binding to PV by previous mutational analyses are marked with an X (5, 14) or a + (10, 30, 35, 40) in the lines above the residue numbers. The boxed residues donate amino acids mutated for this study. The numbering of amino acids corresponds to V-domains of hCD155.

On analysis of the CD155 homologue alignments, we found six amino acids, Q80, S85, S87, K90, N105, and V115, which have been implicated in viral binding as determined by cryoelectron microscopy (16). The corresponding residues have been substituted in the loops and the β-sheet of both tCD155 and nCD155 (Fig. 7). Based on previous mutagenesis studies, we can eliminate from consideration three of the six amino acid positions, K90, N105, and V115, because they did not influence binding and replication (5). It was previously shown that a K90/D mutation in hCD155 does not affect either PV binding or replication (5). Therefore, the corresponding K90/E substitution found in NWM CD155 is also unlikely to affect PV binding and replication. Similarly, an N105/D substitution, which eliminates an N-glycosylation site, is also present in AGM1, a functional receptor (Fig. 7). In addition, it has been shown that hCD155 lacking two N-glycosylation sites (N105/D and N120/S) has an enhanced binding affinity to the virus (5). We doubt, therefore, that a change of the N105 site will significantly influence binding to the NWM receptors. Mutation of E116DE to AAA in the EF loop abolished virus binding, likely due to the loss of consecutive charged residues in this region of hCD155 (5). Since a V115/A substitution does not result in charge disruption of the EF loop, we predict that this substitution would also not significantly influence binding of PV.

This leaves us with three mutations that may affect the binding of PV to the receptors nCD155 and tCD155. The amino acid mutations Q80, S85, and S87 in hCD155 all result in either a decrease or loss of PV1 binding (5, 14, 40). Therefore, we changed the corresponding amino acids in NWM receptors to H80/Q for both NWM receptors and N85YP/SYS and N85YL/SYS for nCD155 and tCD155, respectively (Fig. 7). The cDNAs of mutant and wild-type NWM CD155 receptors were then used to create stable mouse Ltk^– cell lines expressing the proteins. As indicated earlier, mouse Ltk^– cells lack a receptor for PV and only become sensitive to infection if a functional receptor is expressed or viral RNA is transfected into the cells. Interestingly, stably transformed mouse Ltk^– cell lines expressing an nCD155 mutant (L-nCD155mt) or a tCD155 mutant (L-tCD155mt) were competent to bring about a productive infection with significant titers of PV1 in both cell lines (Fig. 8A).

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FIG. 8. Infection and virus binding of stably transfected Ltk– cell lines. (A) Viral titers of PV1 growing in Ltk– cells stably expressing human CD155 (L-hCD155), marmoset CD155 (L-nCD155), mutant nCD155 (L-nCD155mt), tamarin CD155 (L-tCD155), and mutant tCD155 (L-tCD155mt) were determined at 0 h and 24 h. (B) The binding of [35S]methionine-labeled PV1 was measured as described in Materials and Methods.

Since L-nCD155mt and L-tCD155mt cells were permissive to PV1 infection, we performed binding assays on the cells to ascertain the amount of virus-receptor complexes. L-nCD155mt cells showed a very low amount of PV1 receptor complexes at 4°C; however, there was a threefold increase in binding from 4°C to 25°C (Fig. 8B). Interestingly, PV1-receptor complexes were found on NZP-60 cells, whereas there were no complexes on L-nCD155 cells (Fig. 5A and 8B). Both L-tCD155 and L-tCD155mt cells exhibited a two- to threefold increase in binding to PV1 after a temperature change from 4°C to 25°C; however, L-tCD155mt cells had a higher amount of PV1-receptor complexes than L-tCD155 cells at both temperatures (Fig. 8B). Although L-nCD155mt cells bound PV1 with concomitant infection, neither PV2 nor PV3 bound to or caused infection of these cells.

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We have identified CD155 homologues (tCD155 and nCD155) to the human poliovirus receptor (hCD155) in cells of tamarin and marmoset monkeys (TMX and NZP-60), two species of NWMs. The proposed structures of tCD155 and nCD155, which display amino acid identities of 74% and 75%, respectively, with hCD155, place these proteins into the new Ig superfamily (Fig. 1) of which hCD155 is the founding member. In general, CD155 homologues show a high degree of sequence divergence across different species. For example, hCD155 is 90% similar to AGM1, a member of the OWMs, but only 38% to Tage4, the rodent orthologue of CD155 (49). Yet, Tage4 also expresses Tctex-1 and the tyrosine-containing binding motif of the µ1B subunit in its C-terminal domain, and its expression in the gastrointestinal tract in rodents (49) is very similar to that of CD155 in the human gastrointestinal tract (24). It is tempting to speculate that members of the new CD155 Ig family, whether of human, monkey, or rodent origin, perform important and related functions for their respective organisms. This function, however, does not include affinity to PV: the interaction between PV and CD155 is not to the advantage of the host and, thus, PV binding affinity is not conserved among all CD155 homologues or orthologues (44).

