Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA
J Virol. 2018 Dec 10;93(1). doi: 10.1128/JVI.01046-18. Print 2019 Jan 1.
The incoming influenza A virus (IAV) genome must pass through two distinct barriers in order to establish infection in the cell: the plasma membrane and the nuclear membrane. A precise understanding of the challenges imposed by the nuclear barrier remains outstanding. Passage across is mediated by host karyopherins (KPNAs), which bind to the viral nucleoprotein (NP) via its N-terminal nuclear localization sequence (NLS). The binding affinity between the two molecules is low, but NP is present in a high copy number, which suggests that binding avidity plays a compensatory role during import. Using nanobody-based technology, we demonstrate that a high binding avidity is required for infection, though the absolute value differs between cell types and correlates with their relative susceptibility to infection. In addition, we demonstrate that increasing the affinity level caused a decrease in avidity requirements for some cell types but blocked infection in others. Finally, we show that genomes that become frustrated by low avidity and remain cytoplasmic trigger the type I interferon response. Based on these results, we conclude that IAV balances affinity and avidity considerations in order to overcome the nuclear barrier across a broad range of cell types. Furthermore, these results provide evidence to support the long-standing hypothesis that IAV's strategy of import and replication in the nucleus facilitates immune evasion. We used intracellular nanobodies to block influenza virus infection at the step prior to nuclear import of its ribonucleoproteins. By doing so, we were able to answer an important but outstanding question that could not be addressed with conventional tools: how many of the ∼500 available NLS motifs are needed to establish infection? Furthermore, by controlling the subcellular localization of the incoming viral ribonucleoproteins and measuring the cell's antiviral response, we were able to provide direct evidence for the long-standing hypothesis that influenza virus exploits nuclear localization to delay activation of the innate immune response.
为了在细胞中建立感染，进入的甲型流感病毒（IAV）基因组必须通过两个截然不同的屏障：质膜和核膜。对核屏障所带来的挑战的精确理解仍然存在。通过宿主核孔蛋白（KPNAs）介导穿过核膜，核孔蛋白（NP）通过其 N 端核定位序列（NLS）与病毒结合。这两个分子之间的结合亲和力较低，但 NP 以高拷贝数存在，这表明结合亲和力在导入过程中发挥了补偿作用。我们使用纳米体技术证明，尽管结合亲和力在不同细胞类型之间存在差异，但与它们对感染的相对易感性相关，高结合亲和力对于感染是必需的。此外，我们还证明，增加亲和力水平会降低某些细胞类型对亲和力的要求，但会阻止其他细胞类型的感染。最后，我们表明，由于亲和力低而滞留在细胞质中的基因组会触发 I 型干扰素反应。基于这些结果，我们得出结论，IAV 在广泛的细胞类型中平衡亲和力和亲和力的考虑因素，以克服核屏障。此外，这些结果提供了证据支持长期以来的假设，即 IAV 在细胞核中导入和复制的策略促进了免疫逃避。我们使用细胞内纳米体在其核糖核蛋白进入核内之前的步骤阻断流感病毒感染。通过这样做，我们能够回答一个重要但尚未解决的问题，这个问题无法用传统工具来解决：建立感染需要多少个约 500 个可用的 NLS 基序？此外，通过控制传入病毒核糖核蛋白的亚细胞定位并测量细胞的抗病毒反应，我们能够为流感病毒利用核定位来延迟先天免疫反应激活的长期假设提供直接证据。
