Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Cátedra de Inmunología-IDEHU (UBA-CONICET), Junín 956, C1113AAD Buenos Aires, Argentina; W.M. Keck Laboratory for Structural Biology, University of Maryland Institute for Bioscience and Biotechnology Research, Rockville, MD 20850, USA.
Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Cátedra de Inmunología-IDEHU (UBA-CONICET), Junín 956, C1113AAD Buenos Aires, Argentina; Universidad de Buenos Aires, Facultad de Medicina, Departamento de Microbiología, Parasitología e Inmunología-IMPAM (UBA-CONICET), Paraguay 2155, C1121ABG Buenos Aires, Argentina.
Biochim Biophys Acta Gen Subj. 2023 Dec;1867(12):130483. doi: 10.1016/j.bbagen.2023.130483. Epub 2023 Oct 4.
During viral infections, nucleic acid sensing by intracellular receptors can trigger type I interferon (IFN-I) production, key mediators in antiviral innate immunity. However, many flaviviruses use non-structural proteins to evade immune sensing favoring their survival. These mechanisms remain poorly characterized. Here, we studied the role of Zika virus (ZIKV) NS4B protein in the inhibition of IFN-I induction pathway and its biophysical interaction with host proteins.
Using different cell-based assays, we studied the effect of ZIKV NS4B in the activation of interferon regulatory factors (IRFs), NF-κB, cytokines secretion and the expression of interferon-stimulating genes (ISG). We also analyzed the in vitro interaction between recombinant ZIKV NS4B and TANK-binding kinase 1 (TBK1) using surface plasmon resonance (SPR).
Transfection assays showed that ZIKV NS4B inhibits IRFs activation involved in different nucleic acid sensing cascades. Cells expressing NS4B secreted lower levels of IFN-β and IL-6. Furthermore, early induction of ISGs was also restricted by ZIKV NS4B. For the first time, we demonstrate by SPR assays that TBK1, a critical component in IFN-I production pathway, binds directly to ZIKV NS4B (K of 3.7 × 10 M). In addition, we show that the N-terminal region of NS4B is directly involved in this interaction.
Altogether, our results strongly support that ZIKV NS4B affects nucleic acid sensing cascades and disrupts the TBK1/IRF3 axis, leading to an impairment of IFN-β production.
This study provides the first biophysical data of the interaction between ZIKV NS4B and TBK1, and highlights the role of ZIKV NS4B in evading the early innate immune response.
在病毒感染期间，细胞内受体对核酸的感知可以触发 I 型干扰素（IFN-I）的产生，这是抗病毒先天免疫的关键介质。然而，许多黄病毒利用非结构蛋白来逃避免疫感应，从而有利于它们的存活。这些机制仍未得到很好的描述。在这里，我们研究了 Zika 病毒（ZIKV）NS4B 蛋白在抑制 IFN-I 诱导途径及其与宿主蛋白的生物物理相互作用中的作用。
我们使用不同的基于细胞的测定法，研究了 ZIKV NS4B 在干扰素调节因子（IRFs）、NF-κB、细胞因子分泌和干扰素刺激基因（ISG）表达的激活中的作用。我们还使用表面等离子体共振（SPR）分析了重组 ZIKV NS4B 与 TANK 结合激酶 1（TBK1）之间的体外相互作用。
转染实验表明，ZIKV NS4B 抑制了参与不同核酸感应级联的 IRFs 激活。表达 NS4B 的细胞分泌的 IFN-β 和 IL-6 水平较低。此外，ZIKV NS4B 还限制了早期 ISG 的诱导。我们首次通过 SPR 测定法证明，TBK1 是 IFN-I 产生途径中的关键组成部分，它直接与 ZIKV NS4B 结合（Kd 为 3.7×10-6 M）。此外，我们还表明 NS4B 的 N 端区域直接参与了这种相互作用。
总的来说，我们的结果强烈支持 ZIKV NS4B 影响核酸感应级联并破坏 TBK1/IRF3 轴，导致 IFN-β 产生受损。
本研究提供了 ZIKV NS4B 与 TBK1 相互作用的第一个生物物理数据，并强调了 ZIKV NS4B 在逃避早期先天免疫反应中的作用。
