Haartman Institute, Department of Virology, PO Box 21, University of Helsinki, FIN-00014 Helsinki, Finland1
Department of Virology and MediCity Research Laboratories, University of Turku, FIN-20520 Turku, Finland2
National Public Health Institute, Enterovirus Laboratory, Mannerheimintie 166, FIN-00300, Helsinki, Finland3
Author for correspondence: Päivi Joki-Korpela. Fax +358 9 19126491. e-mail paivi.joki-korpela{at}helsinki.fi
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Abstract |
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Introduction |
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To study the immunological properties of this new picornavirus genus, we carried out an antigenic analysis of HPEV1 capsid proteins. One specific object of interest was the region containing the tripeptide motif arginineglycineaspartic acid (RGD) located near the C terminus of the capsid protein VP1. The RGD-containing extension to the VP1 capsid polypeptide of coxsackievirus A9 (CAV9; an enterovirus) has previously been shown to be antigenic (Pulli et al., 1998a , b
). Such motifs are known to participate in cellcell and cellmatrix interactions (Ruoslahti & Pierschbacher, 1987
) and are also utilized by several viral pathogens in their attachment and entry. It has been shown that the RGD-recognizing integrin
v
3 functions as a receptor for CAV9 and that HPEV1 competes with CAV9 for cell surface binding (Roivainen et al., 1994
). Another aim of the study was to recognize immunogenic peptides located elsewhere in the capsid which could be used, for instance, as antigens in HPEV-specific serological assays.
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Methods |
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Antigens and antibodies.
HPEV1, CAV9 and CBV4 antisera were obtained by immunizing rabbits and mice with virus purified in sucrose gradients (Abraham & Colonno, 1984 ). Rabbits were immunized with three sequential 20100 µg doses injected at 2- to 4-week intervals using the popliteal lymph-node method (Leinonen, 1985
), including the use of Freunds complete adjuvant in the first dose. The sera were collected 2 weeks after the last dose. Mice were immunized subcutaneously with three sequential doses of purified virus at 2-week intervals. Freunds complete adjuvant was used at the first dose. The peptides used in immunoassays and for the production of the antisera were synthesized in an Applied Biosystems 433A peptide synthesizer using Fmoc chemistry. They were purified in reverse phase liquid chromatography and analysed for correct molecular mass in a matrix-assisted laser desorption ionization/time-of-flight mass spectrophotometer (FinneganMatt). Peptides P208 and P209 were selected on the basis of peptide scanning results, whereas P151 was chosen for this study because it contained the RGD region. Peptide P154 (VP0 N terminus) was used as a negative control for HPEV1 and CAV9 antisera as this region was not reactive against HPEV1 rabbit antiserum in peptide scanning. In HPEVs, there is an apparent N-terminal extension to the VP3 capsid protein which is not seen in other picornaviruses and peptide P152, which represents this region, was therefore chosen for this study.
Peptide scanning.
Partially overlapping peptides covering the HPEV1 capsid were synthesized in 96-spot format onto cellulose membranes (Frank, 1992 ) according to the manufacturers instructions (SPOTs; Genosys). The whole amino acid sequences of the capsid proteins VP0, VP3 and VP1 were scanned using a 12 aa window moved in three residue shifts. A method previously described for CAV9 (Pulli et al., 1998a
) was used. The membranes containing the HPEV1 peptides were incubated overnight in blocking buffer (Genosys) and rinsed once with TBSTween (Tris-based saline; pH 8·0, 0·05% Tween 20). After this, the membranes were incubated for 30 min with rabbit or mouse antiserum diluted 1:1000 in the blocking buffer. The membranes were washed 5x5 min with TBSTween, and incubated for 30 min with peroxidase-conjugated swine anti-rabbit or rabbit anti-mouse immunoglobulin (DAKO) diluted 1:1500 in the blocking buffer. After washing again 5x5 min with TBSTween, the membranes were incubated for 1 min in ECL detection reagent (Amersham). Chemiluminescence was counted in a 1450 MicroBeta liquid scintillation counter (Wallac) for 1 s per spot. The final results were calculated as an average of the four spots where the same amino acid residue was present. The results were also visualized by exposing the membranes for 1 min on an X-ray film. The visual results correlated well with the LCPS (luminescence counts per s). The membranes were regenerated by treating them 5x10 min in 8 M urea/1% SDS/0·1%
-mercaptoethanol followed by 5x10 min in 50% ethanol/10% acetic acid. Finally, the membranes were washed 3x5 min with TBSTween and stored at 4 °C.
