1 Institute for Animal Science and Health (ID-Lelystad), Division of Infectious Diseases and Food Chain Quality, PO Box 65, 8200 AB Lelystad, The Netherlands
2 Federal Research Centre for Viruses Diseases of Animals, Tübingen, Germany
3 Virology Division, Utrecht University, Utrecht, The Netherlands
Correspondence
Esther Wissink
e.h.j.wissink{at}id.wag-ur.nl
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ABSTRACT |
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Present address: Department of Otorhinolaryngology, UMC Nijmegen, Geert Grooteplein 10, 6525 GA Nijmegen, The Netherlands.
Present address: AMT, Meibergdreef 61, 1105 BA Amsterdam, The Netherlands.
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INTRODUCTION |
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PRRSV neutralization is correlated with antibodies directed against the GP5 protein, both in vivo (Gonin et al., 1999; Kwang et al., 1999
; Pirzadeh & Dea, 1998
; Yoon et al., 1995
) and in vitro (Pirzadeh & Dea, 1997
; Weiland et al., 1999
; Yang et al., 2000
; Zhang et al., 1998
). Monoclonal antibodies (mAbs) against the GP4 protein have also been found to be neutralizing (Meulenberg et al., 1997
), although mAbs against the GP5 protein appeared to be much more effective (Weiland et al., 1999
).
The GP5 protein is a glycoprotein of approximately 200 amino acids with an apparent molecular mass of 25 kDa. The GP5 protein is the most variable protein among PRRSV isolates, with only 5155 % amino acid sequence identity between European and North American isolates (Indik et al., 2000; Kapur et al., 1996
; Mardassi et al., 1995
), with the largest differences observed in the N terminus. Despite these differences, the hydropathy profiles of the GP5 proteins of the American and European isolates are very similar. For North American strains, it has been demonstrated that the GP5 protein is present as part of a disulphide-linked heterodimer with the M protein in the virion (Mardassi et al., 1996
). The N terminus of the GP5 protein contains a predicted signal peptide of about 32 amino acids according to the prediction of von Heijne (1986)
, which is followed by a hydrophilic stretch of about 40 amino acids. This domain contains two or three potential N-linked glycosylation sites at residues 37, 46 and 53 (Indik et al., 2000
; Meulenberg et al., 1995
; Stadejek et al., 2002
) and a highly conserved core sequence (aa 3855) potentially involved in heterodimer formation with the M protein (Verheije et al., 2002
). This N-terminal domain is presumed to be exposed on the outside of the virion and is therefore designated the ectodomain. The ectodomain is followed by a long hydrophobic region of about 60 amino acids that is presumed to span the membrane either one or three times. Whether the N-terminal ectodomain constitutes the only exposed part of the protein or whether a second ectodomain positioned more C-terminally exists is presently unclear (Stadejek et al., 2002
). The last 70 C-terminal amino acids are thought to form the endodomain (Meulenberg et al., 1995
). The GP5 protein, possibly as a heterodimeric complex with the M protein, is presumed to play a role in attachment to host-cell receptors (Dea et al., 2000
; Delputte et al., 2002
; Snijder & Meulenberg, 1998
) and in virus assembly (Verheije et al., 2003).
PRRSV GP5 is the homologue of the GL protein of EAV and the VP-3P protein of LDV. For EAV and LDV, neutralizing epitopes have been mapped to the ectodomain of their respective GP5 homologues (Balasuriya et al., 1995; Chirnside et al., 1995
; Glaser et al., 1995
; Li et al., 1998
). Furthermore, disruption of the disulphide bonds between the GP5 and the M protein of LDV resulted in loss of viral infectivity, suggesting that the heterodimers are involved in receptor binding (Faaberg et al., 1995
). In addition, the ectodomain of the LDV GP5 protein has been reported to be involved in LDV persistence and pathogenicity (Chen et al., 2000
). On the other hand, the importance of GP5 in receptor binding, at least for EAV, has been questioned (Dobbe et al., 2001
).
