Involvement of ß2-microglobulin and integrin {alpha}vß3 molecules in the coxsackievirus A9 infectious cycle

Martha Triantafilou1, Kathy Triantafilou1, Keith M. Wilson b,1, Yoshikazu Takada2, Nelson Fernandez1 and Glyn Stanway1

Department of Biological Sciences, Central Campus, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, UK1
Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037, USA2

Author for correspondence: Martha Triantafilou.Fax +44 1206 872592. e-mail mtrian{at}essex.ac.uk


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
It is becoming apparent that many viruses employ more than one cell surface molecule for their attachment and cell entry. In this study, we have tested the role of integrin {alpha}vß3 and MHC class I molecules in the coxsackievirus A9 (CAV-9) infectious cycle. Binding experiments utilizing CHO cells transfected and expressing human integrin {alpha}vß3, revealed that CAV- 9 particles were able to bind to cells, but did not initiate a productive cell infection. Antibodies specific for integrin {alpha}vß3 molecules significantly reduced CAV-9 infection in susceptible cell lines. Moreover, MAbs specific for ß2- microglobulin (ß2-m) and MHC class I molecules completely inhibited CAV-9 infection. To assess the effect of these antibodies on virus binding, we analysed CAV-9 binding by flow cytometry in the presence of ß2-m- or integrin {alpha} vß3-specific antibodies. The results showed a reduction in CAV-9 binding in the presence of integrin {alpha}vß3- specific antibodies while there was no reduction in the presence of ß2-m-specific MAb. Taken together, these data suggest that integrin {alpha}vß3 is required for CAV-9 attachment but is not sufficient for cell entry, while ß2 -m, although not directly involved in CAV-9 binding, plays a post- attachment role in the CAV-9 infectious process, possibly being involved in virus entry.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
Coxsackievirus A9 (CAV-9) is a non-enveloped RNA virus, a member of the Enterovirus genus of the family Picornaviridae. Enteroviruses are associated with a wide spectrum of clinical symptoms such as flaccid paralysis, respiratory disease and exanthema, and have been implicated in chronic dilated cardiomyopathy and juvenile-onset diabetes. CAV-9 is also one of the most frequent causes of aseptic meningitis (Grist & Reid, 1988 ).

A critical step in virus replication is the interaction of the virus with cell surface molecules on host cells. Approaches that use MAbs to screen for molecules that could play a role in the life-cycle of the virus have allowed the identification of several receptor molecules that appear on the surface of cells. For instance, ICAM-1 was identified as a receptor for 90% of rhinoviruses (Greve et al. , 1989 ) and several coxsackie A viruses (Shaffren et al. , 1997a , b ), whereas several members of the echovirus group utilize the cell surface molecule CD55 (Bergelson et al., 1994 ; Ward et al., 1994 ). Integrins are also known to function as receptors for some enteroviruses; integrin {alpha}2ß1 is a receptor molecule for echoviruses 1 and 8 (Bergelson et al., 1992 , 1993 ), whereas integrin {alpha}vß3 is recognized by the aphthovirus foot-and-mouth disease virus (FMDV) (Berinstein et al., 1995 ; Neff et al., 1998 ) and also CAV-9 (Roivainen et al., 1994 ).

Since the {alpha}v integrins are known to recognize the arginine–glycine–aspartic acid (RGD) motif (Hynes, 1992 ), this is consistent with the presence of such a motif on the G–H loop of the VP1 capsid protein of FMDV (Acharya et al. , 1989 ; Logan et al., 1993 ) and near the C-terminus of the capsid protein VP1 on CAV-9 (Chang et al. , 1989 , 1992 ). However, in contrast to FMDV, where it has been shown that the RGD motif is essential for virus interaction with its cellular receptor (Baxt & Becker, 1990 ; Fox et al., 1989 ; Mateu et al. , 1996 ; Leippert et al., 1997 ; Mason et al., 1994 ; McKenna et al., 1995 ), removal of the RGD motif from CAV-9 either enzymatically or by mutagenesis, yields viable viruses with impaired growth on GMK cells but has no effect on RD cells (Roivainen et al., 1991 , 1996 ; Hughes et al., 1995 ). This suggests that CAV-9 can either utilize other binding domains on integrin {alpha}vß3 or additional receptor molecules to infect cells.

A recent report has also suggested that ß2- microglobulin 2-m) may play a role in the infectious cycle of echoviruses, which are viruses genetically related to CAV-9, and CAV-9 itself (Ward et al., 1998 ). ß2 -m is an invariant, non-glycosylated protein of about 12 kDa that associates non-covalently with the heavy chain to form functional MHC class I molecules at the cell surface (Bjorkman et al., 1987 ; Madden et al., 1995 ).

In an attempt to gain a better understanding of CAV-9 cellular interactions and to determine the significance of ß2-m and integrin {alpha}vß3 in the CAV-9 infectious cycle, we have used MAbs directed against these molecules. The results show that CAV-9 binds to integrin {alpha}vß3 on host cells but binding is not sufficient for internalization, whereas ß2 -m, although it has no direct physical association with CAV-9, seems to play a role in a post-attachment step of the virus infectious process.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Cell lines.
The green monkey kidney cell line (GMK) was maintained in minimal essential medium (MEM) containing 1% (v/v) non-essential amino acids, 10% (v/v) heat-inactivated foetal bovine serum and 100 µg/l gentamicin.

A human rhabdomyosarcoma cell line (RD) was maintained in MEM containing 2% (v/v) non-essential amino acids, 2% (v/v) vitamin solution (Gibco), 10% (v/v) non-heat-inactivated foetal bovine serum and 100 µg/l gentamicin.

