III型干扰素介导的抗汉坦病毒天然免疫机制研究

The mechanism of Hantavirus (HV) in innate immune is unclear. The newly identified type III interferon (IFN-λ) has antiviral activity against a broad spectrum of viruses, including HBV and HCV. Supported by National Nature Science Foundation, we found that IFN-λ inhibited HIV-1 infection of macrophages. Invesigation of the mechanism(s) responsible for IFN-λ action showed that INF-λ induced the expression of CC-chemokines, the ligands for CCR5, and upregulated intracellular expression of type I IFNs and APOBEC3G/3F, the newly identified anti-HIV-1 cellular factors. More importantly, we showed that IFN-λ could inhibit HV replication in vero E6 cells. This inhibitory effect on HV by IFN-λ was dose and time dependent. These data provide direct and compelling evidence that IFN-λ has a critical role in innate immunity against HV infection. So this project through the real-time quantitative RT-PCR, immunofluorescence technique will further prove that IFN-λ could suppress replication of HV in target cells (HUVEC or A549) in order to determine the activity of IFN-λ against HV infection. We will also examine the mechanism(s) of involved in IFN-λ action on HV. We are particularly interested in the role of IFN-λ in the modulation of β3 integrin receptor and Toll-like receptor (TLR) for HV entry into target cells. At both mRNA and protein level in target cells, we will explore the expression of interferon regulatory factors (IRFs) and signal transducer and activator of transcription (STAT). Type I IFNs (IFN-α/β) are well known for their ability to inhibit a wide range of viruses, including HV. Thus, the induction of intracellular IFN-α/β expression by IFN-λ provides an additional mechanism for the anti-HV action of IFN-λ in target cells. Several cellular factors, such as microRNAs, OAS1, ISG15, have recently been identified as type I IFN-inducible anti-HV elements in HV-infected target cells. Therefore, it is important to determine whether IFN-λ has the ability to enhance the expression of these newly identified innate anti-HV factors in target cells. In conclusion, these studies provide the first experimental evidence at cellular and molecular levels that IFN-λ has the ability to suppress HV infection in target cells, which would be of importance not only for our understanding of HV immunopathogenesis but also for the design and development of IFN-λ-based intervention strategies for treating HV infected patients.

抗汉坦病毒（HV）天然免疫机制尚不清楚。新型λ干扰素（IFN-λ）具有抗病毒作用，如HBV、HCV等。在国家基金资助下我们研究发现IFN-λ通过上调CC-趋化因子、Ⅰ型干扰素、APOBEC3G/3F抑制HIV-1在人巨噬细胞中的感染。我们还发现，IFN-λ能抑制HV在Vero E6中的复制，且呈剂量和时间效应，据此推测IFN-λ可能介导了抗HV的天然免疫。本项目通过应用实时定量RT-PCR、免疫荧光等技术进一步验证IFN-λ抑制HV对靶细胞（HUVEC或A549）的感染，确定IFN-λ抗HV作用；并探讨IFN-λ对靶细胞上β3整合素受体、Toll样受体（TLR）、干扰素激活因子（IRF）以及STAT信号转导途径的影响，同时明确IFN-λ激活了靶细胞内Ⅰ型干扰素介导的抗HV病毒因子，如IFN-α、IFN-β、microRNA等，阐明IFN-λ介导的抗HV天然免疫作用机制。

汉坦病毒（Hantavirus）是引起人类致命性疾病肾综合征出血热（HFRS）和汉坦病毒心肺综合征的病原体。然而目前尚无针对汉坦病毒的特效药物。III型干扰素（IFN-λ）是一种新发现的干扰素，其具有抗病毒作用。研究发现普马拉病毒（PUUV）感染引起的HFRS患者血清中IFN-λ的水平在急性期是降低的，体外实验发现IFN-λ预处理的A549细胞能抑制汉滩病毒（HTNV）的复制，但IFN-λ抗HTNV的机制尚不清楚。为此我们研究IFN-λ对HTNV感染A549细胞的作用，并从天然免疫的角度揭示了IFN-λ介导的抗HTNV天然免疫机制。主要结果显示：1）IFN-λ能抑制HTNV的感染与复制，并可维持较长时间的抗病毒状态；IFN-λ1/λ2抑制HTNV作用与其剂量和作用时间呈正相关，且IFN-λ1/λ2抗HTNV作用无显著差异；2）IFN-λ对人A549细胞中β3整合素表达影响较小，但IFN-λ能增加A549细胞中TLR-3、9表达；3）IFN在宿主天然免疫中起重要作用，IFN-λ功能的发挥须与靶细胞上IFN-λ功能性受体复合物作用。研究发现A549细胞上确实存在IFN-λ受体，而且IFN-λ抗HTNV作用是通过激活JAK-STAT信号通路，诱导多种抗病毒蛋白，从而发挥抗病毒作用；进一步研究发现并证实MxB具有抗HTNV的潜能；4）JAK-STAT信号通路中重要的负性调节因子细胞因子信号转导抑制因子（suppressor of cytokine signaling，SOCS）蛋白家族成员SOCS1、SOCS2和SOCS3在IFN-λ处理后表达均明显下调，证实了SOCS在IFN-λ抗HTNV介导的JAK-STAT信号通路中起负调控作用；5）HTNV感染A549细胞以后，用IFN-λ维持处理细胞（病毒感染后），结果显示IFN-λ1/λ2能明显抑制HTNV感染与复制。本课题证实了IFN-λ具有抗HTNV感染作用，阐明了IFN-λ介导的抗HTNV感染天然免疫机制，为未来抗HTNV药物的研究提供了新靶点。该课题已发表论文1篇，SCI论文接收1篇，在投SCI论文1篇，申报专利2项，培养研究生6名。