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

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.

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。

伪狂犬病毒（pseudorabies virus, 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，PRV UL31主要定位在细胞核中，但其亚细胞定位信号和定位的分子机制至今尚未明确。本研究中，我们首先对PRV UL31进行了分子特性和密码子偏爱性分析；紧接着，将PRV UL31氨基端27个氨基酸与增强型黄色荧光蛋白（EYFP）和His标签融合，进行重组蛋白的原核表达、纯化并制备其相应的抗体；利用制备的抗体通过间接免疫荧光技术确定了UL31在病毒感染时的定位情况，实验结果显示，感染前期UL31主要在细胞核中呈现弥散样分布，感染后期UL31被移位到核脊（核周）；通过构建UL31与EYFP的融合表达质粒pUL31-EYFP并转染COS-7活细胞实验证实，在其它病毒蛋白不存在的情况下，UL31-EYFP蛋白在转染的活细胞中主要呈现细胞核中弥散样分布，证实它是一个核靶向蛋白；利用Ran-GTP、importin α/β显性负突变体及免疫共沉淀实验等进一步研究显示，PRV UL31在RanGTP提供能量情况下通过importin α1、α3、α5、 α7、 β1和transportin-1多种受体介导的方式进入细胞核；将pUL31-EYFP瞬时转染COS-7细胞，并将表达UL31-EYFP的COS-7细胞与NIH3T3细胞融合，在放线菌酮存在的情况下证实UL31在细胞内不能进行核质穿梭；通过转染一系列与EYFP融合的PRV UL31缺失或定点突变体并分析它们的定位情况，我们鉴定到了PRV UL31含有一个功能性的bipartite核输入信号（NLS）和一个功能性的PY基序NLS，它们分别是4RRRLLRRKSSAARRKTL20和21TRAARDRYAPYFAY34；此外，我们也证实预测的核输出信号（NES）不起作用。因此，这些研究结果将为阐明UL31在PRV感染过程中的生物学功能奠定基础，也为进一步研制治疗和预防PRV感染的药物和新疫苗提供理论依据。