鸭类寡腺苷酸合成酶基因(OASL)抗流感病毒分子机理研究

2’-5’ oligoadenylate synthetase (OAS) and 2’-5’ oligoadenylate synthetase like (OASL) proteins are nucleic acid sensors in the cytosol, which are induced by Interferons (IFNs). The applicant has found that: 1. more than 46 birds belonging to the carinatae (i.e. duck and chicken) have a contractive OAS family, which consists of only one member (OASL); 2. the expressions of the OASL in lung tissues of ducks infected by the highly (DK/49) or the weakly (GS/65) pathogenic H5N1 virus were significantly increased from 237- to 1030-fold when compared to the corresponding of control individuals; 3. the expression of duck OASL proteins in DF1 (chicken embryonic fibroblasts) cells significantly reduced replications of the DK/49 H5N1 virus. However, the molecular mechanism about whether and how avian OASL (i.e duck OASL) involve into immune response to viruses is largely unclear. In this proposed project, we will perform the following works to define the antiviral profile, identify functional domains and determine the tertiary structure of duck OASL protein: 1. predict the molecular regulatory model of duck OASL through integrating the information of evolutional pattern, co-expression model and predicted tertiary structure; 2. create duck embryonic fibroblasts (DEF) cells, which are over-expressed mutations and truncations of duck OASL or deficient duck OASL using gene-editing technology, such as piggyBac transposon-mediated gene transfer system or CRISPR/Cas9 system; 3.calculate viral growth curve of several RNA viruses (i.e. influenza A viruses, new castle virus and infectious bursal disease virus) and DNA viruses (fowlpox virus), count RNA integrity number and construct gene expressional profiles of the above DEF cells before and after infected by viruses; 4. examine the 2-5A activities of full length, mutations and truncations of duck OASL protein through a heat-inactivated 2-5A synthetase reaction; 5. identify interactors of duck OASL proteins and conform their interactions through molecular biological experiments, such as co-immunoprecipitation and yeast two-hybrid analysis; 6. construct recombinant plasmids, purify recombinant protein and try to determine the tertiary structure of duck OASL. These efforts will provide new insights in the molecular regulatory model for duck OASL’s role in immune response to viral infections and it will be informative to the breeding of duck lines showing resistance to diseases, such as influenza.

寡腺苷酸和类寡腺苷酸合成酶基因(OAS和OASL)是一类干扰素诱导表达的核苷酸感受分子。申请人发现鸭等家禽OAS家族仅存在一个家族成员—OASL基因,鸭OASL基因在感染H5N1流感病毒的肺组织的表达量极显著上调237-1030倍，且该基因在DF1细胞的过表达极显著抑制H5N1流感病毒增殖。然而，鸭等禽类OASL基因的抗病分子机理尚不清楚。在预测鸭OASL基因分子调控模型基础上，申请人拟通过基因修饰技术获得该基因突变体及不同结构域肽段过表达或敲除的DEF细胞系；测定上述细胞系流感病毒等多种RNA和DNA病毒生长曲线、RNA完整度及基因表达图谱等数据；检测鸭OASL各类蛋白合成2-5A的量和类型；筛选并验证OASL基因与其网络基因的互作；过表达、纯化鸭OASL蛋白，并尝试解析其蛋白结构。该研究目标的实现可确定鸭OASL基因抗病毒范围，鉴定其功能结构域，解析其抗病分子机理，以助力鸭抗病优良品系的培育。

摘要.寡腺苷酸(OAS)基因家族包括寡腺苷酸和类寡腺苷酸合成酶基因（OASL）。OAS家族成员的数量在不同物种呈现巨大变化(1-12个)。在哺乳类中，OAS和OASL通过OAS-RNase L、OAS-IRF7或OASL-RIG-I信号通路广泛参与宿主的免疫应答反应。项目申请人前期发现鸭等家禽OAS家族仅存在一个家族成员─OASL基因，且该基因在DF1细胞的过表达极显著抑制H5N1流感病毒的增殖。然而，鸭等家禽OASL基因的抗病分子机理尚不清楚。项目研究人员首先构建了后生动物的OAS基因家族进化树，理清了OAS成员之间的进化关系，预测了功能分化的模型，鉴定了关键的功能结构域和位点；在此基础上，研究人员构建了鸭等鸟类OASL基因、3D和3K突变体以及截短体的过表达载体，纯化获得了鸭等鸟类OASL基因、3D和3K突变体以及截短体重组蛋白，通过比较过表达OASL基因、OASL突变体和截短体细胞与OASL敲除细胞系和对照组细胞系的病毒含量与生长曲线，发现鸭等鸟类OASL基因依赖于UBL结构域合成2-5A产物、激活RNase L酶降解病毒dsRNA，广泛抑制单股负链、单股正链和双链RNA病毒的增殖，但对DNA病毒增殖的抑制有限；进一步分析表明，OASL的3D和3K突变体均丧失OAS酶活性，不能激活和放大RNase L，但OASL的3D突变体与人OASL类似，能通过其OLD结构域、以UBL结构域依赖的模式提高RIG-I抑制流感病毒增殖的活性。综上所述，项目的研究揭示鸭等鸟类OASL基因是抗流感病毒的重要免疫基因，可利用三个保守天冬氨酸的突变选择性激活和放大OAS-RNase L和OASL-RIG-I信号通路，广泛抑制RNA病毒的增殖。此外，研究人员还结合转录组图谱和免疫共沉淀分析，鉴定了133个鸭OASL蛋白的互作蛋白，结合进化分析，揭示鸭等鸟类可能利用OASL-ANKFY1信号通路替补OASL-MBD1信号通路抑制病毒的增殖。上述研究成果系统阐明了鸭等鸟类OASL基因抗病的分子机制，为鸭等家禽的抗病育种提供了重要信息。研究成果以4篇SCI论文发表在Frontiers in immunology, Journal of Integrative Agriculture, Science China Life Sciences和BMC Evolutionary Biology杂志。

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