The Role Of VP16 Protein Of Duck Plague Virus In Virus Life Cycle And Its Relationship With Us3 Protein

Duck plague（DP）,a highly contact infectious disease caused by duck plague virus（DPV）,is one of the important outbreaks currently seriously threatening the development of duck farming industries.In this study,VP16,the encoding protein of UL48 gene of duck plague virus,was taken as the research object,and its influence on the virus life cycle and its relationship with viral kinase Us3were studied.The following results were obtained:1.The protein type of VP16The duck embryo’s fibroblast（DEF）was infected with DPV-BAC strain of duck plague virus at 0.1MOI.The m RNA and protein levels of VP16 at different time points were detected by q RT-PCR and Western Blot.The results showed that the change trend of its transcription and protein expression level was basically the same,which was similar to the late protein UL47.At the same time,PCR was used to detect the inhibitory effects of ganciclovir（GCV）and cycloheximide（CHX）.The results showed that the expression of VP16 was sensitive to the inhibition of both GCV and CHX,which further confirmed that VP16 was a late protein of DPV.2.The effect of VP16 protein on the life cycle of duck plague virusUL48 deletion virus（DPV-BAC-?UL48）,the revertant virus（DPV-BAC-?UL48R）and its parent virus（DPV-BAC）constructed in laboratory were used to study the effect of VP16 protein on the life cycle of DPV.（1）q RT-PCR was used to detect the genome copy number of the virus infected with DEF.The results showed that the copy number of the deletion virus was significantly lower than that of the parent virus and the revertant virus,indicating that VP16 protein affected the adsorption of the virus on the cell surface,the invasion and the DNA replication process.The virus titer released into the supernatant after DEF infection was detected.It was found that the virus titer in the absent virus supernatant was significantly lower than that of the parent virus and the revertant virus,indicating that VP16 protein affected the virus release process.（2）The genome copy number and TCID₅₀of the virus infected with DEF were detected.The results showed that the efficiency of producing infectious virions by the deletion virus was significantly reduced,and more genomes were needed to cause infection comparable to that of the parent virus,indicating that VP16 protein played an important role in the generation of DPV infectious virions.（3）The effect of VP16 on IE gene transcription in DPV was detected by q RT-PCR.The results showed that the m RNA level of IE gene in the deletion virus was significantly decreased compared with that of the parent virus,indicating that the deletion of VP16 would significantly inhibit the IE gene transcription in DPV.3.Identification of key sites of VP16 protein to activate immediate early geneMass spectrometry analysis showed that Ser-101,Ser-407 and Ser-413 are phosphorylated sites of VP16.The VP16 point mutant plasmids were constructed,and the effect of each point on the activation of IE gene by VP16 was tested using the dual luciferase reporting system.The results showed that the activation of IE gene promoter by VP16 was significantly weakened after serine at sites 101,407 and 413 was mutated to alanine.The results showed that the three phosphorylation sites of VP16 protein were the key sites for its activation of IE gene.4.The effect of viral protein Us3 on VP16（1）Co-immunoprecipitation and indirect immunofluorescence experiments showed that VP16and Us3 proteins were colocalized and interacted within cells.（2）The instantaneous expression of VP16 was detected by Western Blot,and it was found that Us3 could promote the protein expression of VP16 in a dose-dependent manner;q RT-PCR detected that Us3 upregulated the m RNA level of VP16,and by infection with the duck plague virus strong strains CHv and Us3 deletion virus,the VP16 m RNA level of the Us3 deletion virus strain was significantly reduced.（3）The diluciferase reporter system detected that Us3 significantly activated the promoter activity of VP16.These results indicated that viral protein Us3 promoted the m RNA level of VP16 to be up-regulated by activating the promoter activity of VP16,thus increasing its protein expression.（4）The diluciferase reporter system detected that Us3 was able to inhibit the activation of the promoter of the IE gene by VP16.5.The region where VP16 interacts with Us3Truncated proteins of VP16 and Us3 with different lengths were constructed.Co-immunoprecipitation and Western Blot were used to screen the interaction regions of VP16 and Us3.The results showed that VP16 interacted with Us3 138-256aa through its 200-298aa and 310-407aaIn conclusion,VP16 is a late protein of DPV,and the deletion of its encoding gene will lead to the decrease of viral replication and the formation efficiency of infectious virions.In addition,VP16can interact with viral protein Us3,which can promote the up-regulation of m RNA level by activating VP16 promoter activity,thus increasing the protein expression of VP16..