CHO-pBJ (CHO cells transfected with the empty vector pBJ) and CHO- {alpha}vß3 [CHO cells transfected with {alpha}v and ß3 cDNAs and expressing human integrin {alpha}v ß3 (Takagi et al., 1997 )] were maintained in 1:1 DMEM/F12 mix supplemented with 10% (v/v) non-heat inactivated foetal bovine serum and 100 µg/ml geneticin. All cell lines were maintained at 37 °C in a 7% CO2 atmosphere.

{blacksquare} CAV-9 plaque assay.
For the production of virus plaques, the cells were infected with virus and a plaquing overlay was used, which consisted of the appropriate medium to which 0·5% (w/v) carboxymethylcellulose was added. The CAV-9 plaque assays were also repeated without the presence of overlay. Plaques were visualized by staining with 0·2% (w/v) crystal violet in 1% (v/v) ethanol.

{blacksquare} Antibodies.
The integrin {alpha}vß3-specific MAb MCA757G which recognizes a domain between the {alpha}v and ß3 molecules, including the RGD motif recognition sequence (Horton et al., 1985 ; Davies et al., 1989 ), and the ß2-m-specific MAb MCA1115 were obtained from Serotec. The HLA-A, -B, -C-specific MAb W6/32, which recognizes a monomorphic epitope complexed by the heavy chain and ß2- m of MHC class I, was obtained from the ATCC. The integrin {alpha}v ß3-specific MAb MAB1976, which recognizes the vitronectin receptor complex of integrin {alpha}vß3 (Cheresh & Spiro, 1987 ), was obtained from Chemicon. CAV-9 neutralizing rabbit polyclonal serum was obtained from the Public Health Laboratories (Colindale, UK). FITC-conjugated rabbit anti-mouse IgG and FITC-conjugated swine anti-rabbit Ig were obtained from Dako.

{blacksquare} Virus blocking assays.
GMK and RD cells were grown as a monolayer in six-well plates (Nunc). MAbs (10 µg) were added in 1 ml serum-free media and incubated at room temperature for 50 min before the addition of approximately 250 p.f.u. CAV-9 virus particles and further incubation at room temperature for 50 min. The monolayer was washed with culture medium and overlaid with 0·5% (w/v) carboxymethylcellulose in culture medium. The incubation was continued for 48–72 h in a 7% CO2 humidified incubator before plaque visualization with crystal violet. Control plates with isotype control IgG were similarly treated.

{blacksquare} Assay of CAV-9 particles produced in cells.
CHO-{alpha}vß3, CHO-pBJ and RD cells (control), were grown in 25 cm2 flasks. CAV-9 virus particles (250 p.f.u.) were added and flasks were incubated for 1 h. Unbound virus was removed by washing three times with binding buffer (PBS supplemented with 2 mM CaCl2 and 1 mM MgCl2 ). The cell monolayers were overlaid with 0·5% (w/v) carboxymethylcellulose in culture medium and incubated for different time periods, 24, 48, 72 and 96 h. At each time period, the cells were frozen and thawed to release virus particles that may have been produced. The cell lysate produced was then added to RD cells that were assayed for the presence of virus by plaque assay.

{blacksquare} Immunofluorescence staining of integrin {alpha}vß3 and MHC class I molecules.
Cells were harvested by gentle agitation and washed with blocking buffer [PBS containing 0·02% (v/v) donor calf serum (DCS), 0·02% (w/v) NaN3]. After washing twice with PBS, cells (typically 0·5x106 per reaction) were incubated with 1 µg MCA1115, 1 µg W6/32 or 1 µg MCA757G in PBS for 1 h at 4 °C. Three washes with PBS followed. The cells were then incubated with an appropriate dilution of FITC-conjugated rabbit anti-mouse IgG for 1 h at 4 °C. Fluorescent staining was analysed by flow cytometry using a FACscan (Becton-Dickinson) counting 10000 cells per sample.

{blacksquare} Virus binding assays by flow cytometry.
Cells were harvested by gentle agitation and washed with blocking buffer [PBS containing 0·02% (v/v) DCS, 0·02% (w/v) NaN 3] at 4 °C for 15 min. CAV-9 (250 p.f.u.), diluted in binding buffer (PBS supplemented with 2 mM CaCl2 and 1 mM MgCl2), was added onto the cells (typically 0·5x106) which were then incubated for 1 h at room temperature. Unbound viruses were removed by washing three times in binding buffer. Cells were then incubated with an appropriate dilution of CAV-9 neutralizing rabbit serum. Staining was visualized with FITC-conjugated swine anti-rabbit Ig. Fluorescent staining was analysed as described above.

{blacksquare} Virus binding assays in the presence of integrin {alpha}vß3 or MHC class I antibodies.
Cells were harvested by gentle agitation and washed with blocking buffer at 4 °C for 15 min. Tubes containing 0·5x106 cells were then incubated with either 1 µg MCA1115, 1 µg W6/32, 1 µg MAB1976 or 1 µg MCA757G in PBS for 1 h at 4 °C. After washing three times in binding buffer (PBS supplemented with 2 mM CaCl2 and 1 mM MgCl2), virus binding was assayed as described above.

{blacksquare} RNA transfection.
RNA was transcribed from CAV-9 cDNA using the Promega large yield transcription method. RNA (0·1 µg) was mixed with 190 µl OPTI-MEM serum-free medium (Gibco) and 10 µl lipofectin reagent (Gibco), incubated at room temperature for 15 min and overlaid onto CHO monolayers, which had been pre-washed with OPTI-MEM. Cells were incubated for 15 h at 37 °C, the supernatant was removed and replaced with fresh culture medium. At 24 h post-transfection, the cell medium was assayed for the presence of virus by the ability to form plaques on RD cells.


   Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Integrin {alpha}vß3 and ß2-m are molecules which have been implicated in the CAV-9 infectious cycle (Roivainen et al., 1994 ; Ward et al., 1998 , respectively). In order to further determine their significance, we tested cell lines susceptible to virus infection, such as RD and GMK, by flow cytometry to assess whether they express significant quantities of ß2-m and integrin {alpha}vß3. The results showed that both molecules were present on the cell surface of these cell lines as detected by flow cytometry (Fig. 1). Approximately the same levels of ß2-m were seen on the RD (Fig. 1B) and GMK cells (Fig. 1E). However, integrin {alpha}v ß3 was expressed in higher quantities on GMK (Fig. 1F ) than on RD cells (Fig. 1C). To determine the significance of integrin {alpha}vß3 and whether its presence alone was sufficient to allow CAV-9 infection, we utilized the cell lines CHO-{alpha}vß3 (CHO cells transfected with {alpha}v and ß3 cDNAs and expressing human integrin {alpha}vß3) and CHO-pBJ cells (transfected with the empty vector). Equal amounts of infectious virus (250 p.f.u.) were added to RD, CHO-pBJ and CHO-{alpha}vß3 and these cells were assayed for plaque formation (Fig. 2). In RD cells (Fig. 2A), plaques developed but in contrast, no plaques were formed in CHO-pBJ (Fig. 2B) or CHO-{alpha}vß3 (Fig. 2C). To ensure that the CHO cells do not support fully the infectious cycle of CAV-9, the amounts of infectious virus were increased (1000 p.f.u.) and added to RD, CHO-pBJ and CHO-{alpha}v ß3 cells, without the presence of plaquing overlay. In 12 h, the RD cells were killed, while the CHO-pBJ and CHO-{alpha}v ß3 cells were incubated for up to 96 h, without formation of plaques.



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Fig. 1. Flow cytometric analysis of ß2-m and integrin {alpha}vß3 expression on RD and GMK cells. Control RD cells were incubated with FITC-conjugated rabbit anti-mouse IgG (A), ß2-m-specific MAb MCA1115 (B), integrin {alpha} vß3-specific MAb MCA757G (C). Control GMK cells were incubated with FITC-conjugated rabbit anti-mouse IgG (D), ß2 -m-specific MAb MCA1115 (E), integrin {alpha}vß3- specific MAb MCA757G (F). The histograms display relative cell numbers as a function of relative fluorescence intensities.

 


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Fig. 2. Results of CAV-9 plaque assay on RD cells (A), CHO- pBJ cells (B) and CHO-{alpha}vß3 cells (C).

 
To exclude the possibility that CHO-{alpha}vß3 and CHO-pBJ cells were infected by the virus but no plaques were formed, CAV-9 virus particles (250 p.f.u.) were added to CHO-{alpha}v ß3, CHO-pBJ and also RD cells which were used as a control. These cells were incubated for different time periods and, for each time period, the cells were frozen and thawed to release CAV-9 virus particles that may have been produced. The cell lysate produced was added to RD cells, which were then assayed for the presence of virus by plaque formation. The data showed no plaque formation on RD cells when CHO-{alpha}vß3 or CHO-pBJ cell lysates had been added. In contrast, plaques formed on RD cells when RD cell lysate had been added (data not shown).

To resolve whether the failure of CHO-{alpha}vß3 cells to support plaque formation resulted from a deficiency in the cell entry process and not a defective expression of integrin molecules at the cell surface, flow cytometric analysis of integrin {alpha}vß3 on the surface of CHO-{alpha}v ß3 was performed, using MAb MCA757G specific for integrin {alpha} vß3. The results demonstrated that CHO-{alpha}v ß3 expressed sufficient levels of integrin {alpha}v ß3 (Fig. 3B). Therefore, it follows that there is no lack or general down-regulation of integrin {alpha}v ß3 expression on the surface of CHO-{alpha}v ß3 cells. To verify whether the CHO cell lines were able to support growth of CAV-9, we transfected the CHO-pBJ, CHO-{alpha}v ß3 and RD cells with viral RNA. The culture medium from transfected cells was plated onto RD cells and subsequently plaques were observed; the plaques produced from CHO and RD cells were comparable, thus suggesting that the failure of CHO cells to support plaque formation was due to a defect in the cell entry process and not from an intracellular deficiency in virus replication.



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Fig. 3. Flow cytometric analysis of integrin {alpha}v ß3 expression on CHO-{alpha}vß3 cells. CHO- {alpha}vß3 cells were incubated with FITC-conjugated rabbit anti-mouse IgG (A) and with integrin {alpha}vß3 MAb MCA757G on CHO-{alpha}vß3 cells (B).

 
Flow cytometric analysis was utilized to test whether CAV-9 was able to bind to CHO-pBJ and CHO-{alpha}vß3 and to compare binding to RD cells (Fig. 4). These experiments showed that CAV-9 was not able to bind to the CHO-pBJ cells (Fig. 4E), but the virus was able to bind to CHO-{alpha}vß3 cells, (Fig. 4F). These results suggest that although CAV-9 binds to integrin {alpha}vß3, the virus may require the presence of additional molecules not present on CHO cells for internalization.



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Fig. 4. Flow cytometric analysis of CAV-9 binding to cells. RD cells (A), CHO-pBJ cells (B) and CHO-{alpha}vß3 cells (C) were incubated without CAV-9 particles (controls). CAV-9 binding on RD cells (D), CHO-pBJ cells (E) and CHO-{alpha}v ß3 cells (F) was then assayed. Virus binding was assayed by incubation with CAV-9 neutralizing serum and developed using an appropriate dilution of FITC-conjugated swine anti-rabbit Ig. The histograms display relative cell numbers as a function of relative fluorescence intensities.

