1Department of Microbiology and Immunology, Rega Institute, KU Leuven, Leuven, Belgium.
2Experimental Oncology and Immunology, Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy.
J Gen Virol. 2019 Apr;100(4):583-601. doi: 10.1099/jgv.0.001235. Epub 2019 Feb 14.
The possible resistance of influenza virus against existing antiviral drugs calls for new therapeutic concepts. One appealing strategy is to inhibit virus entry, in particular at the stage of internalization. This requires a better understanding of virus-host interactions during the entry process, including the role of receptor tyrosine kinases (RTKs). To search for cellular targets, we evaluated a panel of 276 protein kinase inhibitors in a multicycle antiviral assay in Madin-Darby canine kidney cells. The RTK inhibitor Ki8751 displayed robust anti-influenza A and B virus activity and was selected for mechanistic investigations. Ki8751 efficiently disrupted the endocytic process of influenza virus in different cell lines carrying platelet-derived growth factor receptor β (PDGFRβ), an RTK that is known to act at GM3 ganglioside-positive lipid rafts. The more efficient virus entry in CHO-K1 cells compared to the wild-type ancestor (CHO-wt) cells indicated a positive effect of GM3, which is abundant in CHO-K1 but not in CHO-wt cells. Entering virus localized to GM3-positive lipid rafts and the PDGFRβ-containing endosomal compartment. PDGFRβ/GM3-dependent virus internalization involved PDGFRβ phosphorylation, which was potently inhibited by Ki8751, and desialylation of activated PDGFRβ by the viral neuraminidase. Virus uptake coincided with strong activation of the Raf/MEK/Erk cascade, but not of PI3K/Akt or phospholipase C-γ. We conclude that influenza virus efficiently hijacks the GM3-enhanced PDGFRβ signalling pathway for cell penetration, providing an opportunity for host cell-targeting antiviral intervention.
流感病毒对现有抗病毒药物的可能耐药性要求新的治疗概念。一种有吸引力的策略是抑制病毒进入,特别是在内化阶段。这需要更好地了解病毒-宿主相互作用在进入过程中的作用,包括受体酪氨酸激酶(RTKs)的作用。为了寻找细胞靶标,我们在 Madin-Darby 犬肾细胞中的多轮抗病毒测定中评估了 276 种蛋白激酶抑制剂的试剂盒。RTK 抑制剂 Ki8751 对甲型和乙型流感病毒显示出强大的抗病毒活性,并被选为机制研究的选择。Ki8751 有效地破坏了携带血小板衍生生长因子受体β(PDGFRβ)的不同细胞系中流感病毒的内吞过程,PDGFRβ 是一种已知在 GM3 神经节苷脂阳性脂筏上起作用的 RTK。与野生型祖先(CHO-wt)细胞相比,CHO-K1 细胞中更有效的病毒进入表明 GM3 的积极作用,GM3 在 CHO-K1 中丰富,但在 CHO-wt 细胞中不存在。进入的病毒定位于 GM3 阳性脂筏和含有 PDGFRβ 的内体区室。PDGFRβ/GM3 依赖性病毒内化涉及 PDGFRβ 磷酸化,Ki8751 可强烈抑制该磷酸化,以及病毒神经氨酸酶对激活的 PDGFRβ 的去唾液酸化。病毒摄取与 Raf/MEK/Erk 级联的强烈激活同时发生,但不与 PI3K/Akt 或磷酯酶 C-γ 同时发生。我们的结论是,流感病毒有效地劫持了 GM3 增强的 PDGFRβ 信号通路以进行细胞穿透,为宿主细胞靶向抗病毒干预提供了机会。