Molecular Mechanism Of The Duck Hepatitis A Virus Type 1 Non-structural Protein 3CD Regulates RIG-I/MDA5 Antiviral Signaling Pathway

Duck hepatitis A virus type 1(DHAV-1)is one of the important pathogens that harm the duck breeding industry.Previous laboratory studies have revealed that DHAV-1 uses its 3C protease to degrade Interferon regulatory factor 7(IRF7)and inhibit the IFNβ upstream signaling pathway,providing a favorable condition for viral replication.At present,there are no reports on the mechanism of other viral proteins of DHAV-1 regulating the upstream signaling pathway of IFNβ.In this study,the mechanism of DHAV-1 3CD protein(a precursor of 3C protein)regulating the production of RIG-Ⅰ/MDA5-mediated IFNβ was studied,and the main results are as follows:1.The effect of DHAV-1 3CD protein on the expression of molecules related to the upstream pathway of RIG-Ⅰ/MDA5-mediated IFNβ(1)The 3CD protein was transiently expressed in Duck Embryo Fibroblast(DEF)cells.RT-qPCR revealed that the 3CD protein significantly inhibited the Poly(I:C)-induced upregulation of IFNβ,Mx and OASL mRNA levels,and WB revealed that the expression of IFNβ was also significantly inhibited.(2)Eukaryotic expression plasmids of each protein of the IFNβ upstream pathway and 3CD were co-transfected with DEF cells respectively.WB results showed that 3CD protein inhibited the expression of RIG-Ⅰ and IRF7 proteins in a dose-dependent manner,but had no effect on the protein expression of MDA5,MAVS,and TBK1.(3)Dual luciferase reporter gene assays revealed that 3CD protein inhibited RIG-Ⅰand IRF7-mediated activation of the IFNβ promoter.The above results suggest that the 3 CD protein can inhibit the IFNβ upstream signaling pathway by suppressing the expression of RIG-Ⅰ and IRF7 proteins.2.Interaction of DHAV-1 3CD protein with RIG-Ⅰ protein and its mechanism(1)Indirect Immunofluorescence Assay(IFA)and Co-immunoprecipitation(Co-IP)assays showed co-localization and interaction between RIG-Ⅰ protein and 3CD protein.(2)Co-IP assay was used to screen the regions where 3CD protein interacted with RIG-Ⅰ protein,and the results showed that RIG-Ⅰ protein could interact with 3CD protein through its Nterminal structural domain;both 3C and 3D structural domains of 3CD protein could interact with RIG-Ⅰ protein.(3)Co-IP assay showed that the duck-derived MAVS protein interacted with the RIG-Ⅰ protein,but no interaction was detected in the presence of the 3CD protein,indicating that the 3 CD protein prevented the formation of the RIG-Ⅰ-MAVS complex.3.Interaction of DHAV-1 3CD protein with MDA5 protein and its mechanism(1)IFA and Co-IP results showed co-localization and interaction between 3CD protein and MDA5 protein,and further assays showed that 3CD protein interacted with MDA5 protein C-terminal 900-1003aa via 3C structural domain N-terminal 1-28aa.(2)DEF cells were transfected with 3CD and MDA5 expression plasmids and infected with DHAV-1,and RNA Immunoprecipitation(RIP)results showed that both 3CD and MDA5 proteins were able to bind to DHAV-1 RNA.(3)MDA5 was co-transfected with 3CD and 3CD(△1-28aa)respectively and infected with DHAV-1.The RIP results showed that the MDA5 protein in the 3CD(△1-28aa)transfected group was able to bind more DHAV-1 RNA,indicating that the interaction between 3CD protein and MDA5 protein interfered with the binding of DHAV-1 RNA by MDA5.Meanwhile,the dual luciferase reporter gene assay revealed that the 3CD(△1-28aa)transfected group had higher IFNβ promoter activity than the 3CD transfected group,indicating that the interaction of 3CD protein with MDA5 protein inhibited IFNβ promoter activation.The above results suggest that 3CD protein can inhibit MDA5-mediated signaling by interacting with MDA5 protein.4.Interaction of DHAV-1 3CD protein with IRF7 protein and effect on IRF7 protein localization(1)The results of IFA and Co-IP assays showed that 3CD protein could interact with IRF7 protein.(2)pCAGGS-3CD-HA and pCAGGS-IRF7-Flag were co-transfected and assayed after IFA and nucleocytoplasmic separation,resulting in no direct effect of 3CD protein on the localization and distribution of IRF7 protein.5.Effect of RIG-Ⅰ and MDA5 proteins on DHAV-1 replicationAfter infection with DHAV-1 by overexpression of RIG-Ⅰ and MDA5 in DEF cells,RTqPCR and WB results showed that both RIG-Ⅰ and MDA5 significantly inhibited the replication of DHAV-1.Further,3CD and RIG-Ⅰ/MDA5 eukaryotic expression plasmids were co-transfected with DEF cells and then infected with DHAV-1 to test the effect of the interaction between 3CD and RIG-Ⅰ/MDA5 on viral replication,and the results showed that the interaction between 3CD protein and RIG-Ⅰ/MDA5 protein facilitated viral replication.In summary,the 3CD protein inhibits the expression of RIG-Ⅰ protein by interacting with the N-terminal structural domain of RIG-Ⅰ protein,interfering with the formation of the complex between RIG-Ⅰ and MAVS and thus promoting viral replication;the 3CD protein interacts with the C-terminal 900-1003aa of MDA5 protein through the N-terminal 1-28aa of the 3 C structural domain,interfering with the binding of MDA5 to DHAV-1 RNA and facilitating viral replication;the 3CD protein interacts with the IRF7 protein and inhibits the expression of IRF7 protein but does not affect the localization of IRF7 protein.