Study On The Role Of IRF3 Mediating PBLD Inhibition Of BPIV3,VSV And HSV-1 Replication

PBLD is a phenoazine biosynthesis-like domain protein,which is abnormally expressed in a variety of cancer diseases,pretending to be the signature protein of cancer occurrence.Overexpression of PBLD in human intestinal epithelial cells can inhibit NF-κB,thereby inhibiting TNF-αinduced inflammatory response and improving intestinal barrier.In addition,PBLD can induce apoptosis in cancer cells and maintain the healthy state of host cells.In conclusion,there are no reports on the involvement of PBLD in virus replication andⅠFN-Ⅰsignaling.IRF3 is not only a key molecule regulatingⅠFN-Ⅰsignal transduction pathway,also can induce cell apoptosis.This study was the first to discover that PBLD activated antiviralⅠFN-Ⅰresponse and induced apoptosis by regulating IRF3,and further explored its molecular mechanism.The experimental results are as follows:（1）PBLD up-regulated the expression of IRF3 under virus infectionHe La cell lines with PBLD knockout and stable expression were constructed,infection with BPIV3,VSV,HSV-1,and treated with RNA virus analog and DNA virus analog.Cell samples were detected by RT-q PCR and Western Blot.It was found that overexpression of PBLD promoted the expression of IRF3 protein,while knockout of PBLD inhibited the expression of IRF3 protein.The results indicated that PBLD up-regulated the expression of IRF3 after virus infection.（2）PBLD promotes virus-mediated IFN-Ⅰby activating phosphorylation of IRF3（S385/386）HSV-1 and DNA viruses analog processed expression or knockout PBLD He La,by RT-q PCR and Western Blot detection,found expression PBLD can PromoteⅠFN-Ⅰresponse,and suppress ICP0 of viral protein replication,Knockout of PBLD inhibitedⅠFN-Ⅰresponse,suggesting that PBLD could promote theⅠFN-Ⅰresponse against HSV-1.In order to investigate the relationship between IRF3 and PBLD,a key molecule of IFN-ⅠPromote,BPIV3,VSV,HSV-1 were used to infect cells that overexpressed PBLD and si IRF3,cell samples were detected by RT-q PCR and Western Blot.It was found that after si IRF3,PBLD inhibited the up-regulation of IFNβand ISGs expression,indicating that theⅠFN-Ⅰresponse activated by PBLD was dependent on IRF3.A mutant at S385/386 and S396,the key site for phosphorylation of IRF3,was constructed.PBLD,wild-type IRF3 and its mutant were transferred into He La cell line with IRF3 knocked out.Western Blot detection showed that the S385/386 mutation of IRF3 inhibited theⅠFN-Ⅰresponse and the expression of ISGs.The results indicate that PBLD activatesⅠFN-Ⅰthrough phosphorylation of S385/386 of IRF3,not S396.（3）PBLD promotes virus-induced apoptosis by recruiting PUMA localization mitochondria via IRF3VSV and HSV-1 were used to infect He La cell lines with PBLD overexpression or knocked out.Western Blot showed that the expression of C-PARP was up-regulated by overexpression of PBLD.Knockout of PBLD inhibited the expression of C-PARP.These results indicated that PBLD could promote apoptosis induced by virus infection.The virus infected He La cells overexpressing IRF3.Western Blot detection showed that the overexpression of IRF3 up-regulated the expression of C-PARP.Then,si IRF3 in He La cells,and it was found that si IRF3 inhibited the expression of C-PARP,indicating that IRF3 could promote apoptosis induced by viral infection.In order to investigate the relationship between apoptosis induced by PBLD and apoptosis induced by IRF3,si IRF3 in He La cell lines with stable PBLD expression.It was found that PBLD-induced apoptosis was significantly inhibition after si IRF3.The results indicated that PBLD induced apoptosis through IRF3.Mutant expression plasmid was constructed to mutate the K193and K313/315 of IRF3,and PBLD was cotransferred with wild-type IRF3 and its apoptotic mutant to He La cell line with IRF3 knocked out.Western Blot showed that the K313/315 mutation of IRF3 inhibited the expression of C-PARP.The results indicated that PBLD promoted virus-induced apoptosis mainly through K313/315 of IRF3,not K193 of IRF3.In order to investigate the specific mechanism of PBLD inducing apoptosis through IRF3,it was first found that IRF3 could interact with PUMA through Co-Ip.Subsequently,the protein expression of PBLD was increased in He La cells at dose,and it was found that PBLD could enhance the interaction between IRF3 and PUMA.Through mitochondrial isolation experiments,the results showed that PBLD could promote the transfer of IRF3 and PUMA from cytoplasm to mitochondria.Through immunofluorescence experiments,PBLD was found to enhance the interaction between IRF3 and PUMA and transfer to mitochondria,while knockout of PBLD inhibited the interaction between IRF3 and PUMA.He La cell line with PUMA knockout was constructed that IRF3 and PBLD were overexpressed in the cell line.The expression of C-PARP was inhibited by Western Blot detection,indicating that PUMA knockout inhibited IRF3 and promoted virus-induced apoptosis.These results suggest that PBLD promotes virus-induced apoptosis by enhancing the binding of PUMA and IRF3.（4）PBLD inhibits viral replication by activating IRF3-mediated IFN-Ⅰand apoptosisApoptosis induced by PBLD inhibits viral replication.After overexpression of PBLD in He La cells,the cells were treated with apoptosis inducer and apoptosis inhibitor.Western Blot and TCID₅₀ were used to detect the change of virus titer.It was found that after apoptosis inducer treatment,the overexpression of PBLD promoted the expression of C-PARP and inhibited the replication of virus.Inhibit the expression of C-PARP protein and promote the replication of virus.PBLD inhibits viral replication through IRF3.When PBLD was overexpressed in IRF3knockout cell lines,PBLD showed a weaker ability to inhibit viral replication when IRF3 was knocked out compared with the control group.Overexpression of IRF3 in cells with stable overexpression of PBLD significantly inhibited viral replication when PBLD and IRF3 were co-expressed compared to the control group.PBLD inhibits viral replication by promotingⅠFN-Ⅰresponse and inducing apoptosis through IRF3.The S385/386A mutant that phosphorylates IRF3 and K313/315N mutant that induces apoptosis of IRF3 were co-expressed with PBLD in He La cells,respectively,and detected by TCID₅₀.It was found that PBLD promotedⅠFN-Ⅰresponse through IRF3 and PBLD inhibited viral replication through apoptosis induced by IRF3.In summary,from the perspective of PBLD promoting antiviralⅠFN-Ⅰresponse and virus inducing cell apoptosis,this study took the regulation ofⅠFN-Ⅰresponse and cell apoptosis pathway by PBLD through IRF3 as the main line,discover new functions of PBLD for virus replication via IRF3.The molecular mechanism of PBLD activation of antiviralⅠFN-Ⅰresponse and induction of apoptosis by regulating IRF3 was revealed,providing candidate target molecules and new clues for antiviral drug research.