Identification and characterization of influenza virus activators in the human lung

流感流行和间歇性大流行威胁人类健康。由于流感病毒的性质不断变化，目前可用的疫苗和抗病毒药物无法充分应对这一威胁。疫苗需要不断地重新配制，以匹配目前流行的病毒，病毒基因组中的耐药性突变可能使抗病毒药物无效。鉴定病毒传播所需但细胞存活所需的宿主细胞因子可能会定义新的、有吸引力的干预靶点，并可能对流感发病机制产生重要的见解。病毒血凝素（HA）被宿主细胞蛋白酶激活是病毒感染性所必需的。我们已经表明，HA激活蛋白酶TMPRSS 2的表达是H1N1甲型流感病毒（IAV）在小鼠中传播所必需的。此外，我们和其他人已经获得证据表明，TMPRSS 2相关酶，称为替代激活剂，可以支持IAV在细胞培养和小鼠中的传播。然而，目前尚不清楚TMPRSS 2和/或替代活化剂是否有助于IAV在人类中的传播，并且不知道这些酶中的哪些结构域控制HA活化。最后，HA激活蛋白酶的mRNA的二级结构及其对蛋白质表达和抗病毒干预的影响尚未被研究。这些问题将在拟议的项目中得到解决。为此，我们将使用最先进的方法，包括SHAPE用于确定编码HA激活蛋白酶的mRNA的二级结构，精密切割肺切片（PCLS）和用于人类严重流感的猕猴模型。拟议的研究将大大受益于项目合作伙伴的共同背景及其互补的专业知识：波兰的合作伙伴是SHAPE分析的专家，德国的合作伙伴可以获得评估灵长类呼吸道上皮中IAV激活的关键技术，包括PCLS和IAV感染的灵长类动物模型。

Influenza epidemics and intermittent pandemics threaten human health. This threat is not adequately met by currently available vaccines and antivirals due to the ever changing nature of influenza viruses. Vaccines need to be reformulated constantly to match the currently circulating viruses and resistance mutations in the viral genome can render antivirals ineffective. The identification of host cell factors that are required for viral spread but dispensable for cellular survival might define novel, attractive targets for intervention and may yield important insights into influenza pathogenesis. Activation of the viral hemagglutinin (HA) by a host cell protease is essential for viral infectivity. We have shown that the expression of the HA-activating protease TMPRSS2 is essential for H1N1 influenza A virus (IAV) spread in mice. Moreover, we and others have obtained evidence that TMPRSS2-related enzymes, termed alternative activators, can support IAV spread in cell culture and mice. However, it is currently unclear whether TMPRSS2 and/or alternative activators contribute to IAV spread in humans, and it is unknown which domains in these enzymes govern HA activation. Finally, the secondary structures of the mRNAs of HA-activating proteases and their implications for protein expression and antiviral intervention have not been examined. These questions will be addressed in the proposed project. For this, we will use state-of-the-art methodology, including SHAPE for determination of the secondary structure of mRNAs encoding HA-activating proteases, precision-cut lung slices (PCLS) and a macaque model for severe influenza in humans. The proposed studies will greatly benefit from the joint background of the project partners and their complementary expertise’s: The partners in Poland are experts in SHAPE analyses and the partners in Germany have access to key technology for assessment of IAV activation in the primate respiratory epithelium, including PCLS and a primate model for IAV infection.