| Duck plague virus（DPV）belongs to Alphaherpesvirinae subfamily.UL41 gene is expressed with late kinetics and only exists in Alphaherpesvirinae subfamily.At present,there are few reports about DPV UL41 protein.To explore the characteristic and role of UL41 protein in viral life cycle,immune inhibition,UL41 gene deletion and revertant viruses were constructed using a markerless two-step Red recombination system implemented on DPV CHv genome cloned into a bacterial artificial chromosome（BAC-CHv）,named DPV CHv-BAC-ΔUL41,DPV CHv-BAC-UL41R,respectively.The results as follows:1.The structural properties and subcellular localization of UL41 protein（1）The UL41-his fusion protein（76 k Da）,was expressed in a prokaryotic expression system and identified using rabbit-anti DPV polyclonal antibody by western blotting.In addition,the purified UL41-his fusion protein was used to immunize mice and obtained mouse anti-UL41 protein polyclonal antibody,which could detect the UL41 protein and DPV.（2）The allantoic fluid was collected from DPV infected 9-day-old duck embryos and ultracentrifugated through a 30%（wt/vol）sucrose cushion to obtain purified DPV particles.And UL41 protein was existed in purified DPV particles using mouse anti-UL41 protein polyclonal antibody by WB and mass spectrometry,these results indicated UL41 protein is a viral structural protein.（3）UL41 protein only localized in cytoplasm of p EGFP-C2-UL41transfected DEF cells by indirect immunofluorescence assay（IFA）.（4）DPV-infected DEF cells were collected at 7-,12-,24-,36-,48-,60-and 72-hour post infection（hpi）,and the IFA results showed UL41 protein was distributed throughout in DPV-infected DEF cells,mainly distributed in cytoplasm and a small amount in the nucleus showing a dotted distribution at 7～36 hpi,and widely distributed in the nucleus and cytoplasm at 48～72 hpi.2.The UL41 protein localized in the nucleus mainly depends on the nuclear localization signal of DPV UL47 proteinIn order to explore the mechanism of UL41 protein nuclear entry,（1）Bioinformatics analysis found UL41 protein don’t include nuclear localization signal（NLS）,and the residues 40～50 and 768～777 of DPV UL47 gene encoding protein could function as NLS.（2）IFA found UL47 protein was mainly distributed in cytoplasm when the residues 40～50（pc DNA3.1-UL47Δ40-50）and 768～777（pc DNA3.1-UL47Δ768-777）of UL47 protein were deleted respectively or simultaneously.But UL47 protein mainly distributed in nucleus in pc DNA3.1-UL47 transfected DEF cells.The residues 40 to 50 and 768 to 777 of UL47protein were fused with enhanced green fluorescent protein（EGFP）or beta-galactosidase（β-gal）,which were transported into nucleus.In addition,we co-transfected the pc DNA3.1-UL47Δ40-50,pc DNA3.1-UL47Δ768-777 with p EGFP-C2-UL41 into DEF cells,IFA found UL41 protein couldn’t enter nucleus.3.UL41 protein inhibits DNA dependent RNA polymerase（pol）II-transcribed translatable RNA to induce host shutoff（1）UL41 protein induced host shutoff:WB found the global protein synthesis was significantly reduced in early stage of DPV BAC-CHv and DPV CHv-BAC-UL41R recombinant viruses infected DEF cells,but not changed in the DPV CHv-BAC-ΔUL41mutant virus infected DEF cells using mouse anti-puromycin monoclonal antibody.Collectively,UL41 protein played a prominent role in DPV-induced protein synthesis shutoff.（2）UL41 protein selectively inhibited DNA dependent RNA pol II-transcribed translatable RNA:UL41 protein dramatically inhibited the RNA level of RNA pol II transcripts in pcaggs-UL41-HA transfected HEK293T cells using RT-q PCR.The RNA level of pol II transcripts was increased in DPV BAC-CHv-ΔUL41 mutant virus infected DEF cells.The EGFP RNA of pol II promoter transcribed transcripts was inhibited by UL41 protein.Bioinformatics analysis the residues E229,D231,D232 of UL41 protein were conserved.And mutation of E229,D231,D232 of UL41 protein to“A”increased the UL41 protein transcription and expression using RT-q PCR and WB,and did not obviously affect the RNA level of pol II transcribed translatable RNA.These results found the residues E229,D231,D232 of UL41 protein were active sites.4.DPV UL41 protein participated in the orderly expression of viral genes,DNA replication,release and cell to cell spread（1）UL41 protein participated in viral DNA replication and release:viral multi-step growth curve showed that UL41 protein was dispensable for viral replication,but it could promote the release of virions into cell supernatant.UL41 protein promoted the viral DNA replication and release by TCID50 and RT-q PCR.