Dengue Virus NS Proteins Inhibit RIG-I/MAVS Signaling by Blocking TBK1/IRF3 Phosphorylation: Dengue Virus Serotype 1 NS4A Is a Unique Interferon-Regulating Virulence Determinant.
Dengue Virus NS Proteins Inhibit RIG-I/MAVS Signaling by Blocking TBK1/IRF3 Phosphorylation: Dengue Virus Serotype 1 NS4A Is a Unique Interferon-Regulating Virulence Determinant.
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DOI：
10.1128/mbio.00553-15
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发表时间：
2015-05-12
期刊：
影响因子：
6.4
通讯作者：
Mackow ER
中科院分区：
文献类型：
--
作者：
Dalrymple NA;Cimica V;Mackow ER
预先加入I型干扰素或RIG-I激动剂可抑制登革病毒(DENV)的复制，RIG-I激动剂可诱导依赖于RIG-I/MAVS/TBK1/IRF3的保护反应。DENV感染原代人内皮细胞后，病毒滴度迅速升高，提示DENV抑制内皮细胞内复制限制性RIG-I/干扰素-β(β)的诱导途径。我们的研究结果表明，DENV4型非结构蛋白NS2a和NS4B能抑制RIG-I、MDA5、MAVS和Tbk1/IKKε诱导的干扰素-β转录(80%)，但不能抑制刺激性或成分活性IRF3-5D诱导的干扰素-β。NS2A和NS4B的表达呈剂量依赖性地抑制Tbk1和IRF3的磷酸化，提示它们在Tbk1复合体的激活水平上发挥作用。来自DENV1/2/4的NS2a和NS4B以及西尼罗河病毒NS4B蛋白共同抑制Tbk1的磷酸化和干扰素-β的诱导。对不同DENV的NS4A蛋白的比较分析表明，DENV1，而不是DENV2或DENV4，NS4A蛋白唯一地抑制了TBK1。这些发现表明，DENV包含保守的(NS2A/NS4B)和DENV1特异的(NS4A)机制，可以抑制RIG-I/TBK1导向的干扰素反应。总而言之，我们的结果定义了DENV NS蛋白，这些蛋白限制了IRF3和干扰素的反应，从而促进了DENV的复制和毒力。在四价DENV1-4疫苗接种过程中，DENV1特异的NS4A对干扰素诱导的独特调节可能是一个毒力决定因素，有助于增加DENV1感染的严重性和DENV1反应的免疫优势。我们的发现表明，登革病毒血清1、2和4型的NS2a和NS4B蛋白是RIG-I/MDA5导向的干扰素-β诱导的抑制物，它们通过阻断Tbk1的激活来实现这一点。我们确定，干扰素抑制在西尼罗河病毒和DENV1、-2和-4病毒的NS4B蛋白中功能上是保守的。相反，DENV1独特地编码一种额外的干扰素调节蛋白NS4A，它可以抑制TBK1诱导的干扰素。DENV1与重症患者疾病的增加有关，由DENV1 NS4A蛋白增加的干扰素调节可能有助于增加DENV1的复制、免疫优势和毒力。非结构蛋白(NS)对干扰素诱导的调节表明它们在促进病毒复制和传播方面具有潜在的作用，并且是病毒减毒的潜在蛋白质靶标。在疫苗策略中需要考虑DENV1特异性的干扰素调节，如果增强的DENV1复制可能干扰联合接种的四价DENV1-4疫苗中的DENV2-4血清转换。
Dengue virus (DENV) replication is inhibited by the prior addition of type I interferon or by RIG-I agonists that elicit RIG-I/MAVS/TBK1/IRF3-dependent protective responses. DENV infection of primary human endothelial cells (ECs) results in a rapid increase in viral titer, which suggests that DENV inhibits replication-restrictive RIG-I/interferon beta (IFN-β) induction pathways within ECs. Our findings demonstrate that DENV serotype 4 (DENV4) nonstructural (NS) proteins NS2A and NS4B inhibited RIG-I-, MDA5-, MAVS-, and TBK1/IKKε-directed IFN-β transcription (>80%) but failed to inhibit IFN-β induction directed by STING or constitutively active IRF3-5D. Expression of NS2A and NS4B dose dependently inhibited the phosphorylation of TBK1 and IRF3, which suggests that they function at the level of TBK1 complex activation. NS2A and NS4B from DENV1/2/4, as well as the West Nile virus NS4B protein, commonly inhibited TBK1 phosphorylation and IFN-β induction. A comparative analysis of NS4A proteins across DENVs demonstrated that DENV1, but not DENV2 or DENV4, NS4A proteins uniquely inhibited TBK1. These findings indicate that DENVs contain conserved (NS2A/NS4B) and DENV1-specific (NS4A) mechanisms for inhibiting RIG-I/TBK1-directed IFN responses. Collectively, our results define DENV NS proteins that restrict IRF3 and IFN responses and thereby facilitate DENV replication and virulence. Unique DENV1-specific NS4A regulation of IFN induction has the potential to be a virulence determinant that contributes to the increased severity of DENV1 infections and the immunodominance of DENV1 responses during tetravalent DENV1-4 vaccination. Our findings demonstrate that NS2A and NS4B proteins from dengue virus serotypes 1, 2, and 4 are inhibitors of RIG-I/MDA5-directed interferon beta (IFN-β) induction and that they accomplish this by blocking TBK1 activation. We determined that IFN inhibition is functionally conserved across NS4B proteins from West Nile virus and DENV1, -2, and -4 viruses. In contrast, DENV1 uniquely encodes an extra IFN regulating protein, NS4A, that inhibits TBK1-directed IFN induction. DENV1 is associated with an increase in severe patient disease, and added IFN regulation by the DENV1 NS4A protein may contribute to increased DENV1 replication, immunodominance, and virulence. The regulation of IFN induction by nonstructural (NS) proteins suggests their potential roles in enhancing viral replication and spread and as potential protein targets for viral attenuation. DENV1-specific IFN regulation needs to be considered in vaccine strategies where enhanced DENV1 replication may interfere with DENV2-4 seroconversion within coadministered tetravalent DENV1-4 vaccines.