Gerlach Thomas, Hensen Luca, Matrosovich Tatyana, Bergmann Janina, Winkler Michael, Peteranderl Christin, Klenk Hans-Dieter, Weber Friedemann, Herold Susanne, Pöhlmann Stefan, Matrosovich Mikhail
Institute of Virology, Philipps University, Marburg, Germany.
Deutsches Primatenzentrum, Göttingen, Germany.
J Virol. 2017 May 12;91(11). doi: 10.1128/JVI.00246-17. Print 2017 Jun 1.
The replication and pathogenicity of influenza A viruses (IAVs) critically depend on their ability to tolerate the antiviral interferon (IFN) response. To determine a potential role for the IAV hemagglutinin (HA) in viral sensitivity to IFN, we studied the restriction of IAV infection in IFN-β-treated human epithelial cells by using 2:6 recombinant IAVs that shared six gene segments of A/Puerto Rico/8/1934 virus (PR8) and contained HAs and neuraminidases of representative avian, human, and zoonotic H5N1 and H7N9 viruses. In A549 and Calu-3 cells, viruses displaying a higher pH optimum of HA-mediated membrane fusion, H5N1-PR8 and H7N9-PR8, were less sensitive to the IFN-induced antiviral state than their counterparts with HAs from duck and human viruses, which fused at a lower pH. The association between a high pH optimum of fusion and reduced IFN sensitivity was confirmed by using HA point mutants of A/Hong Kong/1/1968-PR8 that differed solely by their fusion properties. Furthermore, similar effects of the viral fusion pH on IFN sensitivity were observed in experiments with (i) primary human type II alveolar epithelial cells and differentiated cultures of human airway epithelial cells, (ii) nonrecombinant zoonotic and pandemic IAVs, and (iii) preparations of IFN-α and IFN-λ1. A higher pH of membrane fusion and reduced sensitivity to IFN correlated with lower restriction of the viruses in MDCK cells stably expressing the IFN-inducible transmembrane proteins IFITM2 and IFITM3, which are known to inhibit viral fusion. Our results reveal that the pH optimum of HA-driven membrane fusion of IAVs is a determinant of their sensitivity to IFN and IFITM proteins. The IFN system constitutes an important innate defense against viral infection. Substantial information is available on how IAVs avoid detection by sensors of the IFN system and disable IFN signaling pathways. Much less is known about the ability of IAVs to tolerate the antiviral activity of IFN-induced cellular proteins. The IFN-induced proteins of the IFITM family block IAV entry into target cells and can restrict viral spread and pathogenicity. Here we show for the first time that the sensitivity of IAVs to the IFN-induced antiviral state and IFITM2 and IFITM3 proteins depends on the pH value at which the viral HA undergoes a conformational transition and mediates membrane fusion. Our data imply that the high pH optimum of membrane fusion typical of zoonotic IAVs of gallinaceous poultry, such as H5N1 and H7N9, may contribute to their enhanced virulence in humans.
甲型流感病毒（IAV）的复制和致病性严重依赖于其耐受抗病毒干扰素（IFN）应答的能力。为了确定IAV血凝素（HA）在病毒对IFN敏感性中的潜在作用，我们通过使用2:6重组IAV研究了IFN-β处理的人上皮细胞中IAV感染的限制情况，这些重组IAV共享A/波多黎各/8/1934病毒（PR8）的六个基因片段，并包含代表性禽源、人源以及人畜共患的H5N1和H7N9病毒的HA和神经氨酸酶。在A549和Calu-3细胞中，具有较高HA介导膜融合最佳pH值的病毒，即H5N1-PR8和H7N9-PR8，相较于具有来自鸭和人病毒HA（在较低pH值下融合）的对应病毒，对IFN诱导的抗病毒状态敏感性更低。通过使用仅在融合特性上存在差异的A/香港/1/1968-PR8的HA点突变体，证实了融合最佳pH值高与IFN敏感性降低之间的关联。此外，在以下实验中观察到病毒融合pH对IFN敏感性有类似影响：（i）原代人II型肺泡上皮细胞和人气道上皮细胞的分化培养物；（ii）非重组人畜共患和大流行IAV；（iii）IFN-α和IFN-λ1制剂。膜融合的较高pH值以及对IFN敏感性降低与病毒在稳定表达IFN诱导跨膜蛋白IFITM2和IFITM3的MDCK细胞中的较低限制相关，已知这些蛋白可抑制病毒融合。我们的结果表明，IAV的HA驱动膜融合的最佳pH值是其对IFN和IFITM蛋白敏感性的决定因素。IFN系统构成了针对病毒感染的重要先天性防御。关于IAV如何避免被IFN系统的传感器检测到并使IFN信号通路失效，已有大量信息。关于IAV耐受IFN诱导的细胞蛋白抗病毒活性的能力，了解得要少得多。IFN诱导的IFITM家族蛋白可阻止IAV进入靶细胞，并能限制病毒传播和致病性。在这里，我们首次表明IAV对IFN诱导的抗病毒状态以及IFITM2和IFITM3蛋白的敏感性取决于病毒HA发生构象转变并介导膜融合的pH值。我们的数据表明，诸如H5N1和H7N9等鸡源家禽人畜共患IAV典型的膜融合高最佳pH值，可能有助于其在人类中增强的毒力。