 
A recent report has suggested a role for ß2-m in the infectious cycle of echoviruses and CAV-9 itself (Ward et al., 1998 ). We therefore proceeded to further investigate this role in the CAV-9 infectious process. We used a MAb specific for ß 2-m (MCA1115) and a MAb specific for MHC class I molecules (W6/32), which recognizes a conserved epitope formed by complexed heavy chain and ß2-m, in order to test whether these antibodies prevented cell infection on RD and GMK cells (Fig. 5). When MCA1115 was used, it reduced plaque formation by 90% on RD (Fig. 5A) and by 85% on GMK (Fig. 5K) compared to control wells treated with isotype control MAbs (Fig. 5F, O). The use of W6/32 prevented infection by 80% on both RD (Fig. 5B) and GMK cells (Fig. 5L) and addition of a mixture of W6/32 and MCA1115 completely inhibited plaque formation (Fig. 5C, J). This antibody mixture was probably more effective because it provided saturation of more epitopes on the ß2-m molecules.



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Fig. 5. Blocking experiments on RD and GMK cells were performed. The following were used on RD cells: ß2-m- specific MAb, MCA1115 (A); MHC class I-specific MAb, W6/32 (B); a mixture of MCA1115 and W6/32 (C); integrin {alpha}v ß3-specific MAb, MAB1976 (D); integrin {alpha} vß3-specific MAb, MCA757G (E); isotype control MAb (F); infection with CAV-9 particles (G); and a mixture of MAB1976 and MCA757G (H). The following were used on GMK cells: integrin {alpha}vß3-specific MAb, MCA757G (I); a mixture of MCA1115 and W6/32 (J); ß2-m-specific MAb, MCA1115 (K); MHC class I- specific MAb, W6/32 (L); integrin {alpha}vß3-specific MAb, MAB1976 (M); a mixture of MAB1976 and MCA757G (N); isotype control MAb (O); and infection with CAV-9 particles (P). These plates are representative of a number of independent experiments.

 
To confirm the role that integrin {alpha}vß3 plays in virus infection, we used integrin {alpha}vß3- specific MAbs MCA757G and MAB1976. The use of MCA757G prevented cell infection by 40% on RD (Fig. 5E) and by 50% on GMK (Fig. 5I). The MAB1976 prevented cell infection by 50% on both cell lines (Fig. 5D, M). A mixture of MCA757G and MAB1976 prevented cell infection by 50% (Fig. 5H, N).

To determine whether ß2-m or integrin {alpha}v ß3 act as a cellular receptor for CAV-9 or whether these molecules are associated with a post-binding stage of the virus life- cycle we tested whether MHC class I-specific, ß2-m- specific and integrin {alpha}vß3-specific antibodies shared the same binding epitopes with CAV-9 particles and thus inhibit virus binding on the cell surface. We quantified the degree of virus binding in the presence of MHC class I-, ß2-m- and integrin {alpha}vß3-specific antibodies. Flow cytometric analysis showed that, in the presence of integrin {alpha} vß3-specific antibody MCA757G, virus binding was reduced by 30% on RD cells (Fig. 6D) and by 40% on GMK cells (Fig. 7D) compared to that observed in the absence of integrin {alpha}vß3-specific antibody (Figs 6B and 7B). In the presence of MAB1976, virus binding was reduced by 40% on RD cells (Fig. 6E) and by 50% on GMK cells (Fig. 7E). A combination of both antibodies reduced virus binding by 50% (Figs 6F and 7F).



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Fig. 6. Flow cytometric analysis of CAV-9 binding to RD cells in the presence of antibodies. RD cells were first incubated without CAV-9 particles (A). CAV-9 binding to RD cells in the absence of these antibodies is shown in (B). CAV-9 binding to RD cells in the presence of MAb MCA1115 (C), MAb MCA757G (D), MAb MAB1976 (E) and a mixture of MAb MCA757G and MAb MAB1976 (F) is shown. Binding was assayed by incubation with CAV-9 neutralizing serum and developed using an appropriate dilution of FITC-conjugated swine anti-rabbit Ig. The histograms display relative cell numbers as a function of relative fluorescence intensities. Percentage inhibition of CAV-9 binding to RD cells using MAb MCA757G (open bars) and MAB1976 (hatched bars) at 0·5, 1·0 and 2·0 µg is shown in (G). The error bars are calculated from the SD over a number of independent experiments.

 


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Fig. 7. Flow cytometric analysis of CAV-9 binding to GMK cells in the presence of antibodies. GMK cells were first incubated without CAV-9 particles (A). CAV-9 binding to GMK cells in the absence of these antibodies is shown in (B). CAV-9 binding to GMK cells in the presence of MAb MCA1115 (C), MAb MCA757G (D), MAb MAB1976 (E) and a mixture of MAb MCA757G and MAb MAB1976 (F). Binding was assayed by incubation with CAV-9 neutralizing serum and developed using an appropriate dilution of FITC-conjugated swine anti-rabbit Ig. The histograms display relative cell numbers as a function of relative fluorescence intensities. Percentage inhibition of CAV-9 binding to GMK cells using MAb MCA757G (open bars) and MAB1976 (hatched bars) at 0·5, 1·0 and 2·0 µg is shown in (G). The error bars are calculated from the SD over a number of independent experiments.

