Contributions of the S2 spike ectodomain to attachment and host range of infectious bronchitis virus

Abstract

The spike protein is the major viral attachment protein of the avian coronavirus infectious bronchitis virus (IBV) and ultimately determines viral tropism. The S1 subunit of the spike is assumed to be required for virus attachment. However, we have previously shown that this domain of the embryo- and cell culture adapted Beaudette strain, in contrast to that of the virulent M41 strain, is not sufficient for binding to chicken trachea (Wickramasinghe et al., 2011). In the present study, we demonstrated that the lack of binding of Beaudette S1 was not due to absence of virus receptors on this tissue nor due to the production of S1 from mammalian cells, as S1 proteins expressed from chicken cells also lacked the ability to bind IBV-susceptible embryonic tissue. Subsequently, we addressed the contribution of the S2 subunit of the spike in IBV attachment. Recombinant IBV Beaudette spike ectodomains, comprising the entire S1 domain and the S2 ectodomain, were expressed and analyzed for binding to susceptible embryonic chorio-allantoic membrane (CAM) in our previously developed spike histochemistry assay. We observed that extension of the S1 domain with the S2 subunit of the Beaudette spike was sufficient to gain binding to CAM. A previously suggested heparin sulfate binding site in Beaudette S2 was not required for the observed binding to CAM, while sialic acids on the host tissues were essential for the attachment. To further elucidate the role of S2 the spike ectodomains of virulent IBV M41 and chimeras of M41 and Beaudette were analyzed for their binding to CAM, chicken trachea and mammalian cell lines. While the M41 spike ectodomain showed increased attachment to both CAM and chicken trachea, no binding to mammalian cells was observed. In contrast, Beaudette spike ectodomain had relatively weak ability to bind to chicken trachea, but displayed marked extended host range to mammalian cells. Binding patterns of chimeric spike ectodomains to these tissues and cells indicate that S2 subunits most likely do not contain an additional independent receptor-binding site. Rather, the interplay between S1 and S2 subunits of spikes from the same viral origin might finally determine the avidity and specificity of virus attachment and thus viral host range.

The avian coronavirus spike protein

“…Specifically, M41 S1 had a greater binding avidity than that of the attenuated vaccine strain H120 on trachea and lung (Wickramasinghe et al, 2011). For Beaudette S1 no obvious binding could be observed on chicken trachea (Wickramasinghe et al, 2011), but also not on CAM (Promkuntod et al, 2013). The S1 of the nephropathogenic B1648 did not show any appreciable staining of the trachea and the kidney, while for the S1 of QX, a strain with reported reproductive tract tropism, only mild patchy staining to the oviduct was observed.…”

“…6B), extension of this protein with the S2 part of the spike ectodomain resulted in binding to BHK21 cells ( Fig. 6B) and CAM (Promkuntod et al, 2013). Interestingly, extension of the M41 spike, now comprising the complete ectodomain, resulted in an increased affinity and extended binding profile to the chicken trachea (Fig.…”

“…4A; Wickramasinghe et al, 2011) and CAM ( Fig. 4B; Promkuntod et al, 2013). Differences in amount of staining between laboratories might be explained by the use of 20-fold higher protein concentrations by Shahwan and coworkers (Shahwan et al, 2013) compared to that used by Wickramasinghe et al Nevertheless, it is clear that the multimerization state of the spike affects the binding characteristics.…”

“…In contrast to some other coronaviruses, no endosomal proteases have been elucidate to contribute to the infection of IBV (see also Section 6.5). Although the S2 domain is not principally involved in binding to a host cell receptor, the interplay between S1 and S2 might synergistically determine the avidity and specificity of virus attachment (de Haan et al, 2006;Promkuntod et al, 2013).…”

“…Likely important binding properties also reside in this domain of other avian gammacoronaviruses. The contribution of domains outside the S1 became clear when studying Beaudette spike binding (Promkuntod et al, 2013). While Beaudette S1 was not sufficient for binding to chicken trachea (Fig.…”