Unlike OWMs, NWMs are not susceptible to oral infection with PV. NWMs, however, can be infected by intracerebral injection but, surprisingly, this is dependent not only upon the NWM species but also upon the PV serotype. Among the serotypes, PV1 is clearly favored in its ability to infect the NWM cells, tamarin and brown capuchin. PV3 can also infect tamarin cells, whereas PV2 is excluded from interaction with those NWM CD155 molecules that have been tested. These include the black-tailed marmoset, tamarin, and brown capuchin, all members of subfamilies of the family Cebidae (our studies) (23, 27-29). In their study on rapid sequence changes of the CD155 gene during evolution, Ida-Hosonuma et al. showed that the V-domain of the brown capuchin CD155, if exchanged for the V-domain of hCD155, could serve as a receptor for PV1 only, an observation that was not further investigated (23).

Unlike TMX cells, NZP-60 cells are resistant to all three serotypes. Our experiments have clearly shown that the inability of the three PV serotypes investigated here to infect marmoset NZP-60 cells is related to the earliest step in infection: lack of ability to form virus-receptor complexes. If the cell membrane barrier is bypassed by transfection of virion RNA, replication and virus maturation occur just as efficiently as in mouse Ltk^– cells, a highly permissive substrate for intracellular PV replication. Fittingly, on transfection of the PV-Luc replicon, the NZP-60 cells showed significant levels of translation and replication of replicon RNA.

The resistance of NZP-60 cells to PV infection cannot be explained by a lack of nCD155 expression on these cells. Flow cytometry of the NWM cells showed that expression of nCD155 on NZP-60 cells under the conditions of the experiments was nearly twofold higher than that of hCD155 on HeLa cells or tCD155 on TMX cells. Together, these data show that the block to infection of NZP-60 cells occurs at the stage of receptor binding, a conclusion supported by binding studies of PV serotypes to these cells. We cannot determine whether the nCD155 protein can alter virions, since the amount of virus-receptor-bound complexes was very low on the NZP-60 cells.

Previous experiments have shown that the receptor-virus interaction follows biphasic kinetics (reference 36 and references therein). The initial binding step involving an electrostatic interaction is fully reversible and temperature independent. The second step requires near-physiological temperatures for an increase in "breathing" of the virion structure, thereby exposing higher-affinity binding sites (36). This additional binding leads to irreversible structural changes of the (bound) virion and is hypothesized to, in turn, result in additional contacts with the north rim of the "canyon," leading to the uncoating of the virus (16, 17, 36). Our experiments reported here are in accordance with this mechanism. They show an increase in the binding of the virions when the temperature of binding was increased from 4°C to 25°C. Interestingly, increased binding, in turn, covaries with a productive infection, regardless of the level of virus-receptor complexes. Specifically, PV1 and PV3 both showed an increase in the amount of bound virus complexes from 4°C to 25°C and a concomitant replication in TMX cells. Moreover, the amount of receptor-bound complexes to PV1 was higher than that to PV3, which correlated with higher titers of PV1 in TMX cells. In contrast, PV1 showed no growth phenotype in NZP-60 cells, and there was no appreciable increase in virus-receptor complexes from 4°C to 25°C.

Since successful infection requires structural changes of the virion associated with an increase in bound complexes from 4°C to 25°C, we assessed the abilities of TMX and NZP-60 cells to alter PV1 virion particles after binding. In support of our infection and binding data, TMX cells converted the 160S native particles to 135S particles, whereas the NZP-60 cells were deficient in binding and thus unable to alter virion particles. Therefore, we made an attempt to change the amino acid composition of the V-domains of tCD155 and nCD155 by mutagenesis with the aim of affecting PV1 binding. Previous studies have shown that mutation of three amino acids in hCD155 (Q80, S85, and S87) causes a loss in viral binding and replication of PV1 (5, 40). By alignment of hCD155, tCD155, and nCD155, these amino acids were predicted to be involved in virion binding if engineered into the NWM CD155. Accordingly, the mutant receptors were expressed in mouse fibroblast cells (L-tCD155mt and L-nCD155mt), and their abilities for virus binding and uncoating were determined.