 
When virus binding was tested on CHO-{alpha}vß3 cells in the presence of MCA757G or MAB1976, the CAV-9 binding was reduced by 70% and 90% respectively (Fig. 8C, D). A combination of both antibodies completely inhibited virus binding (Fig. 8E).



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Fig. 8. Flow cytometric analysis of CAV-9 binding to CHO- {alpha}vß3 cells in the presence of antibodies. CHO- {alpha}vß3 cells were first incubated without CAV-9 particles (A). CAV-9 binding to CHO-{alpha}vß3 cells in the absence of these antibodies is shown in (B). CAV-9 binding to CHO- {alpha}vß3 cells in the presence of MAb MCA757G (C), MAb MAB1976 (D) and a mixture of MAb MCA757G and MAb MAB1976 (E). Binding was assayed by incubation with CAV-9 neutralizing serum and developed using an appropriate dilution of FITC conjugated swine anti- rabbit Ig. The histograms display relative cell numbers as a function of relative fluorescence intensities.

 
No difference in virus binding on RD and GMK cells was observed in the presence of MAb MCA1115 compared to that observed in the absence of this antibody (Figs 6C and 7C). Similar results were obtained when W6/32 was utilized (data not shown). Overall, these results show that the inhibitory effect that these MCA1115 and W6/32 antibodies had on the CAV-9 infectivity is not associated with CAV-9 binding. However, integrin {alpha}v ß3, although involved in virus binding, is unlikely to be the only receptor molecule utilized by CAV-9 for binding on RD and GMK cells, since the integrin-specific antibodies did not completely inhibit binding on these cell lines as they did on CHO-{alpha}v ß3. We conclude that integrin {alpha}vß3 is involved in the initial virus binding step, but other receptor molecules may also be involved in binding and internalization, whereas ß2-m is probably involved in a post-binding stage of CAV-9 infection.


   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
Entry of viruses into cells is a complex, multi-step process and, for several viruses, cell attachment and internalization have been shown to be distinct steps requiring different cell surface receptors. In addition, it is possible that viruses can enter cells by alternative pathways, again requiring different molecules. For example, CAV-21 binds to CD55 but requires ICAM-1 for cell entry (Shaffren et al. , 1997a ). Haemagglutinating echovirus strains bind to CD55 but can also interact with other cellular receptors (Powell et al., 1998 ). The coxsackie B viruses have been reported to use CD55 (Bergelson et al. , 1995 ; Shaffren et al., 1995 ), CAR protein (Bergelson et al., 1997 ; Shaffren et al., 1997c ) and possibly a 100 kDa nucleolin-related membrane protein (De Verdugo et al. , 1995 ). Human immunodeficiency virus type 1 also provides an example of a complex cell entry process. The initial contact is with CD4 (Maddon et al., 1986 ) or galactosyl ceramide (Bhat et al., 1991 ) while many other surface molecules, such as the chemokine receptor fusin (Feng et al., 1996 ) or CC-CKR-5 (Deng et al., 1996 ; Dragic et al., 1996 ), are required for virus entry.

CAV-9 has been reported to utilize the integrin {alpha}v ß3 in binding to cells. We wished to test whether the presence of integrin {alpha}vß3 alone was sufficient to allow CAV-9 infection. Thus, we tested whether CAV-9 could form plaques on CHO cells transfected with human integrin {alpha}vß3. Infection with CAV-9 did not give rise to plaques on CHO-{alpha}v ß3 (Fig. 2C) or on the control cells CHO-pBJ (Fig. 2B). The possibility of virus infection without production of plaques was also examined, but our experiments revealed that virus was not present. To verify that failure of the CHO cell lines to support plaque formation was due to a defect in cell entry and was not as a result of an intracellular deficiency in virus replication, we transfected these cells with viral RNA. Infectious particles resulted, showing that the CHO cell lines could support virus replication after delivery of viral RNA inside the cells.

Binding experiments using CHO-{alpha}vß3 revealed that although CAV-9 particles could not initiate a productive infection, CAV-9 particles could bind on CHO-{alpha}vß3 cells, thus showing that although CAV-9 binds on integrin {alpha}v ß3, this molecule is not sufficient for cell entry.

We further investigated the role of integrin {alpha}v ß3 in the CAV-9 infectious cycle by the use of the anti-integrin {alpha}vß3 MAb MCA757G and MAB1976. These data demonstrated that MCA757G and MAB1976 were able to partially block virus binding to both GMK and RD cells but could not completely abolish it (Figs 6 and 7). Similarly, these antibodies did not fully inhibit infectivity in plaque reduction assays (Fig. 5). A mixture of both antibodies inhibited binding by approximately 50% on both RD and GMK cells. This is consistent with previous findings that showed CAV-9 mutants lacking the RGD motif were still infectious (Roivainen et al., 1991 ; Hughes et al., 1995 ). Although our results show that these MAbs have approximately the same protective effect on both RD and GMK cell lines, Roivainen et al. (1996) have shown that a polyclonal antibody to the vitronectin receptor of integrin {alpha}vß3 had a drastic effect on GMK cells and only a marginal protective effect on RD cells. This discrepancy could be due to the fact that we are not using identical CAV-9 strains, since our CAV-9 stock has been grown and isolated from RD cells, whereas the CAV- 9 stock of Roivainen et al. (1996) had been grown in GMK cells. The virus could have evolved according to the cellular environment. Another explanation could be that they might not have used saturating amounts of antibody on RD cells.

The partial blocking observed by MCA757G and MAB1976 antibodies on RD and GMK cells, may be due to the fact that although these antibodies bind to integrin {alpha}vß3, they do so at epitopes not directly involved in CAV-9 attachment and therefore have only a partial effect on CAV-9 infection and binding. However, an alternative explanation is that the virus can also utilize other cell molecules for attachment on RD and GMK cells.

