Evaluation Of Duck Plague Virus UL15 Protein NLS As A Potential Antiviral Drug Target

In herpesvirus,the UL15 protein is a terminase subunit and is an important member involved in the packaging process of viral DNA.Duck plague virus?DPV?is a member of the herpesvirus subfamily.Studies have shown that the UL15 protein is ultimately localized in the nucleus in cells infected with DPV.However,the UL15protein can only be localized in the cytoplasm when transiently transfected expression plasmid pEGFP-N1-UL15 or pcDNA3.1-UL15.Currently,it has not been elucidated how the pUL15 enters the nucleus.Therefore,this article aims to explore the nuclear import process of DPV UL15 protein,in order to preliminarily clarify the nuclear import mechanism of DPV UL15 protein,which establish the foundation for studying the function of DPV UL15 protein and the packaging process of DPV genome.In addition,it also provides a theoretical basis for the development of new anti-DPV drugs.Based on the above objectives,the main research contents and results obtained in this paper are as follows:1.Identification of assistant protein for DPV UL15 protein into nuclearIn this study,UL15 protein and its related proteins,UL6,UL28,UL33,were studied to constructed eukaryotic expression plasmids pcDNA3.1-His-UL15,pCMV-C-Flag-UL6,pCMV-HA-UL28 and pCMV-Myc-UL33.The recombinant plasmids were alone transfected,or double-transfected and triple-co-transfected into DF-1,and the localization of UL15 protein was detected by indirect immunofluorescence assay.The results showed that UL15 and UL28 proteins could only be expressed in the cytoplasm when they were transfected alone,UL6 protein could be completely localized in the nucleus,and UL33 protein diffused throughout the cells.The UL15protein can only be localized in the cytoplasm when co-expressed with the UL28 or UL33 protein,and the UL15 protein can be partially localized in the nucleus when co-expressed with the UL6 protein.However,the UL15 protein can be fully localized to the nucleus when it is co-expressed with UL28 and UL33 proteins.But the UL15protein cannot enter the nucleus when it is co-expressed with UL6 and UL28 proteins or co-expressed with UL6 and UL33 proteins.Based on the above results,it was found that the UL6 protein can promote the partial entry of the UL15 protein into the nucleus,but the UL15 protein can completely enter the nucleus when both the UL28and UL33 proteins are present.This indicates that the localization of the UL15 protein in cells is closely related to the UL28 and UL33 proteins.2.Identification of UL15 protein NLSIn this experiment,the nuclear localization signal?NLS?of UL15 protein was analyzed.The results showed that there is no strong NLS in UL15 protein,but there are two weak NLS,NLS1 and NLS2.After deletion and multi-point mutation of NLS1 and NLS2,it was found that the deletion and mutation of NLS1 had a significant effect on the localization of UL15 protein.To further confirm the effect of NLS1 and NLS2 on UL15 protein localization.we construct expression plasmids,pcDNA3.1-His-UL15_1-345-345 and pcDNA3.1-His-UL15_346-740,which contained NLS1 and NLS2,respectively.The results showed that UL15_1-345-345 and UL15_346-74046-740 proteins were not localized to the nucleus when they are co-expressed with UL28 and UL33proteins.This indicates that the entry into the nucleus of the UL15 protein requires not only NLS1 but also the full length of the UL15 protein.In order to further identify the effect of amino acids in NLS1 on the nuclear import of UL15 protein,the K,R and P amino acids in NLS1 were mutated to Q,respectively.The results showed that the mutations of amino acids at K185,R188 and P194 had no significant effect on the localization of UL15 protein,however,the localization of the UL15 protein was significantly affected by mutation of amino acid K187 or K190.To detect the function of NLS1,NLS1 was expressed by fusion with foreign protein GFP,GFP-GFP or GFP-?-Gal,and the eukaryotic plasmids pEGFP-C2-NLS1,pEGFP-GFP-C2-NLS1and pEGFP-C2-?-Gal-NLS1 were constructed.The results showed that NLS1 did not have the ability to alter the localization of the protein GFP-?-Gal,but to some extent NLS1 made the fluorescent signal stronger of GFP and 2×GFP in the nucleus.These results illustrated that DPV the NLS1 has a weak NLS function.