鸭圆环病毒ORF3蛋白C端核定位信号调控细胞凋亡和病毒致病性的分子机制

Duck Circovirus (DuCV), an important pathogen of immunosuppressive diseases in ducks, can be divided into two genetypes: DuCV I and DuCV II. But it is still unclear about the relationship between the function of viral proteins and pathogenicity of DuCV. Previous study shows that obvious lesion can be found in the spleen, thymus and bursa of Fabricius in ducks infected with DuCV, suggesting DuCV pathogenicity is related to the lymphocytic apoptosis. ORF3 protein of DuCV was proved to induce apoptosis. Our research found that ORF3 proteins from two genotypes of DuCV were significantly different. T236A mutation occurred in ORF3 gene of DuCV I resulted in the translation terminating ahead, 20 amino acids missing in the C-terminal and lost the ability of nuclear localization, which suggest that nuclear localization signal (NLS) is under the different C-terminal region. The nuclear localization is closely related to the apoptosis activity. Previous study also shows that the ability of inducing apoptosis of the ORF3 protein of DuCV II was stronger than that of DuCV I. According to another evidence that the positive rate of DuCV II is much higher than that of DuCV I in dead ducklings, we hypothesized that the 20 amino acids in C-terminal of DuCV II ORF3 protein may play an important role in inducing apoptosis and further affect the pathogenicity of DuCV. Therefore, through the eukaryotic expression system, reverse genetic system and rescue of duck circovirus in ducklings, we aim to illustrate the role of the nuclear localization signal under C-terminal of DuCV II ORF3 protein in regulating the apoptosis activity and viral proliferation and pathogenicity. This research will reveal the virological and biological roles of the differences of C-terminal amino acids between DuCV I and II ORF3 protein.

鸭圆环病毒(Duck circovirus, DuCV)引起鸭的免疫抑制性疾病，分为2个基因型，该病毒蛋白功能和致病性关系尚不清楚。DuCV感染鸭在脾脏、胸腺和法氏囊形成明显病灶，与淋巴细胞凋亡相关。两基因型ORF3蛋白(凋亡蛋白)差异显著，I型ORF3基因发生T236A突变， C端缺失20个氨基酸，胞核定位改变，提示此区域存在核定位信号。核定位与凋亡活性密切相关，II型ORF3蛋白诱导凋亡能力高于I型，结合病死鸭中II型DuCV检出率高于I型的结果，推测II型ORF3蛋白 C端20个氨基酸有促细胞凋亡的功能，继而影响DuCV致病性。本课题拟采用真核表达系统、反向遗传操作系统和雏鸭体内病毒拯救模型，研究两型ORF3蛋白C端差异区域的核定位信号在调控细胞凋亡活性以及病毒复制和致病性中的作用，以期阐明基因I型和II型DuCV ORF3蛋白C端氨基酸差异的病毒学和生物学意义。

鸭圆环病毒(Duck circovirus, DuCV)引起鸭的免疫抑制性疾病，但其致病机制和病毒复制调控机制目前尚不清楚。DuCV感染鸭在脾脏、胸腺和法氏囊形成明显病灶，与淋巴细胞凋亡相关。ORF3蛋白是DuCV唯一具有凋亡活性的病毒蛋白。DuCV分为2个基因型，前期发现两基因型ORF3蛋白C端存在20个氨基酸及胞核定位差异，提示此区域存在核定位信号并可能调控细胞凋亡活性。我们研究发现基因I型和II型DuCV ORF3 T236A差异导致C端翻译终止和20个氨基酸差异，此差异在两基因型间非常保守。生物信息预测和功能验证证实此区域含有1个核定位信号，而其可促进ORF3蛋白核转位而抑制其促细胞凋亡的活性。另外，通过基因组比较分析我们发现DuCV存在特有的4拷贝串联重复序列（QTR），且QTR在基因I型和II型DuCV毒株中显著不同，并非常保守。研究发现QTR作为转录的下游反应原件维持病毒mRNA稳定性。进一步利用亚基因组模型和反向遗传模型进行QTR功能分析，发现QTR在DuCV Rep和Cap基因转录表达和病毒基因组复制中发挥重要作用。本研究系统阐明了基因I型和II型ORF3和QTR序列差异和功能调控机制，明确了其在DuCV基因分型、病毒凋亡活性和病毒复制调控中的重要作用，为理解 DuCV 致病机理和动物圆环病毒的生命角色提供帮助。