I型单纯疱疹病毒UL2蛋白核质转运信号的鉴定及其在病毒感染中功能的研究

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. UL2, a protein that plays vital roles in the process of HSV-1 DNA replication, has been previously shown to target predominantly to the nuclei of transfected cells using indirect immunofluorescence assay (IFA). However, neither the accurate subcellular localization, subcellular localization signals, mechanisms of its subcellular localization and transport, nor its biological function was well understood. . In this study, IFA was firstly conducted to investigate the exact subcellular localization of UL2 in HSV-1-infected cells. To further investigate the subcellular distribution of UL2 in transfected living cells, enhanced yellow fluorescent protein (EYFP)-tagged UL2 variants and fluorescence microscopy were applied. Then, the functional nuclear localization signal (NLS) and nuclear export signal (NES) were mapped and identified. . To explore the nuclear import mechanisms of UL2, 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 UL2. Further, to identify the cellular receptor responsible for UL2 nuclear targeting and further characterizing the nuclear import pathway of UL2, 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 UL2. To investigate the contribution of CRM1 pathway to the nuclear export of UL2 and to determine whether UL2 is a nucleocytoplasmic shuttling protein, COS-7 cells were transfected with UL2-EYFP expressing plasmid. Then, co-immunoprecipitation assay was employed to verify the interactions of UL2 with cellular receptor importin α, importin β and/or CRM1. Subsequently, heterokaryon assays were performed, with the exception that 3 h prior to and following heterokaryon formation, the cells were treated with leptomycin B to inhibit CRM1 function. . To verify whether the NLS or NES is functional during infection, recombinant viruses with mutations of the NLS and/or the NES in UL2 were constructed, and then IFA was performed to investigate the exact subcellular localization of UL2 in cells infected with wildtype HSV-1 or recombinant virus. To observe whether the subcellular localization of UL2 affects viral replication of HSV-1, the plaque formation and viral proliferation characteristics were researched for each recombinant virus. 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.

I型单纯疱疹病毒（HSV-1）是人群中极为常见的病毒，可引起多种疾病，已成为严重危害人类健康的重要病原之一，目前尚无有效的治疗药物和疫苗。UL2是HSV-1复制过程中一个非常重要的蛋白，单独转染时主要定位于细胞核，但是关于UL2的准确定位、转运机制、定位信号及其生物学功能尚不清楚。本项目拟采用病毒感染及构建UL2荧光蛋白表达质粒来确定UL2的准确定位；构建一系列UL2突变体来确定UL2的核定位信号（NLS）和核输出信号（NES）；通过Ran-GTP、importinα/β突变体和leptomycin B药物抑制实验及免疫共沉淀实验，探讨该蛋白的核质转运机制；运用异源核融合技术研究UL2能否进行核质穿梭；进一步构建含UL2 NLS和NES突变的病毒，研究它们在病毒复制中的作用，从而为阐明UL2在HSV-1感染中的生物学功能奠定基础，也为进一步的抗病毒药物靶点设计和疫苗研制提供理论依据。

I型单纯疱疹病毒（HSV-1）是人群中极为常见的病毒，可引起多种疾病，已成为严重危害人类健康的重要病原之一，目前尚无有效的治疗药物和疫苗。UL2是HSV-1复制过程中一个非常重要的蛋白，单独转染时主要定位于细胞核，但是关于UL2的准确定位、定位信号和定位的分子机制至今尚未明确。本研究中，我们首先将UL2的全长ORF片段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UL2在病毒感染时的定位情况，实验结果显示，在病毒感染的8、12和24小时后，UL2都主要呈现细胞核定位；通过构建UL2与EYFP的融合表达质粒pUL2-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，UL2-EYFP蛋白在转染的活细胞中主要定位在细胞核中，证实它是一个核靶向蛋白；利用Ran-GTP、importin α/β显性负突变体和抑制剂及免疫共沉淀实验等进一步研究显示，UL2是在RanGTP提供能量情况下通过importin α1、α5、α7、β1和transportin-1多种受体介导的方式进入细胞核；将pUL2-EYFP瞬时转染COS-7细胞，并将表达UL2-EYFP的COS-7细胞与NIH 3T3细胞融合，在放线菌酮存在的情况下证实UL2在细胞内能进行核质穿梭；通过转染一系列与EYFP融合的UL2缺失或定点突变体并分析它们的定位情况，我们鉴定到了UL2含有一个功能性的核输入信号（NLS，1MKRACSRSPSPRRRPSS17），它的关键氨基酸是12RRR14；同时，我们也证实预测的核输出信号（NES）不起作用。此外，根据鉴定到的UL2 NLS及其关键氨基酸，我们构建了UL2缺失和NLS关键氨基酸突变的HSV-1相关重组病毒BAC，并且发现UL2的核输入可能对于病毒的有效复制是至关重要的。因此，这些研究结果将为阐明UL2在HSV-1感染中的生物学功能奠定基础，也为进一步的抗病毒药物靶点设计和疫苗研制提供理论依据。