Mechanistic Studies On The Regulation Of NDV Replication By The Mitochondrial Protein CHCHD10

Newcastle disease virus(NDV)is an RNA virus that can exploit host systems to promote its own replication.It has been shown that NDV can promote its own replication by affecting mitochondrial function(e.g.,mitochondrial fusion)in host cells to prevent interferon-beta(IFN-β)production.However,the mechanism by which NDV affects mitochondrial function and then prevents the activation of antiviral signals is not fully understood.Therefore,revealing the molecular mechanism by which NDV affects the mitochondrial function of host cells and then regulates its own replication in the host is important for elucidating the interaction between NDV and host cells,as well as for the further prevention and control of Newcastle Disease(ND).To this end,our study first revealed a significant decrease in the protein level of the mitochondrial protein coiled-coil-helix-coiled-coil-helix domain containing 10(CHCHD10)in the late stage of NDV infection by proteomic sequencing analysis.Then the effects of CHCHD10 on host cell mitochondrial dynamics,as well as downstream RIG-I-like receptor(RLR)and nuclear factor kappa B(NF-κB)signalling pathways were explored by quantitative real-time PCR(q PCR),Western Blot(WB),immunofluorescence and flow cytometry.It was finally revealed that in the late stage of NDV infection,the down-regulation of host cell CHCHD10 protein level inhibited the mitochondrial fusion process,altered the spatial distribution of the mitochondrial antiviral signaling protein(MAVS)and reduced its protein level,impeding the activation of RLR and NF-κB antiviral signalling pathways,thereby favouring viral replication.The main content is as follows:1.Study of host cell proteomics in the late stage of NDV infectionIn order to investigate the mitochondria-associated proteins in host cells that are involved in NDV infection,primary chicken embryo fibroblasts(CEFs)were first isolated from 10-day-old chicken embryos in this study.An infection model was established using NDV-infected CEF cells,followed by proteomics sequencing,analysis and validation.The results revealed that the protein content of CHCHD10 was significantly reduced in the late stage of NDV infection.2.A significant reduction in host CHCHD10 protein during the late stages of NDV infectionIn order to verify that the reduction of host CHCHD10 protein level was generalised during the late stage of NDV infection,we re-verified this by in vitro and in vivo experiments,respectively.The results revealed that gene transcription and protein expression of CHCHD10 were significantly down-regulated in the late stage of NDV infected chicken fibroblast cell line DF-1 or human non-small cell lung cancer cell line A549.Meanwhile,after NDV infection of SPF chickens,the viral load in the heart,liver,spleen,lung,duodenum,brain,bursa of fabricius and trachea of the attacking group gradually increased,finally until death.Due to the differences of the organs,the CHCHD10 gene transcript levels in different organs varied during the infection,but all organs showed a decline in the late stage of infection.Among them,the liver,spleen,lung and duodenum showed a decline earlier,while the heart,brain,bursa of fabricius and trachea showed the latest decline.These results suggest a significant decrease of CHCHD10 expression levels in both susceptible cells and susceptible hosts during the late stage of NDV infection.3.CHCHD10 regulates mitochondrial dynamics equilibrium in the late stage of NDV infectionIn order to confirm that the reduction of CHCHD10 protein expression level in the late stage of NDV infection is favourable to NDV replication,we used NDV-infected A549 cells silenced or overexpressing CHCHD10,and found that the expression of CHCHD10 protein level was negatively correlated with the viral load in the infected cells.To investigate whether the reduced CHCHD10 protein disrupts host mitochondrial fusion and subsequently favours self-replication during the late stage of NDV infection,we first examined the effect of CHCHD10 protein level on mitochondrial membrane potential by flow cytometry.It was found that interfering with CHCHD10 expression further aggravated the reduced mitochondrial membrane potential in the late stage of NDV infection,whereas overexpression of CHCHD10 significantly alleviated this phenomenon.Subsequently,the regulatory effects of CHCHD10 protein levels on the expression levels of mitochondrial fusion-associated proteins Mitofusin 1(Mfn1),Mitofusin 2(Mfn2),and Optic Atrophy 1(OPA1),as well as on the gene transcription levels of mitochondrial fission-associated proteins dynamin related protein 1(Drp1),fission 1(Fis1),and mitochondrial fission factor(Mff),were examined by q PCR and WB after NDV infection.The results revealed that when NDV infected the