Foot-and-mouth disease virus can utilize the C-terminal extension of coxsackievirus A9 VP1 for cell infection

Martina Leippert1 and Eberhard Pfaff1

Federal Research Centre for Animal Virus Diseases, Paul-Ehrlich-Strasse 28, D-72076 Tübingen, Germany1

Author for correspondence: Eberhard Pfaff. Fax +49 7071 967 303. e-mail eberhard.pfaff{at}tue.bfav.de


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
Foot-and-mouth disease virus (FMDV) is known to employ the conserved Arg–Gly–Asp (RGD) tripeptide located on the variable {beta}G–{beta}H loop of the VP1 capsid protein for binding to cells. Coxsackievirus A9 (CAV9) also carries an RGD sequence, but on a short C-terminal extension of its VP1 and in a different amino acid context. This apparent relationship raised the question of whether insertion of the heterologous CAV9 sequence into FMDV would influence infection by the genetically modified FMDV. Four VP1 mutants were generated by PCR mutagenesis of a full-length FMDV cDNA plasmid. After transfection of BHK-21 cells, viral protein synthesis and virus particle formation could be detected. Two of the four mutants, mV9b and mV9d, could be propagated in BHK-21 cells, but not in CV-1 cells. Both of these mutants contained 17 amino acids of the C terminus of CAV9 VP1. Infection of BHK cells could be specifically inhibited by rabbit immune serum raised against a synthetic peptide representing the amino acid sequence of the C-terminal extension of CAV9 VP1. This demonstrated the direct involvement of the inserted sequence in cell infection. In fact, genetically modified FMDV O1K was capable of employing the VP1 C-terminal RGD region of CAV9 for infection of BHK cells. In addition, these results show that, even in cell culture-adapted viruses, the RGD-containing {beta}G–{beta}H loop plays an important role in virus infectivity.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
Attachment of virus to target cells is the first step of virus infection. This specific interaction is responsible for the host range and tissue tropism of viruses. Various cell surface molecules, including proteins, carbohydrates and lipids, can serve as virus receptors. Binding to cells can be a single- or multi-step process, requiring one or more accessory molecules. Among the viruses of the Picornaviridae (Bachrach, 1977 ; Rueckert & Wimmer, 1984 ; Sangar, 1979 ; Stanway & Hyypiä, 1999 ), a variety of cell surface molecules are involved in cell entry (Evans & Almond, 1998 ). Rhinoviruses use the intracellular adhesion molecule type 1 (ICAM-1; major group) (Greve et al., 1989 ; Staunton et al., 1989 ) or low-density lipoprotein (LDL; minor group) (Hofer et al., 1994 ). Cardioviruses (encephalomyocarditis virus) employ the vascular cellular adhesion molecule 1 (VCAM-1) (Huber, 1994 ), while enteroviruses, which are the most heterogeneous group, can use the poliovirus receptor (PVR; PVR 1–3) (Mendelsohn et al., 1989 ), decay-accelerating factor (DAF) (Bergelson et al., 1995 ; Karnauchow et al., 1996 ; Powell et al., 1998 ; Shafren et al., 1995 , 1997 ; Ward et al., 1994 ) and the coxsackievirus–adenovirus receptor (CAR) (Bergelson et al., 1997 ; Tomko et al., 1997 ). With the exception of aphthoviruses, receptor binding occurs at the bottom of so-called ‘canyons’ on the virus surface (Colonno et al., 1988 ; Hogle et al., 1985 ; Luo et al., 1987 ; Rossmann et al., 1985 ). There are no canyons on the virus capsid surface of foot-and-mouth disease virus (FMDV), the only member of the genus Aphthovirus. Binding to target cells is mediated by a conserved Arg–Gly–Asp (RGD) tripeptide located on the protruding flexible {beta}G–{beta}H loop of the capsid protein VP1 (Acharya et al., 1989 ; Fox et al., 1989 ; Logan et al., 1993 ). This relates to adhesive glycoproteins such as fibronectin and vitronectin, which contain RGD motifs, recognition of which is through RGD-binding cell receptors called integrins. These heterodimers are composed of an {alpha}- and a {beta}-subunit, of which 16 {alpha}- and eight {beta}-subunits are known, combining to form 21 receptors for different ligands (Hynes, 1992 ; Pierschbacher & Ruoslahti, 1987 ; Ruoslahti & Pierschbacher, 1987 ; Tamkun et al., 1986 ; Yamada & Olden, 1978 ). FMDV interacts with the RGD-binding integrin {alpha}v{beta}3 (vitronectin receptor) (Neff et al., 1998 , 2000 ).

