| Infectious bursal disease is an acute, highly contagious and lethal infectious disease of young chickens caused by infectious bursal disease virus. Its pathogeny—infectious bursal disease virus (IBDV)—ainly damage the bursa of Fabricius, a central immune organ of chicken, leaving them immunosuppressive. IBDV is an Avibirnavirus which belongs to the Birnaviridae family. Its genome contains two separate dsRNAs, namely segment A with a length of about3200bp and B about2800bp. VP2and VP3are both structural proteins of IBDV, and they together account for more than90%of total viral protein. VP2—the only component of viral caspsid—is the major host protective antigen of the virus and mainly contributes to cell tropism and virulence of IBDV. VP3, another important structural protein of IBDV, can interact with VP1, VP2and virus dsRNA. It is the compenent of ribonucleoprotein (RNP) and plays a critical role as scaffold protein in the assembly of virus capsid. The occurrence of infectious bursal disease is the result of the game between pathogeny and host. The mechanism of IBDV could be more fully understood by studying from the angles of virus, host and their interaction. Previous reports showed that certain natural immune moleculars, limiting factors and its signaling pathways palyed a critical role in IBDV infection. Whereas, it also needs continuous effort before the interaction mechanism between IBDV and host be completely understood.Immunoprecipitation (IP) and Mass spectrometry (MS) were amplified in this study to screen host cell proteins interacting with IBDV structural protein VP2and VP3by the usage of susceptible DF1cells.Ribosome protein L4(RPL4) was identified as a host proteins to interact with IBDV VP3by IP and MS analysis. Its interaction was further confirmed by co-IP (co-immunoprecipitation) and the result of confocal microscopy showed that RPL4and VP3co-localized in the nuclus of DF1cells. It was reported that ribosome proteins play an important role of protein synthesis in cells and it also participated in life events of molecular regulation and stress response. It was reported that RPL4regulated the assembly of reverse transcription virus. The role of the interaction between RPL4and VP3in IBDV life cycle needs further investigation.Casein kinase la (CKla) is a kinase of Ser/Thr. It participates in cell signal pathways, such as Wnt/β-Catenin (Wnt), Hedgehog (Hh) and NF-κB, and plays an important role in cell cycle, cell apoptosis and differentiation, and vesicular transport. Besides, CKla also play an important role of regulation in the replication of yellow fever virus (YFV), hepatitis C virus (HCV), Rotavirus (RV) Human herpesvirus (HHV). However, few reports about interaction between CK1α and animal viruses are available till now. In this study, VP2monoclonal antibody (McAb) was used to react with capsid protein VP2of IBDV in IP assay and the result of MS showed that CKla maybe one of host cell proteins that interacted with VP2. This interaction was further confirmed by co-immunoprecipitation (co-IP), and the confocal microscopy revealed that CK1α and VP2co-localized in the cytoplasm of DF1cells. CKla of chicken is highly conserved and its homology with human CKla is100%. CKla is speculated to influence the replication of IBDV due to the importance of human CKlα in virus infection. To prove this, we test the role of CKla inhibitor D4476in IBDV infection and replication. The inhibition of CKla led to the increase of quantity of viral structual proteins in DF1cells in a dose-dependent manner.Our research identified for the first time that CKlα and RPL4could interact with structural protein VP2and VP3of IBDV respectively. Preliminary study showed that CKla restricted IBDV replication. This study benefits to further elucidate the pathogenic mechanism of IBDV. |
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