To determine whether the virus utilizes other binding domains on integrin {alpha}vß3 or whether it can also attach on other receptor molecules, and therefore the binding is not completely inhibited, CAV-9 binding experiments in the presence of MCA757G or MAB1976 on CHO-{alpha}vß3 cells were performed (Fig. 8). These experiments showed that, in the presence of MCA757G, CAV-9 binding was inhibited by 70%, whereas in the presence of MAB1976, CAV-9 binding was inhibited by 90%.

Taken together, these data show that since a mixture of these antibodies completely inhibited virus binding on CHO-{alpha}v ß3, in contrast to RD and GMK cells, additional surface molecules may be involved for CAV-9 attachment, internalization and cell entry on RD and GMK cells.

Since integrin {alpha}vß3 was unlikely to be the only molecule involved in the CAV-9 attachment and also internalization processes, we initiated a search for other candidate molecules. It has been reported that ß2-m plays a role in the infectious cycle of the echoviruses which are genetically similar to CAV-9 and CAV- 9 itself, since a ß2-m-specific antibody blocked infection of RD cells by enterovirus-7, -8, -11 and -25 and CAV-9 (Ward et al., 1998 ). We therefore proceeded to determine the role of ß2-m in the CAV-9 infectious cycle by using MAbs directed against ß2-m (MCA1115) and MHC class I molecules (W6/32). The MAb MCA1115 inhibited virus infection by 90%, whereas using a combination of MCA1115 and W6/32 in an attempt to saturate all epitopes on the ß2-m molecules, completely inhibited virus infection. Although these antibodies were able to inhibit CAV-9 infection, they had no direct effect on CAV-9 binding (Fig. 6B, C) and (Fig. 7B, C). This argues against a physical association between CAV-9 and ß2-m being responsible for the inhibitory effect of the MAbs on virus infection.

It has been previously shown that MAbs to MHC class I and ß2-m can block infection of viruses such as simian virus 40 (Stang et al., 1997 ), adenovirus 2 and adenovirus 5 (Hong et al., 1997 ). These pathogens can utilize MHC class I molecules as a receptor, while in the case of CAV-9 we have shown that ß2-m does not play a direct role in CAV-9 binding. Therefore, we postulate that ß2-m plays a role in a post-binding event of the CAV-9 infectious cycle, possibly associated with virus internalization. Alternatively, the MAbs might down-regulate cellular activities that are modulated by MHC class I molecules and are necessary for CAV-9 entry. These results are in good agreement with those of Ward et al. (1998) which have shown that ß2-m is not a receptor molecule for echoviruses, but may play a post-attachment role in echovirus infection.

In conclusion, this study shows that CAV-9 attachment and subsequent cell entry might be a more complex event than previously thought and require different cell surface molecules. Although integrin {alpha}vß3 is one of the molecules involved in virus attachment, it is not sufficient for cell entry, while ß2-m may play a subsequent role in virus internalization.


   Acknowledgments
 
We would like to thank Mary Ruth and Jenny List from the Department of Haematology at Colchester General Hospital, for their invaluable assistance with flow cytometry.


   Footnotes
 
b Present address: GlaxoWellcome Research and Development, Biotechnology Analytical Laboratories, Langley Court, South Eden Park Road, Beckenham BR3 3BS, UK.


   References
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Abstract
Introduction
Methods
Results
Discussion
References
 
Acharya, R. , Fry, E. , Stuart, D. , Fox, G. , Rowlands, D. & Brown, F. (1989). The three dimensional structure of foot and mouth disease virus at 2·9 resolution. Nature 337, 709-716.[Medline]

Baxt, B. & Becker, Y. (1990). The effect of peptides containing the arginine-glycine-aspartic acid sequence on the adsorption of foot and mouth disease virus to tissue culture cells. Virus Genes 4, 73-83.[Medline]

Bergelson, J. M. , Shepley, M. P. , Chan, B. M. C. , Hemler, M. E. & Finberg, R. W. (1992). Identification of integrin VLA-2 as a receptor for echovirus 1. Science 255, 1718-1720 .[Medline]

Bergelson, J. M. , St John, N. F. , Kawaguchi, S. , Chan, M. , Stubdal, H. , Modlin, J. & Finberg, R. W. (1993). Infection by echoviruses 1 and 8 depends on the {alpha}2 subunit of human VLA-2. Journal of Virology 67, 6847-6852 .[Abstract]

Bergelson, J. M. , Chan, M. , Solomon, K. R. , St John, N. F. , Lin, H. & Finberg, R. W. (1994). Decay-accelerating factor (CD55), a glycosylphosphatidyl-inositol-anchored complement regulatory protein, is a receptor for several echoviruses. Proceedings of the National Academy of Sciences, USA 91, 6245-6249 .[Abstract]

Bergelson, J. M. , Mohantry, J. G. , Crowell, R. L. , St John, N. F. , Lublin, D. M. & Finberg, R. W. (1995). Coxsackievirus B3 adapted to growth on RD cells binds to decay-accelerating-factor (CD55). Journal of Virology 69, 1903-1906 .[Abstract]

Bergelson, J. M. , Cunningham, J. A. , Droguett, G. , Kurt-Jones, E. A. , Krithivas, A. , Hong, J. S. , Horwitz, M. S. , Crowell, R. L. & Finberg, R. W. (1997). Isolation of a common receptor for coxsackie B viruses and adenoviruses 2 and 5. Science 275, 1320-1323 .[Abstract/Free Full Text]