Echoviruses 22 and 23, recently assigned to the genus Parechovirus (Stanway et al., 1994 ; Stanway & Hyypiä, 1999 ), echovirus 9 strain Barty (Zimmermann et al., 1996 ) and coxsackievirus A9 (CAV9) (Chang et al., 1992 ) also possess an RGD motif known to play a role in cell attachment and infection (Fig. 1). The motif is a C-terminal extension of the VP1 of these viruses. As shown for CAV9, the RGD region can bind to the vitronectin receptor ({alpha}v{beta}3) (Roivainen et al., 1991 , 1994 ) and therefore serves as an additional receptor-binding site.



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Fig. 1. Amino acid sequences of the C termini of VP1 from different enteroviruses and the loop region of FMDV type O1K. Dashes represent gaps included to optimize the alignment of the sequences. Amino acid positions of FMDV type O1K VP1 are indicated below. Abbreviations: CAV, coxsackievirus A; CBV, coxsackievirus B; PV, poliovirus; Echo, echovirus; O1K, FMDV O1Kaufbeuren.

 
In previous work, we investigated the influence on virus growth and infectivity of single amino acid exchanges within the RGD region of FMDV type O1K. Almost all of the amino acid exchanges concerning the RGD tripeptide generated non-infectious virus particles. Certain flanking regions were also found to be involved in cell infection. The present work sought to determine the influence of an exchange of the heterologous RGD region in the C-terminal extension of CAV9 VP1 for that of the {beta}G–{beta}H loop of FMDV in the infectious process of the latter. It was noted that such an exchange retained virus infectivity, but the specificity was now for the inserted CAV9 sequence.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Cell line and plasmids.
Baby hamster kidney (BHK-21), African green monkey kidney (CV-1), human cervix carcinoma (HeLa) and human rhabdomyosarcoma (RD) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1 mM glutamine and 10% (v/v) foetal calf serum in a humidified atmosphere of 5% (v/v) CO2 at 37 °C. The full-length cDNA clone pSPffPolyC (Zibert et al., 1990 ), which represents the complete genome of FMDV type O1K (Forss et al., 1984 ), was obtained from E. Beck (Giessen, Germany). The plasmid pSFV-P1, which contains the P1-coding region (structural proteins) of FMDV type O1K, was used for in vitro transcription and subsequent co-transfections of BHK-21 cells. For subcloning of PCR products, a modified pEMBL-19 vector (pEMBL-P1) was used. The plasmids pSFV-P1 and pEMBL-P1 have been described previously (Leippert et al., 1997 ).

{blacksquare} Construction of virus mutants.
Transcription of the full-length cDNA clone pSPffPolyC and subsequent transfection of BHK-21 cells with the corresponding RNA results in infectious FMDV particles (termed ‘Vpsp’). In order to create recombinant mutated virus particles (mV), synthetic oligonucleotides (Table 1) were used in a PCR to amplify the desired sequences (Ehrlich, 1989 ). The resulting PCR products, representing the C-terminal extension of CAV9 VP1, were cloned into the full-length cDNA plasmid pSPffPolyC, as described previously (Leippert et al., 1997 ). The mutated plasmids were transcribed in vitro and the resulting RNA was transferred into BHK-21 cells by electroporation. Co-transfection of cells was carried out together with an in vitro-transcribed RNA derived from the P1-coding plasmid pSFV-P1, as described previously (Leippert et al., 1997 ).


