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Journal of Virology, August 2007, p. 8648-8655, Vol. 81, No. 16
0022-538X/07/$08.00+0     doi:10.1128/JVI.01569-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Enterovirus 70 Receptor Utilization Is Controlled by Capsid Residues That Also Regulate Host Range and Cytopathogenicity

Melissa Stewart Kim and Vincent R. Racaniello^*

Department of Microbiology, Columbia University College of Physicians & Surgeons, 701 W. 168th St., New York, New York 10032

Received 21 July 2006/ Accepted 17 May 2007

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Enterovirus type 70, an etiologic agent of acute hemorrhagic conjunctivitis, may bind different cellular receptors depending on cell type. To understand how EV70-receptor interaction is controlled, we studied two variants of the virus with distinct receptor utilization. EV70-Rmk, derived by passage in rhesus monkey kidney cells, replicates poorly in HeLa cells and does not cause cytopathic effects. Decay accelerating factor (DAF) is not a cell receptor for EV70-Rmk. Passage of EV70-Rmk in HeLa cells lead to isolation of EV70-Dne, which does not replicate in rhesus monkey kidney cells but grows to high titers in HeLa cells and causes cytopathic effects. DAF is sufficient for cell entry of EV70-Dne. EV70-Rmk replicates in human eye and brain-derived cell lines, whereas the Dne strain replicates only in HeLa cells and in conjunctiva-derived 15C4 cells. The two EV70 strains differ by five amino acid changes in the viral capsid. Single substitution of four of the five EV70-Rmk amino acids with the residue from EV70-Dne leads to lytic replication in HeLa cells. Conversely, substitution of any of the five EV70-Dne amino acids with the EV70-Rmk amino acid does not alter replication in HeLa cells. Three of these capsid amino acids are predicted to be located in the canyon encircling the fivefold axis of symmetry, one amino acid is found at the fivefold axis of symmetry, and one is located the interior of the capsid. The five EV70 residues define a region of the capsid that controls viral host range, DAF utilization, and cytopathogenicity.

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Acute hemorrhagic conjunctivitis (AHC) is a viral infection of the eye that was first reported in Ghana in 1969 (5). This highly contagious infection is transmitted through direct or indirect contact with eye secretions. An incubation period of 24 to 48 h is followed by rapid onset of ocular pain, swelling of the eyelids, excessive tearing, pain, and subconjunctival hemorrhage. The disease usually resolves in 3 to 7 days. Enterovirus type 70 (EV70), a member of the Picornaviridae, is one of the etiologic agents of AHC. This recently emerged virus (25) has caused two global AHC pandemics (4, 24), underscoring its highly transmissible nature.

EV70 is an unusual enterovirus because AHC is not initiated through the alimentary tract but upon introduction of the virus directly into the eye. The pathogenesis of AHC has not been studied due to lack of a suitable animal model. EV70 has been shown to cause conjunctivitis in the rabbit (21), but little progress has been made with this animal model because there are few immunological reagents or defined mutants available. In humans, paralysis occurs in approximately 1 of 10,000 symptomatic EV70 infections (41). Unlike poliomyelitis, this complication is transient and involves both acute flaccid paralysis of the lower limbs and cranial nerve palsies. The mechanism by which EV70 enters the central nervous system (CNS) and the associated switch from eye to CNS pathogenesis has not been studied.

Several mouse models for viral diseases have been established by producing transgenic mice expressing cell receptors for viruses. These animal models have proven valuable for studying the pathogenesis of poliomyelitis (15, 34), measles (3, 9, 16, 28, 33), and echovirus paralysis and myocarditis (17). At least one cell receptor for EV70 is decay accelerating factor (DAF), also known as CD55 (19). CD55 is a member of the family of regulators of complement activation and is an important modulator of the complement system (23). Treatment of cells with neuraminidase blocks binding of EV70 (1, 18, 39); however, this effect is not due to removal of sialic acid on CD55. Rather, a second sialylated cell surface protein may be required for EV70 binding. The finding that EV70 can infect human leukocyte cell lines that do not produce CD55 at detectable levels also suggests the presence of a second cell surface receptor for the virus (10).

