Harnessing microRNAs to explore influenza virus immunity

描述（由申请人提供）：为了更好地理解流感病毒嗜性与抗病毒免疫发展之间的关系，我们开发了以细胞特异性方式限制病毒复制的新平台。这涉及将内源性细胞特异性microRNA靶位点插入流感病毒mRNA中，导致仅在miRNA存在下减毒。使用限制在免疫系统的所有细胞中复制的病毒允许发现先天性细胞因子应答依赖于免疫细胞的感染。然而，这所需的精确免疫细胞仍然未知。该提议将检验树突状细胞和巨噬细胞的直接感染对于抗病毒细胞因子应答的协调是必需的这一假设。为了解决这一假设，Aim 1将产生细胞特异性microRNA靶向的流感病毒文库，所述细胞特异性microRNA包括限于以下细胞的那些：骨髓细胞（树突状细胞和巨噬细胞）、淋巴样细胞（B和T细胞）、内皮细胞和纤毛上皮细胞。作为对照，病毒将被设计为在所有哺乳动物细胞中靶向，而非靶向的乱序病毒作为阴性对照。这些病毒将被用来确定感染的细胞类型所需的协调抗病毒细胞因子的反应。除了直接感染流感病毒的细胞外，未感染的细胞也通过旁观者激活参与免疫应答。为了更好地定义直接感染的作用，Aim 2将产生一组表达siRNA的流感病毒，这些siRNA旨在靶向宿主细胞因子。因此，特异性细胞因子仅在直接感染流感病毒的细胞中钝化。这些重组病毒将用于确定体内诱导特异性细胞因子的直接感染要求。该提案的目的是通过利用microRNA使用互补方法来定义病毒嗜性在诱导抗病毒免疫中的作用。这些研究将阐明流感病毒的免疫应答和发病机制的关键见解，并为未来合理的疫苗和抗病毒设计提供基础。

DESCRIPTION (provided by applicant): In an effort to better understand the relationship between influenza virus tropism and the development of antiviral immunity we developed novel platform to restrict virus replication in a cell-specific manner. This involved insertion of endogenous, cell-specific microRNAs target sites into influenza virus mRNA, resulting in attenuation only in the presence of the miRNA. Using a virus restricted from replication in all cells of the immune system allowed for the discovery that the innate cytokine response relies on infection of immune cells. However the precise immune cells required for this remain unknown. This proposal will test the hypothesis that direct infection of dendritic cells and macrophages wil be required for the orchestration of antiviral cytokine responses. To address this hypothesis Aim 1 will generate a library of influenza viruses targeted by cell-specific microRNAs including those restricted to: myeloid cells (dendritic cells and macrophages), lymphoid cells (B and T cells), endothelial cells, and ciliated epithelial cells. As a control, viruses will be designed to be targeted in all mammalian cells, and an untargeted scrambled virus as a negative control. These viruses will be used to define the infected cell types required for orchestrating antiviral cytokin responses. In addition to cells directly infected with influenza, non-infected cells also participae in the immune response through bystander activation. To better define the role of direct infection Aim 2 will generate a panel of influenza viruses that express siRNAs designed to target host cytokines. Therefore, specific cytokines will be blunted only in cells directly infected with influenza virus. These recombinant viruses will be used to define the direct infection requirements for the induction of specific cytokines in vivo. The aims of this proposal use complementary approaches, through the exploitation of microRNAs, to define the role of virus tropism on the induction of antiviral immunity. These studies will elucidate key insights into the immune response and pathogenesis of influenza viruses and provide the basis for future rational vaccine and antiviral design.