The A20 Deubiquitinase Activity Negatively Regulates LMP1 Activation of IRF7
The A20 Deubiquitinase Activity Negatively Regulates LMP1 Activation of IRF7
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DOI：
10.1128/jvi.00364-10
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发表时间：
2010-06-01
影响因子：
5.4
通讯作者：
Pagano, Joseph S.
中科院分区：
文献类型：
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作者：
Ning, Shunbin;Pagano, Joseph S.
A20具有去泛素化酶（DU B）和泛素E3连接酶活性，这是终止导致NF-κ B活化的Toll样受体（TLR）信号传导和阻断肿瘤坏死因子（TNF）诱导的细胞毒性和细胞凋亡所需的。A20由EB病毒（EBV）癌蛋白LMP 1诱导。然而，其双重泛素编辑活动尚未在EBV感染或IRF 7应答的背景下进行研究。A20和IRF 7都具有致癌特性。我们最近发现LMP 1通过K63连接的泛素化激活IRF 7，这需要RIP 1和TRAF 6，但这种泛素化事件如何调节尚未研究。在这里，我们表明，A20负调控IRF 7的转录活性LMP 1诱导。A20 C端锌指基序的缺失或突变对IRF 7活性的抑制没有影响，而DUB缺陷的截短或点突变消除了A20抑制IRF 7的能力。相应地，A20 N-末端DUB结构域，而不是C-末端E3连接酶结构域，与IRF 7物理相互作用。A20的瞬时表达减少了体内IRF 7的K63连接的泛素化，但是用纯化的成分进行的体外去泛素化测定显示IRF 7不作为A20 DUB活性的底物。此外，A20与IRF 7在潜伏性EBV感染的3型Raji细胞中内源性相互作用，其中A20和IRF 7的表达由相当水平的内源性LMP 1组成性诱导。通过表达A20短发夹RNA（shRNA）载体在Raji细胞中敲低内源性A20增加了内源性IRF 7活性和泛素化，以及IRF 7的靶标LMP 1的蛋白水平。因此，A20负调节LMP 1刺激的IRF 7泛素化和EBV潜伏期的活性，其DUB活性对于该功能是必不可少的。最后，我们讨论了IRF对EB病毒潜伏期的调节和作用。
A20 possesses both deubiquitinase (DUB) and ubiquitin E3 ligase activities that are required for termination of Toll-like receptor (TLR) signaling leading to NF-kappa B activation and for blockage of tumor necrosis factor (TNF)-induced cytotoxicity and apoptosis. A20 is induced by the Epstein-Barr virus (EBV) oncoprotein LMP1. However, its dual ubiquitin-editing activities have not been investigated in the context of either EBV infection or IRF7 responses. Both A20 and IRF7 have oncogenic properties. We have recently shown that LMP1 activates IRF7 through K63-linked ubiquitination which requires RIP1 and TRAF6, but how this ubiquitination event is regulated has not been studied. Here, we show that A20 negatively regulates IRF7 transcriptional activity induced by LMP1. Deletion or mutation of A20 C-terminal zinc finger motifs had no effect on the inhibition of IRF7 activity, whereas DUB-deficient truncation or point mutation ablated the ability of A20 to inhibit IRF7. Correspondingly, the A20 N-terminal DUB domain, but not the C-terminal E3 ligase domain, interacts physically with IRF7. Transient expression of A20 reduced K63-linked ubiquitination of IRF7 in vivo, but an in vitro deubiquitination assay with purified constituents shows that IRF7 did not act as a substrate for A20 DUB activity. Moreover, A20 interacts with IRF7 endogenously in latently EBV-infected type 3 Raji cells, in which expression of both A20 and IRF7 is constitutively induced by the considerable level of endogenous LMP1. Knockdown of endogenous A20 in Raji cells by expression of A20 short hairpin RNA (shRNA) vectors increases endogenous IRF7 activity and ubiquitination, as well as the protein level of LMP1, a target of IRF7. Thus, A20 negatively regulates LMP1-stimulated IRF7 ubiquitination and activity in EBV latency, and its DUB activity is indispensable for this function. Finally, we discussed the regulation and function of IRFs in EBV latency.