To facilitate genetic analysis of EV70 receptor utilization, the first infectious DNA copy of the EV70 genome was constructed and used to study two strains of the virus with a distinct host range. One virus isolate, EV70-Rmk, replicates in rhesus monkey kidney cells and poorly in HeLa cells and does not cause cytopathic effects. Here we show that DAF does not serve as a cell receptor for EV70-Rmk entry. Passage of EV70-Rmk in HeLa cells lead to isolation of EV70-Dne, which does not replicate in rhesus monkey kidney cells but grows to high titers in HeLa cells and causes cytopathic effects. DAF is sufficient for cell entry of EV70-Dne. The two EV70 strains differ by five amino acid changes in the viral capsid. Single substitution of four of the five EV70-Rmk amino acids with the corresponding residue from EV70-Dne lead to lytic replication in HeLa cells. Conversely, substitution of any of the five EV70-Dne amino acids with the corresponding residue from EV70-Rmk did not alter replication in HeLa cells. The five EV70 residues define a region of the capsid that controls viral host range, DAF utilization, and cytopathogenicity.

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Cell lines. HeLa S3 (human cervical carcinoma), LLC-MK2 (rhesus monkey kidney), 15C4 (human Chang conjunctiva), L (L2929, murine fibroblast), NIH 3T3 (murine fibroblast), T98-G (human glioblastoma multiforme), U373-MG (human glioblastoma), SY5Y (human neuroblastoma), and RK13 (rabbit kidney) cell lines were propagated in Dulbecco modified Eagle growth medium (DMEM) containing 10% bovine calf serum and 1% penicillin-streptomycin (Gibco). These cell lines were obtained from American Type Culture Collection (Manassas, VA) with the exception of the U373-MG and T98-G, which were obtained from the laboratory of C. S. Young at Columbia University. The HCE cell line (for human cornea fibroblast, obtained from Peter Reinach, SUNY State College of Optometry) was propagated in 50% DMEM-50% F-12 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum, 250 ng of mouse epidermal growth factor (Sigma-Aldrich, St. Louis, MO)/ml, and 2.5 µg of bovine insulin (Sigma-Aldrich, St. Louis, MO)/ml.

The stable L cell line L-hDAF was isolated after cotransformation of cells with a plasmid carrying DNA of the GPI-linked form of DAF (pDAFBOS, provided by D. Lublin, Washington University, St. Louis, MO) with a plasmid encoding the puromycin resistance gene (pRSVPR puro-R). Transformants were selected in 10 µg of puromycin/ml for 2 weeks. DAF production was determined by flow cytometry using anti-human CD55 (DAF) SCR1 domain antibody (BD Biosciences Pharmingen, San Diego, CA) and anti-human CD55 (DAF) SCR3 (clone BRIC 216, MCA914; Serotec, Raleigh, NC). The cell line used in these experiments, L-DAF8H4, was obtained after four cycles of sorting for high DAF SCR1 domain expression. This cell line is maintained in DMEM, 10% bovine calf serum, 1% penicillin-streptomycin, and 10 µg of puromycin/ml.

Viruses. EV70 prototype strain J670/71 was obtained from the American Type Culture Collection (Manassas, VA) and propagated in rhesus monkey kidney LLC-MK2 cells at 37°C. The virus was plaque purified three times in LLC-MK2 cells and designated EV70-Rmk. This virus was passaged nine times in HeLa cells, after which cytopathic effects became evident 8 h postinfection. The virus was plaque purified three times in HeLa cells and designated EV70-Dne. Other virus stocks were obtained by transfection of cell monolayers with RNA produced by in vitro transcription of cloned genomic DNA, including EV70-Rmk (from plasmid pRMK14), EV70-Dne (from plasmid pDNE9), and poliovirus P2/Lansing (from plasmid PT7L) (32). RNA transcripts were produced by in vitro transcription of a linearized template using T7 RNA polymerase (Invitrogen) as previously described (26). Virus titers were determined by plaque assay in HeLa cells or LLC-MK2 cells as described previously (26).

Isolation of viral RNA. Virion RNA for the production of DNA clones was isolated by ultracentrifugation of infected cell supernatants in an SW28 rotor at 28,000 rpm for 3 h at 4°C. Supernatants were discarded, and pellets were pooled and suspended in 500 µl of DMEM. An equal volume of TRIzol reagent (Invitrogen) was added to the suspended pellet in 1.5-ml Eppendorf tubes. This mixture was centrifuged at 14,000 rpm for 10 min to induce phase separation. The top, aqueous phase was harvested, an equal volume of isopropanol was added, and the mixture was centrifuged for 10 min at 14,000 rpm. The supernatant was aspirated, leaving the RNA pellet to air dry. The RNA was resuspended in H₂O and incubated for 10 min at 65°C, and then 10 mM dithiothreitol and 5% (vol/vol) RNasin (Promega, Madison, WI) were added.

