真菌病毒AfPV的分类地位及其对Aspergillus flavus影响的分子机理研究

Aspergillus flavus is a fungal pathogen that not only induces diseases in human, animals and plants, but also produces aflatoxins with strong carcinogenic and mutagenic effects. However, antifungal antibiotics are toxic and easily confer to the fungal resistance. Therefore, there are lack of safe and efficient prevention measures against A. flavus. Mycoviruses with high host specificity can host fungal growth and development as well as pathogenic regression, therefore they have potential to inhibit fungal infection. In our previous study, a novel mycovirus was isolated from 417 A. flavus strains. Analysis of sequence indicated that this virus and other 4 viruses might be a new genus in Partitiviridae., but coat protein sequence of the virus encoded a unknown protein. Virus removal assay demonstrated that the virus infection significantly changed some biological characters on A. flavus, but the mechanism was unclear. Therefore, this project intends to determine the classification of the virus by further cloning and analysis of virus genome sequence and structure, and by research of the virus particle morphology and characteristics of coat protein sequences and so on. This project will aslo investigate the mechanism of the virus influence on its host, by mining the genes induced by AfPV by comparing the gene expression profiles in the presence of the virus and in the absence of the virus, and by confirming the function of candidate genes in A. flavus using the routine gene manipulation techniques such as gene knock-out, gene complementation. The research results will enrich classification of the genera in Partitiviridae, and also provide theoretical foundation for exploitation of mycoviruses as a tool to combat A. flavus infection in the future.

黄曲霉不仅是人、动物和植物的致病菌，还产生强烈致癌、致畸作用的黄曲霉素，危害极大。抗真菌药物毒性大、副作用强，容易引起真菌耐药性，因此目前缺乏安全、有效的黄曲霉防治措施。真菌病毒宿主专一性强，能使宿主真菌生长发育及致病力衰退，在治疗真菌感染方面潜力大。我们前期从417株黄曲霉中分离出一种新型真菌病毒。序列分析显示该病毒与其他四种病毒可能组成双分病毒科中一个新病毒属，但病毒外壳蛋白为未知蛋白序列。病毒脱除实验表明，该病毒侵染对黄曲霉性状产生明显影响，但机理未知。因此项目拟进一步克隆和分析病毒全基因组序列和结构，研究病毒粒子形态及外壳蛋白序列等特点，明确该病毒的分类地位。比较病毒存在和不存在的情况下黄曲霉基因表达差异，并采用基因敲除和互补技术对病毒诱导的相关基因进行功能验证，解析该病毒对黄曲霉影响的分子机理。研究结果将丰富双分病毒科中病毒属的分类，为今后利用病毒治疗黄曲霉感染提供理论基础。

黄曲霉菌是人的条件致病菌，其感染的治疗主要依赖抗真菌药物，但化学药物毒性大、副作用强，并且容易引起耐药性。真菌病毒是一类寄主为真菌的病毒，有些真菌病毒的侵染能够影响宿主真菌生长发育，并导致致病力衰退，具有治疗真菌感染的潜力。我们从异常的黄曲霉菌株中分离得到真菌病毒AfPV。通过病毒的水平传染，我们得到AfPV的感染能够引起黄曲霉菌落异常，生长速度减缓，孢子产量减少，但对黄曲霉在小鼠肺部的定殖似乎没有影响。我们对AfPV基因组全长进行克隆，发现其基因组为3条双链RNA片段（dsRNA1全长1763bp，dsRNA2全长1422bp和dsRNA3全长1186bp），分别含有一个开放阅读框并编码62kDa，42kDa和32kDa的蛋白。其中序列分析显示dsRNA1编码病毒的复制酶（RdRp），dsRNA2编码的蛋白与NCBI数据库中其他病毒的外壳蛋白（CP）没有相似性，但透射电子显微镜、SDS-PAGE和多肽片段质谱（PMF-MS）分析显示dsRNA2编码病毒CP蛋白，dsRNA3编码蛋白在NCBI数据库中没有相似性。系统发育树分析显示AfPV与其他一些相关的病毒组成双方病毒科中一个新的病毒属，我们将之命名为Epsilonpartitivirus。通过转录组分析，我们得到AfPV感染后，寄主上调表达的基因1864个，下调表达基因2263个。通过GO和KEGG富集分析，我们得到AfPV感染后寄主显著上调的基因富集在Non-homologous end-joining、Homologous recombination和Mismatch repair等代谢通路，他们主要为寄主基因组DNA复制，校正，DNA损失断裂修复系统，因此我们推测被AfPV感染后，寄主的基因组DNA可能受到不同程度损伤。DNA依赖的蛋白激酶催化亚基Ku70，是黄曲霉Non-homologous end-joining代谢通路中的重要基因，我们将AfPV传染到ku70敲出突变体黄曲霉菌株CA14（Δku70尿嘧啶营养缺陷型）中得到携带AfPV的菌株CA14-b。通过形态学观察比较，我们发现被真菌病毒AfPV感染后Δku70突变体菌株同样出现了菌落生长异常的现象。由此我们推测Ku70可能是病毒AfPV与黄曲霉互作调控的下游或者不直接参与AfPV对寄主的影响的调控。