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

['Ashley M. Campbell', 'Department Of Molecular Genetics', 'University Of Toronto', 'Toronto', 'Carlos F. De La Cruz-Herrera', 'Edyta Marcon', 'Donnelly Centre', 'Jack Greenblatt', 'Lori Frappier']

Date: 2022-02 

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.

Funding: This work was supported by grants 153014 to L.F. and FDN-154338 to J.G. from the Canadian Institute of Health Research (CIHR; https://cihr-irsc.gc.ca/e/193.html ), as well as by a CIHR postdoctoral fellowship (15817) to C.F.D.L.C-H. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

To better understand the mechanisms of action of BGLF2, we used affinity-purification coupled to mass-spectrometry (AP-MS) to profile the host protein interactions of BGLF2. This revealed a previously unknown interaction with the RISC, a miRNA-mediated regulatory complex, implicating BGLF2 as a miRNA regulator. Here, we show that BGLF2 interferes with let-7 miRNAs function, including its role in regulating the expression of SUMO proteins. As a result, BGLF2 increases free SUMO levels and thereby drives SUMOylation, providing a mechanism for the induction of SUMOylation previously observed for BGLF2.

BGLF2 is an EBV tegument protein that is expressed late in EBV lytic infection [ 29 ]. BGLF2 interacts with EBV tegument components BBLF1 and BKRF4 and is required for efficient production of virions [ 29 – 31 ]. In addition, BGLF2 was found to activate AP-1 promoters, leading to upregulation of the mitogen-activated protein kinase (MAPK) pathway and increased transcription of the EBV BZLF1 lytic switch gene [ 31 , 32 ]. BGLF2 has also been reported to inhibit interferon (IFN) signaling and NF-κB activity by preventing p65 phosphorylation [ 32 – 35 ]. In addition to these roles in inhibiting immune signaling, we had previously found that BGLF2 upregulated p21 levels and promoted G 1 /S cell cycle arrest through interactions with the cellular NIMA-related protein kinase (HNEK9) and GEM-interacting protein (GMIP) [ 27 ]. Finally, BGLF2 was found to globally upregulate the SUMOylation of cellular proteins by an unknown mechanism [ 23 ]. The modification of proteins by the addition of the Small Ubiquitin-like Modifiers (SUMOs) is an important mechanism of regulating both viral and cellular proteins that can affect their activity, localization, or stability, and can promote protein-protein interactions. SUMOylation of cellular proteins is often associated with antiviral and stress responses and viruses are known to encode proteins that induce SUMOylation or target SUMOylated cellular proteins for degradation [ 23 , 36 – 41 ].

Herpesviruses express a large number of proteins (~80–200) in lytic infection, many of which are known or likely to function to alter the cellular environment to promote infection. Accumulating studies indicate that even proteins with well characterized direct roles in viral infection have additional role in manipulating cellular processes [ 22 – 25 ], although many have yet to be investigated. We have previously screened a library of EBV proteins for cellular changes associated with EBV lytic infection, including disruption of PML nuclear bodies [ 26 ], induction of G 1 /S arrest [ 27 ], inhibition of the DNA damage response [ 28 ] and modulation of cellular SUMOylation [ 23 ]. The EBV protein BGLF2 was found to promote G 1 /S cell cycle arrest and to induce global SUMOylation of cellular proteins, indicating that it has some capacity to modulate cellular processes [ 23 , 27 ].

miRNAs are evolutionarily conserved across eukaryotes as small non-coding RNAs (21–25 nts) important for mRNA regulation [ 6 , 7 ]. Cellular- and EBV-derived pri-miRNAs are cleaved by DROSHA/DGCR8 to produce precursor miRNAs (pre-miRNAs) [ 8 , 9 ]. Pre-miRNAs are then exported from the nucleus to the cytoplasm by exportin 5 (XPO5; [ 10 – 12 ]). Cytoplasmic pre-miRNAs are further processed by Dicer to produce a mature ~22-nucleotide miRNA duplexes. Dicer associates with the transactivation-responsive RNA-binding protein (TRBP; also called TARBP2) which bridges the interaction between Dicer and Argonaute (Ago1, -2, -3, or -4) proteins [ 13 ]. Argonaute is the main component of the microribonucleoprotein complex called the miRNA-Induced Silencing Complex (RISC or miRISC), which also contains TNRC6 proteins. TNRC6A (also called GW182), TNRC6B, and TNRC6C are functionally redundant [ 14 – 17 ], and all can interact any Ago protein to form different versions of RISC. One strand of the miRNA duplex (guide strand), bound by Dicer, is loaded into an Ago protein, and RISC is recruited to targeted mRNAs by the partial complementarity of the miRNAs loaded into Ago [ 14 , 18 , 19 ]. Ago2 is the only human Argonaute protein with an endonuclease activity that cleaves mRNA when a small non-coding RNA has complete complementarity to the target mRNA [ 20 , 21 ]. miRNA-mediated degradation is also promoted by the TNRC6 component of RISC, which recruits the CCR4-NOT deadenylation complex and the decapping activator DDX6 [ 14 ]. TNRC6 also recruits eIF4E2 to inhibit translation initiation [ 14 , 18 ].

