Ebola virus VP35 protein binds double-stranded RNA and inhibits alpha/beta interferon production induced by RIG-I signaling
Ebola virus VP35 protein binds double-stranded RNA and inhibits alpha/beta interferon production induced by RIG-I signaling
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DOI：
10.1128/jvi.02199-05
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发表时间：
2006-06-01
影响因子：
5.4
通讯作者：
Basler, Christopher F.
中科院分区：
文献类型：
--
作者：
Cardenas, Washington B.;Loo, Yueh-Ming;Basler, Christopher F.
埃博拉病毒（EBOV）VP 35蛋白阻断病毒诱导的干扰素调节因子3（IRF-3）的磷酸化和活化，所述干扰素调节因子3是诱导α/β干扰素（IFN-α/β）表达的关键转录因子。然而，这种阻塞发生的机制仍然没有完全确定。我们现在提供的证据表明，VP 35具有双链RNA（dsRNA）结合活性。具体地，VP 35结合到聚（rI）-聚（rC）包被的琼脂糖凝胶珠，但不结合到对照珠。相反，发现两个VP 35点突变体R312 A和K309 A的dsRNA结合活性大大受损。竞争分析表明，VP 35与poly（rI）- poly（rC）、poly（rA）- poly（rU）或来自EBOV序列的体外转录的dsRNA特异性相互作用，而不与单链RNA（ssRNA）或双链DNA相互作用。然后，我们筛选了野生型和突变型VP 35，以确定它们靶向激活IRF-3的信号通路的不同组分的能力。这些实验表明，VP 35阻断了由RIG-I过表达诱导的IRF-3的活化，RIG-I是一种细胞解旋酶，最近参与了病毒或dsRNA对IRF-3的活化。有趣的是，在这些测定中，dsRNA结合受损的VP 35突变体具有降低但可测量的IFN拮抗剂活性。此外，发现野生型和dsRNA结合突变体VP 35具有相同的能力来抑制由IPS-1（最近鉴定的RIG-I下游的信号传导分子）的过表达或由IRF-3激酶IKKF-1和TBK-1的过表达诱导的IFN-β启动子的活化。这些数据支持dsRNA结合可能有助于VP 35 IFN拮抗剂功能的假设。然而，在IRF-3激酶附近的点处，很可能还存在其他抑制机制。
The Ebola virus (EBOV) VP35 protein blocks the virus-induced phosphorylation and activation of interferon regulatory factor 3 (IRF-3), a transcription factor critical for the induction of alpha/beta interferon (IFN-alpha/beta) expression. However, the mechanism(s) by which this blockage occurs remains incompletely defined. We now provide evidence that VP35 possesses double-stranded RNA (dsRNA)-binding activity. Specifically, VP35 bound to poly(rI) - poly(rC)-coated Sepharose beads but not control beads. In contrast, two VP35 point mutants, R312A and K309A, were found to be greatly impaired in their dsRNA-binding activity. Competition assays showed that VP35 interacted specifically with poly(rI) - poly(rC), poly(rA) - poly(rU), or in vitro-transcribed dsRNAs derived from EBOV sequences, and not with single-stranded RNAs (ssRNAs) or double-stranded DNA. We then screened wild-type and mutant VP35s for their ability to target different components of the signaling pathways that activate IRF-3. These experiments indicate that VP35 blocks activation of IRF-3 induced by overexpression of RIG-I, a cellular helicase recently implicated in the activation of IRF-3 by either virus or dsRNA. Interestingly, the VP35 mutants impaired for dsRNA binding have a decreased but measurable IFN antagonist activity in these assays. Additionally, wild-type and dsRNA-binding-mutant VP35s were found to have equivalent abilities to inhibit activation of the IFN-beta promoter induced by overexpression of IPS-1, a recently identified signaling molecule downstream of RIG-I, or by overexpression of the IRF-3 kinases IKKF epsilon and TBK-1. These data support the hypothesis that dsRNA binding may contribute to VP35 IFN antagonist function. However, additional mechanisms of inhibition, at a point proximal to the IRF-3 kinases, most likely also exist.