（2）The effect of UL41 protein in viral cell-to-cell spread:the plaque size was decreased in DPV CHv-BAC-ΔUL41 infected DEF cells using plaque morphology assay,which showed UL41 protein participated in viral cell-to-cell spread.（3）UL41 protein participated in orderly expression of viral genes:compared with the m RNA level of viral genes in DPV CHv-BAC infected DEF cells,the m RNA level of many viral genes was changed in DPV CHv-BAC-ΔUL41 infected DEF cells,many genes were significantly upregulated,such as UL55,LORF4,UL54,UL51,UL50,UL49,UL44,UL43,UL39,UL38,UL35,UL34,UL33,UL32,UL31,UL29,UL28,UL27,UL26,UL24,UL22,UL20,UL19,UL18,UL13,UL12,UL11,UL7,LORF2,US3,US6,US7,US8 and US10;some genes were slightly upregulated,such as UL37,UL36,UL23,UL16,UL14,UL9,SORF3,ICP22 and ICP4;some viral genes were downregulated,such as UL53,UL47,UL46,UL45,UL42,UL8 and US2;some viral genes were unchanged,such as UL40,UL21,UL17,UL10,UL4,UL3,UL2,LORF3,US4 and US5.And the transcription level of viral genes was similar between DPV CHv-BAC parent viruses and DPV CHv-BAC-UL41R revertant viruses.5.UL41 protein inhibited the RIG-I/MDA5 immune pathway in DEF cells to mediate immune evasion（1）UL41 protein inhibited the RIG-I/MDA5 immune pathway in DEF cells:UL41protein significantly inhibited the IFN-β,ISG,IFN-β-Luc production stimulated by poly（I:C）in DEF cells.The transcription of IFN-β,Mx,OASL was increased in DPV BAC-CHv-ΔUL41 mutant virus infected DEF cells.Co-transfected pcaggs-UL41-HA with RIG-I,MDA5,MAVS,STING,TBK1 and IRF7 eukaryotic expression plasmids into DEF cells,respectively,UL41 protein inhibited the IFN-β-Luc activity stimulated by RIG-I,MDA5,MAVS,STING,TBK1 and IRF7.Mutation of the residues E229,D231,D232 of UL41protein to“A”couldn’t inhibit the IFN-β,ISG or IFN-β-Luc production.（2）UL41 protein significantly decreased the m RNA level of adaptors:UL41 protein significantly decreased the m RNA level of adaptors stimulated by poly（I:C）in DEF cells,especially RIG-I,MDA5,STING,and IRF7.Co-transfected pcaggs-UL41-HA with RIG-I,MDA5,MAVS,STING,TBK1 and IRF7 eukaryotic expression plasmids into HEK293T cells,respectively,UL41protein significantly decreased the m RNA level of RIG-I,MDA5,MAVS,STING,TBK1and IRF7.Mutation of the residues E229,D231,D232 of UL41 protein to“A”couldn’t inhibit the m RNA level of RIG-I,MDA5,MAVS,STING,TBK1 or IRF7.（3）Knockdown the expression of IRF7 protein in DEF cells promoted the DPV CHv-BAC-ΔUL41 mutant virus replication:knockdown the expression of IRF7 protein in DEF cells by sh RNA,RT-q PCR and WB found the knockdown efficiency was 50%.TCID50 found knockdown the expression of IRF7 protein in DEF cells facilitated the replication of DPV CHv-BAC-ΔUL41 mutant virus.Above all,UL41 protein as a viral structural protein,mainly localized in the cytoplasm and nucleus of DPV-infected DEF cells.UL41 protein couldn’t enter the nucleus from cytoplasm independently.Another viral protein UL47 could transport the UL47,UL41,EGFP,β-gal into nucleus mainly depends on its NLS,residues 40～50 and 768～777.Further studies found UL41 protein selectively degrades RNA pol II-transcribed translatable RNA to induce the host protein synthesis shutoff.UL41 protein was dispensable for viral replication,but promoted viral DNA replication,release,cell-to-cell spread by regulating orderly expression of viral genes.In addition,UL41 protein inhibited the RIG-I/MDA5immune pathway by broadly inhibiting the m RNA level of important adaptor molecules,including RIG-I,MDA5,MAVS,STING,TBK1 and IRF7.And the conserved residues E229,D231 and D232 of UL41 protein played a crucial role in degrading m RNA and innate immune inhibition. |
