Institute for Animal Health, Ash Road, Pirbright, Surrey GU24 0NF, UK.
School of Biomedical Sciences, University of Ulster, Cromore Road, Coleraine BT52 1SA, Northern Ireland.
J Gen Virol. 2013 Feb;94(Pt 2):298-307. doi: 10.1099/vir.0.048322-0. Epub 2012 Nov 7.
The ovarian tumour (OTU) domain of the nairovirus L protein has been shown to remove ubiquitin and interferon-stimulated gene 15 protein (ISG15) from host cell proteins, which is expected to have multiple effects on cell signalling pathways. We have confirmed that the OTU domain from the L protein of the apathogenic nairovirus Dugbe virus has deubiquitinating and deISGylating activity and shown that, when expressed in cells, it is highly effective at blocking the TNF-α/NF-κB and interferon/JAK/STAT signalling pathways even at low doses. Point mutations of the catalytic site of the OTU [C40A, H151A and a double mutant] both abolished the ability of the OTU domain to deubiquitinate and deISGylate proteins and greatly reduced its effect on cell signalling pathways, confirming that it is this enzymic activity that is responsible for blocking the two signalling pathways. Expression of the inactive mutants at high levels could still block signalling, suggesting that the viral OTU can still bind to its substrate even when mutated at its catalytic site. The nairovirus L protein is a very large protein that is normally confined to the cytoplasm, where the virus replicates. When the OTU domain was prevented from entering the nucleus by expressing it as part of the N-terminal 205 kDa of the viral L protein, it continued to block type I interferon signalling, but no longer blocked the TNF-α-induced activation of NF-κB.
该卵巢肿瘤（OTU）领域的纳罗病毒 L 蛋白已被证明去除泛素和干扰素刺激基因 15 蛋白（ISG15）从宿主细胞蛋白，预计将有多个影响细胞信号通路。我们已经证实，从致病性纳罗病毒 Dugbe 病毒的 L 蛋白的 OTU 域具有去泛素化和去 ISGylating 活性，并表明，当在细胞中表达时，它是非常有效的在阻断 TNF-α / NF-κB 和干扰素/JAK/STAT 信号通路，即使在低剂量。OTU 的催化位点的点突变[C40A、H151A 和双突变]都消除了 OTU 域去泛素化和去 ISGylating 蛋白的能力，并大大降低了其对细胞信号通路的影响，证实了这种酶活性是负责阻断两条信号通路。高表达失活突变体仍能阻断信号转导，表明病毒 OTU 即使在其催化位点发生突变时，仍能与其底物结合。纳罗病毒 L 蛋白是一种非常大的蛋白，通常局限于细胞质，病毒在细胞质中复制。当 OTU 域通过表达为病毒 L 蛋白的 N 端 205 kDa 的一部分而被阻止进入核内时，它继续阻断 I 型干扰素信号，但不再阻断 TNF-α 诱导的 NF-κB 激活。