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Table 1. Primers used in PCR mutagenesis

 
{blacksquare} Detection of viral protein synthesis and electron microscopy of mutated virus particles.
Viral protein synthesis was demonstrated by indirect immunofluorescence (Harlow & Lane, 1988 ) 4 h after electroporation of BHK cells by using an FMDV VP1-specific monoclonal antibody (MAb 37; Pfaff et al., 1989 ). The existence of virus particles was shown by electron microscopy, as described previously (Leippert et al., 1997 ).

{blacksquare} Sequence determination of mutated viral RNA.
Infectious virus mutants were grown on BHK-21 cells for 2–3 h. Total cellular RNA was isolated (Chomczynski & Sacchi, 1987 ) and the viral P1-coding sequence was amplified by RT–PCR for subsequent sequence analysis. In addition, the virus mutants were plaque-purified. Sequence analysis of each of eight plaque-purified virus populations was carried out as described above.

{blacksquare} Rabbit immune sera and MAbs.
Two peptides representing the C terminus of CAV9 VP1 were synthesized by using a Tecan RSP 5000 peptide synthesizer. The amino acid sequences of the synthetic peptides are KVPVAQSRRRGDMSTLPRS (P1) and KNTVTTVAQSRRRGDMSTRS (P2). Prior to immunization, the peptides were coupled to keyhole limpet haemocyanin (Pfaff et al., 1982 ). Rabbits were immunized three times, each time with 400 µg conjugated peptide (equivalent to 100 µg uncoupled peptide) in Freund’s complete (first injection) or incomplete (subsequent injections) adjuvant, at intervals of 2 weeks. Rabbit antisera (rP1, rP2) were further tested in Western and slot-blot analyses. MAbs 37 and 99 have been described previously (Pfaff et al., 1988 , 1989 ).

{blacksquare} Western blotting.
The capsid proteins of cDNA-derived infectious virus particles (wild-type Vpsp and mutants mV9b and mV9d) were separated on a 15% (w/v) SDS–polyacrylamide gel (Laemmli, 1970 ) and transferred to a nitrocellulose membrane by the semi-dry technique (Trans-Blot SD electrophoretic transfer cell, Bio-Rad). Immunological detection of the proteins was done as described by Towbin et al. (1979) with modifications. Briefly, the nitrocellulose membrane was saturated with 2% (w/v) BSA in Tris-buffered saline (TBS; 10 mM Tris–HCl, 150 mM NaCl, pH 7·6) at room temperature, followed by incubation with rabbit antiserum diluted 1:1000 with 0·2% (w/v) BSA in TBS or with MAb 99 (1:10) for 2 h. The appropriate alkaline phosphatase-conjugated second antibody was bound for 1 h and the colour reaction was developed by using BCIP substrate containing NBT.

{blacksquare} Slot blot with synthetic peptides.
Aliquots (5, 1 and 0·5 µg) of each peptide were bound to a nitrocellulose filter and washed with TBS. Blocking and subsequent treatment were done as described above for the Western blotting.

{blacksquare} Plaque-reduction assay.
Dilutions of Vpsp and the mutated virus preparations containing about 100 p.f.u. were mixed with serial dilutions (1:4, 1:8, 1:16, 1:32, 1:64 and 1:128) of heat-inactivated rabbit immune serum, pre-immune serum, MAb 99 or a non-FMDV-reactive MAb for 30 min at room temperature, with gentle agitation. Confluent BHK-21 cell monolayers grown in 6-well plates were washed with DMEM and then incubated with the virus–antibody dilutions. After 30 min at 37 °C, the inoculum was removed and the cells were washed once. The cells were overlaid with 2% (w/v) Sephadex G200 in DMEM and incubated for 30–48 h. Following fixation with 5% (v/v) formaldehyde, the cells were stained with GIEMSA solution and plaques were counted. Plaque reduction was determined by comparing the results of the immune serum- or MAb 99-treated samples with those for the pre-immune serum-or non-FMDV-reactive MAb-treated samples.

