流感病毒NS1蛋白抑制干扰素刺激基因表达及其机制研究

Mammalian cells produce type I interferon in response to virus infection. Viral proteins and viral nucleic acids are detected by pathogen recognition receptors (PRRs) as pathogen-associated molecular patterns(PAMPs).Pathogens such as influenza A viruses (IAV) have to overcome a number of barriers defined and maintained by the host, to successfully establish an infection. One of the initial barriers is collectively characterized as the innate immune system. This is a broad anti-pathogen defense program that ranges from the action of natural killer cells to the induction of an antiviral cytokine response.The innate immune response is the first unspecific defense of the host activated by pathogens like IAV. Once initiated, proinflammatory cytokines and chemokines are released as part of an inflammatory response, which attract immune cells such as macrophages or neutrophils to the source of infection. Among the cytokines, which are induced, the activated type I interferon (IFN) system represents the most powerful innate immune defense mechanism against IAV replication and spread. To establish productive infection, IAV have to cross the cellular membrane to enter the cell, where they abuse the cellular machinery to support their own reproduction. In addition, IAV start to counteract the host immune response against viral infection that comprises not only the innate, but also the adaptive immune systems. In recent years a number of findings helped to unravel some of the cellular recognition mechanisms and signaling responses to IAV infection as mechanisms to counteract viral replication. A plethora of studies has increased our knowledge on viral proteins interfering with cellular signaling cascades to support viral replication as it was demonstrated for HA and NS1, or to suppress antiviral immune response as known for NS1. The project intends to use the influenza virus reverse genetics to build the replacement of the NS gene reassortant viruses and gene mutation of the virus, through the comparison and analysis of the biological characteristics of the rescue strain, pathogenicity, such as the use of DNA microarray analysis the strength of strain infected mice and cells (interferon stimulation) differentially expressed genes by RNA interference and overexpression of the technology to explore the infection-related genes, to carry out the NS1 protein on interferon-stimulated gene transcription levels, and further reveal the influenza virusinfection of immune-related molecular mechanisms, and to provide more technical ideas and theoretical basis for the prevention of influenza virus, treatment.

流感病毒感染宿主后，会利用宿主细胞产生的蛋白质来完成自己的生命周期，其与宿主相互作用的感染免疫机制仍未完全明了。NS1蛋白是流感病毒的重要毒力因子, 可通过对干扰素的拮抗作用影响机体的抗病毒活性，但NS1蛋白对干扰素刺激基因（ISGs）及所表达蛋白的拮抗作用仍不明确。本项目拟利用流感病毒反向遗传操作技术，构建NS基因置换重配病毒和基因突变病毒，通过对拯救毒株的生物学特性，致病性等的比较和分析，利用基因表达谱芯片比较分析强弱毒株感染小鼠与细胞（加干扰素刺激）后差异表达的基因，通过RNA干扰和过表达技术发掘感染相关基因，开展NS1蛋白对干扰素刺激基因转录水平影响的研究，进一步揭示流感病毒感染免疫相关分子机制，为流感病毒的预防、治疗提供更多的技术思路和理论依据。

流感病毒感染宿主后，会利用宿主细胞产生的蛋白质来完成自己的生命周期，NS1蛋白是流感病毒的重要毒力因子, 在调节流感病毒的致病性及毒力方面发挥着重要的作用。本研究通过分析其NS1蛋白相关毒力关键基因区段和关键位点，利用流感病毒反向遗传操作技术，对NS1不同氨基酸分别进行定点突变，拯救毒力减弱株，对重组病毒致病性开展研究。利用表达谱测序比较分析强弱毒株感染小鼠后差异表达的基因，通过发掘感染相关基因，进一步揭示流感病毒感染免疫相关分子机制。成功拯救出PR8/NS1-A149V及PR8F/NS1不同位点突变毒株。通过小鼠攻毒实验证明PR8F/NS1蛋白第81-92位氨基酸缺失降低了对BALB/c小鼠的致病性。确定NS1第149位丙氨酸是毒力关键位点，证明NS1/A149V突变株能降低PR8感染哺乳动物致病能力。同时，利用数字基因表达谱比较强弱毒株感染小鼠肺组织差异表达的基因，分析特异性上调或下调的基因及通路。由于两者IFN-β及ISGs基因表达水平基本一致，初步推测其感染小鼠致病能力减弱与拮抗干扰素作用无关。发现在人肺癌细胞系A549中，在干扰素诱导的条件下，IFITM1、2、3基因转录水平显著升高。而当细胞中转染NS蛋白基因后，IFITM1、2、3基因转录水平同步下降，下降幅度随NS蛋白基因的增多而增大。表明流感NS蛋白的表达可显著降低IFITM2、3蛋白表达水平。siRNA干扰和病毒感染实验显示，干扰IFITM1、2和3表达后，H5N1亚型禽流感病毒引起的细胞病变效应（CPE）增强，表明，IFITM1、2和3可以抵御H5N1亚型禽流感病毒感染。利用稳定表达细胞系进行病毒感染，通过q-PCR方法分析结果显示，IFITM3可抑制H5N1早期感染。本流感病毒的预防、治疗提供更多的技术思路和理论依据。