Production of infectious viral DNA clones. DNA copies of the EV70-Rmk and EV70-Dne viral genomes were cloned into the plasmid pATEVL. Viral RNA was reverse transcribed at 42°C for 1 h by using Superscript II reverse transcriptase (Invitrogen) and a 3' primer which encoded (5' to 3') a BamHI restriction site, 20 polyadenine bases, and the last 15 bases at the 3' end of the EV70 genome. After 1 h, 1 U of RNase H was added, and the sample was incubated for 30 min at 37°C. Viral DNA was amplified by PCR using the 3' primer described above, and a 5' end primer comprising from 5' to 3' an AscI restriction site, a T7 promoter, and the first 12 bases of the viral RNA. EV70 primers were derived from the EV70 reference sequence D00820 (National Center for Biotechnology Information). PCR products were cloned into plasmid pATEVL. This vector was produced by introducing a polylinker containing cleavage sites for AscI, SpeI, ApaI, BsteII, and PacI into the EcoRI and HindIII sites of plasmid pAT153. RNA transcripts were produced by in vitro transcription of a BamHI-linearized template using T7 RNA polymerase (Promega) and introduced into LLC-MK2 cells using DEAE-dextran (40). EV70-Rmk virus produced after transfection with RNA from clone 14 was selected for further studies and named EV70-Rmk14.

Nucleotide sequence analysis of subgenomic DNA clones produced from EV70-Dne RNA revealed that it differed from the genome of EV70-Rmk by five coding changes. A full-length infectious DNA copy of the EV70-Dne genome was produced from the Rmk14 full-length DNA clone by using PCR mutagenesis to change five amino acids in the RMK polyprotein: E14K, I557M, F694L, R719P, and N787D. Mutagenic primers were annealed to both strands of the parental supercoiled plasmid, and then gaps between sites of hybridization were filled in during the elongation cycle of the PCR. After thermocycling, 1 µl of DpnI restriction enzyme (New England Biolabs, Beverly, MA) was added to each reaction to digest parent double-stranded plasmids. After heat inactivation at 80°C for 20 min, samples were introduced into XL10Gold bacteria (Stratagene, La Jolla, CA). The sequence of the entire EV70-Dne DNA was determined to ensure that no other mutations were present. RNA transcripts were produced by in vitro transcription of a BamHI-linearized template using T7 RNA polymerase and introduced into HeLa cells using DEAE-dextran. EV70-Dne virus produced after transfection with RNA from clone 9 was selected for further studies and named EV70-Dne.

Single amino acid coding changes were introduced into the genome of EV70-Dne and EV70-Rmk by PCR mutagenesis of cloned DNA as described in the preceding paragraph. The presence of the desired nucleotide was confirmed by nucleotide sequencing of full-length viral cDNA derived from plasmid DNA isolated from individual bacterial colonies. (The nucleotide sequence of EV70-Rmk has been deposited in GenBank under accession number 76496395.)

Quantitation of viral RNA. Slot blot hybridization analysis was performed by extracting total cytoplasmic RNA from infected cells and vacuum blotting samples onto a GeneScreen membrane (New England Nuclear, Boston, MA). Samples were cross-linked to the membrane by using UV irradiation in a Stratalinker (Stratagene, La Jolla, CA). Membranes were prehybridized for 1 h prior to the addition of the probe overnight. Membranes were washed six times under increasingly stringent conditions and then quantitated using a PhosphorImager instrument and ImageQuant software (Amersham Radiochemicals, Piscataway, NJ). Positive-strand amplification was detected using a negative-strand RNA produced by in vitro-transcribed RNA probe complementary to nucleotides 5832 to 7221 of the EV70 genome. The hybridization probe was radiolabeled with [-³²P]UTP (Amersham Biosciences, Piscataway, NJ) during an in vitro transcription reaction using T7 RNA polymerase.

EV70 plaque assay. Titers of EV70-Dne were determined by plaque assay on HeLa cell monolayers as described previously (26). This procedure was modified to determine titers of EV70-Rmk by plaque assay on LLC-MK2 cells. After virus adsorption, 2 ml of an overlay composed of 0.9% agar noble and 1x DMEM supplemented with 10% bovine calf serum (BCS), 2% penicillin-streptomycin, and 0.5% sodium bicarbonate was added. After solidification of the overlay, 2 ml of DMEM (supplemented with 10% BCS, 1% penicillin-streptomycin) was added to further nourish the cell monolayer. The plates were incubated for 72 h at 37°C and then stained as described previously (26).

