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
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Abstract |
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Introduction |
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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
2ß1 is a receptor molecule for echoviruses 1 and 8 (Bergelson et al., 1992
, 1993
), whereas integrin
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 v integrins are known to recognize the arginineglycineaspartic acid (RGD) motif (Hynes, 1992
), this is consistent with the presence of such a motif on the GH 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
vß3 or additional receptor molecules to infect cells.
A recent report has also suggested that ß2- microglobulin (ß2-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 vß3 in the CAV-9 infectious cycle, we have used MAbs directed against these molecules. The results show that CAV-9 binds to integrin
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.
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Methods |
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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- vß3 [CHO cells transfected with
v and ß3 cDNAs and expressing human integrin
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.
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.
Antibodies.
The integrin vß3-specific MAb MCA757G which recognizes a domain between the
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
v ß3-specific MAb MAB1976, which recognizes the vitronectin receptor complex of integrin
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.
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 4872 h in a 7% CO2 humidified incubator before plaque visualization with crystal violet. Control plates with isotype control IgG were similarly treated.
Assay of CAV-9 particles produced in cells.
CHO-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.
Immunofluorescence staining of integrin
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.
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.
Virus binding assays in the presence of integrin
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.
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.
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Results |
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To resolve whether the failure of CHO-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
vß3 on the surface of CHO-
v ß3 was performed, using MAb MCA757G specific for integrin
vß3. The results demonstrated that CHO-
v ß3 expressed sufficient levels of integrin
v ß3 (Fig. 3B
). Therefore, it follows that there is no lack or general down-regulation of integrin
v ß3 expression on the surface of CHO-
v ß3 cells. To verify whether the CHO cell lines were able to support growth of CAV-9, we transfected the CHO-pBJ, CHO-
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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To determine whether ß2-m or integrin 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
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
vß3-specific antibodies. Flow cytometric analysis showed that, in the presence of integrin
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
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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Discussion |
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CAV-9 has been reported to utilize the integrin v ß3 in binding to cells. We wished to test whether the presence of integrin
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
vß3. Infection with CAV-9 did not give rise to plaques on CHO-
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-vß3 revealed that although CAV-9 particles could not initiate a productive infection, CAV-9 particles could bind on CHO-
vß3 cells, thus showing that although CAV-9 binds on integrin
v ß3, this molecule is not sufficient for cell entry.
We further investigated the role of integrin v ß3 in the CAV-9 infectious cycle by the use of the anti-integrin
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
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 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 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-
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-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 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 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.
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Acknowledgments |
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Footnotes |
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References |
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Received 18 March 1999;
accepted 21 June 1999.