The aim of this study was to determine the location of a neutralizing epitope in the GP5 protein of European type PRRSV. Neutralizing mAbs described by Weiland et al. (1999) were found to be specific for a plaque-purified virus (PPV) subpopulation of the Dutch PRRSV isolate I-10. Hence the nucleotide sequence of the ORF5 gene of PPV was determined and compared with that of PRRSV LV, which does not react with these mAbs. Site-directed mutagenesis was used to identify the amino acid residues essential for recognition of the native protein by the neutralizing mAbs. Pepscan analysis further defined the boundaries of the epitope bound by these mAbs.
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METHODS |
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Monoclonal antibodies and antisera.
Three mAbs, P10/a46, P10/b38 and P4/a2-19, directed against the GP5 protein of the PPV strain, belong to a set of 15 mAbs that all react against the same antigenic region on the GP5 protein (Weiland et al., 1999). Monoclonal antibody 3AH9 was raised against aa 170201 of European type PRRSV (Rodriguez et al., 2001
). Peptide serum p703 was raised against an LV-specific peptide consisting of aa 145161 of the GP5 protein (Meulenberg et al., 1995
).
Construction of mutant ORF5 genes in the pCIneo mammalian expression vector.
Plasmid p5a6 containing the ORF5 gene of the PRRSV PPV strain (Conzelmann et al., 1993) was generously provided by K. Conzelmann and has been described previously (Weiland et al., 1999
). The ORF5 sequences of pABV437, the PacI mutant of the genome-length cDNA clone of LV (Meulenberg et al., 1998
), and of plasmid p5a6 were amplified using oligonucleotides LV275 and LV282, located upstream and downstream of ORF5, respectively (Table 1
). The nucleotide sequence directly upstream of the start codon of ORF5 was modified to a consensus Kozak sequence (Kozak, 1987
). In addition, the restriction sites XbaI and NotI were added upstream and downstream of ORF5, respectively. The PCR fragments were digested with XbaI and NotI and ligated into the corresponding sites of the pCIneo mammalian expression vector (Promega). This resulted in plasmids pABV786 and pABV789 containing the ORF5 genes of LV and p5a6, respectively.
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The fourth difference between both ORF5 genes, codon 158 (LysArg), was introduced in pABV786 by exchange of a XcmIBlpI fragment between plasmids pABV786 and pABV789. This resulted in construct pABV806.
Construction of a full-length neutralization-sensitive PRRSV cDNA clone.
In order to introduce a proline at aa 24 into pABV437, a fusion PCR was performed with primers LV46 and LV286 and with primers LV285 and LV107, respectively. The mutated fragments were then digested with BstXI and NheI and ligated into the similarly digested plasmid pABV651, a cDNA clone encompassing the structural genes of PRRSV LV. From the resulting clones, the AatIIHpaI fragment was excised and introduced into pABV437, resulting in full-length construct pABV911. Plasmid constructs were amplified and purified using the Qiagen plasmid mini kit. Recombinant DNA techniques were performed essentially as described by Sambrook et al. (1989).
Sequence analysis.
Fragments generated by PCR were analysed by nucleotide sequencing. Sequences were determined with the PRISM Ready Dye Deoxy Terminator cycle sequencing kit and the ABI PRISM 310 Genetic Analyser (Perkin Elmer).
DNA transfection.
BHK-21 cells seeded in 24-well plates were transfected with plasmids using Lipofectamine (Gibco BRL). Transfection mix was removed after 4 h and replaced with complete BHK-21 medium, and cells were incubated for another 20 h at 37 °C in a CO2 incubator.
Production of infectious virus from full-length genomic cDNA clones.
To obtain infectious virus, the full-length genomic cDNA clones pABV437 and pABV911 were transcribed in vitro. The RNAs were then transfected into BHK-21 cells using Lipofectin (Gibco BRL). The culture supernatant of BHK-21 cells was harvested 24 h after transfection and the supernatant was subsequently used to inoculate PAMs. After 1 h, the inoculum was removed and fresh culture medium was added. At 24 h post-infection (p.i.), the supernatant was harvested and virus titres (expressed as TCID50 ml-1) were determined on PAMs.
Virus neutralization assay.
