The Roles And Molecular Mechanisms Of Host Protein CK1α And CSGalNAcT2 Interacting With VP2 In The Replication Of Infectious Bursal Disease Virus

Infectious bursal disease(IBD) caused by infectious bursal disease virus(IBDV) is an acute and highly contagious disease, which mainly destroys the central immune organs, bursa of fabricius, of young chickens(3-6 weeks), so IBD is also called chicken "AIDS". With the evolution and variation of IBDV, the classic IBDV(cIBDV), variant IBDV(vIBDV), and very virulent IBDV(vvIBDV) successively appeared in the world. The high lethality of vvIBDV brought huge economic losses to world poultry industry. Recently, based on the deepening of epidemiology and mature of reverse genetics technique, VP2 protein as the sole component of outer layer capsid of IBDV has been confirmed to be the basis of the virulence and cell tropism of IBDV. However, the fact of occurrence and development of IBD is the battle process between IBDV and its host. Although VP2 has been proved to have important effect on virulence and cell tropism in the aspect of the virus, the specific role of VP2 in communicating with host cell is still unclear. Therefore, deep understanding of interaction between VP2 and host has become the current research focus, which contributes to reveal the different pathogenic and immunological mechanism of IBDV.In this study, from the point of virus and host interaction, different techniques involved in protein-protein interaction was adopted to screen the host proteins interacting with VP2 of IBDV, and their roles and molecular mechanism in IBDV replication was further investigated. Firstly, using the immunoprecipitation coupled with mass spectrum(IP/MS), a putative interacting protein called casein kinase l alpha(CK1α) was identified from the IBDV infected cells using the specific monoclonal antibody of VP2. The interaction of CK1α and VP2 during IBDV infection was further confirmed by the co-immunoprecipitation and laser confocal microscopy assay. At the same time, CK1α was down-regulated during IBDV replication. Through the gene regulation assay, we found the down-regulation of CK1α inhibited IBDV growth and up-regulation of CK1α promoted IBDV replication. Additionally, IBDV replication ability was also decreased by D4476, the inhibitor of CK1α, which suggested that the effect of CK1α on IBDV replication was dependent on its kinase activity. Above all, the downregulation of CK1α might stands for an antiviral response of host to IBDV infection. To further uncover the underlying molecular mechanism exploited by host to limit IBDV replication through down regulating CK1α expression, after our exploration, we found IBDV infection could induce IFN-β production, and the overexpression of CK1α could impair the antiviral effect of IFN-β. However, this antagonistic action was resulted from the negative regulation of CK1α on IFNAR1 rather than on the IFN-β expression, because the intracellular C terminus of IFNAR1 contains a conserved degron, which closely related with ubiquitylation degradation of IFNAR1. And the key of initiation of IFNAR1 ubiquitylation might be the S545, which is the potential site phosphorylated by CK1α. Through site directed mutagenesis, we found that S545 A reduced the ubiquitylation level of IFNAR1 mediated by CK1α. Furthermore, the amount of IFNAR1 on the cell membrane was turn out to be increased by IBDV infection through flowcytometry assay. Therefore, we suggested that CK1α as a host senor was down regulated by IBDV infection, which further caused the up-regulation of IFNAR1 and finally enhanced the antiviral ability against IBDV replication.However, under the antiviral pressure of host, viruses also evolve to adopt many strategies to facilitate own replication via interaction with host proteins. In this study, we also identified a Golgi apparatus membrane proptein, chondroitin sulphate N-acetylgalactosaminyltransferase-2(CSGalNAcT2) which is beneficial for IBDV replicaction. CSGalNAcT2 was able to interact with VP2 at Golgi apparatus. From the results of gene chip and real time RT-PCR, we confirmed the CSGalNAcT2 was up-regulated during the IBDV replication. Moreover, we proved the up-regulation of CSGalNAcT2 was beneficial to IBDV replication, and this promotion effect relied on the integrity of Golgi apparatus structure. Gogli apparatus is the location of replication fatory(ribonucleoprotein complexes) of IBDV, the interaction of VP2 and CSGalNAcT2 in this study further indicated IBDV could utilize the CSGalNAcT2 to recruit VP2 to Gogli apparatus for the IBDV assembly and replication.In summary, this study firstly showed the different effects of host proteins on IBDV replication through the interaction with VP2, including the antiviral mechanism triggered by CK1α and the molecular mechanism of CSGalNAcT2 promoting IBDV replication, which are very significant for us to understand the pathogenesis and immunological mehcanism of IBDV.