Institute of Virology, Philipps University, Marburg, Germany.
Center for Tumor Biology and Immunology (ZTI), Core Facility Cellular Imaging, Philipps University, Marburg, Germany.
J Virol. 2019 Dec 12;94(1). doi: 10.1128/JVI.01223-19.
Previous studies revealed that certain avian influenza A viruses (IAVs), including zoonotic H5N1 and H7N9 IAVs, infect cultured human lung microvascular endothelial cells (HULEC) more efficiently than other IAVs and that tropism to HULEC is determined by viral hemagglutinin (HA). To characterize mechanisms of HA-mediated endotheliotropism, we used 2:6 recombinant IAVs harboring HAs from distinctive avian and human viruses and found that efficient infection of HULEC correlated with low conformational stability of the HA. We next studied effects on viral infectivity of single-point amino acid substitutions in the HA of 2:6 recombinant virus A/Vietnam/1203/2004-PR8 (H5N1). Substitutions H8Q, H103Y, T315I, and K58I (K58I in the HA2 subunit), which increased stability of the HA, markedly reduced viral infectivity for HULEC, whereas substitutions K189N and K218Q, which altered typical H5N1 virus-like receptor specificity and reduced binding avidity of the HA, led to only marginal reduction of infectivity. None of these substitutions affected virus infection in MDCK cells. We confirmed the previous observation of elevated basal expression of IFITM3 protein in HULEC and found that endosomal acidification is less efficient in HULEC than in MDCK cells. In accord with these findings, counteraction of IFITM3-mediated restriction by amphotericin B and reduction of endosomal pH by moderate acidification of the extracellular medium enhanced infectivity of viruses with stable HA for HULEC without significant effect on infectivity for MDCK cells. Collectively, our results indicate that relatively high pH optimum of fusion of the HA of zoonotic H5N1 and H7N9 IAVs allows them to overcome antiviral effects of inefficient endosomal acidification and IFITM3 in human endothelial cells. Receptor specificity of the HA of IAVs is known to be a critical determinant of viral cell tropism. Here, we show that fusion properties of the HA may also play a key role in the tropism. Thus, we demonstrate that IAVs having a relatively low pH optimum of fusion cannot efficiently infect human endothelial cells owing to their relatively high endosomal pH and increased expression of fusion-inhibiting IFITM3 protein. These restrictions can be overcome by IAVs with elevated pH of fusion, such as zoonotic H5N1 and H7N9. Our results illustrate that the infectivity of IAVs depends on an interplay between HA conformational stability, endosomal acidification and IFITM3 expression in target cells, and the extracellular pH. Given significant variation of levels of HA stability among animal, human, and zoonotic IAVs, our findings prompt further studies on the fusion-dependent tropism of IAVs to different cell types in humans and its role in viral host range and pathogenicity.
先前的研究表明，某些禽流感病毒（IAV），包括人畜共患的 H5N1 和 H7N9 IAV，比其他 IAV 更有效地感染培养的人肺微血管内皮细胞（HULEC），并且对 HULEC 的趋向性由病毒血凝素（HA）决定。为了描述 HA 介导的内皮趋向性的机制，我们使用了带有独特的禽源和人源病毒 HA 的 2:6 重组 IAV，并发现 HULEC 的高效感染与 HA 的低构象稳定性相关。接下来，我们研究了 2:6 重组病毒 A/Vietnam/1203/2004-PR8（H5N1）HA 单点氨基酸取代对病毒感染力的影响。增加 HA 稳定性的取代 H8Q、H103Y、T315I 和 K58I（HA2 亚基中的 K58I）显著降低了病毒对 HULEC 的感染力，而改变典型的 H5N1 病毒样受体特异性并降低 HA 结合亲和力的取代 K189N 和 K218Q 仅导致感染性略有降低。这些取代均不影响 MDCK 细胞中的病毒感染。我们证实了先前在 HULEC 中观察到的 IFITM3 蛋白基础表达升高的现象，并发现 HULEC 中的内体酸化效率低于 MDCK 细胞。与这些发现一致，两性霉素 B 对抗 IFITM3 介导的限制以及通过适度降低细胞外培养基的 pH 来降低内体 pH，增强了具有稳定 HA 的病毒对 HULEC 的感染力，而对 MDCK 细胞的感染力没有明显影响。总的来说，我们的结果表明，人畜共患的 H5N1 和 H7N9 IAV 的 HA 融合的相对较高 pH 最佳值允许它们克服人类内皮细胞中低效的内体酸化和 IFITM3 的抗病毒作用。HA 的受体特异性是决定病毒细胞趋向性的关键决定因素。在这里，我们表明 HA 的融合特性也可能在趋向性中起关键作用。因此，我们证明了具有相对较低融合 pH 最佳值的 IAV 由于其相对较高的内体 pH 和融合抑制 IFITM3 蛋白的表达，无法有效地感染人内皮细胞。具有升高的融合 pH 的 IAV 可以克服这些限制，例如人畜共患的 H5N1 和 H7N9。我们的结果表明，IAV 的感染性取决于靶细胞中 HA 构象稳定性、内体酸化和 IFITM3 表达之间的相互作用，以及细胞外 pH。鉴于动物、人类和人畜共患 IAV 之间 HA 稳定性水平的显著差异，我们的发现促使进一步研究 IAV 对人类不同细胞类型的依赖融合的趋向性及其在病毒宿主范围和致病性中的作用。