The sensitivity of viruses vABV437 and vABV911 to neutralization was determined in an immunoperoxidase monolayer assay (IPMA) 24 h p.i. PAMs were plated into 96-well microtitre plates and incubated overnight at 37 °C. To determine the neutralization index, twofold serial dilutions of culture supernatant of mAb P10/a46 were mixed with 100 TCID50 of the viruses, incubated at 37 °C for 1 h and subsequently transferred to the PAMs. At 24 h p.i., cells were washed with PBS, dried and stored at -20 °C until an IPMA was performed. Monoclonal antibody 122.17 was used to detect the expression of the PRRSV N protein.
Immunoperoxidase monolayer assay.
Cells were fixed with cold 96 % methanol for 25 min and the GP5 protein expressed by the various ORF5 constructs was detected with mAb P10/a46, P10/b38 or P4/a2-19 in an IPMA as described by Wensvoort et al. (1986).
Radioimmunoprecipitation.
Peptide serum 703 (p703) and mAb P10/a46 were used to precipitate Tran35S-labelled GP5 proteins from lysates of BHK-21 cells transfected with the different ORF5 constructs. BHK-21 cells transfected with the pCIneo vector were used as negative control. Cellular proteins were labelled with Tran35S label in MEM-E without methionine and cysteine (Gibco BRL), supplemented with 1 % L-glutamine, 100 U penicillin ml-1 and 100 U streptomycin ml-1 for 4 h after starving with the same medium without label for 30 min. Cells were lysed in PBS containing 1 g SDS l-1, 10 ml Triton X-100 l-1 and 5 g sodium desoxycholate l-1 (PBS-TDS) for 10 min on ice. Cell lysates were mixed with either an equal volume of hybridoma supernatant or 15 µl of p703 and incubated overnight at 4 °C. Subsequently, protein ASepharose (Amersham Pharmacia Biotech) was added and lysates were incubated at 4 °C for another 2 h. Immunoprecipitates were then washed with PBS-TDS, resuspended in loading buffer and samples analysed by 14 % SDS-PAGE. Gels were dried and immunoprecipitated proteins were visualized by autoradiography.
Pepscan analysis.
Pepscan analysis was performed using overlapping 12-mer peptides as described in Slootstra et al. (1997). Optical densities (OD) were determined with a ccd-camera. The values are logarithmic values in a range of 0 to 4000.
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RESULTS |
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In conclusion, a proline residue at position 24 enables recognition of a neutralization epitope that is located in the N-terminal ectodomain.
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DISCUSSION |
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Remarkably, the reactivity of the three neutralizing mAbs in the Pepscan system was independent of the presence of a proline at position 24, since the set of 12-mers containing a Cys24 and the 12-mers containing a Pro24 gave comparable OD values in the Pepscan analysis. Moreover, residue 24 is located within the signal peptide predicted by the SignalP computer algorithm (Nielsen et al., 1997). Several authors have reported that signal peptide cleavage is predicted to occur between aa 32 and 33 (Meulenberg et al., 1995
; Rodriguez et al., 2001
), i.e. in the core of the epitope. In view of our results, however, it is more likely either that the signal peptide is not cleaved off and the amino acid residue at position 24 is itself part of the epitope in the native protein, or that signal peptide cleavage occurs more N-terminally.
Examples of non-cleaved predicted signal peptides exist (Gewurz et al., 2002). Assuming that the signal peptide of PRRSV GP5 is not cleaved, both residue 24 and the part of the epitope recognized by the Pepscan analysis would be present in the GP5 protein. A proline at position 24, a helix-breaking residue, might be responsible for exposing the epitope, whereas a cysteine at position 24 might hide the epitope, possibly by forming a disulphide bond with another cysteine residue in the GP5 protein.
However, sequence data from one of the escape mutant viruses showed that the GP5 protein of this virus possessed a leucine at position 24, emphasizing the importance of the proline (E. Weiland, unpublished data). Therefore, a more likely explanation might be that the signal peptide might be cleaved but that Pro24 affects the site of this signal peptide cleavage. Nothwehr & Gordon (1989) introduced proline residues at various positions in the signal peptide of human pre(
pro)apolipoprotein A-II and observed that the site of cleavage was affected by the location of a proline, i.e. there was a tendency to maintain a distance of four to five residues between the proline and the site of cleavage. Furthermore, they reported that two or more potential cleavage sites might compete for recognition by the signal peptidase, although one site is preferred. Using the SignalP computer algorithm for both the Cys24 and the Pro24 GP5 proteins, probable cleavage sites are located between residues 28 and 29, 30 and 31, and 32 and 33 (Fig. 1B
). In the Cys24 protein, the preferred cleavage site is located between residues 32 and 33. In the Pro24 protein, however, the proline promotes the signal peptidase to cleave between residues 28 and 29. This would result in a mature glycoprotein that was two to three amino acids larger and thus the presence of the epitope in the Pro24 protein, in contrast to the Cys24 protein. Obviously, N-terminal sequencing of both mature glycoproteins should give the definitive answer.
