Epstein-Barr Virus BGLF2 commandeers RISC to interfere with cellular miRNA function.

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. Author summary: 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. [ABSTRACT FROM AUTHOR]
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