One-step growth analysis. Cells (5 x 10⁵ cells in a 3.5-cm plate) were infected with virus at a multiplicity of infection (MOI) of 5. After adsorption for 30 min at 37°C, the inoculum was aspirated, and 1 ml of fresh growth medium was added. At each time point, monolayers were scraped into the medium and frozen and thawed three times, and cell debris was removed by centrifugation at 2,000 rpm for 10 min. Viral supernatants were titrated on HeLa monolayers for Dne virus and LLC-MK2 monolayers for Rmk virus as described in the previous section. Each one-step growth analysis was repeated three times, and one representative graph is presented.

Measurement of cytopathic effect. Virus-induced cell killing was quantitated by staining with trypan blue. HeLa cells were infected at an MOI of 3, the inoculum was aspirated, and the monolayer washed twice, and then 0.5 ml of growth medium was added. At different times after infection, cells and medium were collected and the cells were pelleted by low-speed centrifugation. Pellets were resuspended in phosphate-buffered saline, trypan blue dye was added, and the cells were examined by light microscopy. Approximately 150 to 200 cells were counted for each sample, and the percent viability was determined by dividing the number of cells that excluded dye by the number of cells examined.

Treatment of cells with antibodies or enzymes. Monoclonal antibodies (0.5 µg) were added to monolayers of 10⁶ HeLa cells, followed by incubation for 20 min at room temperature. The monoclonal antibodies used were mouse immunoglobulin G2a (IgG2a) anti-human SCR1 DAF (CD55; clone IA10; BD Biosciences Pharmingen), mouse IgG1 anti-human SCR3 DAF (CD55; clone BRIC 216, MCA914; Serotec), and mouse IgG2a anti-trinitrophenol hapten (BD Biosciences Pharmingen). Antibodies were aspirated from the wells prior to infection at an MOI of 3. At 24 h postinfection, the monolayers and their supernatants were harvested and subjected to RNA slot blot analysis. Positive-strand RNA replication was determined using a negative-strand RNA hybridization probe as described above. The replication signal for each sample was normalized to the signal for mock-infected cells.

Enzymatic blocking of viral replication was performed as described above, except that cells were incubated with enzymes diluted in DMEM without serum at 37°C for 30 min. The quantity of enzyme used per 10⁶ HeLa cells was: 0.1 U of phosphoinositol-specific phospho-lipase C (PI-PLC from Bacillus cereus; Sigma-Aldrich, St. Louis, MO), and 0.01 U of neuraminidase (from Vibrio cholerae; Biochemika, St. Louis, MO).

Structural location of amino acid changes in the EV70 capsid. EV70 strain-specific amino acid changes were mapped onto the capsid of bovine enterovirus (BEV) type 1 (GenBank accession number NC_001859) as follows. Amino acid alignments of BEV and EV70 were performed independently for each of the four structural proteins using the maximum matching tool of MacVector (Accelerys, Inc.), and the BEV residue that corresponds to each changed amino acid in EV70 was identified. The BEV capsid structure was visualized by using MolScript. Protomer coordinates were used to produce pentamers using Virus Particle Explorer (VIPER) (36).

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Isolation of two strains of EV70 and comparison of host range and DAF receptor requirements. It has been reported that the cell surface protein DAF (or CD55) serves as a receptor for EV70 (19). To confirm this observation, a mouse L-cell line was isolated that synthesizes human DAF on the cell surface (L-hDAF cells). Neither L cells nor L-hDAF cells are susceptible to infection with the J670/71 prototype strain of EV70 passaged in LLC-MK2 (hereafter called EV70-Rmk). Mouse L cells are permissive for EV70-Rmk infection, as demonstrated by the release of infectious virus after transfection by viral RNA (data not shown). These findings suggest that DAF is not sufficient for entry of EV70-Rmk into cells.

EV70-Rmk replicates in the rhesus monkey kidney cell line LLC-MK2 and also forms plaques in these cells. However, the virus grows poorly in HeLa cells and does not form plaques on this cell line. After nine sequential passages of EV70-Rmk in HeLa cells, a viral variant called EV70-Dne was isolated that forms plaques on monolayers of HeLa cells but no longer replicates in LLC-MK2 cells. Infection of HeLa cells with EV70-Dne virus leads to cell lysis, whereas infection of the same cells with EV70-Rmk does not cause cytopathic effect. Yields of EV70-Dne from infected HeLa cells are significantly higher (10⁷ PFU/ml) compared to yields from HeLa cells infected with EV70-Rmk (10⁵ PFU/ml). EV70-Dne virus replicated in L-hDAF but not in L cells, suggesting that DAF serves as a cell receptor for this virus isolate.