Our data indicated that the identified neutralization epitope is located at the N terminus of the GP5 ectodomain, comprising residues 2935 (Fig. 1B). The presence of both linear and conformation-dependent neutralization epitopes in the GP5 protein of PRRSV has been described by others, although their locations in the protein have not been determined (reviewed by Dea et al., 2000
). Recently, both a non-neutralization and a neutralization epitope located at the N terminus of the GP5 of North American PRRSV strains were identified by Ostrowski et al. (2002)
, comprising residues 2731 and residues 3745, respectively. The neutralization epitope is located in an area that is conserved among PRRSV isolates (Fig. 1B
) and between PRRSV and LDV. The VP-3P protein of LDV also contains a neutralization epitope in its ectodomain that is mapped between residues 37 and 60 (Li et al., 1998
). Interestingly, this epitope comprises the residues that form the main recognition site in the epitope described by Ostrowski et al. (2002)
. The ectodomain of the EAV ORF5 protein, GL, is much longer and shows far less homology with the LDV and PRRSV GP5 ectodomains. Nevertheless, it also contains three overlapping neutralization epitopes, just upstream of the first transmembrane segment (Balasuriya et al., 1995
; Chirnside et al., 1995
; Glaser et al., 1995
). The presence of neutralizing epitopes in the GP5 ectodomains of PRRSV, LDV and EAV leads to the suggestion that the GP5 ectodomains play an important role in arterivirus infection. Another argument that pleads for such a role of the GP5 ectodomain is the fact that the disulphide bonds between the LDV GP5 and M protein are essential for LDV infectivity, suggesting that the heterodimeric GP5M complexes might be involved in receptor binding (Faaberg et al., 1995
). In addition, N-glycans associated with the ectodomain of the LDV GP5 protein determine LDV neuropathogenicity and sensitivity to antibody neutralization (Chen et al., 2000
).
However, the strongest argument against such a role for GP5 is that the exchange of the putative ectodomain of the EAV equivalent of GP5 with that of PRRSV GP5 does not alter its tropism (Dobbe et al., 2001). Moreover, recent research in which the ectodomain of the M protein of PRRSV was replaced by that of other arteriviruses revealed that the tropism of the resulting chimeric viruses had remained unchanged (Verheije et al., 2002
). These authors concluded that the heterodimeric GP5M complexes are essential for arterivirus assembly, but that they do not determine host-cell specificity.
To explain these contradictory results, the role of GP5 in PRRSV infection of PAMs should be further investigated. Recently, Delputte et al. (2002) reported that PRRSV binds to glycosaminoglycans at the cell surface of PAMs and that this binding is probably mediated by the heterodimeric GP5M complexes. These complexes might thus initiate PRRSV infection of PAMs by attachment to a low-affinity receptor, followed by interaction of other PRRSV glycoprotein(s) with a high-affinity receptor that determines tropism. Our data suggest that neutralizing antibodies that recognize the ectodomain of GP5 may interfere with this initial step of the PRRSV infection. It is unlikely that the neutralization epitope identified in this study plays an important role in PRRSV infection, because the sequence is variable (Stadejek et al., 2002
) and it is easily lost from the population (Weiland et al., 1999
). The neighbouring highly conserved region, however, containing the neutralization epitope identified by Ostrowski et al. (2002)
and implicated in heterodimerization with the M protein (Verheije et al., 2002), might play a role in PRRSV infection. Further studies will be necessary to clarify the role of GP5 in PRRSV infection in more detail.
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ACKNOWLEDGEMENTS |
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Received 6 November 2002;
accepted 18 February 2003.