The Molecular Mechanism Of Infectious Bronchitis Virus Negatively Regulates Interferon Pathway

Infectious bronchitis virus(IBV),a member of the genus gammacoronavirus of the family coronaviridae.It is reported that papain-like proteases encoded by many alpha and beta coronaviruses showed deubiquitination(DUB)activity that interfere the host's IFN signaling pathway.However,the function of PLP encoded by IBV was poorly reported,and the interaction between IBV and host is unclear.IBV co-infected with other respiratory viruses especially H9 subtype avian influenza virus(AIV)with high morbidity and mortality rates in many countries continues to be reported.IBV blocking the host antiviral immune response may be related to such phenomenon.We conducted the following researches to explore these issues.1.The papain-like protease of avian infectious bronchitis virus has deubiquitinating activity.Ubiquitination and deubiquitination are critically involved in regulation of virus-induced type I IFN signaling pathways.The activation of related receptors and the transduction of the cell signaling pathway in the innate immune response require ubiquitination.In this study,we detected the DUB activity of IBV PLP and identified the core domain of DUB activity.Firstly,pRK5-HA-Ub,pRK5-HA-K48 and pRK5-HA-K63 were transfected into DF1 cells that infected with IBV,respectively.Found that IBV exhibits DUB activity with significant reductions of the levels of Ub,Ub-K48 and Ub-K63 conjugated proteins.The DUB activity exhibited a clear time dependence,with stronger DUB activity in the early stage of viral infection.PLP is the catalytic domain of IBV Ck/CH/JS/2010/12 encoded by the region from nucleotides 4243 to 5553,and the transmembrane(TM)domain is encoded by the region from nucleotides 5781 to 6252 downstream of PLP.PLP and PLP-TM eukaryotic expression plasmids were constructed and co-transfected in DF1 cells with ubiquitin molecules,respectively.The result showed that IBV replicase-encoded PLP,including the downstream TM domain,is a DUB enzyme and dramatically reduced the level of Ub-conjugated proteins while processing both Ub-K48 and Ub-K63 linked polyubiquitin chains.By contrast,PLP did not cause any reduction of HA-Ub-conjugated proteins.It shows that the transmembrane domain plays a key role in the DUB activity of PLP-TM.In addition,mutations of the catalytic residues of PLP-TM,Cys1274Ser and His1437Lys,have a slight effect on DUB activity against Ub,Ub-K48 and Ub-K63 conjugated proteins,indicated that the DUB activity of the PLP-TM wild-type protein is not completely dependent on its catalytic activity.2.IBV antagonistic host interferon expression in the early stage of infectionMelanoma differentiation associated gene-5(MDA5)is the main pattern recognition receptor for host cells to recognize IBV,and belongs to the retinoic acid-inducible gene ?(RIG-?)-like receptor family together with RIG-1.MDA5 and RIG-1 induce mitochondrial antiviral signaling gene(MAVS)activation through their N-terminal CARD domain interacting with the caspase activation and recruitment domains(CARD)domain downstream of the MAVS.Activated MAVS recruits downstream interferon regulatory factor 3/7(IRF3/7)and nuclear transcription factor ?B(NF-?B)to induce type ? interferon expression.Whether IBV antagonistic host interferon expression related to MDA5 and MAVS remains to be studied,RIG-? is considered to be absent in chicken.However,the absence raises the question that whether this protein influences the antiviral immune response against IBV infection.Chicken embryo infected with IBV showed that the chIFN-? transcription level was lower than the control group in the early infection stage(12 h after infection)and up regulated from 36 h,to the peak at 60 h post-infection.The chIFN-(3 level of infected group showed significant higher than that of control group from 48 h to 72 h post infection,The expression of cytokines(chIFN-? and chMx)were similar to that of chIFN-?.MDA5-specific stimulant poly(I:C)were transfected into DF1 cells after IBV infection.It showed that IBV inhibit the expression of IFN-? induced by poly I:C in the early stage of infection(12 h after infection).These results showed that antagonism of the IBV response to the host interferon mainly occurred in the early stage of infection.We have cloned chicken MDA5(chMDA5)and domestic goose RIG-?(dgRIG-?)and demonstrated that they act as positive regulators in the expression of chIFN-? induced by IBV,and neither the expression of chMDA5 nor dgRIG-? in DF1 cells affected viral replication.The cleavage of chMAVS was induced by IBV at early stage post-infection but was rarely cleaved after 24 h.A gene silencing technique was also used to identify whether chMDA5,chTLR3 and chMAVS influence the antiviral immune response mediated by IBV,Compared with the control group,silencing of chMAVS or chMDA5 in DF1 cells significantly reduced IBV-induced chIFN-P transcription.The simultaneous silencing of chMDA5,chTLR3 and chMAVS reduced the transcription of chIFN-? most.These results suggest that IBV inhibits chMDAS dependent type ?IFN signaling pathway by the cleavage of adaptor protein chMAVS.3.The synergistic pathogenesis of IBV and H9 subtype AIVThis study explored the synergistic pathogenic mechanism of IBV and H9 subtype AIV from the perspective of IBV antagonistic to host interferon.More severe lesions such as growth retardation,subcutaneous hyperemia and many petechial to ecchymotic hemorrhages were found in the co-infection group compared with the single infection group.The expression level of interferon in IBV infection group and mixed infection group was lower than control group at 12 h after infection,indicating that IBV inhibited the expression of interferon.Co-infected with IBV,H9 subtype AIV showed higher accumulation of genome load in the allantoic fluid,trachea,lung and intestinal compared with embryos that received H9 subtype AIV alone at 72 h of infection.The SPF chickens co-infected with IBV and H9 subtype AIV showed respiratory symptoms after 3 days,and death appeared at 7 day of infection.The chicken survival rate of co-infection group,H9 subtype AIV and IBV infection group are 80%,95%and 90%respectively.Higher expression of IFN-?,IFN-?,IL-1? and IL-13 in trachea,lung and intestinal had been detected in co-infection group compared with chickens that received H9 subtype AIV or IBV alone.Thus,we come to a conclusion that IBV enhance the replication of H9 subtype AIV as a result of IBV inhibit interferon expression in early stage of infection.Virus replication increased the expression level of inflammatory factors,creating a cytokine storm and causing more severe tissue damage.