{blacksquare} Plaque assay with different cell lines.
Viruses were propagated on BHK-21 cells. Cell debris was removed and dilutions of the clarified lysates (10-1–10-7) were added to 6-well plates containing confluent monolayers of BHK-21, CV-1, HeLa or RD cells. After incubation for 30 min at 37 °C, the cells were treated as described for the plaque-reduction assay.


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Generation of mutated virus particles
Four different virus mutants were constructed by utilizing the full-length cDNA plasmid pSPffPolyC. Fig. 2 shows the amino acid sequences of the {beta}G–{beta}H loops of the virus mutants and the FMDV O1K wild-type sequence. The CAV9-derived sequences VAQSRRRGDMST (12 amino acids) and NTVTTVAQSRRRGDMST (17 amino acids), which represent the C-terminal extension of CAV9 VP1, were inserted in two different sites within the {beta}G–{beta}H loop of type O1K VP1. As shown in Fig. 2, the insertion sites are located between amino acids 142–151 and 142–159, resulting in mutants mV9a and mV9c for the shorter, 12 amino acid sequence and mutants mV9b and mV9d for the 17 amino acid sequence. The in vitro-transcribed RNAs of the recombined full-length cDNA plasmids were transferred into BHK-21 cells by electroporation. Viral intracellular protein synthesis could be detected by indirect immunofluorescence after 4 h. In addition, virus particles could be isolated from cell culture supernatants with all four mutants 24 h after electroporation, as shown by electron microscopy (data not shown). However, no virus propagation could be observed after transfer of cell culture supernatant to BHK-21 cells, except for the wild-type Vpsp. Despite electroporation of ten times the amount of the mutated RNA, no virus propagation took place.



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Fig. 2. Amino acid sequences of the {beta}G–{beta}H loop of FMDV strain O1K and the derived mutants. The O1K sequence is shown twice, with the amino acid positions indicated above. For the mutants, only the inserted CAV9 sequences are shown. The insertion site for mutants mV9a and mV9b is between positions 142 and 151; for mutants mV9c and mV9d, it is between positions 142 and 159. The lengths of the mutated {beta}G–{beta}H loops compared with the wild-type virus are: mV9a, +4 aa; mV9b, +9 aa; mV9c, -4 aa; mV9d, +1 aa.

 
In order to overcome this problem, a similar experiment was performed with BHK cells co-transfected with both the in vitro-transcribed recombined RNA and an in vitro-transcribed Semliki Forest virus (SFV) RNA encoding the O1K wild-type P1 only. Supernatants from electroporated cells were transferred to BHK cell monolayers, as a result of which, virus proliferation of the two mutants mV9b and mV9d was observed. Both mutants possess the 17 amino acid C-terminal RGD sequence of CAV9. In contrast, mutants mV9a and mV9c did not result in any plaque formation or cytopathic effect, even at 96 h after infection. The mutants mV9a and mV9c possess the shorter CAV9 C-terminal RGD sequence of 12 amino acids, lacking the 5'-terminal amino acids NTVTT. This indicates a role for these 5'-terminal amino acids during infection of the cells.

Sequence analyses of the infectious mutants after several passages
The virus mutants mV9b and mV9d were passaged six times on BHK cells. After isolation of total RNA from infected cells, RT–PCR was performed. Subsequent sequencing analysis confirmed that the CAV9-derived RGD sequences were present in both mutants. These results showed that no recombination event had taken place after co-transfection of the in vitro-transcribed recombined RNA together with the wild-type P1 coding RNA and that the infectivity of the mutants was preserved.