Virus produced from cloned DNA copies of the genomes of EV70-Rmk and EV70-Dne display the host range phenotype of the parent viruses. EV70-Rmk virus replicated in LLC-MK2 cells and produced low yields in HeLa cells, whereas EV70-Dne virus replicated to high titers in HeLa cells but not in LLC-MK2 cells. (Fig. 1). EV70-Rmk is cytolytic in LLC-MK2 cells and forms plaques in this cell line but is not cytolytic and does not form plaques in HeLa cells. EV70-Dne forms plaques on HeLa cells but not on LLC-MK2 cells. EV70-Dne virus, but not EV70-Rmk virus, productively infects L-hDAF cells but not L cells, suggesting that DAF serves as a receptor for EV70-Dne but is not sufficient for entry of EV70-Rmk into cells.

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FIG. 1. Host range of EV70-Rmk14 and EV70-Dne in cultured cells. One-step growth analysis of viruses was performed in human (HeLa), monkey (LLC-MK2), and murine (L, L-hDAF) cell lines with EV70-Rmk14 (top) or EV70-Dne (bottom) virus at an MOI of 5. Infections were halted at different times postinfection, and virus titers were determined by plaque assay.

A variety of cell lines were examined for susceptibility to infection with EV70-Rmk and EV70-Dne. All of these cell lines were susceptible to infection by EV70-Rmk14, although viral yields are low (Fig. 2). Of the human cell lines tested, EV70-Dne virus replicates only in HeLa cells and in the human conjunctival cell line 15c4, but the virus yields per cell are higher than observed after infection with EV70-Rmk (Fig. 2).

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FIG. 2. Host range in cell lines derived from human eye and brain. One-step growth analysis of viruses was performed using EV70-Rmk14 (top) or EV70-Dne (bottom) virus to infect (MOI = 5) the following eye and brain derived cell lines: HeLa, T98, LLCMK2, U373MG, 15C4, SY5Y, or HCE. Infections were halted at different times postinfection, and virus titers were determined by plaque assay.

Blockade of cell receptors for EV70-Rmk and EV70-Dne. To provide information on the molecules that serve as cell receptors for EV70-Rmk and EV70-Dne, HeLa cells were treated with neuraminidase, phosphoinositol phospholipase C (PI-PLC), or antibodies to DAF, followed by virus infection. Viral entry was assayed by measuring levels of positive-strand RNA production in cells. Antibodies to SCR1 or SCR2 of DAF blocked infection by EV70-Dne but did not inhibit infection by EV70-Rmk (Fig. 3). Treatment of cells with PI-PLC also inhibited infection by EV70-Dne but had minimal effect on replication of EV70-Rmk (Fig. 3). Neuraminidase treatment inhibits of cells blocked replication of EV70-Rmk but had no effect on replication of EV70-Dne (Fig. 3). These results show that HeLa cell receptors for EV70-Rmk and EV70-Dne are distinct.

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FIG. 3. Effect on viral replication of enzyme or antibody treatment of cultured cells. HeLa cells were incubated with one of the following as indicated along the x axis: phosphate-buffered saline (PBS [mock treated]), neuraminidase, PI-PLC, or antibodies specific for hapten or the SCR1 or SCR2 domains of the human DAF molecule. Cells were washed and infected with EV70-Rmk14 or EV70-Dne at an MOI of 3. At 24 h postinfection, the total RNA was isolated from infected cells and transferred onto a nitrocellulose membrane for slot blot analysis. Positive-strand viral replication was assessed by hybridization with a radiolabeled negative-strand EV70 RNA probe. The amount of hybridized probe was determined with a PhosphorImager and ImageQuant software and reported as the optical density. The data were normalized to the mock (PBS)-treated sample (PBS treatment = 100% replication).

