The infection of the avian coronavirus infectious bronchitis virus (IBV) is initiated by the binding of the spike glycoprotein S to sialic acids on the chicken host cell. In this study we identified the receptor-binding domain (RBD) of the spike of the prototype IBV strain M41. By analyzing the ability of recombinantly expressed chimeric and truncated spike proteins to bind to chicken tissues, we demonstrate that the N-terminal 253 amino acids of the spike are both required and sufficient for binding to chicken respiratory tract in an α-2,3-sialic acid-dependent manner. Critical amino acids for attachment of M41 spike are present within the N-terminal residues 19-69, which overlap with a hypervariable region in the S1 gene. Our results may help to understand the differences between IBV S1 genotypes and the ultimate pathogenesis of IBV in chickens.

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.

High-level, inducible expression of heterologous genes in the cyanobacterium Synechococcus sp. strain PCC 7942 was obtained using the Escherichia coli trc promoter and lac/ repressor. The pet€ gene of Anabaena sp. strain PCC 7937 encoding plastocyanin precursor protein and the €. coli uidA gene encoding )?-glucuronidase were initially placed under the control of the trc promoter and lac/ repressor by cloning into the E. coli pTrc99C expression vector and were introduced into the chromosomal platform for integration in metF (PIM) of the Synechococcus R2-PIM9 recipient strain. These pTrc99C-derived constructs often gave rise to transformants that did not contain a complete insert gene, probably because of gene conversion events. Selection of the desired Synechococcus R2-PIM9 transformants was vastly improved using the new pTrclS vector that contains the aadA gene encoding streptomycin resistance as an extra antibiotic resistance marker. The influence of IPTG concentration and induction time on gene expression with the €. coli trc/lac/ system in Synechococcus was determined using Pglucuronidase as a reporter. The Anabaena PCC 7937 pet€ gene in Synechococcus was expressed to a high level upon induction with IPTG as shown by RNA and immunoblot analysis. The general usability of pTrclS as a cloning vector for inducible heterologous gene expression in Synechococcus was confirmed by the introduction of several more genes.

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The effect of copper on the levels of plastocyanin (PC) and cytochrome c553 (cyt-c)-specific transcripts from Anabaena sp. PCC 7937 was investigated. The addition of copper resulted in a marked increase in PC mRNA levels, and a decrease in cyt c mRNA levels. Thus the functional exchange between PC and cyt c seems to be regulated at the mRNA level. The copper-dependent increase in PC and decrease in cyt c mRNA levels was abolished when chloramphenicol was added to the cells. This suggests that de novo synthesis of at least one trans-acting element is required to regulate PC and cyt c mRNA levels. Both PC and cyt c mRNA stability was found to be unaltered under varying Cu2+ regimes. This leads to the conclusion that expression of both genes is regulated at the level of initiation of transcription.