Nevertheless, several point mutations could be detected in different parts of the capsid protein, as shown in Fig. 3. With mV9b, three mutations resulting in amino acid exchanges were noted. The first was at position 6 of the inserted CAV9 sequence (Val->Glu). Two additional amino acid exchanges were found in the capsid protein VP2, at positions 134 (Lys->Asn) and 173 (Val->Leu). The latter Val->Leu exchange was also be detected with mutant mV9d, which had no other amino acid exchanges up to that point.



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Fig. 3. Mutations in the capsid protein P1 of the mutants mV9b and mV9d after propagation on BHK cells. In mutant mV9b, two amino acid exchanges occurred in the capsid protein VP2, at position 134 from Lys to Asn and at position 173 from Val to Leu (nt 2365 and 2480); a third exchange occurred in VP1, at position 6 of the recombinant CAV9 sequence, from Val to Glu. Mutant mV9d showed one amino acid exchange in VP2, at position 173, the same as for mutant mV9b. The second mutation in VP1, at position 10 of the recombinant CAV9 sequence from Arg to Gly, was detected later after plaque purification.

 
Sequence analyses of plaque-purified virus mutants
In order to analyse the mutations in detail, virus mutants mV9b and mV9d were plaque-purified after seven passages on BHK cells. Total RNA was isolated and RT–PCR was performed. Subsequent sequencing analyses of eight plaque-purified virus populations confirmed the CAV9 insertions in all cases. In addition, the single mutations described above were present.

Interestingly, a new mutation (Arg->Gly) could be detected in mV9d at position 10 of the inserted CAV9 sequence (Fig. 3). Of the eight plaque-purified populations, only one had the original triplet encoding arginine (AGA), four were mixed populations and, in three of them, the glycine-coding triplet (GGA) was clearly predominant. This points to a development process of mV9d resulting in increases in stability and fitness during further cell culture passages.

Susceptibility of different cell lines to the virus mutants
CV-1, HeLa, RD and BHK-21 cells were tested for their susceptibility to infection by the two virus mutants mV9b and mV9d and the wild-type Vpsp in a plaque assay. Each of these cell lines was infected with serial virus dilutions and incubated for at least 30 h. As shown in Fig. 4, only BHK cells were susceptible to mV9b, mV9d and Vpsp. In addition, no significant differences in progeny titre were observed between the viruses. Thus, no change in cell tropism was noted for the mutated viruses. Nevertheless, the virus mutants showed a different plaque morphology on BHK cells. Compared with the <=3 mm plaque diameter for the wild-type Vpsp, plaques produced by mV9b were up to 5 mm in diameter, whereas mV9d produced plaques that were never larger than 1·5 mm in diameter (Fig. 4).



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Fig. 4. Plaque assay with BHK-21 and CV-1 cells. cDNA-derived Vpsp and the mutants mV9b and mV9d were titrated on confluent cell monolayers in 6-well plates. At 30 h after infection, cells were stained and plaques were counted. Only BHK cells could be infected with Vpsp, mV9b and mV9d; there was no significant difference in virus titre. CV-1 cells (as well as HeLa and RD cells; data not shown) were not susceptible. On BHK cells, the plaque sizes of the mutants were different when compared with Vpsp, ranging up to 5 mm in diameter for mV9b and up to 1·5 mm for mV9d, in comparison with up to 3 mm in diameter for Vpsp.

 
Inhibition of virus infection by specific rabbit antisera
Rabbit antisera (rP1, rP2) were raised against two synthetic peptides, P1 and P2, representing the amino acid sequence of the C-terminal extension of CAV9 VP1 (see Methods). The binding characteristics of the antisera were first tested by slot-blot analysis with the peptides used for immunization (P1 and P2), as well as with a synthetic peptide containing residues 142–151 of the FMDV O1K {beta}G–{beta}H loop and a synthetic RGD peptide known to inhibit cell attachment to fibronectin and vitronectin. As shown in Fig. 5, the antisera rP1 and rP2 recognized only the corresponding synthetic peptide. No cross-reaction was found, even though 12 of the 17 amino acids were identical between the two peptides. These antisera did not react with any of the other peptides tested.