Genetic and functional analysis of differences in host range. To delineate the genetic basis for the host range difference of EV70-Rmk and EV70-Dne viruses, the nucleotide sequence of both viral genomes was determined from cloned DNA copies of viral RNA. The results of this analysis indicate that five amino acid changes distinguish the two strains of EV70: one in VP4, one in VP3, and three in VP1 (Table 1) . The contribution of these amino acid changes to EV70 viral growth in HeLa cells was determined using viruses with a single amino acid change at each of these positions. EV70-Rmk does not form plaques and grows poorly in HeLa cells (Fig. 4A). Introduction of any single amino acid change from the EV70-Dne strain into the EV70-Rmk background produced viruses that form plaques on a HeLa cell monolayer and replicate to higher titers in these cells (10 to 100 PFU/cell, Fig. 4A). Virus RRRRD was not included in this analysis because its titers were too low to conduct one-step growth analysis at the required MOI. Introduction of any single amino acid change from the EV70-Rmk strain into the EV70-Dne background had little effect on lytic growth and final yields in HeLa cells (Fig. 4B). Virus yields (PFU/cell) were 119 (DDDDD), 178 (RDDDD) 62 (DRDDD), 125 (DDRDD), 133 (DDDRD), and 35 (DDDDR). These results suggest that at least four of the five amino acid changes in the capsid of EV70-Dne virus are redundant for lytic, high-titer growth in HeLa cells, and no single position is required for this phenotype.

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TABLE 1. Amino acid differences between EV70-Rmk14 and EV70-Dne strains

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FIG. 4. Role of amino acid differences between EV70-Rmk and EV70-Dne viruses in replication in HeLa cells. One-step growth analysis was performed with mutants of EV70-Dne harboring single amino acid substitutions to the EV70-Rmk14 sequence. HeLa cells were infected at an MOI of 5, infections were halted at different times postinfection, and virus titers were determined by plaque assay. (A) Growth analysis of viruses DDDDD (EV70-Dne encoding amino acids K14, M238, L133, P178, and D226), DRRRR (K14K, I238, F133, R178, N226), RDRRR (E14, M238, F133, R178, and N226), RRDRR (E14, I238, L133, R178, and N226), RRRDR (R14, I238, F133, P178, and N226), and RRRRR (EV70-Rmk, E14, I283, F133, R178, and N226). (B) Growth analysis of viruses DDDDD (see above), RDDDD (E14, M238, L133, P178, and D226), DRDDD (K14, I238, L133, P178, and D226), DDRDD (K14, M238, F133, P178, and D226), DDDRD (K14, M238, L133, R178, and D226), and DDDDR (K14, M238, L133, P178, and N226).

The presence of DAF on the surface of L cells is sufficient to allow infection by EV70-Dne but not EV70-Rmk (Fig. 1). To determine which of the five amino acid differences between these two viruses controls this phenotype, L-hDAF cells were infected with viruses bearing single amino acid changes at each position. The results indicate that introduction of any of the five amino acid changes found in the EV70-Dne strain into EV70-Rmk allows growth on L-hDAF cells (Fig. 5A). Based on this finding, it would be expected that substitution of single amino acids in EV70-Dne with residues from EV70-Rmk should not dramatically alter replication in L-hDAF cells. The results of infection of L-hDAF cells with these mutant viruses confirms this expectation (Fig. 5B).

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FIG. 5. Role of amino acid differences between EV70-Rmk and EV70-Dne viruses in hDAF usage. One-step growth analysis was performed using mutants of EV70-Dne virus (described in the legend to Fig. 4). L-hDAF cells were infected at an MOI of 5, infections were halted at different times postinfection, and virus titers were determined by plaque assay.

The contribution of each of these five amino acids to lytic infection of HeLa cells was determined by examining the effect on cell viability of infection with viruses harboring single amino acid changes. Cells infected with EV70-Rmk virus remain nearly 100% viable, as do uninfected cells, as determined by trypan blue exclusion (Fig. 6). In contrast, most cells are killed after infection with EV70-Dne. Cells infected with all other mutant viruses lose viability by 27 h postinfection, although to different extents. These results suggest that each of the five amino acid positions is redundant in regulating the lytic phenotype of EV70.

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FIG. 6. Role of amino acid differences between EV70-Rmk and EV70-Dne viruses in cell killing. HeLa cells were infected at an MOI of 3, using EV70-Dne (DDDDD), EV70-Rmk14 (RRRRR), or mutant viruses described in the legend to Fig. 4. Infections were halted at different times postinfection, and cells were pelleted by low-speed centrifugation, resuspended in PBS with trypan blue dye, and examined by light microscopy. The percent viability was determined by dividing the number of cells that excluded dye by the number of cells examined.

