Institut für Virologie der Universität zu Köln, Fürst-Pückler-Str. 56, 50935 Köln, Germany1
Author for correspondence: Birgit Nelsen-Salz.Fax +49 221 4783902. e-mail birgit.nelsen-salz{at}medizin.uni-koeln.de
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Abstract |
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In earlier studies, it has been proposed that echovirus 9 strains E9/Hill and E9/Barty vary in their capsid surfaces, since they adsorb to GMK cells with different kinetics and strain-induced antibodies exhibit quantitatively different neutralizing capacities (Rosenwirth & Eggers, 1982 ). This hypothesis was supported by our finding that the main difference between the strains is an insertion in the C-terminal part of VP1 of the virulent strain E9/Barty, including an arginineglycineaspartic acid (RGD) motif (Zimmermann et al., 1995
, 1996
). It has already been shown that three members of the Picornaviridae, foot-and-mouth disease virus, coxsackievirus A9 and echovirus 22, interact with their target cells by an RGD
vß3 or RGD
vß1 contact (Neff et al., 1998
; Pulli et al., 1997
; Roivainen et al., 1994
). In the case of E9/Barty, a 310 amino acid segment that includes the RGD motif is responsible for cell attachment and mouse pathogenicity and, furthermore, cell attachment of E9/Barty but not E9/Hill is inhibited specifically by RGD-containing peptides (Zimmermann et al., 1997
). These results allow the presumption that E9/Barty may utilize the RGD motif as its receptor-binding site.
In this communication, we present evidence that E9/Barty binds via the RGD motif to the vß3 integrin (vitronectin receptor; VNR). We demonstrate firstly that E9/Barty and coxsackievirus A9, which is known to bind to VNR (see above), share receptor specificities. Furthermore, we confirm that antiserum to VNR inhibits attachment and plaque formation of E9/Barty and related recombinant constructs. Both shared receptor specificity and inhibition by the antiserum are not true for the apathogenic prototype E9/Hill. It is hypothesized that susceptibility of murine muscle tissue to infection with E9/Barty depends on the developmental stage of the cells.
We performed competition binding experiments and analysed cell attachment of radiolabelled E9/Barty in the presence of unlabelled homologous or heterologous viruses. For that purpose, GMK cell monolayers were simultaneously infected with standard amounts of 35S-labelled E9/Barty and competing virus. After 30 min incubation at room temperature, the supernatant was removed and cell-bound radioactivity was determined. Binding of radiolabelled E9/Barty to GMK cells was reduced to about 50% by either unlabelled E9/Barty or coxsackievirus A9, whereas E9/Hill did not affect binding of 35S-labelled E9/Barty (Fig. 1). These results confirm the hypothesis that E9/Barty utilizes the same cellular receptor as coxsackievirus A9 on GMK cells, i.e. VNR, whereas the apathogenic E9/Hill uses another site.
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Series of experiments were carried out using an inoculum of labelled echovirus 9 strains E9/Hill, E9/Barty, rE9-C1, rE9-C2 and rE9-C4. [35S]Methionine-labelled virus was used to infect GMK cell monolayers that had been pre-incubated for 45 min with the indicated antiserum (1:1000 dilution of the stock solution from Gibco BRL). After an additional 15 min incubation in the presence of labelled virus, the supernatant was removed and cell-bound radioactivity was measured. Cell attachment of E9/Barty, rE9-C1 and rE9-C2 was inhibited markedly by antiserum against human VNR (Fig. 2a). On the other hand, only background inhibition was measurable for E9/Hill and rE9-C4.
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These results strongly suggest (i) that VNR is a candidate receptor for E9/Barty, but not for the prototype E9/Hill, and (ii) that the viruscell interaction takes place via the RGD motif, since only recombinant viruses encoding this motif were inhibited significantly by antibodies against VNR.
The fact that E9/Barty and coxsackievirus A9 recognize the same receptor and, therefore, infect the same cells may help to understand why clinical symptoms induced by these two viruses in newborn mice are indistinguishable. On the other hand, the different receptor specificities of E9/Hill and E9/Barty may explain their different pathogenicity for newborn mice (Eggers & Sabin, 1959 ; Zimmermann et al., 1997
). In further studies, the possible importance of a functional RGD motif for pathogenicity of several isolates of echovirus 9 from humans has been demonstrated (Nelsen-Salz et al., 1999
).
Integrins are supposed to be involved in terminal muscle cell differentiation (Menko & Boettiger, 1987 ); for example, the expression of integrin
vß3 is down-regulated as part of the myogenic differentiation programme of human skeletal muscle in vitro (Blaschuk et al., 1997
). It has already been described for coxsackievirus A13 that productive infection of muscle cells cultured from tissues of foetal mice is limited to the stage of differentiation characterized by rapid cell fusion (Goldberg & Crowell, 1971
). These data and our findings concerning the receptor-binding site are consistent with the fact that clinical symptoms are clearly decreased after infection of suckling mice with E9/Barty 2, 4 or 6 days after birth, as compared with severe disease after infection of newborn mice (Bültmann et al., 1983
). Moreover, the rate of virus replication clearly depends on the age of the infected animal (Fig. 3
). In 1-week-old mice, we observed only a limited increase in infectious titre in the muscle tissue. No virus replication took place in 2-, 3- or 12-week-old mice. This allows the suggestion that the reduced pathogenicity of E9/Barty in older mice is not only due to the increased mass of compensatory muscle tissue, but is also a consequence of the down-regulation of
vß3 integrin.
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Acknowledgments |
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References |
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Received 8 April 1999;
accepted 7 June 1999.