Nuclear Localization Identification Of Duck Tembusu Virus Nonstructural Protein 5 And The Effect Of Motif C On Virus Proliferation

Duck Tembusu virus(DTMUV)belongs to flavivirus genes,flaviviridae family,causing severe economic loss for the Chinese poultry industry since the first outbreak in2010 in China.Nonstructural protein 5(NS5)is the largest and the most conserved protein,and harbors an N-terminal methyltransferase domain(MTase)and a C-terminal RNA dependent RNA polymerase domain(RdRp).RdRp domain,which includes a nuclear localization signal sequence(NLS)and seven motifs which are close related to the RdRp activity,plays a vital role in viral genome replication.However,the subcellular localization of NS5 and the function of RdRp motifs are still elusive.To investigate the partial underlying mechanism of duck Tembusu virus replication,in this study,the subcellular localization and the functional RdRp domain motif C of DTMUV 5 are analyzed.1.Subcellular localization of DTMUV NS5To determine the subcellular localization of DTMUV,NS5 gene was cloned and constructed into prokaryotic expression vector for efficient expression,and the mouse anti DTMUV NS5 polyclonal antiserumwas prepared;duck embryo fibroblast cells(DEF)and baby hamster cells(BHK)were infected with duck Tembusu virus or transfected with NS5 plasmids,samples were harvested at different hours post infection or transfection.The subcellular localization of duck Tembusu virus NS5 was detected by indirect immunofluorescence assay by using mouse anti duck Tembusu virus NS5 polyclonal antiserum as the primary antibody.The result indicated that duck Tembusu virus NS5 localized within the cytoplasm.To exclude the possibility that NS5 transfers quickly in and out of the nucleus,nuclear export protein inhibitor was used,and the result showed that NS5 still localized within the cytoplasm.2.The impact of RdRp motif C to DTMUV proliferationTo understand the influence of different RdRp motifs to DTMUV,motif C which contained the activity center sites was studied.The alanine single site mutation replicons were constructed within motif C and two replicons V671 A and V672 A were selected to decrease viral replication;based on the replicon data,two related infectious clones were constructed and rescued,named r TMUV-NS5-V671 A and r TMUV-NS5-V672 A respectively;and the biological characteristic of these two infectious clones were studied,including the genome stability,growth characteristics in vitro and pathogenicity analysis.The result showed that these two recombinant viruses exhibited stable inheritance till the fifth generation,and there was no other site mutation;these two recombinant viruses had longer time to cause CPE than WT.Compared with the wild type,r DTMUV-NS5-V671 A and r DTMUV-NS5-V672 A caused a delay in the death of duck embryos,as well as caused similar clinical lesions and viral loads in tissue in ducklings.In conclusion,DTMUV NS5 localized within the cytoplasm;selected two sites influenced viral replication in motif C V671 A and V672A;constructed and rescued two infectious clones r TMUV-NS5-V671 A and r TMUV-NS5-V672 A,demonstrated that V671 A and V672 A in RdRp influenced viral replication,as well as viral proliferation.