| Porcine reproductive and respiratory syndrome(PRRS) has become one of the most common and economically significant infectious diseases in the swine industry worldwide, which is caused by porcine reproductive and respiratory syndrome virus with a tropism for cells of the monocytic lineage. Based on nucleotide differences, PRRSV has been divided into European strains and North American strains. Since May 2006, atypical PRRS(so-called porcine high fever syndrome(PHFS) in china) was pandemic in china, having been confirmed that the causative agent of this outbreak was highly pathogenic North American PRRSV(HP-PRRSV).In the natural host, porcine alveolar macrophages(PAM) are predominant target cell of PRRSV, three receptors for PRRSV have been identified on PAM cell surface, including heparin sulfate(Hs), sialoadhesin(Sn) and CD163. Hs helps PRRSV binding to the cell membrane, Sn mediates the virus attchement and internalization, while CD163 has been described to function as a receptor for PRRSV uncoating, genome release and replication. The expression of CD163 was shown to allow non-permissive cells to become permissive to PRRSV replication with production of progeny infectious virus while the other two cannot, which indicates that CD163 plays a major role in PRRSV entry.As one of the scavenger receptor cysteine-rich superfamily, CD163 takes part in immuno-response to antigen and the inflammations and it is tightly regulated by a variety of factors. As a member of the disintegrin and metalloprotease family that is spread on many kinds of cell surfaces, ADAM17 is demonstrated to mediates human CD163 downregulation and then regulate inflammations. In spite of the central role in many biological processes, whether ADAM17 could mediate porcine CD163 downregulation or not and how this metalloprotease governs PRRSV infection remains unstudied.At first, the c DNA of porcine CD163 complete coding sequence was amplified via RT-PCR from total cellular RNA of PAM cells. The sequence was cloned into pc DNA3.1 vector and sequencing result showed that the cloned sequence was correct. The eukaryotic expressing Plasmid p CDNA3.l-CD163 was constructed and transfected into HEK293 cells. Under the selection of G418 and flow cytometry, the HEK293 cellline stably expressing porcine CD163 was constructed. IFA and westem Blot were performed to verify the CD163 expression in the constructed cell lines in the protein level. The HEK 293/CD163 cells were inoculated with PRRSV and IFA was Performed, confirming PRRSV infection in HEK293/ CD163 cell lines.Secondly, overexpression of ADAM17, ablation of ADAM17 expression using specific small interfering RNA, inhibition and activation of ADAM17 on the surface of HEK293/CD163 and PAM resulted in different regulations of CD163 expression. Using flow cytometry, we demonstrated that the inhibition of ADAM17 upregulates membrane CD163 expression. Furthermore, overexpression of ADAM17 induced downregulation of CD163 expression, whereas ablation of ADAM17 expression using specific small interfering RNA resulted in upregulation of CD163 expression. Overall, these findings indicate that ADAM17 downregulates CD163 expressionTo investigate the role of ADAM17 in altering PRRSV entry by regulating viral cellular receptors, IFA was used to assess PRRSV infection after inoculated with PRRSV in HEK293/CD163. At last, we found that ablation of ADAM17 expression or inhibition of ADAM17 enhances PRRSV entry in HEK293/CD163 and overexpression of ADAM17 or activation of ADAM17 resulted in increase in PRRSV infection. Overall, these findings indicate that ADAM17 downregulates CD163 expression and hinders PRRSV entry.In brief, HEK293 cells stably expressing CD163 was established under selection of G418 using flow cytometry. The HEK 293/CD163 cells were inoculated with PRRSV and IFA was Performed, confirming PRRSV infection in HEK293/ CD163 cell lines. We demonstrated that ADAM17 could mediate downregulation of CD163 and then hinder PRRSV infection of permissive cells. |
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