Reverse transcription, PCR and sequence analysis.
RNA was isolated from clinical samples using the Ultraspec RNA isolation system (Biotex Laboratories). The RNA pellet was dissolved in water and stored at -70 °C until analysed. The primers used for reverse transcription, PCR and sequence analysis amplified a 490 nt region from the VP0 capsid protein gene. The C1+ (5' AAAAACGTCTAGTGGGCC 3') primer represented HPEV1 genomic RNA (Hyypiä et al., 1992 ) at positions 645662, whereas the C2- (5' GGTARTTCNACATGTGTAATCTG 3') primer was complementary to positions 11131135. We also used primers from the VP1 capsid protein gene of HPEV1, which amplify a 374 nt fragment. The C3+ (5' GTTGCACAYACATATGACAC 3') primer was of genomic polarity (positions 27222741) and the C4- (5' GTARAAACCTCTATCTAAATA 3') primer was complementary to the sequence at positions 30763096. cDNA synthesis was performed (37 °C for 1 h) in a reaction mixture (40 µl) containing 5 µl specimen RNA, 8 µl 5xRT buffer (Promega), 2 µl dNTP mix (10 mM dATP, dCTP, dGTP, dTTP; Boehringer Mannheim), 0·1 µl ribonuclease inhibitor RNasin (40 U/µl; Promega), 50 pmol C2- or C4- primer and 0·1 µl MMLV reverse transcriptase (200 U/µl; Promega). For PCR, 5 µl cDNA reaction mixture was combined with 75 µl water, 10 µl 10xPCR buffer (Finnzymes), 2 µl dNTP mix (10 mM dATP, dCTP, dGTP, dTTP; Boehringer Mannheim), 0·5 µl Taq DNA polymerase (2 U/µl; Finnzymes) and 50 pmol primers (C1+/C2- or C3+/C4-). The mixture was amplified through 40 cycles of heating at 95 °C for 2 min, primer annealing at 54 °C for 2 min and DNA synthesis at 72 °C for 4 min. The PCR products were analysed in 1% agarose gels containing ethidium bromide. For sequencing, the PCR amplicons of expected size were purified from the agarose gels using the Qiaex II Gel Extraction kit (Qiagen) and sequenced with fluorescent dye-labelled terminators using an automated DNA sequencer (310 Genetic Analyser; Abi Prism). Primers C1+ and C3+ were used for sequencing.
Plaque neutralization test.
A549 and GMK cells were grown to full confluency in 6-well cell culture dishes (Costar). Sixty microlitres of each antiserum dilution in Hanks balanced salt solution supplemented with 0·6% foetal calf serum (dilution medium) containing approximately 100 p.f.u. HPEV1 was incubated for 1 h at 37 °C. The cells were washed once with Hanks solution and the antiserumvirus mixture was added. After incubation for 15 min at room temperature, the cells were overlaid with 0·5% carboxymethyl cellulose in the cell culture medium (MEM) and the plates were incubated in 5% CO2 atmosphere at 37 °C for 48 h. After this, the medium was removed and the cells were stained with crystal violet solution prior to counting the number of plaques. For the CAV9 and the RGD mutants, 50 µl virus diluted 1:100 in dilution medium was mixed with an equal volume of a 1:4 dilution of HPEV1 or CAV9 antiserum. The mixtures were incubated for 2 h at 37 °C and diluted for subsequent infection for GMK cells from 10-3 to 10-8. The cells (GMK) were infected and stained as above.
Enzyme immunoassay (EIA) and immunoblotting.
Ninety-six-well microtitre plates (Maxisorp; Nalge Nunc International) were coated at room temperature overnight with peptides (5 µg/ml in 50 mM NaHCO3; pH 9·6; 150 µl per well) or with purified HPEV1 or CAV9 (0·3 µg/ml; 50 µl per well). The wells were washed three times with PBSTween (containing 0·1% Tween 20) and blocked with 0·1% BSA in PBS (300 µl per well) for 30 min. The wells were washed as above and sera diluted in PBS supplemented with 1% BSA, 1% foetal calf serum and 0·1% Tween 20 were added into the wells. The plates were incubated for 1 h at 37 °C and washed three times with PBSTween. Horseradish peroxidase (HRP)-conjugated goat-anti rabbit IgG (1:10000 dilution; ICN Biomedicals) was added to the wells and the plates were incubated at 37 °C for 1 h. After washing as above, the H2O2/o-phenylenediamide substrate/chromogen solution was added and the reaction was stopped after 30 min with 1 M H2SO4. The absorbance was measured at 492 nm in a spectrophotometer (Multiscan EX; Labsystems). Proteins in lysates of HPEV1-, CAV9- or CBV4-infected cells were separated by SDSPAGE and blotted onto nitrocellulose membranes (Schleicher and Schuell) which were subsequently incubated in PBS containing 5% milk powder (Valio) (PBSmilk) for 15 min at room temperature to block non-specific binding. The HPEV1 antiserum was diluted 1:1000, CAV9 antiserum diluted 1:1000, CBV4 antiserum diluted 1:1000 and P151 peptide antiserum diluted 1:100 in PBSmilk and, after overnight incubation at room temperature with the antiserum, the membranes were washed as above in PBSTween (containing 0·05% Tween 20). Visualization was achieved by HRP-conjugated anti-rabbit serum (Bio-Rad Laboratories).
