Identification Of The Subcellular Import Signal,Nuclear Import Mechanism Of Herpes Simplex Virus Type 1 UL2 And Construction Of Its Recombinant Virus

Herpes simplex virus 1?HSV-1?,a prevalent human virus that can cause many diseases,has become one of the important pathogen that is severely harmful to human health.However,no effective drug or vaccine has been developed thus far.The HSV-1UL2 gene encoded protein UL2 is a Uracil-DNA glycosylase?UDG?,which can precisely cut inappropriate uracil from the synthetic DNA,this plays an important role in the base repair of virus replication.In addition,although UL2 is not essential for viral replication in vitro,it is indispensable for effective replication in neurons.Therefore,UL2 may take a vital role in viral pathopoiesis,latency and reactivation.Nevertheless,the exact function of UL2 in HSV-1 infection is not well understood to date.In this study,to investigate the subcellular distribution of UL2 in transfected live cells,eukaryotic expression plasmid bearing full length ORF fragment of UL2 fused with enhanced yellow fluorescence protein?EYFP?was constructed?using molecular cloning technology?and transiently expressed in live COS-7 cells,and result showed that UL2 was predominantly target to the nucleus in the absence of other viral proteins,comfirming that UL2 is a nuclear targeting protein.In order to verify whether there is a nuclear localization signal?NLS?in UL2,a series of UL2 deletion mutants fused to EYFP were constructed and transfected into COS-7 cells for fluorescence analysis,results showed a functional NLS of UL2 was identified and mapped to aa 1 to 17?MKRACSRSPSPRRRPSS?,and its key amino acid?aa?was¹²RRR¹⁴.Then,the dominant negative of RanGTP?Ran-Q69L?,importin?,importin?and inhibitors of nuclear transport receport,as well as the co-immunoprecipitation assays,were applied to explore the nuclear import mechanism of UL2.As results,the nuclear import of UL2 was found to be dependent on Ran-,importin?1-,?5-,?7-,?1-and transportin-1-mediated pathway.Additionally,the predicted nuclear export signal?NES?was shown to be nonfunctional.Moreover,the heterokaryon assay confirmed that UL2 is a nuclear-cytoplasmic shuttling protein.Besides,based on the identified of UL2 NLS and its key aa,the related bacterial artificial chromosome?BAC?of HSV-1 recombinant virus,including UL2 NLS mutant?UL2 NLSm?,UL2 deletion??UL2?and UL2 revertant??UL2R?,were constructed using Red homologous recombination technique in HSV-1 BAC Luc contained E.coli GS1783 strain,which were then transfected into Vero cells to obtain corresponding recombinant viruses.Viral titers and growth curves showed that nuclear import of UL2 might be crucial for the effective replication of HSV-1infection.Taken together,these jobs are of significance for the further study of the functions of UL2 in HSV-1 infection,as well as for further insights into the design of medicine target of antiviral infection and development of vaccine against HSV-1.