Berinstein, A. , Roivainen, M. , Hovi, T. , Mason, P. W. & Baxt, B. (1995). Antibodies to the vitronectin receptor (integrin {alpha}vß3) inhibit binding and infection of foot and mouth disease virus to cultured cells. Journal of Virology 69, 2664-2666 .[Abstract]

Bhat, S. , Spitalnid, S. L. , Gonzales-Scarano, F. & Silberberg, D. H. (1991). Galactosyl ceramide or a derivative is an essential component of the neural receptor for human immunodeficiency virus type 1 envelope glycoprotein gp120. Proceedings of the National Academy of Sciences, USA 88, 7131-7134 .[Abstract]

Bjorkman, P. J. , Saper, M. A. , Samraoui, B. , Bennet, W. S. , Strominger, J. L. & Wiley, D. C. (1987). Structure of the human class I histocompatibility antigen, HLA-A2. Nature 309, 506-512.

Chang, K. H. , Auvinen, P. , Hyypiä, T. & Stanway, G. (1989). The nucleotide sequence of coxsackievirus A9: implications for receptor binding and enterovirus classification. Journal of General Virology 70, 3269-3280 .[Abstract]

Chang, K. H. , Day, C. , Walker, J. , Hyypiä, T. & Stanway, G. (1992). The nucleotide sequences of wild- type coxsackievirus A9 strains imply that an RGD motif in VP1 is functionally significant. Journal of General Virology 73, 621-626.[Abstract]

Cheresh, D. A. & Spiro, R. C. (1987). Biosynthetic and functional properties of an ARG-GLY-ASP-directed receptor involved in human melanoma cell attachment to vitronectin, fibrinogen and von Willebrand factor. Journal of Biological Chemistry 262, 17703-17711 .[Abstract/Free Full Text]

Davies, J. , Warwick, J. , Totty, N. , Philp, R. , Helfrich, M. & Horton, M. (1989). The osteoclast functional antigen, implicated in the regulation of bone resorption, is biochemically related to the vitronectin receptor. Journal of Cell Biology 109, 1817-1826 .[Abstract]

Deng, H. , Liu, R. , Ellmeier, W. , Choe, S. , Unutmaz, D. , Burkhart, M. , DiMarzio, P. , Marmon, S. , Sutton, R. E. , Hill, C. M. , Davis, C. B. , Peiper, S. C. , Schall, T. J. , Littman, D. R. & Landau, N. R. (1996). Identification of a major co-receptor for primary isolates of HIV-1. Nature 381, 661-666.[Medline]

De Verdugo, U. R. , Selinka, H.-C. , Huber, M. , Kramer, B. , Kellermann, J. , Hofschneider, P. H. & Kandolf, R. (1995). Characterization of a 100- kilodalton binding protein for the six serotypes of coxsackie B viruses. Journal of Virology 69, 6751-6757 .[Abstract]

Dragic, T. , Litwin, V. , Allaway, G. P. , Martin, S. E. , Huang, Y. , Nagashima, K. A. , Cayanan, C. , Maddon, P. J. , Koup, R. A. , Moore, J. P. & Paxton, W. A. (1996). HIV-1 entry into CD4 cells is mediated by the chemokine receptor CC-CKR-5. Nature 381, 667-673.[Medline]

Feng, Y. , Broder, C. C. , Kennedy, P. E. & Berger, E. A. (1996). HIV-1 entry cofactor: functional cDNA cloning of a seven transmembrane, G protein-coupled receptor. Science 272, 872-877.[Abstract]

Fox, G. , Parry, N. R. , Barnett, P. V. , McGinn, B. , Rowlands, D. & Brown, F. (1989). The cell attachment site on foot-and- mouth disease virus includes the amino acid sequence RGD (arginine- glycine-aspartic acid). Journal of General Virology 70, 625-637.[Abstract]

Greve, J. M. , Davis, G. , Meyer, A. M. , Forte, C. P. , Yost, S. C. , Marlor, C. W. , Kamarck, M. E. & McClelland, A. (1989). The major human rhinovirus receptor is ICAM-1. Cell 56, 839-847.[Medline]

Grist, N. R. & Reid, D. (1988). General pathogenicity and epidemiology. In Coxsackieviruses, A General Update, pp. 221-239. Edited by M. Berdinelli & H. Friedman. New York: Plenum Press.

Hong, S. S. , Karayan, L. , Tournier, J. , Curiel, D. T. & Boulanger, P. A. (1997). Adenovirus type 5 fiber knob binds to MHC class I {alpha}2 domain at the surface of human epithelial and B lymphoblastoid cells. EMBO Journal 16, 2294-2306 .[Abstract/Free Full Text]

Horton, M. A. , Lewis, D. , McNulty, K. , Pringle, J. A. S. & Chambers, T. J. (1985). Monoclonal antibodies to osteoclastomas: definition of osteoclast specific cellular antigens. Cancer Research 45, 5663-5669 .[Abstract]

Hughes, P. J. , Horsnell, C. , Hyypiä, T. & Stanway, G. (1995). The coxsackievirus A9 RGD motif is not essential for virus viability. Journal of Virology 69, 8035-8040 .[Abstract]

Hynes, R. O. (1992). Integrins: versatility, modulation and signaling in cell adhesion. Cell 69, 11-25.[Medline]

Leippert, M. , Beck, E. , Weiland, F. & Pfaff, E. (1997). Point mutations within the ßG- ßH loop of foot and mouth disease virus O1K affect virus attachment to target cells. Journal of Virology 71, 1046-1051 .[Abstract]

Logan, D., Abu-Ghazaleh, R., Blakemore, W., Curry, S., Jackson, T., King, A., Lea, S., Lewis, R., Newman, J., Parry, N., Rowlands, D., Stuart, D. & Fry, E. (1993). Structure of a major immunogenic site on foot and mouth disease virus. Natur e 362, 566–568.