Our data showed an increase in the formation of virus-receptor complexes as well as in viral titers for both L-tCD155mt and L-nCD155mt cells with PV1. The L-nCD155mt cells were able to support PV1 infection, although the virus-receptor complexes in L-nCD155mt cells were 55-fold lower than in HeLa cells at 4°C. However, the PV1 and nCD155mt complexes increased threefold from 4°C to 25°C, seemingly enough to lead to a productive infection of L-nCD155mt cells. Notably, PV1 did not show any virus-receptor complexes on L-nCD155 cells, whereas virus-receptor complexes were found on NZP-60 cells. This apparent discrepancy could be due to the lack of complexes that wild-type nCD155 can form in marmoset cells but not in mouse cells, thus interfering with the already weak binding of the receptor to the virus in L-nCD155 cells. This may also explain why L-nCD155mt cells did not bind and could not be infected with PV2 or PV3 (data not shown). However, the mutations engineered into NWM CD155 molecules were selected based on mutagenesis studies of hCD155 and binding to PV1. Therefore, the mutations may not have significantly increased the binding of the other two serotypes. This would lend support to the hypothesis that there are distinct binding differences between CD155 and PV serotypes. As pointed out before, we do not believe that the differences described here are strain specific rather than serotype specific. The structures of all three PV serotypes show a high degree of similarity in architecture that is necessary for morphogenesis, stability, and receptor binding of the virion. The three serotypes, on the other hand, are defined by a large number of amino acid substitutions mapping over large areas of the virion (38). This leaves little room for variability with respect to strains of a given serotype.

The profound differences by which the three PV serotypes bind to CD155 receptors are surprising. To be sure, the extent of virus-receptor complex formation of the PV strains to hCD155 is very similar (3, 14) and so are corresponding structures of the virus/receptor complexes (6, 16). Originally, the differences were discovered only when mutants of hCD155 were studied (5, 14). For example, cell line 84, containing a mutation at P84SYS/HYSA in hCD155, is deficient in binding PV1 and Sabin 1, but not Sabin 2 or Sabin 3 (14). A hybrid PV1 virus in which the neutralizing antigenic site Ia was exchanged with that of PV2, showed an increase in virus-bound complexes on cell line 84 (14). Therefore, the mutation P84SYS/HYSA in hCD155 leads to loss of binding of PV1, which can be rescued by the exchange of antigenic sites on the virus surface (14).

As with mutant hCD155 molecules, the differences in the binding of the three PV serotypes to the NWM CD155 receptors are remarkable (our studies) (23). When Ltk^– cells were transformed with a chimeric receptor of brown capuchin/hCD155, the cells were susceptible to PV1, but not to PV2 or PV3 (23). Surprisingly, there is only a difference of 4 amino acids in the V-domain of the brown capuchin and marmoset CD155 (data not shown). As mentioned, the brown capuchin/hCD155 chimera is susceptible to PV1, while marmoset cells are not susceptible. The diversity of interactions between the NWM CD155 molecules and the PV serotypes has given rise to differences in serotype susceptibilities of the NWM species.

Evidence has previously been presented that suggested that C-cluster coxsackie A viruses may be the progenitors of the PVs, the critical event being a switch from the ICAM-1 receptor to the CD155 receptor (25). This raises a possibility that a hitherto-hidden poliovirus-like virus may have evolved by adapting to one or the other NWM CD155 for proliferation. Such a poliovirus-like virus may present a reservoir that may seed PVs back into the human population once poliovirus has been eradicated globally and use of a polio vaccine has been permanently terminated, as is the current plan of the World Health Organization.

 
We thank Aniko Paul, Hidemi Toyoda, Jeronimo Cello, and Steffen Mueller for technical suggestions and for help in preparation of the manuscript.

This research was supported by NIH grant AI39486. S.K. was supported by National Research Service Award T32 AI007539.

 
* Corresponding author. Mailing address: Department of Molecular Genetics and Microbiology, Life Science Building Room 213, Stony Brook University, Stony Brook, NY 11794-5222. Phone: (631) 632-8787. Fax: (631) 632-8891. E-mail: ewimmer{at}ms.sunysb.edu

Published ahead of print on 14 May 2008.

Present address: The National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.

Present address: Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.

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Journal of Virology, July 2008, p. 7167-7179, Vol. 82, No. 14
0022-538X/08/$08.00+0     doi:10.1128/JVI.02664-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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