Identification And Mechanism Evaluation Of Nuclear Localization Signal In Tembusu Virus NS5 Protein

In April 2010,after the outbreak of Tembusu virus disease in the southeastern coastal areas of our country(Zhejiang,Fujian province and other places),it quickly spread in China's duck farms,and the infection of ducks caused egg laying decline in laying ducks and neurological symptoms in ducklings,which causing huge economic losses to our country's duck industry.The pathogen of the disease is Tembusu virus(TMUV),a member of Flavivirus.Other flaviviruses of the same genus are Dengue virus(DENV),Japanese encephalitis virus(JEV),West Nile virus(WNV)and Zika virus(ZIKV).Studies have shown that the NS5 proteins of many flaviviruses can block JAK-STAT signaling to antagonize the IFN-I response of the host,thereby evading innate immune response.Previous studies in our laboratory showed that TMUV-NS5 antagonized JAK-STAT signal transduction by interacting with KPNA1 at 37-45 aa.At the same time,nuclear localization signal(NLS)plays an important role in virus replication and viral protein function.On this basis,this study screened and identified the NLS_37-45 of TMUV-NS5 protein,and explored its related functions and mechanism of evaluation deeply.The research contents include the following aspects:1.Activity identification of TMUV-NS5-NLS_37-45 and subcellular localization analysis of TMUV-NS5.In order to further verify the biological activity of this amino acid sequence,the tandem expression plasmid of NLS and 2×GFP was constructed in the study.IFA results showed that NLS_37-45 could carry 2×GFP into the nucleus,and the Co-IP results also showed that NLS_37-45-2×GFP interacted with KPNA1.The biological activity of NLS_37-45 was confirmed.In addition,NLS_37-45 was connected to the front end of the TMUV-NS5-Rd Rp domain.Cell localization results showed that NLS_37-45 could carry the Rd Rp domain originally located in the cytoplasm into the nucleus,which further demonstrated the accuracy of the previous results.Meanwhile,according to amino acid similarity analysis,37-45 amino acids of TMUV-NS5 were highly conserved in TMUV.The basic amino acids in NLS are essential for its activity.In order to determine the functional basic amino acids in 37-45 aa,the basic amino acids in NLS_37-45 were single-point mutated to construct a series of mutant expression plasmids containing NLS_37-45 and two GFP proteins.IFA showed that except for wild-type NLS_37-45-2×GFP,which could be completely localized in the nucleus,the other mutants showed incomplete nuclear localization.To verify the accuracy of the above results,the mutant plasmids and Myc-KPNA1 were co-transfected into HEK293 cells.The Co-IP results showed that the interaction of other mutants with KPNA1 was weakened compared with the NLS_37-45-2×GFP?s co-transfection group.The above experiments confirmed that each basic amino acid in TMUV-NS5-NLS_37-45 played a functional role.After confirming the biological activity of NLS_37-45,whether it can locate the NS5protein in the nucleus was identified in our subsequent study.Therefore,the subcellular localization of TMUV-NS5 protein was analyzed.HA-NS5 plasmid was overexpressed in Hela cells,and the laser confocal results showed that TMUV-NS5 was localized in the cytoplasm and was not affected by IFN-?stimulation time.The use of the nuclear export inhibitor LMB also did not concentrate the NS5 protein in the nucleus,which indicated that the subcellular localization of TMUV-NS5 protein was in the cytoplasm.Although the nuclear localization activity of NLS_37-45 region was confirmed in this study,the nuclear localization of TMUV-NS5 was not found.It is speculated that the nuclear localization of the proteins is closely related to various factors.2.Analysis of nuclear localization ability of NLS_37-45In this study,expression plasmids that can express 2,3,and 4×GFP proteins so as to simulate cargo proteins of different molecular weights were constructed.We transfected Hela cells and combined with laser confocal analysis of their cellular localization.The results showed that when the molecular weight of the protein is small,NLS_37-45 can carry proteins into the nucleus,showing complete nuclear localization;when the molecular weight increases to a certain size,it cannot complete nuclear localization,showing a diffuse distribution.This result indicated that the nuclear localization ability of NLS_37-45 was related to the molecular weight of cargo protein.To further verify the above results,the NS5 protein was truncated in this study,and the cell localization of each truncated body was identified.The results showed that the truncated body containing NLS_37-45 was completely nuclear localization,while the region without NLS_37-45 was located in the cytoplasm.Moreover,NLS_37-45-Rd Rp was completely nuclear localized,and TMUV-NS5 contained NLS_37-45 localized only in the cytoplasm.In this study,allelic substitutions of NLSs with nuclear localization ability of their NS5proteins in flaviviruses were performed to observe whether such substitutions could affect cell localization of TMUV-NS5.The results showed that the mutant TMUV-NS5 proteins were still located in the cytoplasm,which indicated that the localization ability of NLS_37-45 was not limited to the molecular weight of cargo proteins,but also related to the molecular structure of the cargo proteins.Different types of cargo protein also affect the function of NLS.The above results to a certain extent explain the reason why NLS_37-45 has nuclear localization function but does not localize TMUV-NS5 in the nucleus.3.Comparative analysis of flavivirus NLS_37-45 in the same regionBased on the above analysis,this study confirmed that TMUV-NS5-NLS_37-45 had typical nuclear localization signal function.In order to further analyze whether this region of flavivirus members had the same function,five typical flaviviruses were selected to have a similarity analysis of amino acid sequences in the same region.The results showed that the sequence was relatively conserved,and TMUV was highly similar to WNV and JEV with only one difference in basic amino acid species,ZIKV,YFV and DENV-2 were less similar to TMUV-NS5-NLS_37-45.Subsequently,in this study,all sequences were linked with 2×GFP to analyze whether the 37-45 aa(or 38-46 aa)regions of different flaviviruses had the same nuclear localization activity as TMUV-NS5-NLS_37-45.Confocal laser results showed that other flaviviruses in this region could not fully localize 2×GFP in the nucleus,indicating that NLS_37-45 had specificity in TMUV activity.The corresponding basic amino acid in the same region of flavivirus was mutated into the same basic amino acid structure as TMUV-NS5-NLS_37-45.After transfection,cell localization results showed that the mutated sequence could carry 2×GFP and completely locate in the nucleus,indicating that the sequence had nuclear localization ability after mutation.In addition,Co-IP also showed that the mutated sequence interacted with KPNA1.These results suggest that the NS5 protein of other flaviviruses may enhance the inhibition of JAK-STAT signaling pathway and further antagonize the natural immune response if the above-mentioned mutations occur in other flaviviruses.In conclusion,this study successfully identified the nuclear localization signal(NLS_37-45)of TMUV-NS5.We explored its nuclear localization ability.It is clear that the nuclear localization ability of TMUV-NS5-NLS_37-45 is closely related to the molecular weight and structure of the cargo protein.It also revealed the risk of such mutations in other flaviviruses.