Functional Characterization Of Canine Interferon Lambda And The Nuclear Export Characteristics Of Influenza B Virus Nuclear Export Protein(Nep)

In this study, we provide the first comprehensive annotation of canine IFN-λ (CaIFN-λ, type III IFN). Phylogenetic analysis based on genomic sequences indicated that CaIFN-λ is located in the same branch with Swine IFN-λ1 (SwIFN-λ), Bat IFN-λ1 (BaIFN-λ), and human IFN-X1 (HuIFN-λ1). CaIFN-λ was cloned, expressed in Escherichia coli,and purified to further investigate the biological activity in vitro. The recombinant CaIFN-λ (rCaIFN-λ) displayed potent antiviral activity on both homologous and heterologous animal cells in terms of inhibiting the replication of the New Jersey serotype of vesicular stomatitis virus (VSV), canine parvovirus, and influenza virus A/WSN/33 (H1N1), respectively. In addition, we also found that rCaIFN-λ exhibits a significant anti-proliferative response against A72 canine tumor cells and MDCK cells in a dose-dependent manner. Furthermore, CaIFN-λ activated the JAK-STAT signaling pathway.To evaluate the expression of CaIFN-λ induced by virus and the expression of IFN-stimulated genes (ISGs) induced by rCaIFN-λ in the MDCK cells, we measured the relative mRNA level of CaIFN-λ and ISGs(ISG15, Mxl, and 2’5’-OAS) by quantitativereal-time PCR and found that the mRNA level of CaIFN-λ and the ISGs significantly increased after treating the MDCK cells with viruses and rCaIFN-λ protein, respectively. Finally, to evaluate the binding activity of rCaIFN-λ to its receptor, we expressed the extracellular domain of the canine IFN-λ receptor 1(CaIFNλR1-EC) and determined the binding activity via ELISA. Our results demonstrated that rCaIFN-λ bound tightly to recombinant CaIFNλRl-EC (rCaIFNλRl-EC).Influenza virus nuclear export protein (NEP) is encoded by the spliced mRNA from RNA segment 8 and plays crucial roles for the nuclear export of viral ribonucleoprotein complex through CRM1-mediated cellular protein transport system. It also participates in the process of replication and transcription of viral genome and the assembly and budding of mature virions. However, the detailed mechanisms of nucleocytoplasmic traffic of NEP remain incompletely understood. Here, we revealed that there are three CRM1-dependent, hydrophobic NESs existing on NEP of influenza B virus and they are all critical for the effective replication of influenza A virus.