Location in the capsid of EV70 of amino acids that regulate host range. The virus most closely related to EV70 by primary sequence, for which there are structural data, is bovine enterovirus. The structural proteins of BEV-1 and EV70 are very similar (48% identity) (30), and BEV is nearly colinear in tertiary structure even with more distantly related human enteroviruses (38). The known crystallographic structure of bovine enterovirus type 1 (BEV-1) was therefore used to predict the locations in the viral capsid of amino acid residues that differ between EV70-Rmk and EV70-Dne.

Amino acids in the capsid of BEV-1 that are analogous to the five capsid residues that differ between EV70-Rmk and EV70-Dne were identified (Table 1). Amino acid 14 of VP4 could not be located because no structural information on this region of the capsid was obtained (37, 38). This amino acid presumably resides inside the capsid of the native virion, as is the case for VP4 of other picornaviruses. EV70 VP1 amino acid 133 is predicted to be located to the DE loop at the fivefold axis of symmetry (Fig. 7). This position appears to be partially exposed in the BEV capsid structure. It could mediate interprotomer contacts as in the capsids of poliovirus type 2 and coxsackievirus B3 (22, 27), or it might be involved in direct receptor contact as observed for residues of the BC and HI loop of rhinovirus type 2 (13).

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FIG. 7. Predicted location in the viral capsid of EV70 amino acids that influence host range and cell killing. The known crystallographic structure of BEV-1 was used to predict the locations in the viral capsid of amino acid residues that differ between EV70-Rmk14 and EV70-Dne. Capsid protomer proteins are color coded, with VP1 blue, VP2 yellow, and VP3 red. VP4 is omitted for clarity. Amino acid changes are enumerated in white. (A) Exterior view of a pentamer, comprising five copies each of VP1, VP2, VP3, and VP4 (not shown), revealing the locations of four of the five strain-specific amino acid changes: in the VP1 DE loop, amino acid 133 of EV70 VP1 (F1133L, BEV 1128 in figure), and three in the canyon, including 238 of EV70 VP3 (I3238M, BEV 3240), 178 of EV70 VP1 (R1178P, BEV 1151), and 226 of EV70 VP1 (N1226D, BEV 1219). Residues on only one protomer are labeled. (B) Closer exterior view of canyon and fivefold axis of symmetry. Amino acid changes in all five protomers are labeled.

The three remaining amino acids are all exposed on the surface of the capsid, and map to the canyon (Fig. 7), a known site of receptor interaction for enteroviruses, including poliovirus (2, 6, 7, 12), coxsackieviruses A21 (42) and B3 (11), and major group rhinoviruses types 14 and 16 (20, 29, 35). The EV70 residues mapping to the canyon include 238 of VP3 (I3238M), 178 of VP1 (R1178P), and 226 of VP1 (N1226D). Amino acid 238 of VP3, near the carboxy terminus of the protein, protrudes into and helps form the canyon. Residue 178 is located in the EF loop of VP1 on the canyon rim and protrudes out radially from the stem of the fivefold axis. Amino acid 226 of VP1 is located in the H-strand at the wall of the canyon near the fivefold axis of symmetry.

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DAF was previously identified as a receptor for EV70 based on the observation that anti-DAF monoclonal antibodies block EV70 binding to HeLa cells and protect the cells against infection (19). In addition, the production of human DAF in mouse 3T3 cells conferred susceptibility to infection; however, virus yields increased only twofold compared to 3T3 cells lacking DAF (19). The virus used by Karnauchow et al. was derived from isolate J670/71 and propagated in LLC-MK2 cells. We also propagated isolate J670/71 in LLC-MK2 cells; however this virus, called EV70-Rmk, does not utilize DAF to infect cells. Replication of this virus is not affected by anti-DAF antibodies or treatment of cells with PI-PLC and does not occur in L-DAF cells. However, replication of EV70-Rmk is sensitive to treatment of cells with neuraminidase. Passage of EV70-Rmk in HeLa cells lead to isolation of a virus that enters cells in a DAF-dependent manner. In mouse cells, no other human protein is required for entry of EV70-Dne; we conclude that the receptor for this strain of EV70 is human DAF. It seems likely that EV70 entry into cells relies on different cell receptors, depending on the cell type and the passage history. This conclusion is in accord with previous suggestions that EV70 entry into human leukocyte cell lines is not dependent upon DAF but an unidentified sialylated molecule (10).