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Fig. 5. Slot-blot assay with synthetic peptides. Rabbit antisera rP1 and rP2 were raised against the peptides P1 and P2. Both sera were tested in a slot-blot assay against additional synthetic peptides, representing different RGD regions. The peptides were: 1, 2, peptides P1 and P2; 3, {beta}G–{beta}H loop residues 142–151 of FMDV type O1K; 4, competitive peptide that inhibits cell attachment to fibronectin and vitronectin; 5, control peptide. The rabbit antisera recognized only the specific CAV9 peptides against which they were raised.

 
A closer examination of the peptide–antisera reactions used Western blot analysis. As shown in Fig. 6, the capsid protein VP1 of the mutants mV9b and mV9d could be detected by the rabbit immune serum rP2, whereas the wild-type VP1 of Vpsp was not detectable. Furthermore, MAb 99, directed against the RGD sequence of FMDV type O1K, reacted only with the VP1 of Vpsp, and not with the VP1 of the mutants. Thus, these different antibodies allowed us to distinguish between mutated and wild-type VP1. Corresponding to the slot-blot results, the immune serum rP1, directed against the synthetic peptide representing 12 amino acids of CAV9 VP1, did not react with any VP1.



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Fig. 6. Western blot analysis. Capsid proteins of cDNA-derived virus particles of the wild-type Vpsp (wt) and the mutants mV9b (9b) and mV9d (9d) were separated by 15% (w/v) SDS–PAGE and transferred to nitrocellulose. MAb 99, directed against the RGD region (144–159) of FMDV type O1K (Pfaff et al., 1988 ), detected only the VP1 of Vpsp and not that of the mutants. The rabbit immune serum rP2, raised against the CAV9 analogous synthetic peptide P2, detected only the mutated VP1 and not the wt VP1. The different molecular mass of mV9b VP1 is due to the extension of the {beta}G–{beta}H loop by 9 amino acids compared with the wild-type. MAb 37, directed against FMDV O1K (Pfaff et al., 1989 ), recognized both wt and mutant VP1. No reaction was seen with the pre-immune serum (preP2) or with the rabbit immune serum rP1 raised against the synthetic peptide P1 (not shown).

 
In order to elaborate further on the consequences of the CAV9 VP1 insertion, the rabbit immune sera rP1 and rP2 were tested for their potential to inhibit virus infection of BHK cells. Fig. 7 shows that immune serum rP2 gave a clear reduction in numbers of plaques in a plaque-reduction assay, indicating an inhibition of infection by the mutants mV9b and mV9d. The inhibitory effect was dose dependent and decreased with increasing serum dilution. Immune serum rP2 had no inhibitory effect on the cDNA-derived wild-type Vpsp, indicating that the RGD sequence was not recognized in a different amino acid context. This was confirmed with MAb 99, which could reduce the number of plaques of Vpsp only and not of the two mutants. Immune serum rP1, raised against the shorter synthetic peptide, had no inhibitory effect against any of the viruses.



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Fig. 7. Plaque-reduction assay. Virus particles of the mutants mV9b (a) and mV9d (b) and the cDNA-derived wild-type Vpsp (c) were incubated with rabbit immune sera rP1 ({blacksquare}) and rP2 ({bullet}) and with MAb 99 ({blacktriangleup}). The samples were applied to confluent BHK cells and plaque reduction was determined after 30 h. With immune serum rP2, infection of cells with both mutants mV9b and mV9d could be clearly inhibited. At a serum dilution of 1:64, plaque reduction was still over 50%, whereas no plaque reduction could be observed with Vpsp. On the other hand, MAb 99 blocked infection by Vpsp but not by the virus mutants. The immune serum rP1 had no inhibitory effect on the mutants or Vpsp.

