Epstein-Barr Virus BGLF2 commandeers RISC to interfere with cellular miRNA function.
Epstein-Barr Virus BGLF2 commandeers RISC to interfere with cellular miRNA function.
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DOI：
10.1371/journal.ppat.1010235
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发表时间：
2022-01
期刊：
影响因子：
6.7
通讯作者：
Frappier L
中科院分区：
文献类型：
--
作者：
Campbell AM;De La Cruz-Herrera CF;Marcon E;Greenblatt J;Frappier L
EB病毒(Epstein-Barr Virus，EBV)BGLF2蛋白是一种对细胞环境有多种影响的被膜蛋白，包括诱导细胞蛋白的SUMO化。利用亲和纯化与质谱联用的方法，我们鉴定了miRNA诱导的沉默复合体(RISC)是BGLF2的顶级相互作用因子，这是miRNA功能所必需的。我们证实了BGLF2与RISC的Ago2和TNRC6组分在多个细胞系中的相互作用，以及它们在也包含应激颗粒标记G3BP1的细胞质中的共同定位。此外，BGLF2的表达导致多种细胞类型的加工体丢失，表明RISC在mRNA调控中的功能被破坏。与这一观察结果一致，BGLF2中断了Ago2与多个miRNAs的联系。以let-7miRNAs为模型，我们验证了BGLF2干扰RISC在miRNA介导的mRNA沉默中的作用的假设。使用含有let-7a调控位点的3‘UTrs的多个报告构建体，我们发现BGLF2抑制依赖于这些3’UTrs的let-7a miRNA活性，包括已知受let-7miRNAs调控的相扑转录本的miRNA活性。与这些结果一致，我们发现BGLF2增加了细胞中非结合相扑蛋白的水平，而不影响相扑转录本的水平。这种游离相扑的增加是已知的驱动SUMO化的原因，并解释了BGLF2在诱导SUMO化中的作用。BGLF2的表达抑制了Ago2蛋白中let-7miRNAs的装载，相反，与WT EBV感染相比，缺乏BGLF2的EBV裂解感染导致了let-7a和SUMO转录本与Ago2的相互作用增加。因此，我们确定了BGLF2作为miRNA调节因子的一个新角色，并表明这种活性的一个结果是相扑转录本的失调，导致游离相扑蛋白水平和SUMO化水平增加。爱泼斯坦-巴尔病毒(EBV)感染世界上大多数人，终身存在，并与多种癌症有关。为了进行有效的裂解感染，EBV必须操纵多条细胞途径。BGLF2是一种EBV裂解蛋白，已知可以调节几个细胞过程，包括增加细胞蛋白的小泛素样修饰物(SUMO)的修饰，这一过程被称为SUMO化。在这里，我们首次证明了BGLF2与miRNA功能所需的细胞复合体(RISC)相互作用，并通过隔离该复合体干扰一些细胞miRNAs的功能。这种效应的后果之一是相扑蛋白的表达增加，这是由于抑制了通常下调其表达的miRNAs。由此产生的SUMO蛋白的增加驱动SUMO化，为先前报道的BGLF2诱导的细胞蛋白SUMO化提供了一种机制。此外，BGLF2作为miRNA调节器的发现表明，这种EBV蛋白可以通过干扰正常调节它们的细胞miRNAs来控制许多细胞途径。
The Epstein-Barr virus (EBV) BGLF2 protein is a tegument protein with multiple effects on the cellular environment, including induction of SUMOylation of cellular proteins. Using affinity-purification coupled to mass-spectrometry, we identified the miRNA-Induced Silencing Complex (RISC), essential for miRNA function, as a top interactor of BGLF2. We confirmed BGLF2 interaction with the Ago2 and TNRC6 components of RISC in multiple cell lines and their co-localization in cytoplasmic bodies that also contain the stress granule marker G3BP1. In addition, BGLF2 expression led to the loss of processing bodies in multiple cell types, suggesting disruption of RISC function in mRNA regulation. Consistent with this observation, BGLF2 disrupted Ago2 association with multiple miRNAs. Using let-7 miRNAs as a model, we tested the hypothesis that BGLF2 interfered with the function of RISC in miRNA-mediated mRNA silencing. Using multiple reporter constructs with 3’UTRs containing let-7a regulated sites, we showed that BGLF2 inhibited let-7a miRNA activity dependent on these 3’UTRs, including those from SUMO transcripts which are known to be regulated by let-7 miRNAs. In keeping with these results, we showed that BGLF2 increased the cellular level of unconjugated SUMO proteins without affecting the level of SUMO transcripts. Such an increase in free SUMO is known to drive SUMOylation and would account for the effect of BGLF2 in inducing SUMOylation. We further showed that BGLF2 expression inhibited the loading of let-7 miRNAs into Ago2 proteins, and conversely, that lytic infection with EBV lacking BGLF2 resulted in increased interaction of let-7a and SUMO transcripts with Ago2, relative to WT EBV infection. Therefore, we have identified a novel role for BGLF2 as a miRNA regulator and shown that one outcome of this activity is the dysregulation of SUMO transcripts that leads to increased levels of free SUMO proteins and SUMOylation. Epstein-Barr virus (EBV) infects most people worldwide, persists for life and is associated with several kinds of cancer. In order to undergo efficient lytic infection, EBV must manipulate multiple cellular pathways. BGLF2 is an EBV lytic protein known to modulate several cellular processes including increasing the modification of cellular proteins with the Small Ubiquitin-Like Modifier (SUMO), a process referred to as SUMOylation. Here we show for the first time that BGLF2 interacts with a cellular complex (RISC) required for miRNA function and interferes with the function of some cellular miRNAs by sequestering this complex. One of the consequences of this effect is the increased expression of SUMO proteins, due to inhibition of the miRNAs that normally downregulate their expression. The resulting increase in SUMO proteins drives SUMOylation, providing a mechanism for the previously reported BGLF2-induced SUMOylation of cellular proteins. In addition, the discovery of BGLF2 as a miRNA regulator suggests that this EBV protein can control many cellular pathways by interfering with cellular miRNAs that normally regulate them.
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