The Research Of Transcriptional Regulation Function Of Duck Plague Virus ICP4

Duck Plague Virus(DPV)belongs to the Herpesvirus family.It is known that other ICP4 of herpes simplex virus is an immediate early(IE)gene,while the data of ICP4 of DPV is lacking.The study has not been reported.In this paper,the transcription and regulation of the ICP4 gene of DPV were studied and the following results were obtained.1.DPV ICP4 gene typeUsing green fluorescent protein as a reporter gene,the fluorescence distribution of pEGFPICP4 in duck embryo fiber(DEF)cells was observed.It was found that green fluorescence was only distributed in the nucleus,and the ICP4 protein was initially identified in the nucleus;further anti-ICP4 polyclonal antibody was used.The antibody was used in indirect immunofluorescence to detect the location of ICP4 after DPV-infected DEF cells,and it was found that ICP4 protein was detected only in the nucleus,indicating that the ICP4 protein is localized in the nucleus.Fluorescence quantitative PCR was used to detect the transcription level of ICP4 gene at different time after DPV infection of DEF cells.The results showed that the expression of ICP4 was detected 2 h after viral infection,and its expression level continued to increase and peaked at 36 h.Further PCR was used to detect whether the ICP4 transcription of DPV-infected DEF was affected by the nucleic acid synthesis inhibitor GCV and the protein synthesis inhibitor CHX,the results showed that both drugs failed to affect the transcription of ICP4,indicating that ICP4 of DPV has features of alpha herpesvirus IE gene.2.Transcriptional regulation of DPV ICP4 geneThe effect of ICP4 on the transcription level of some viral genes in DPV-infected DEF cells was detected by RT-qPCR.The results showed that the mRNA level of IE gene ICP27 was upregulated 12 h after infection,and the early(E)gene TK was detected 24 h after infection.The mRNA levels of UL28 and late(L)genes gC and UL15 were up-regulated.The cells in the experimental group died at 48 h after infection,so the mRNA levels of each gene were lower than those of the control group,indicating that overexpression of ICP4 accelerated DPV infection.The process of further detecting the promoter of viral gene promoter by ICP4 using dual luciferase reporter system,and found that ICP4 can up-regulate the promoter activities of TK,UL28,gC,UL15 and ICP27,and down-regulate the activity of the promoter of its own gene,indicating ICP4 has a transcriptional activation effect on the IE gene,E gene and L gene of the virus,but has a transcriptional inhibitory effect on the own gene.3.DPV ICP4 gene anti-apoptosis detectionThe dual luciferase reporter system was used to detect the effect of ICP4 against the apoptotic gene US5.It was found that eukaryotic expression of ICP4 up-regulated the promoter activity of the anti-apoptotic gene US5,indicating that ICP4 has transcriptional activation against the apoptotic gene US5;the overexpression of ICP4 by qPCR did not affect the caspase-3 mRNA transcription level of apoptotic cells induced by hydrogen peroxide.In summary,ICP4 of DPV is an IE gene whose protein is localized in the nucleus.ICP4 can activate the transcription of some IE,E and L genes of DPV,but inhibits the transcription of selfgenes;ICP4 does not have direct anti-apoptotic function.