流感病毒PA-X基因与鸡细胞互作蛋白鉴定及影响病毒致病力的机制解析

Influenza virus has a small genome and relies heavily on host cellular machinery to replicate. Influenza virus recruits host proteins mainly by virus-host interactions, therefore identifying which host proteins and complexes come into physical contact with the viral proteins is crucial for a comprehensive understanding of how influenza virus rewires the host’s cellular machinery during the course of infection. PA-X was recently found to have a role of suppressing the replication of influenza virus. In this proposal we will firstly identify host interacting factors of PA-X gene in chicken by AP-MS (Affinity purification-mass spectrum) and construct PA-X—host interacting network. Then we will carry out siRNA screen to classify the effects of identified hosts interacting proteins on influenza virus infection in chicken DF1 cell line. Thirdly, 2-3 key genes that have significant effects on virus infection will be selected for further studies to elucidate the underlying biological mechanisms of the contribution of key PA-X—host interactions to virus virulence. Lastly, we will validate key PA-X—host interactions and their effects in H1N1 influenza virus. Our study will give new insight into the function of PA-X gene, facilitate a more comprehensive understanding of how the host machinery is manipulated during the course of influenza virus infection and provide theoretical basis for chicken anti-flu breeding.

流感病毒的复制和传播依赖于宿主细胞系统且主要通过病毒—宿主蛋白互作的形式募集和雇佣宿主蛋白为己所用，此外宿主对病毒的拮抗也主要通过和病毒蛋白的互作实现。PA-X为新发现的病毒自身的具有抑制病毒复制功能的基因，本研究以H5N1 流感病毒为代表鉴定PA-X基因的宿主互作蛋白并解析PA-X—宿主蛋白互作影响病毒致病力的机制。首先以亲和纯化-质谱技术鉴定PA-X在鸡细胞中的互作蛋白，构建PA-X—宿主蛋白互作网络。其次利用RNA干扰技术筛选基因沉默后对病毒感染有显著影响的宿主基因，鉴定宿主助流感和抗流感因子。随后选取对病毒感染影响最为显著的关键宿主互作基因深入阐明关键PA-X—宿主蛋白互作影响病毒感染的生物学机制。最后在H1N1流感病毒中验证关键PA-X—宿主互作对其他流感病毒感染的影响。本研究将丰富对PA-X基因功能的认识，为禽流感病毒致病机制的解析和家禽抗禽流感育种提供理论支持。

PA-X为是新发现的禽流感病毒自身的具有抑制病毒复制功能的基因，本研究以H5N1 流感病毒为代表鉴定了PA-X基因在鸡细胞中的宿主互作蛋白，构建了PA-X基因-鸡宿主蛋白互作网络，获得56个和PA-X相互作用的高置信互作蛋白。GO分析发现和PA-X有相互作用的宿主基因在营养贮存、脂质转运、RNA加工等生物学功能上明显富集，发现albumin、Duf1899/1900、Anx、cation transporter/ATPase和WD40等结构域的PA-X相互作用蛋白中出现频率较高，表明具有这些结构域的蛋白更可能和PA-X发生相互作用。选取了高置信度互作蛋白UBA52和OTUB1作为候选功能基因进行研究，发现干扰UBA52和OTUB1后可显著抑制H5N1禽流感病毒滴度，表明上述二基因均为促进H5N1禽流感病毒复制的鸡宿主基因。本研究丰富了对PA-X基因功能的认识，为禽流感病毒致病机制的解析和家禽抗禽流感育种提供了理论支持。