Effects Of US3 Protein Kinase Activity On Duck Plague Virus Replication And Subcellular Localization Of UL47 Protein

Duck plague,also known as duck virus enteritis,is an acute,febrile,septic disease that appears in ducks,geese,or swans,with high morbidity and mortality,and has caused serious economic losses in the waterfowl industry.Duck plague virus（DPV）is the pathogen of duck plague and belongs to Alphaherpesvirinae subfamily.Alphaherpesvirus US3 gene encodes a viral Ser/Thr protein kinase,which is a multifunctional protein that plays an important role in viral replication,virulence,and host immune escape.However,there are few reports about the functions of DPV US3protein.Therefore,this thesis describes the functions of DPV US3 protein and its protein kinase activity in viral replication,aiming to further elucidate the pathogenesis of DPV infection,and to provide a candidate for development of attenuated DPV vaccines.The main results are as follows:1.Effects of DPV US3 protein on viral cell-to-cell spread and capsid nuclear egressTo explore the functions of DPV US3 protein,we constructed DPV US3-deleted mutant strain（DPVΔUS3）and its revertant virus strain（DPV RUS3）,tested their biological characteristics,and found:（1）Viral growth curves showed that US3 gene deletion significantly reduced the viral titer(TCID₅₀),indicating that US3 promotes DPV replication.（2）Plaque assays showed that lack of US3 protein caused smaller plaque sizes,indicating that US3 promotes DPV cell-to-cell spread.（3）Electron microscopy assays showed an accumulation of nucleocapsids and primary enveloped virions in DPVΔUS3 infection,indicating that DPV US3 protein promotes capsid nuclear egress.2.Effects of US3 protein kinase activity on viral replication and UL31/UL34localizationA series of experiments were carried out to further explore the functions of DPV US3 protein:（1）We used the Phospho-PKA Substrate（RRXS*/T*）（100G7E）to detect the protein phosphorylation caused by US3 protein.The results showed that there were multiple phosphorylated bands in the parental virus-infected cells,but the bands were disappeared in DPVΔUS3-infected cells,indicating that DPV US3 protein has protein kinase activity.（2）We constructed another DPV mutant virus strain DPV US3K213A,which was identified as having no US3 protein kinase activity,detected viral growth curves,plaque sizes and capsid assembly of DPV WT,DPV US3K213A,DPVΔUS3and DPV RUS3 strains,and found that the effects of US3 on viral replication were dependent on its protein kinase activity,including viral cell-to-cell spread and capsid nuclear egress.（3）Indirect immunofluorescence assays（IFA）showed that an irregular localization of UL31 or UL34 protein in the nucleus was observed in DPVΔUS3-and DPV US3K213A-infected cells,with discrete and a round foci distribution,while UL31or UL34 protein was localized in the nucleus with uniform distribution in the parental virus infection.（4）Co-immunoprecipitation assays showed the interaction of US3 and UL31 proteins.The interaction of US3 and UL31 proteins and the localization of UL31/UL34 protein regulated by US3 protein kinase activity might promote capsid nuclear egress in DPV.3.UL54 protein was a new phosphorylated substrate of US3 protein kinaseTo identify phosphorylated substrates of US3 protein,a series of experiments were carried out as follows:（1）A recombinant virus strain DPV US3-Flag was constructed,and US3 protein was enriched by Flag tag antibody for mass spectrometry analysis.The results showed that there are several viral proteins observed in the enrichment of US3protein,including US10,UL7,UL12,UL31,UL34,UL37,UL47,UL49,UL54,g B,LORF3,LORF4 and LORF5 proteins.（2）UL47 protein was detected to be phosphorylated by Phospho-PKA antibody when US3 protein was co-expressed with UL31,UL34,UL47 or g B protein,and the phosphorylation level of UL47 protein was dependent on US3 protein concentrations,indicating that UL47 protein is a phosphorylated substrate of US3 protein kinase.（3）US3 protein was co-expressed with US10,UL54,LORF3,LORF4 or LORF5 protein,and found UL54 protein was phosphorylated when co-expressed with US3 protein,suggesting that UL54 protein is a phosphorylated substrate of US3 protein,which is first reported inα-herpesvirus.4.The phosphorylation of UL47 protein caused by US3 protein promotes the cytoplasmic localization of UL47As described above,UL47 protein was phosphorylated by US3 protein.To detect the interaction of US3 and UL47 proteins,the phosphorylated sites of UL47 caused by US3 protein,and the effect of US3 protein on UL47 localization,the following experiments were carried out:（1）Co-immunoprecipitation assays showed the interaction of US3 and UL47 proteins in co-expression of US3 and UL47 proteins or DPV WT virus infection.（2）IFA assays showed that US3 protein was localized in the cytoplasm when it was expressed alone;UL47 protein was localized in the nucleus when it was expressed alone;US3 protein interacted with UL47 when they were co-expressed and then co-localized in the cytoplasm;UL47 protein couldn’t be phosphorylated by US3^K213A protein when US3^K213A and UL47 proteins were co-expressed,was localized in the nucleus,while the interaction of UL47 and US3^K213Aproteins was still detected.All results indicated that the cytoplasmic localization of UL47 protein is dependent on its phosphorylation caused by US3 protein rather than protein-protein interaction.（3）Identification of Phospho-PKA antibody,MS analysis and IFA assay showed that six sites of UL47 at T29,S30,S42,T47,S161 and T775were the phosphorylation targets of US3 protein,which provides data to further explore the roles of this phosphorylation in viral replication.In summary,DPV US3 protein promotes viral replication by regulating viral cell-to-cell spread and capsid nuclear egress using its protein kinase activity.DPV US3protein regulates the localization of UL31/UL34 protein,and interacts with UL31protein.In addition,two viral proteins UL47 and UL54 are both phosphorylated substrates of US3 protein,and UL54 protein was first reported to be phosphryalted by US3 protein inα-herpesvirus.US3 protein interacted with UL47 and phosphorylated six sites of UL47 protein,including T29,S30,S42,T47,S161 and T775,facilitating cytoplasmic localization of UL47 protein.