伪狂犬病毒晚期蛋白UL31亚细胞定位信号的鉴定及其生物学功能的研究

伪狂犬病毒（PRV）能够引起多种家畜和野生动物的伪狂犬病，猪是其自然宿主。UL31是PRV编码的晚期蛋白，与PRV早期包膜形成有关。前期的研究表明，UL31主要定位于细胞核，但其准确的定位及定位信号、转运机制和生物学功能尚不清楚。本项目拟采用病毒感染及构建UL31与荧光蛋白融合的表达质粒转染细胞来确定UL31的准确定位；运用异源核融合实验研究UL31是否能够进行核质穿梭；分别通过Ran-GTP和importin α/β突变体抑制实验及leptomycin B药物处理抑制实验，初步探讨该蛋白核输入及核输出的转运机制；构建UL31的缺失及定点突变体来鉴定UL31的核定位信号（NLS）和核输出信号（NES）；进一步构建NLS和NES突变病毒，研究UL31的NLS及NES在病毒复制中的作用，从而为阐明UL31在PRV感染中的生物学功能奠定基础，也为进一步抗病毒药物靶点的选择和疫苗研制提供理论依据。

As a pathogen of swine, pseudorabies virus (PRV) has resulted in devastating diseases in livestocks and wild animals. UL31, a PRV-encoded late protein, is important for primary envelopment formation of PRV virions. UL31 has been previously shown to target predominantly to the nuclei of PRV-infected cells. However, neither the mechanisms of its subcellular localization and transport, nor its biological function are well understood. .In this study, immunofluorescence assays were performed to investigate the exact subcellular localization of UL31 in PRV-infected PK-15 cells firstly. To further investigate the subcellular distribution of UL31 in transfected living cells, enhanced yellow fluorescent protein (EYFP)-tagged UL31 variants and fluorescence microscopy were applied. The plasmid pUL31-EYFP encoding UL31 fused to the N terminus of EYFP was constructed and transfected into COS-7 cells to study the subcellular localization of UL31 in the absence of other viral proteins. Then, a functional nuclear localization signal (NLS) and a nuclear export signal (NES) were mapped and identified. .To explore the nuclear import mechanism of UL31, a dominant negative (DN) RanGTP (Ran-Q69L), which is deficient in GTP hydrolysis, was introduced to determine whether Ran is required for the nuclear transport of UL31. Further, to identify the cellular receptor responsible for UL31 nuclear targeting and further characterizing the nuclear import pathway of UL31, DN importin α and DN importin β, which lack the ability to bind importin β and Ran respectively, were also introduced to determine whether they are required for the nuclear transport of UL31. To investigate the contribution of CRM1 pathway to the nuclear export of UL31 and to determine whether UL31 is a nucleocytoplasmic shuttling protein, COS-7 cells were transfected with pUL31-EYFP. Subsequently, heterokaryon assays were performed, with the exception that 3 h prior to and following heterokaryon formation, the cells were treated with LMB to inhibit CRM1 function. .In order to verify whether the NLS or NES is functional during infection, recombinant viruses with mutations of the NLS and/or the NES in UL31 were constructed, and then immunofluorescence assays were performed to investigate the exact subcellular localization of UL31 in PK-15 cells infected with wildtype PRV or recombinant virus. To investigated whether the subcellular localization of UL31 affects viral replication of PRV, plaque formation and viral proliferation characteristics were observed for each recombinant virus. Together, these works are of significance for the further study of the functions of UL31 in PRV infection, as well as for further insights into the design of medicine target of antiviral infection and development of vaccine.