 

   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
FMDV binds to target cells using the conserved tripeptide RGD, which is located on the {beta}G–{beta}H loop of the capsid protein VP1. Mutations within the RGD motif and adjacent regions often lead to non-infectious virus particles (Mason et al., 1994 ; Leippert et al., 1997 ). This underlines the importance of the RGD sequence and neighbouring amino acid positions in virus binding to cellular integrin receptors such as {alpha}v{beta}3 (vitronectin receptor) (Neff et al., 1998 ). CAV9 is an enterovirus that replicates in the gut and which also possesses an RGD sequence, on a short C-terminal extension of VP1 (Chang et al., 1992 ). Among coxsackieviruses, this extension is unique to CAV9 and provides the possibility of binding to {alpha}v{beta}3 (Roivainen et al., 1991 , 1994 ).

The present work sought to determine the functional relationship of these RGD sequences in the two viruses. To this end, the study focused on genetically engineered FMDV containing the heterologous RGD sequence of CAV9. Although common to FMDV and CAV9, the RGD tripeptide is in a different environmental situation, yet has been shown to bind to the vitronectin receptor ({alpha}v{beta}3) in both cases (Neff et al., 1998 ; Roivainen et al., 1991 , 1994 ). Our intention in this study was to examine whether FMDV was able to use the CAV9 C-terminal extension for infection of cells and, furthermore, to study the influence of the variable amino acid sequences flanking the RGD motif. Therefore, the RGD region located in the {beta}G–{beta}H loop of FMDV O1K VP1 was replaced with the RGD-containing C-terminal extension of CAV9 VP1. Two of four recombinant virus mutants (mV9b, mV9d), containing the identical 17 amino acids from CAV9 at different insertion sites in the {beta}G–{beta}H loop, could be propagated on BHK-21 cells. In contrast, two other mutants (mV9a, mV9c), with insertions of only 12 of the CAV9 amino acids, failed to infect BHK cells.

Subsequent sequence analysis of the mutants mV9b and mV9d revealed three and one amino acid mutations, respectively, which are probably responsible for their infectivity. Proliferation of the two virus mutants was only successful after co-transfection with an in vitro-transcribed SFV P1 wild-type RNA, enabling at least one further replication cycle in which mutations could occur. The inserted CAV9 sequences were present in both virus mutants, showing that no recombination event had taken place. Furthermore, sequence analyses of plaque-purified virus populations confirmed the CAV9 insertions and the mutations described above. The newly detected mutation within the RGD insertion of mutant mV9d indicates that changes had occurred in the flanking amino acid sequence that optimized binding to the cellular receptor. Because mV9d showed a reduced plaque size when compared with the wild-type Vpsp, although the virus titres were almost the same, future examinations will focus on the relationship between these mutations and plaque size.

Mutants mV9a and mV9c failed to infect BHK cells. By comparison with the wild-type virus, the overall length of the {beta}G–{beta}H loop seemed to play only a minor role in virus infectivity. Rather, the localization and, therefore, the accessibility of the RGD triplet seemed to be more critical factors. We assume that the amino acid sequence NTVTT in the longer CAV9 insertion offers an advantage for the virus mutants mV9b and mV9d. Protein secondary structure predictions made according to Chou & Fasmann (1974) indicate that the sequence SRRRGD has an extremely hydrophilic character and does not adopt an {alpha}-helix conformation, in contrast to FMDV O1K (Logan et al., 1993 ). The introduced sequence NTVTT, present in the infectious mutants mV9b and mV9d, seems to fold into a {beta}-sheet as in FMDV O1K (Logan et al., 1993 ). The two non-infectious virus mutants mV9a and mV9c do not possess this NTVTT sequence and, therefore, would not adopt a {beta}-sheet. This conformational shift could be responsible for presenting the RGD in the right position for receptor binding.

