在EB病毒潜伏感染中LMP1和宿主细胞因素对病毒拷贝数的影响与调控机制研究

Epstein-Barr virus (EBV) is etiologically associated with the development of both lymphoid and epithelial origin malignancies, including Burkitt’s lymphomas and nasopharyngeal carcinoma (NPC). The oncogenicity of EBV is still largely unknown. The viral load is among the various infection markers which is related the viral pathogenesis. Our previous study demonstrated that the replacement of the full-length latent membrane protein 1 (LMP1) gene by that of NPC origin in the EBV genome led to the significant difference of EBV copy number. Therefore, we have found that the structure of LMP1 gene is one determinant on the viral copy umber during the latent infection, and we have also obtained several cell lines containing different EBV copy number. In the future study of this proposal, a series of EBV genome mutant will be established by homogenous recombination technique after a precise analysis on the LMP1 gene and protein structure. The crucial function domain(s) or site(s) which determine the EBV latent copy number will be then analyzed precisely. On the other hand, the gene expression profiles will be detected by cDNA array for different cell lines harboring high or low EBV copies. Combined with other previous results, including the interaction of EBV-encoded nuclear antigen (EBNA1) with host proteins and the different expression proteins reported in lymphoblastoid cell lines, special host factors will be chosen for further study. Their function in the regulation of the copy number in the viral latent infection and the function of their own in EBV-infected cells will be identified. The expression and function of them impacted by EBV infection be also evaluated. The results about LMP1 and host factors in the EBV-copy regulation will be verified in clinical specimens. Through the study in this proposal, in-depth knowledge about EBV’s etiological mechanism in the development of related cancers would be achieved, and novel biomarkers for the diagnosis and therapy of EBV-associated cancers due to their regulation on EBV latent copies are to be possibly discovered.

EB病毒（EBV）基因组大，其作为重要肿瘤病因的致病机制仍有待深入研究，病毒载量与致瘤作用直接相关。本项目前期在EBV全基因组中置换鼻咽癌来源潜伏膜蛋白LMP1全基因导致感染细胞中EBV拷贝数差异大，由此发现LMP1基因结构影响EBV潜伏感染的拷贝数高低，并获得含不同EBV拷贝数的细胞系。本项目拟在此基础上，根据LMP1基因和蛋白结构特点与变异位点、应用同源重组技术在EBV全基因组中构建LMP1系列突变体、研究LMP1影响EBV拷贝数高低的精准功能域或位点。对已有含不同拷贝数的细胞系进行差异基因表达谱检测和分析，结合前期其他结果选择宿主因子，研究其对EBV拷贝数的调控与功能，以及EBV感染对其表达和功能的影响。对来自LMP1和宿主因素的研究结果在临床样本中进行检测验证。通过本研究将更深入了解EBV在肿瘤发生中的致病机制，发现调控EBV拷贝数及可用于EBV相关性肿瘤诊断治疗的分子靶标。

EB病毒（EBV）属于疱疹病毒科，具有172kb的基因组，它是第一个被确认的人类肿瘤病毒，而其参与相关性肿瘤发生发展的病因学机制仍有待研究。EBV的DNA基因组在细胞中维持在一定的拷贝数，随着细胞的复制而复制并分离进入子代细胞。在病毒拷贝数的维持和传代中，EBV需要通过自身的能力和与宿主发生相互作用。本项目前期结果已显示，EBV癌蛋白LMP1的基因结构可影响病毒以一定拷贝数形式存在于细胞中，而宿主因素可能与EBV核抗原EBNA1发生作用使病毒拷贝数能维持在细胞中。在本项目的研究中，通过同源重组等技术、构建LMP1的C端功能结构域的缺失突变体，发现激活区CTAR3、而不是CTAR1和CTAR2的缺失导致EBV的拷贝数降低。进一步构建了跨CTAR3的15bp缺失突变体，结果不影响EBV拷贝数。对不同拷贝数的细胞系进行差异基因表达谱检测和分析，并进行了EBV核抗原EBNA1相互作用蛋白的预测和IP联合质谱的检测和分析，获得EBNA1互作蛋白之间的作用网络。亲环素A（CYPA）与EBNA1相互作用被招募到核内、促进EBV复制和拷贝数维持。当CYPA过表达时，能与USP7竞争与EBNA1的结合。鼻咽癌病人血清外泌体中CYPA具有重要的诊断意义。核内BRD7也能与EBNA1互作，其表达受EBV感染调控，并可影响多种细胞蛋白的表达，这可能是其参与EBV潜伏感染和拷贝数维持的作用机制。通过本项目的研究，获得了从多方位了解EBV维持潜伏感染、复制和拷贝数维持的机制，发现了多个可能用于EBV相关性肿瘤诊疗的潜在分子靶标。