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Results |
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Rabbit immune serum recognized clearly one linear immunogenic site in the N-terminal part of the VP0 capsid protein (aa 8299) (Fig. 1). The mouse immune serum pool reacted with an overlapping antigenic region (aa 7996) as well as with another region at the C-terminal part of the VP0 polypeptide (aa 253271). Two regions in the VP3 capsid protein were reactive with the rabbit antiserum. One site was located in the N-terminal part of the polypeptide (aa 1535) and the other one was localized in the C-terminal region (aa 183195). The N terminus of the VP3 capsid protein (aa 214), adjacent to the antigenic site recognized by the rabbit antiserum, showed some reactivity with the murine antiserum pool. The rabbit antiserum did not recognize any clearly reactive antigenic regions in the VP1 polypeptide, although the RGD-containing region in the C-terminal region (aa 218231) showed weak reactivity which exceeded the background.
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Discussion |
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Although immunogenic epitopes of some representatives of picornaviruses have been relatively well characterized, little is known about the detailed antigenic determinants of most of the members of the family. In order to elucidate the antigenic properties of HPEV1, we applied various techniques to study the immunogenicity of the virus and its cross-reactivity with RGD-containing CAV9 and CBV4, which has no RGD motif. By using the peptide scanning technique, an immunodominant epitope was recognized in the N-terminal region of the VP0 capsid polypeptide (positions 7999). When compared with picornaviruses whose three-dimensional virus particle structure is known (e.g. Hogle et al., 1985 ; Rossmann et al., 1985
; Luo et al., 1987
; Acharya et al., 1989
), this region seems to follow the
A2 sheet and by analogy with these viruses, it is predicted to form a loop structure buried between VP2 and VP3 (Stanway et al., 1994
). It is possible, however, that such buried sequences can become exposed to the immune system, as previously shown for a highly immunogenic region in the poliovirus VP1 protein (Roivainen et al., 1993
). Rabbit antibodies raised against the peptide covering the antigenic HPEV1 VP0 sequence inhibited infection partially in cell culture. In sequence comparisons of clinical HPEV1 isolates and the reference strain, this site was highly conserved, and was even closely related to the animal parechovirus-like agent, Ljungan virus. Again, an analogous situation occurs in enteroviruses, where a highly conserved region near to the N terminus of VP1 (Hovi & Roivainen, 1993
) is reactive in peptide scanning (Roivainen et al., 1991b
; Hovi & Roivainen, 1993
; Pulli et al., 1998a
).
