Harnessing microRNAs to explore influenza virus immunity

描述（由申请人提供）：为了更好地了解流感病毒趋向性与抗病毒免疫发展之间的关系，我们开发了一种新的平台，以细胞特异性的方式限制病毒复制。这涉及到将内源性的、细胞特异性的microrna靶向位点插入到流感病毒mRNA中，仅在microrna存在的情况下导致衰减。使用一种在免疫系统的所有细胞中限制复制的病毒，可以发现先天细胞因子反应依赖于免疫细胞的感染。然而，这需要的精确免疫细胞仍然未知。这一提议将验证树突状细胞和巨噬细胞的直接感染将需要抗病毒细胞因子反应的协调这一假设。为了验证这一假设，Aim 1将建立一个由细胞特异性microrna靶向的流感病毒文库，包括那些局限于骨髓细胞（树突状细胞和巨噬细胞）、淋巴细胞（B细胞和T细胞）、内皮细胞和纤毛上皮细胞的病毒文库。作为对照，病毒将被设计成在所有哺乳动物细胞中被靶向，而非靶向的混乱病毒作为阴性对照。这些病毒将用于确定协调抗病毒细胞素反应所需的感染细胞类型。除了直接感染流感的细胞外，未感染的细胞也通过旁观者激活参与免疫应答。为了更好地确定直接感染的作用，Aim 2将产生一组表达靶向宿主细胞因子的sirna的流感病毒。因此，特异性细胞因子只有在直接感染流感病毒的细胞中才会钝化。这些重组病毒将用于确定在体内诱导特定细胞因子的直接感染要求。本提案的目的是利用互补的方法，通过利用microrna，来确定病毒趋向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反应和发病机制，并为未来合理的疫苗和抗病毒药物设计提供基础。

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.