Cyclophilin A Interacts With Viral VP4 And Inhibits The Replication Of Infectious Bursal Disease Virus

Infectious bursal disease(IBD), caused by infectious bursal disease virus(IBDV), is an acute, highly contagious disease in young chickens and is also an important immunosuppressive disease. IBDV is a member of the genus Avibirnavirus belonging to the Birnaviridae family. Its genome consists of two segments of double-stranded RNAs(A and B). Segment A contains two partially overlapping open reading frames(ORFs). The smaller ORF encodes a nonstructural viral protein denoted VP5. The larger ORF encodes the viral proteins VP2, VP3 and VP4. VP1, which is encoded by segment B, is a RNA-dependent RNA polymerase(RdRp). Non-structural protein VP4 is a serine protease of infectious bursal disease virus that can catalyzes the hydrolysis of polyprotein pVP2-VP4-VP3 to form the viral proteins pVP2, VP4, and VP3, and is associated with the tubule-like particles in IBDV-infected cells. In addition, VP4 is involved in the host’s innate immune response.The occurrence and development of infectious disease is the results of the interaction between pathogen and host. The researches of the molecular mechanisms of IBDV infection should include not only the virus aspect but also the host responses. In this study, the bait vector of IBDV VP4 was constructed. 22 kinds of potential interacted proteins were obtained from the expression library of chicken embryo fibroblast(CEF) cells using the Matchmaker Gold Yeast Two-Hybrid System. Cyclophilin A(CypA) is one of the members of the cyclophilins(Cyps) family, which possesses peptidyl-prolyl cis-trans isomerase(PPIase) activity. It is an acceleration factor in protein folding and assembly and is also involved in various physiological processes, such as inflammation, cancer, apoptosis, and immune response. The relationship of CypA and virus is also very closely and involved in the replications of HIV-1, HCV, HBV, SARS, RV and other virus. CypA may also play an important role in the infection of IBDV.In this study, CypA was further confirmed to interact with VP4 of IBDV using Co-IP, GST pull-down, and confocal microscopy assays. Moreover, we found that the overexpression of CypA suppressed IBDV replication, whereas the knock-down of CypA by small interfering RNAs promoted the replication of IBDV through detecting the expression of viral proteins, genomic copy numbers and viral titers by western blot, real time RT-PCR and TCID50.Cyclosporin A(CsA), specifically binding to the PPIase sites of CypA, is often used to research the activity of CypA. In this study, CsA could promote the expression of IBDV structural proteins but did not block the CypA-VP4 interaction, indicating that CypA might be involved in the regulation of IBDV replication via the PPIase activity.In summary, this study provides the first demonstration that the chicken CypA interacts with IBDV VP4 and CypA is a restriction factor for IBDV infection. The PPIase of CypA may be involved in regulating the replication of IBDV. These findings contribute to a further understanding of the molecular mechanism of IBDV infection.