HPEV1 has an RGD tripeptide at the C terminus of capsid protein VP1 and the infection can be blocked by RGD-containing peptides (Stanway et al., 1994 ). The corresponding region has been shown to be functionally important and antigenic in CAV9, an enterovirus with a similar RGD motif in a similar position (Roivainen et al., 1991a
; Chang et al., 1992
). The reactivity of the C-terminal part in HPEV1 VP1 was weak in peptide scanning. However, when EIA was used, rabbit hyperimmune serum against HPEV1 reacted strongly with the RGD-containing peptide. This phenomenon could be due to the different conformation of the epitope when different techniques are used. Antiserum against this peptide was able to inhibit 50% of HPEV1 infectivity in cell culture at 1:30 dilution. Interestingly, remarkable antigenic cross-reactivity was observed between HPEV1 and CAV9. These two viruses share a functional RGD motif recognizing a cell surface integrin and compete for binding to the host cell (Stanway et al., 1994
; Roivainen et al., 1994
; Pulli et al., 1997
). The RGD motifs of these two viruses are located at the C terminus of the VP1 polypeptide, which is exposed on the surface of the CAV9 virion (Hendry et al., 1999
). The observation that HPEV1 immune serum partially neutralizes CAV9 infection in cell culture, but has no significant effect on the growth of CAV9 RGD mutants supports the idea that the RGD motif is a shared epitope between these viruses. Pulli et al. (1998a
) found three major antigenic regions in CAV9. Two of these were located in the capsid protein VP1 (N-terminal region and the RGD-containing C-terminal region) and one was in the N-terminal region of VP3. The VP1 RGD motif itself was rather poorly reactive, whereas antibody-binding sites were located on both sides of the sequence. When rabbits were immunized with peptides covering the RGD region and flanking amino acids, it was found that a 10 aa peptide following the RGD sequence was the most efficient in inducing production of neutralizing antibodies against CAV9 (Pulli et al., 1998b
). Also, another enterovirus, echovirus 9 (EV9) Barty strain, contains a functional RGD sequence located similarly to CAV9 (Zimmermann et al., 1995
, 1996
, 1997
), but the antigenicity of the region in EV9 is currently not known. In an aphthovirus, FMDV, the functional RGD region, located elsewhere in the VP1 polypeptide (residues 146148) in an exposed position (Acharya et al., 1989
), is known to be highly antigenic (Fox et al., 1989
). Monoclonal antibodies against this mobile region block the attachment of FMDV to BHK cells. It has also been shown that the RGD sequence in FMDV interacts directly with neutralizing antibodies (Verdaguer et al., 1995
), although considerable participation of amino acids flanking the RGD motif on immune recognition of the site has also been observed (Fox et al., 1989
).
Antigenic regions of picornaviruses have been previously studied by using synthetic peptides, peptide scanning and neutralization escape mutants. In poliovirus 1 (PV1), PV3, CAV9, human rhinovirus 14 (HRV14) and FMDV, the major antigenic regions frequently contain amino acid sequences from the capsid protein VP1, whereas VP2 and VP3 are less often involved in the antigenic structures (Minor et al., 1986 ; Sherry et al., 1986
; Roivainen et al., 1991b
; Usherwood & Nash, 1995
; Pulli et al., 1998a
). In light of these results, HPEV1 capsid protein VP1 was surprisingly poorly reactive in peptide scanning and the localization of the major antigenic region in VP0 differs from most of the other picornaviruses studied. As a method, peptide scanning is ideal for mapping linear epitopes, but discontinuous antigenic sites can also be identified by this method (Roivainen et al., 1991b
). However, a negative result in peptide scanning does not exclude the possibility, that the studied region is immunogenic, especially when strictly conformational epitopes are concerned.
The antigenic region identified in HPEV1 is located approximately 25 aa after the site corresponding to VP2/VP4 cleavage in enteroviruses, whereas the major antigenic regions described previously for other picornaviruses (e.g. PV1, CAV9, FMDV, HRV2, HRV14) are situated mainly in the BC and EF loops of VP2 (Rossmann et al., 1985 ; Minor et al., 1986
; Sherry et al., 1986
; Appleyard et al., 1990
; Mateu, 1995
; Pulli et al., 1998a
). In Theilers murine encephalomyelitis virus, a picornavirus causing demyelinating disease in mice, a predominant antigenic region at the N terminus of VP2 (aa 214) appears to be more close to the VP4/VP2 cleavage site than the HPEV1 antigenic region (Kim et al., 1992
). Detailed structural data of HPEV1 particles would be helpful in understanding these differences.
In conclusion, our studies revealed an immunodominant epitope in the VP0 polypeptide, in a region which is not found to be antigenic in other previously studied picornaviruses. Both rabbit and mouse immune systems recognized this antigenic region when the peptide scanning technique was used. Remarkable conservation of this region was observed when sequences obtained from clinical specimens from different geographic locations were compared. Although the C-terminal region of CAV9 VP1, which also contained the functionally analogous RGD motif, has been shown to be highly immunogenic by peptide scanning (Pulli et al., 1998a , b
), no significant reactivity was seen in this region in HPEV1. However, the RGD-containing peptide was antigenic when EIA was used and this peptide was able to induce production of neutralizing antibodies in a rabbit. Also, this region was relatively well conserved among HPEV1 strains. The antigenic cross-reactivity between HPEV1 and CAV9 suggests that cross-neutralization between picornaviruses sharing only short common epitopic sequences can occur.
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Acknowledgments |
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References |
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Received 19 October 1999;
accepted 14 March 2000.