牛流行热病毒α1基因在病毒复制过程中的作用及其机制研究

Bovine ephemeral fever virus (BEFV) is the causative agent of bovine ephemeral fever, and little is known of viral replication mechanism, which restricts the development of antiviral drugs. Earlier studies from our team have demonstrated that BEFV alpha 1 protein interacted with the host AnxA2 protein, moreover, each of the genes expression promoted the replication of BEFV and there was an obvious synergistic effects on viral replication when alpha 1 and AnxA2 gene were co-transfected. In this study, the alpha 1 gene deleted BEFV strain will be rescued via reverse genetics technology, then the proliferation and morphology of rescued virus particles will be tested to determine whether alpha 1 gene is required for the replication of BEFV. In addition, the viral proteins combining with BEFV alpha 1 protein and their binding sites will be screened and identified by using coimmunoprecipitation, then replication ability and virus morphology of the rescued BEFV with the corresponding binding site mutation of alpha 1 will be detected, and the viral gene that could be synergistic with the alpha 1 gene will be found. Furthermore, the binding sites between alpha 1 protein and AnxA2 protein will be located and mutated, and then the effects on replication of infectious virus strains based on binding site-specific mutagenesis will be analyzed.What's more, other candidate genes involved in the process of alpha 1 promoting virus replication will be screened and identified by high-throughput sequencing, and then signal transduction pathway of alpha 1 promoting viral replication will be confirmed through regulating the expression of candidate genes. Taken together, it will be the first to reveal the function of BEFV alpha 1 and its molecular mechanism of viral infection from the perspective of the relationship between alpha 1 and viral gene or the host AnxA2, which will provide new target molecule for the research and development of antiviral drugs.

牛流行热病毒（BEFV）是引起牛流行热的病原，其复制机制研究滞后，制约了抗病毒药物的研发。本团队研究发现，病毒α1蛋白与宿主AnxA2蛋白结合，二者编码基因均促进BEFV的复制，且共转染后有显著的叠加效应。本研究拟利用反向遗传技术拯救α1基因缺失的病毒，检测其增殖及病毒粒子形态是否异常，分析α1基因是否是BEFV复制所必需的基因；利用免疫共沉淀技术筛选与α1结合的病毒其他蛋白，并拯救α1基因上相应结合位点变异的病毒，检测其复制能力及病毒形态，发现与α1协同作用的病毒基因；定位α1上与AnxA2结合的位点，检测相应位点变异对病毒复制的影响；高通量筛选并验证参与α1促进病毒复制的其他候选基因，检测并分析α1促进病毒复制的信号通路；本研究拟以α1基因与病毒其他基因及宿主AnxA2基因的关系为切入点，率先发现α1基因功能，揭示其调控病毒复制的分子机制，以期为抗BEFV药物研发提供靶标分子。

牛流行热病毒(BEFV)是引起牛流行热的病原，其复制机制研究滞后，制约了抗病毒药物的研发。BEFV非结构蛋白α1在病毒复制中的作用及其分子机制未知。本项目建立了过表达或部分沉默α1基因的稳定细胞系，发现α1基因促进BEFV复制；利用反向遗传技术拯救缺失α1基因的重组病毒，TCID50检测α1基因缺失降低BEFV感染细胞上清中的滴度；电镜下观察α1基因缺失胞内病毒粒子增多，而正常BEFV感染细胞胞内病毒分布少。免疫共沉淀技术筛选发现α1蛋白与BEFVM蛋白结合；过表达M基因显著地促进BEFV释放；应用RT-qPCR和WesternBlot检测发现BEFV感染促进AnxA2mRNA和蛋白的表达，AnxA2促进BEFV的释放。免疫共沉淀实验发现AnxA2与BEFVα1蛋白结合，共表达α1与AnxA2基因后，显著提高病毒的滴度。GSTPull-down和免疫共沉淀实验发现，BEFV的基质蛋白M与AnxA2蛋白相互作用，且M蛋白与AnxA2蛋白的第五个结构域（AnxA2-V，268-334aa）结合。通过质膜分离实验发现，AnxA2促进M蛋白在细胞膜上的定位。进一步研究发现AnxA2通过其V结构域与M蛋白结合促进M蛋白在细胞膜上的定位，进而促进BEFV病毒粒子的释放。转录组数据分析并验证发现过表达α1下调ACBD3的表达，ACBD3抑制BEFV复制，ACBD3抑制细胞凋亡，揭示了BEFVα1通过下调ACBD3促进细胞凋亡的分子机制；酵母双杂交系统筛选发现BEFVα1通过与Nix蛋白互作诱导细胞自噬，促进病毒复制。本研究以α1基因与病毒M基因及宿主AnxA2、ACBD3和Nix基因的关系为切入点，率先发现了α1基因功能，揭示其调控病毒复制的分子机制，为抗BEFV药物研发提供靶标分子。本项目共发表SCI论文6篇，获得山东省科技进步一等奖1项。圆满完成预期指标。