The two strains of EV70 described here both infect HeLa cells but with different outcomes. EV70-Rmk grows to a low titer and does not cause cytopathic effect in HeLa cells. In contrast, EV70-Dne replicates to 10-fold-higher titers in HeLa cells and induces a complete cytopathic effect. Although EV70-Rmk replicated in all of the human (corneal, glial, and conjunctival) cell lines tested, replication of EV70-Dne was restricted to HeLa cells, the cell line used for passage and selection, and the human conjunctival line 15C4. Analysis of cell surface expression of DAF by flow cytometry revealed that these two cell lines produce at least 10 times more DAF than other nonsusceptible cell lines such as HCE, SY5Y, U-373MG, and T-98G (data not shown). The level of surface DAF may therefore determine susceptibility to infection by EV70-Dne.

EV70-Rmk and EV70-Dne differ by five amino acids in the viral capsid. Four of the five residues contribute individually in a sufficient and redundant manner to growth in HeLa and L-DAF cells. Each residue alone similarly engenders a cytopathic phenotype in HeLa cells. Comparative structural analysis suggests that all five changes map to the capsid in regions that have been shown to mediate virus receptor interactions for other enteroviruses (reviewed in reference 31). A simple explanation for why the EV70-Dne strain utilizes DAF as a cell receptor is that the capsid residues of former strain allow interaction with DAF. The capsid residues identified in the present study might delineate a region of the capsid that regulates DAF binding. Consequently, substitution of any single amino acid into the EV70-Rmk strain allows the virus to bind DAF. EV70 VP1 amino acids 133, 178, and 226, and VP3 amino acid 238, all could be involved in direct receptor contact. VP4 amino acid 14 is located in the interior of the virion, and hence cannot be involved in direct contact with the cellular receptor. It has been suggested that the poliovirus capsid must undergo structural changes to bind to its cellular receptor (8). Amino acid changes in VP4 could influence receptor binding of EV70 by modulating the ability of the capsid to undergo structural changes. An alternative hypothesis is that the amino acid changes identified in the present study do not directly influence binding, but rather uncoating of the viral RNA. For example, because expulsion of VP4 is a crucial step in the release of picornavirus RNA (14), it is possible that changes at VP4 amino acid 14 regulate EV70 host range by affecting uncoating. EV70 VP1 amino acid 133, which could mediate interprotomer contacts, might also act at a post-receptor-binding step.

There are several possible explanations for the difference in cytopathogenicity between EV70-Rmk and EV70-Dne in HeLa cells. The receptor utilized by EV70-Rmk in HeLa cells might not permit efficient cell entry, resulting in low levels of genome replication and poor induction of cell killing. The difference in cell killing might also be independent of receptor utilization. EV70-Dne amino acids in each of the five capsid positions might more efficiently induce cell killing, perhaps by inducing apoptosis.

Our results suggest that passage of EV70 in LLC-MK2 cells selects for viruses that utilize a sialylated molecule other than DAF as a cell receptor. In contrast, passage of EV70 in HeLa cells selects for viruses that are dependent upon DAF for cell entry. To provide clues about which receptor might be utilized by clinical isolates of EV70, we compared the amino acid sequences of 37 different clinical isolates obtained during epidemics of the period from 1970 to 1990 (data not shown). Only the sequence of VP4 was available for 14 clinical isolates; all of these isolates have the Rmk amino acid E at VP4 position 14. The remaining 23 isolates have the Rmk amino acid F at VP1 position 133; 20 of these have the Rmk amino acid N at VP1 position 226 (the residue in the other 3 isolates could not be determined). Of these 23 isolates, 6 carry the Rmk amino acid R at VP1 position 178, and the remaining 17 have the Dne amino acid P. These observations suggest that clinical isolates and EV70-Rmk might utilize a similar, non-DAF receptor. Furthermore, DAF-dependent cell entry may be characteristic of isolates adapted to growth in cell cultures.

 
This study was supported by Public Health Service grant AI20017 from the National Institute of Allergy and Infectious Diseases to V.R.R.

 
* Corresponding author. Mailing address: Department of Microbiology, Columbia University College of Physicians & Surgeons, 701 W. 168th St. New York, NY 10032. Phone: (212) 305-5707. Fax: (212) 305-5106. E-mail: vrr1{at}columbia.edu

Published ahead of print on 30 May 2007.

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Journal of Virology, August 2007, p. 8648-8655, Vol. 81, No. 16
0022-538X/07/$08.00+0     doi:10.1128/JVI.01569-06
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