Molecular Characteristics And Function Of Duck Enteritis Virus US1 Protein

Duck enteritis virus(DEV)belongs to Herpesvirales,and there is no report on the DEV US1 gene and its encoded protein(ICP22).In this paper,the molecular characteristics and functions of duck enteritis virus US1 protein were studied,and the following results were obtained:1.Molecular characteristics of US1 gene and encoded proteinThe pet32a(+)-ICP22 prokaryotic expression plasmid was constructed,and the ICP22 recombinant protein was purified to prepare mouse anti-ICP22 antibody.The antibody specificity was then checked and the protein molecular weight was verified.The results showed that the molecular weight of ICP22 protein produced a molecular weight migration of approximately 22 kDa in both prokaryotic and eukaryotic expression systems,and a protein of approximately 57 kDa was obtained,indicating that ICP22 protein is a post-translationally modified protein.Subsequently,fluorescence quantitative and immunoblotting were used to detect changes in US1 gene transcription and ICP22 protein expression levels.The results showed that the two trends were basically the same.They were detected at 0.5 h and 2 h after infection,respectively,and the expression level increased with time.It gradually rises,peaks at 24 h or 36 h,and then declines with time.In addition,gene type identification test results show that the US1 gene transcription does not depend on the synthesis of DNA and other viral proteins and is an immediate early gene.2.Construction and identification of US1 gene single and double copy deletion strainsBased on the artificial chromosome platform of duck enteritis virus bacteria constructed in this laboratory,this article uses two-step homologous recombination technology to sequentially knock out 2 copies of the DEV US1 gene,and PCR amplification and RFLP digestion to identify single and double copies of the US1 gene It has been successfully knocked out and named GS1783-p BAC-DEV-?US1,2?US1respectively.The plasmid of the US1 gene-deleted strain was extracted,transfected into DEF cells,and rescued by recombinant virus.After the fluorescent spots appeared,the viruses were collected and identified by PCR,indirect immunofluorescence and immunoblotting.After confirmation,the harvested recombinant viruses were named DEV CHv-BAC-G-?US1 and DEV CHv-BAC-G-2?US1.3.The effect of US1 gene on virus proliferation and gene transcriptionThrough virus titer and copy number determination,it was determined that the deletion of US1 gene can significantly reduce viral genome replication and virus titer.Further electron microscopy results show that the deletion of the US1 gene can affect the budding process of the nucleus of the virions,the proportion of the inner shell of the nucleus increases,the proportion of virions that complete the primary encapsulation around the nucleus is reduced,but the completion of coating can be observed in the cytoplasm Mature virion viruses indicate that the US1 gene plays an important role in the virion maturation process,but it is not essential.Dynamic transcriptome analysis of US1 deleted strains and parental strains showed that after the deletion of US1 gene,viral gene transcription was generally suppressed,while cellular gene transcription levels were mostly up-regulated.Further analysis showed that ICP22 could participate in many biological processes,such as signal transduction,cell cycle regulation,RNA polymerase transcription,involving Toll-like receptors,MAPK,Wnt,RNA POL ?,cytoplasmic DNA sensing and other signaling pathways.The results of RT-qPCR showed that the deletion of US1 gene caused the upregulation of the expression levels of basic transcription factors related to RNA POL ?,such as MAT1 A,MED23,TAF7 L,EAF1,GTF2H2,while the mRNA levels of DNA repair molecule BRCA1,transcription negative regulator HEXIM1,chaperone protein Hsp40C5 B,cyclin E and other genes decreased significantly.The results of RT-qPCR validation were consistent with the change trend of transcriptome data,which further illustrated the reliability of transcriptome data.In conclusion,the DEV US1 gene plays an important regulatory role in cell gene transcription,and its regulation of viral and host gene transcription may be achieved through transcriptional common links such as basic transcription factors.4.Intracellular localization and nuclear localization signal identification of ICP22 proteinThe results of bioinformatics analysis showed that ICP22 protein had a strong ability to actively enter the nucleus.The results of intracellular localization and NLS biological function identification showed that ICP22 protein could actively enter the nucleus and completely localize in the nucleus under viral infection and plasmid transfection.Among them,amino acids at position 305-314 were a classical mononuclear localization signal,and ICP22 without this part could not actively enter the nucleus.Further point mutation test results showed that ICP22 protein could not actively enter the nucleus after 309 R single mutation and 308-312 KRKRP all mutations.To this end,we constructed 309 R and308-312 KRKRP mutant viral strains and observed the intracellular localization of ICP22 protein under the infection of the mutant viral strain.The results showed that both mutations could affect the efficiency of ICP22 protein into the nucleus but did not change the characteristics of the final localization to the nucleus.