McKenna, T. St C. , Lubroth, J. , Rieder, E. , Baxt, B. & Mason, P. W. (1995). Receptor binding site- deleted foot and mouth (FMD) virus protects cattle from FMD. Journal of Virology 69, 5787-5790 .[Abstract]

Madden, D. R. (1995). The three dimensional structure of peptide–MHC complexes. Annual Reviews in Immunology 13, 587-590.[Medline]

Maddon, P. J. , Dalgleish, A. G. , McDougal, J. S. , Clapham, P. R. , Weiss, R. A. & Axel, G. (1986). The T4 gene encodes the AIDS virus receptor & is expressed in the immune system and the brain. Cell 47, 333-348.[Medline]

Mason, P. W. , Rieder, E. & Baxt, B. (1994). RGD sequence of foot and mouth disease virus is essential for infecting cells via the natural receptor but can be bypassed by an antibody-dependent enhancement mechanism. Proceedings of the National Academy of Sciences, USA 91, 1932-1936 .[Abstract]

Mateu, M. G. , Valero, M. L. , Andreu, D. & Domingo, E. (1996). Systematic replacement of amino acid residues within the Arg-Gly-Asp, containing loop of foot and mouth disease virus and effect on cell recognition. Journal of Biological Chemistry 271, 12814-12819 .[Abstract/Free Full Text]

Neff, S. , Sa-Carvalho, D. , Rieder, E. , Mason, P. W. , Blystone, S. D. , Brown, E. J. & Baxt, B. (1998). Foot and mouth disease virus virulent for cattle utilizes the integrin {alpha}vß3 as its receptor. Journal of Virology 72, 3587-3594 .[Abstract/Free Full Text]

Powell, R. M. , Schmitt, V. , Ward, T. , Goodfellow, I. , Evans, D. J. & Almond, J. W. (1998). Characterization of echoviruses that bind decay accelerating factor (CD55): evidence that some haemagglutinating strains use more than one cellular receptor. Journal of General Virology 79, 1707-1713 .[Abstract]

Roivainen, M. , Hyypiä, T. , Piirainen, L. , Kalkkinen, N. , Stanway, G. & Hovi, T. (1991). RGD-dependent entry of coxsackievirus A9 into host cells and its bypass after cleavage of VP1 protein by intestinal proteases. Journal of Virology 65, 4735-4740 .[Medline]

Roivainen, M. , Piirainen, L. , Hovi, T. , Virtanen, I. , Riikonen, T. , Heino, J. & Hyypiä, T. (1994). Entry of Coxsackievirus A9 into host cells: specific interactions with {alpha} vß3 integrin, the vitronectin receptor. Virology 203, 357-365.[Medline]

Roivainen, M. , Piirainen, L. & Hovi, T. (1996). Efficient RGD independent entry of coxsackievirus A9. Archives of Virology 141, 1909-1919 .[Medline]

Shaffren, D. R. , Bates, R. C. , Agrez, M. V. , Herd, R. L. , Burns, G. F. & Barry, R. D. (1995). Coxsackievirus B1, B3 and B5 use decay accelerating factor as a receptor for cell attachment. Journal of Virology 69, 3873-3877 .[Abstract]

Shaffren, D. R. , Dorahy, D. J. , Ingham, R. A. , Burns, G. F. & Barry, R. D. (1997a). Coxsackievirus A21 binds to decay-accelerating-factor but requires intracellular adhesion molecule 1 for cell entry. Journal of Virology 71, 4736 -4743.[Abstract]

Shaffren, D. R. , Dorahy, D. J. , Greive, S. J. , Burns, G. F. & Barry, R. D. (1997b). Mouse cells expressing human intracellular adhesion molecule-1 are susceptible to infection by coxsackievirus A21. Journal of Virology 71, 785 -789.[Abstract]

Shaffren, D. R. , Williams, D. T. & Barry, R. D. (1997c). A decay accelerating factor-binding strain of coxsackievirus B3 requires the coxsackievirus–adenovirus receptor protein to mediate lytic infection of rhabdomyosarcoma cells. Journal of Virology 71, 9844 -9448.[Abstract]

Stang, E. , Kartenbeck, J. & Parton, R. G. (1997). Major histocompatibility complex class I molecules mediate association of SV40 with caveolae. Molecular Biology of the Cell 8, 47-57.[Abstract]

Takagi, J. , Kamata, T. , Meredith, J. , Puzon-McLaughlin, W. & Takada, Y. (1997). Changing ligand specificities of {alpha}vß1 and {alpha}vß3 integrins by swapping a short diverse sequence of the ß subunit. Journal of Biological Chemistry 272, 19794-19800 .[Abstract/Free Full Text]

Ward, T. , Pipkin, P. A. , Clarson, N. A. , Stone, D. M. , Minor, P. D. & Almond, J. W. (1994). Decay accelerating factor (CD55) identified as the receptor for echovirus 7 using CELICS, a rapid immuno-focal cloning method. EMBO Journal 13, 5070-5074 .[Abstract]

Ward, T. , Powell, R. M. , Pipkin, P. J. , Evans, D. J. , Minor, P. D. & Almond, J. W. (1998). Role of {alpha}2 microglobulin in echovirus infection of rhabdomyosarcoma cells. Journal of Virology 72, 5360-5365 .[Abstract/Free Full Text]

Received 18 March 1999; accepted 21 June 1999.