cells silencing CHCHD10 at the late stage,the gene transcript levels of Mfn1,Mfn2 and OPA1 were significantly down-regulated,and the gene transcript levels of Drp1,Fis1 and Mff were significantly up-regulated,whereas the results of the overexpression group were the opposite.WB and q PCR results were largely consistent,that is,the reduced CHCHD10 protein allowed NDV to significantly inhibit the expression of Mfn1,while accelerating the cleavage of long isoform of OPA1(L-OPA1)into short isoform of OPA1(S-OPA1),leading to the inactivation of OPA1,whereas overexpression of CHCHD10 significantly attenuated the effects of NDV on Mfn1 and OPA1 proteins.Finally,immunofluorescence revealed that silencing CHCHD10 resulted in significant fragmentation of the mitochondrial skeleton during the late stages of NDV infection.These results suggest that the reduced CHCHD10 protein favours self-replication by inhibiting the host mitochondrial fusion process during the late stage of NDV infection.4.CHCHD10 regulates the RLR and NF-κB pathways in the late stage of NDV infectionStudies have shown that mitochondrial fusion is required for enhanced RLR signalling and activation of the NF-κB pathway.Therefore,we went on to explore the effect of CHCHD10 protein levels on the activation of RLR and NF-κB signalling pathways during the late stage of NDV infection.By dual luciferase reporter gene,immunofluorescence,q PCR and WB,we verified the role of CHCHD10 protein level on the regulation of key molecules of RLR and NF-κB signalling pathways during the late stage of NDV infection.Firstly,the effect of CHCHD10 protein level on IFN-β and NF-κB activation was detected in the late stage of NDV infection using a dual luciferase reporter gene,and the gene transcription level of IFN-β was detected by q PCR.The results revealed that in the late stage of infection,interfering with the expression of CHCHD10 significantly reduced the activation of IFN-β and NF-κB,as well as down-regulated the transcriptional level of IFN-β,whereas overexpression of CHCHD10 significantly enhanced the activation of the two and increased the transcriptional level of IFN-β.Immunofluorescence observation of interferon regulatory factor 3(IRF3)and NF-κB nuclear localisation showed consistent results,that is,interfering with CHCHD10 expression significantly attenuated the nuclear localisation ability of IRF3 and NF-κB,while overexpression enhanced the nuclear entry ability of both.Meanwhile,we further verified,by WB and q PCR,the effect of CHCHD10 protein level on the phosphorylation degree of key proteins TANK-binding kinase 1(TBK1),IRF3,and NF-κB of RLR and NF-κB pathways,as well as on the gene transcription level of the subsequent antiviral factors(ISG56,PKR,Mx1,and IL-6).The results showed that interfering with CHCHD10 protein expression both significantly reduced the phosphorylation level of each key protein and the gene transcription level of antiviral factors at the late stage of NDV infection.Finally,we inhibited both antiviral pathways using BX795 and BAY 11-7082 inhibitors to detect the gene transcript levels of viral load,IFN-β and antiviral factors.It was finally confirmed that NDV hinders the activation of the RLR pathway and the NF-κB pathway by decreasing the protein level of CHCHD10,thus antagonizing the production of IFN-β and antiviral factors,which ultimately favours its own replication.In order to verify whether the activation of CHCHD10-regulated RLR and NF-κB pathways is related to MAVS,we examined the effects of CHCHD10 protein levels on the spatial distribution and expression levels of MAVS in the late stage of NDV infection by immunofluorescence,WB and q PCR experiments.It was found that in the late stage of NDV infection,MAVS in cells interfering with CHCHD10 mainly showed scattered dot-like distribution compared with other groups.Meanwhile,both the transcript level and protein level of MAVS underwent a significant decrease in the late stage of infection.This suggests that the reduction of CHCHD10 protein expression in the late stage of NDV infection can alter the spatial distribution of MAVS as well as inhibit its expression level,thus hindering the activation of RLR and NF-κB pathways.In summary,this study found that in the late stage of NDV infection,the reduction of the protein level of the mitochondrial protein CHCHD10 disrupted the mitochondrial fusion process,thereby altering the spatial distribution of MAVS,inhibiting the protein expression of MAVS,impeding the activation of the RLR and the NF-κB pathway,and decreasing the secretion of IFN-β,which ultimately promoted viral replication.In contrast,overexpression of CHCHD10 attenuated the imbalance in mitochondrial dynamics caused by NDV infection,thereby inhibiting viral proliferation.This study provides new ideas to gain insight into the mechanism of NDV replication.