Characterizing the role of selected host factors in replication and pathogenicity of clinically-relevant influenza A viruses in the human lung

甲型流感病毒(IAV)是一种编码多达13种病毒蛋白的RNA病毒。由于编码能力有限，IAV需要使用宿主细胞机制来完成其感染周期。高通量筛选已经确定了数百种对IAV复制必不可少的人类蛋白质。我们的研究小组进行了全基因组RNAi筛查，确定了早期IAV复制所需的295个细胞辅助因子。在随后对所有IAV蛋白进行的亲和纯化质谱分析中，检测到大约60个宿主因子，这些因子与已发表的siRNA筛选数据直接重叠。这些不仅在IAV复制中起作用，而且直接与IAV蛋白相互作用的宿主因子将是我们研究的对象。我们的目标是进一步从宿主因素列表中选择正交验证的候选毒株，了解它们在三种临床相关IAV毒株-季节性H3N1、大流行性H1N1和高致病性H5N1的复制和致病性中的作用。这些病毒的毒力程度不同，这使我们能够进一步研究影响病毒致病的宿主因素。然而，像A549这样被广泛用于IAV研究的永生化人类上皮细胞可能存在潜在的缺陷，例如与非致瘤细胞相比，宿主调控网络存在差异。因此，需要更多的模型来对影响IAV感染结局的人类宿主因素进行全面的研究。人的体外肺组织可以被IAV感染，以分析病毒复制、细胞趋向性和细胞因子诱导等，使其成为一个特别有用的模型系统。与细胞培养系统相比，它的优势是这种模型应用了真实的人体组织，其中不同类型的细胞仍然在3D结构中连接在一起，并能够相互作用和交流。IAV在肺组织中的复制与其在人类中的致病性相关，这使得该模型也成为研究IAV毒力所涉及的宿主因素的理想模型。我们计划通过使用专为复杂组织设计的名为Vivo-Morpholinos(Vms)的特殊分子来沉默肺组织中选定的候选基因。VMS已经成功地用于沉默不同组织中的基因，以及老鼠等活着的动物。不再表达所选基因的肺组织随后将被分析病毒复制、细胞免疫反应和其他终点，与非靶标对照VM治疗的肺组织相比。总之，我们的项目集中在生理相关的人肺组织模型中涉及临床相关IAV复制和致病的宿主因素的表征。这些研究不仅将加深我们对这些宿主因素在IAV感染循环中的作用的理解，还将为未来抗IAV新药的开发提供重要的见解。

Influenza A virus (IAV) is an RNA virus encoding up to 13 viral proteins. Due to this limited coding capacity IAV needs to use the host cellular machinery for completing its infection cycle. High-throughput screens have identified hundreds of human proteins indispensable for IAV replication. Our research group performed a genome-wide RNAi screen that identified 295 cellular cofactors required for early-stage IAV replication. In a subsequent affinity-purification mass spectrometry analysis of all IAV proteins about 60 host factors were detected that directly overlap with data from published siRNA screens. These host factors that not only play a role in IAV replication but also directly interact with IAV proteins will be subject of our investigation. We aim to further characterize a selection of orthogonally validated candidates from the list of host factors regarding their roles in replication and pathogenicity of three clinically relevant IAV strains - seasonal H3N2, pandemic H1N1 and highly-pathogenic H5N1. These viruses differ in their degrees of virulence, which enables us to further examine host factors affecting viral pathogenesis. However, immortalized human epithelial cells like A549, which are widely used for IAV research, can have potential drawbacks such as differences in host regulatory networks compared to non-tumorigenic cells. Therefore, additional models are needed to perform comprehensive studies on human host factors that influence the outcome of an IAV infection. Human ex vivo lung tissue can be infected with IAVs to analyze e.g. virus replication, cell tropism and cytokine induction making it a particularly useful model system. Its advantage compared to cell culture systems is that this model applies authentic human tissue in which different cell types are still connected in the 3D structure and able to interact and communicate with each other. Replication of IAVs in the lung tissue correlates with their pathogenicity in humans, which makes this model ideal to also investigate host factors involved in IAV virulence. We plan to silence selected candidate genes in lung tissue by making use of specialized molecules designed to work in complex tissues named Vivo-Morpholinos (VMs). VMs have been successfully used to silence genes in different tissues as well as in living animals like mice. The lung tissue that no longer expresses the selected gene will then be analyzed for virus replication, cellular immune responses and other endpoints compared to lung tissue treated with non-target control VMs. In summary, our project concentrates on the characterization of host factors involved in replication and pathogenicity of clinically relevant IAVs in a physiologically-relevant human lung tissue model. These studies will not only improve our understanding of the roles of these host factors in the IAV infection cycle but also provide critical insight into the future development of novel antiviral drugs against IAV.