Although the amino acid sequences flanking the RGD motifs of the mutants mV9b and mV9d were different from the original wild-type FMDV RGD region, binding to and infection of BHK cells by the mutants was possible. Despite the fact that CAV9 interacts with the vitronectin receptor {alpha}v{beta}3 (Roivainen et al., 1991 , 1994 ), BHK cells are not susceptible to infection by this virus (Fortmüller, 1997 ). Yet, the virus mutants mV9b and mV9d were equally infectious for BHK cells in comparison with the wild-type Vpsp. These results are in line with the hypothesis that additional cell surface components are necessary to mediate infection.

Because of the observation that the RGD sequence within the C-terminal extension of CAV9 VP1 interacts with the vitronectin receptor ({alpha}v{beta}3) on green monkey kidney cells (Roivainen et al., 1994 ), the susceptibility of CV-1, HeLa and RD cells to infection by the virus mutants mV9b and mV9d was tested. Both HeLa and RD cells are susceptible to CAV9, although the RGD motif is not involved in the infection (Roivainen et al., 1991 , 1996 ). As expected, the mutants failed to infect RD and HeLa cells. However, no infection occurred in CV-1 cells, despite their susceptibility to CAV9 infection (Fortmüller, 1997 ). Therefore, there was no change in cell tropism compared with the cDNA-derived wild-type Vpsp.

For functional characterization of the role of the CAV9 insertions, rabbit antisera were raised against two synthetic peptides, P1, representing 12 amino acids, and P2, representing 17 amino acids, within the VP1 C terminus of CAV9. The antisera rP1 and rP2 recognized only the corresponding peptide used for immunization and did not react with other RGD-containing synthetic peptides. As demonstrated in Western blot analyses, the capsid protein VP1 of both mutants could be detected with the immune serum rP2. Nevertheless, infection of BHK cells by the mutants mV9b and mV9d was blocked specifically by the rP2 serum but not by MAb 99, showing that the CAV9 RGD region was involved directly in the mutant FMDV infection. Plaque reduction was more efficient with mutant mV9b than with mV9d. This was probably due to the different positions of the CAV9 insertion within the {beta}G–{beta}H loop, making it more accessible to specific antibodies in mutant mV9b. Furthermore, this experiment shows that the essential role of the RGD region on the {beta}G–{beta}H loop in cell infection extends to cell culture-adapted viruses.

It seems that FMDV can tolerate variation of amino acid sequences in the virus capsid allowing it to interact with different cellular receptors. These amino acid exchanges can occur in the RGD sequence itself. The virus therefore retains the possibility of entering cells by different ways in response to environmental changes (Baranowski et al., 2000 ). There is evidence that the virus mutants were able to regain and optimize cell-receptor binding as a result of modifications in the virus capsid. Mutants mV9b and mV9d failed to infect BHK cells after transfection of RNA. However, after co-transfection with an in vitro-transcribed SFV P1 wild-type RNA, virus replication occurred several times, making a sequence modification possible. Therefore, virus binding might happen in an RGD-independent way. Nevertheless, infection of BHK cells was blocked by peptide-specific antibodies that recognized only the amino acid sequence of the CAV9 insertion. Finally, infection of cells with these mutants took place by interaction with the inserted C-terminal VP1 region from the coxsackievirus.

In summary, the present results provide evidence that genetically engineered FMDV can utilize the RGD-containing VP1 C terminus of CAV9 for cell infection. The CAV9 insertion was involved directly in cell entry, since infection could be inhibited by antisera against a synthetic peptide from the CAV9 VP1 C terminus. Since CAV9 cannot infect BHK cells, in contrast to the mutants, the susceptibility of BHK cells requires a second virus–cell interaction that is different from that used by CAV9.


   Acknowledgments
 
We thank Kenneth McCullough and W. Bodemer for careful and critical reading of the manuscript, F. Weiland for electron microscope work, Sandra Bordel and Karina Mildner for skilful technical assistance and Ulrike Fortmüller and Angelika Oehmig for helpful and stimulating discussions. This work was supported in part by EU grant PL97-3665.


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Abstract
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References
 
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Received 14 January 2000; accepted 2 March 2001.



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