Department of Molecular Pathology, Institute for Pathology, University of Tübingen, Liebermeisterstr. 8, D-72076 Tübingen, Germany1
Author for correspondence: Hans-Christoph Selinka. Fax +49 7071 29 5334. e-mail hans-christoph.selinka{at}med.uni-tuebingen.de
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With regard to these findings, a study was performed to elucidate the interplay of DAF and CAR on HeLa cells. To address this topic, we selected two different strains of serotype CBV3, one of which (CBV3-HA; ATCC VR-688) is able to haemagglutinate human group O erythrocytes, whereas the other, cDNA-generated CBV3 (Kandolf & Hofschneider, 1985 ; Klump et al., 1990
), is not. Comparing virusreceptor interactions of these strains of CBV3, we present data supporting the model of a putative DAF/CAR complex on human HeLa cells.
To investigate the different binding properties of haemagglutinating and non-haemagglutinating CBV3, virus-overlay protein-binding assays (VOPBAs) were performed. Octylglucoside-solubilized cellular proteins of human HeLa and hamster CHO cells, prepared as described by Krah (1989) , were separated by 10% SDSPAGE, and subsequently blotted onto PVDF membranes. The filters were exposed to 35S-labelled viruses (300000 c.p.m.) for 2 h and membrane-bound radioactivity was detected by autoradiography. As shown in Fig. 1(a)
, the cDNA-generated CBV3 bound to a single protein of 46 kDa whereas the haemagglutinating CBV3-HA attached to two proteins with molecular masses of 46 kDa and 70 kDa. Using the protocol of Xu et al. (1995)
for VOPBAs, as well as sucrose gradient-purified preparations of infectious CBV virions (160S) depleted of highly abundant 125S provirions, the previously described 100 kDa binding protein for CBV (Raab de Verdugo et al., 1995
) was not observed in the present study. So far, the role of 125S provirions interacting with a 100 kDa membrane protein for the pathogenesis of infectious disease is not known. The VOPBA presented in Fig. 1(a)
demonstrates that infectious virions of the cDNA-generated CBV3 strain primarily bind to CAR (46 kDa), whereas virions of the haemagglutinating CBV3-HA variant recognize determinants of DAF (70 kDa) and CAR proteins.
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To perform binding inhibition assays, HeLa cells were preincubated for 30 min with receptor-specific antibodies prior to exposure to [35S]methionine-labelled virions. As shown in Fig. 2(a), adherence of the non-haemagglutinating CBV3 strain to HeLa cells was significantly reduced in the presence of antibody RmcB as well as antibody IF7 (1:100 dilutions of ascites fluids), indicating a steric inhibitory effect of anti-DAF antibodies towards this non-DAF-binding strain. By combining anti-DAF and anti-CAR antibodies, binding was completely suppressed in an additive manner. Whereas attachment of the haemagglutinating CBV3-HA was reduced to 40% by use of anti-DAF antibodies, binding of this virus was not significantly inhibited using a 1:100 dilution of RmcB. Although anti-CAR antibodies alone did not cause any detectable inhibitory effect towards haemagglutinating CBV3-HA, they augmented the inhibitory effect of anti-DAF antibodies, resulting in complete inhibition of virus binding in the presence of both anti-CAR and anti-DAF antibodies (Fig. 2a
). For further analysis of this antibody-mediated inhibitory effect, plaque reduction assays were performed. As shown in Fig. 2(b
), CBV3 and CBV3-HA exhibited clear differences in their plaque phenotypes. Infection of HeLa cells with the haemagglutinating CBV3-HA revealed a small plaque phenotype with plaques only 3050% of the diameter of those of CBV3, without affecting the growth characteristic and titre of this virus (see Fig. 3a
). Minimal amounts of CAR-specific and DAF-specific antibodies (dilution 1:1000) were used to study the interplay of haemagglutinating and non-haemagglutinating CBV with CAR and DAF with regard to infection of HeLa cells (Fig. 2c
). Cells were grown in 24-well plates, preincubated with antibodies IF7, RmcB or a combination of both antibodies, followed by exposure to CBV3 or CBV3-HA. Monolayers were overlaid with agarose and surviving cells were stained with crystal violet after 48 h incubation at 37 °C. Regarding infectivity, plaque formation by both viruses required 10-fold higher virus titres in the presence of minimal amounts of CAR-specific RmcB antibodies (Fig. 2c
). In contrast to the study of Shafren et al. (1997b
) with CBV3/New, the presence of DAF-specific antibody IF7 alone did not cause any inhibitory effect on CBV3 and CBV3-HA infections. However, in accordance with the CBV3/New study (Shafren et al., 1997b
), the combination of both IF7 and RmcB resulted in a synergistic inhibitory effect with respect to infection of HeLa cells with both CBV3 strains. These data further support the model that DAF and CAR are closely associated on the membrane of human HeLa cells.
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The additive effects of minimal amounts of anti-CAR and anti-DAF antibodies on virus binding and infection presented in this paper favour a model of specific interactions of both haemagglutinating and non-haemagglutinating strains of CBV3 with a DAF/CAR complex on the surface of HeLa cells, with CAR being the infection-mediating component. In this model, the cDNA-generated, non-haemagglutinating CBV3 primarily binds to CAR whereas the haemagglutinating strain CBV3-HA contacts both components of the putative DAF/CAR complex. The haemagglutinating CBV3-HA virus has the ability to bind to human RD cells but, in contrast to the haemagglutinating CBV3-RD variant of Crowell and colleagues (Reagan et al., 1984 ), the host range of CBV3-HA is not extended to these cells (data not shown) but is still dependent on CAR. The presence of a DAF/CAR complex on HeLa cells is also supported by recently published data of Martino et al. (1998)
, reporting the observation that pretreatment of HeLa cells with DAF-binding viruses or antibodies sterically blocks the interaction of adenovirus type 2 with CAR. Data reported for coxsackie A virus 21 (CAV21), which binds to DAF but requires the DAF-adherent ICAM-1 molecule for cell entry (Shafren et al., 1997a
), might well be interpreted accordingly.
Since the CBV canyon harbouring the receptor-binding site is not as pronounced as the canyon of polioviruses and rhinoviruses (Muckelbauer et al., 1995 ), a multicomponent receptor complex may substantially increase the efficiency of virus binding. However, despite different binding properties, the growth characteristics of CBV3 and CBV3-HA did not significantly differ in HeLa cells (Fig. 3a
). Therefore, the positive role of DAF as a CBV3 sequestration site, as postulated by Shafren et al. (1997b
), could not be verified for the haemagglutinating CBV3-HA strain and may vary within DAF-binding CBV strains. However, in cells or tissues where CAR expression is very low, e.g. smooth muscle cells, the proposed DAF/CAR complex might well select for DAF-binding CBV3-HA, due to its affinity for both components, DAF and CAR. Supporting this model, a high prevalence of DAF-binding CBV3 strains among clinical isolates has been reported (Bergelson et al., 1997b
). Therefore, it is tempting to speculate whether a tissue-specific pattern of DAF and CAR molecules might control selection of DAF-binding CBV3 strains in vivo. Consequently, investigations should be pursued to determine the distribution of DAF and CAR molecules in different tissues as well as their interplay with regard to CBV susceptibility and virus resistance.
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References |
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Received 5 May 1999;
accepted 7 September 1999.