Epstein-Barr virus (EBV) is a γ-herpesvirus that infects ~90% of the global population and is a causative agent for several kinds of lymphomas, nasopharyngeal carcinoma, and ~10% of gastric carcinomas. Like all herpesviruses, EBV can alternate between latent and lytic forms of infection. Latent infection involves the expression of a small subset of EBV proteins, cell immortalization and maintenance of EBV episomes at a constant copy number. Lytic infection involves expression of an ordered cascade of ~80 proteins, EBV genome amplification and production of virions. In addition, EBV encodes 25 primary microRNAs (pri-miRNAs) which produce 44 mature miRNAs [ 1 , 2 ]. These miRNAs, which have been mainly studied in the context of EBV-induced cancers, regulate the functions of both cellular and viral mRNAs, at least in part to inhibit innate and adaptive immune responses (reviewed in [ 3 – 5 ]).

Results

BGLF2 interacts with components of RISC To better understand the cellular effects of BGLF2, we performed affinity purification-mass spectrometry (AP-MS) with BGLF2 to identify the cellular proteins it targets. To this end, FLAG-tagged BGLF2 was expressed in 293T cells, recovered on anti-FLAG resin, and co-purifying proteins were trypsinized and analyzed by liquid chromatography-tandem mass spectroscopy (LC-MS/MS). Peptide recovery (“spectral counts”) from two independent experiments with BGLF2 were compared to empty FLAG plasmid controls and to the average peptide recovery in the Contaminant Repository for Affinity Purification (CRAPome), a database of peptide recovery in over 400 AP-MS experiments [42] to identify nonspecific interactors. The top 30 interactors that were obtained in both experiments, but not in the empty plasmid control, and at levels significantly higher than the CRAPome, are shown in Table 1. Among the top interactors was the previously characterized interaction with HNEK9 that is associated with p21 induction by BGLF2 [27]. In addition, prominent interactions were discovered for proteins and complexes important for RNA regulation. These proteins include eIF4E2 (also called 4EHP), GIGYF1/2, and ZNF598, which form complexes that function in translation silencing and ribosome-associated quality control (RQC; [43–46]). Other prominent interacting RNA regulatory proteins include all of the trinucleotide repeat containing 6 (TNRC6) paralogs (A, B, C), as well as Argonaute (Ago) proteins 1 and 2, which are the main protein components of the miRNA-Induced Silencing Complex (RISC). RISC is typically found in processing-bodies (p-bodies), where it functions in miRNA regulation and, under stress conditions, also localizes with stalled mRNA-protein complexes in stress granules. In keeping with the BGLF2-RISC interaction, several proteins recovered with BGLF2 were components of stress granules and p-bodies. These BGLF2 interactions suggests a role for BGLF2 in miRNA regulation. PPT PowerPoint slide

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TIFF original image Download: Table 1. Affinity Purification-Mass Spectrometry Performed with BGLF2 Reveals an Interaction with RISC. https://doi.org/10.1371/journal.ppat.1010235.t001 To validate the interaction with RISC, we expressed BGLF2-FLAG in 293T cells, then performed FLAG immunoprecipitations (IP) and Western blotting for endogenous Ago2 and TNRC6A. Since RISC interacts with RNA, we performed these IPs with and without RNase A treatment to determine if the interaction is RNA-mediated. As shown in Fig 1A, BGLF2 recovered Ago2 and TNRC6A, and these interactions were not affected by RNase A treatment, indicating that they were not RNA-mediated. We then performed similar BGLF2-FLAG IPs (with RNase A treatment) in AGS gastric carcinoma cells, commonly used to study EBV lytic infection, and again confirmed recovery of Ago2 and TNRC6A (Fig 1B). We also confirmed the interaction of BGLF2-FLAG with Ago2 in EBV lytic infection in AGS-EBV cells (Fig 1C), which contain a Tet-inducible BZLF1 lytic switch protein enabling induction of lytic infection in all of the cells by the addition of doxycycline (Dox; AGS-EBV-Z cells described in [22]). Similarly, BGLF2-FLAG was confirmed to interact with Ago2 in Raji Burkitt’s lymphoma cells, reactivated to the lytic cycle by sodium butyrate/TPA treatment (Fig 1D). PPT PowerPoint slide

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TIFF original image Download: Fig 1. BGLF2 interacts with RISC. (A) 293T cells were transfected with pCMV3FC-BGLF2 (BGLF2) or pCMV3FC empty plasmid (EP) control and lysed 48 hours later. Cell lysates were treated with 20 μg/ml RNase A for 30 min or left untreated, as indicated, followed by IP using anti-FLAG resin. Western blotting was performed using antibodies against Ago2, TNRC6A, FLAG, or actin. 5% of the samples were run as inputs. Arrowhead indicates the Ago2 protein band. (B) AGS were transfected and processed as in A, except all samples include an RNase A treatment. 2.5% of the samples were run as inputs. (C) AGS-EBV-Z cells were transfected with pCMV3FC-BGLF2 or pCMV3FC then treated with Dox to induce EBV reactivation. Cells were lysed 24 hours later and FLAG IPs were performed followed by Western blotting for FLAG and Ago2. 2% of the lysates were run as inputs. A BZLF1 blot is also shown as proof of reactivation, where the upper band is the Tet-induced recombinant BZLF1 and the lower band is BZLF1 produced from the EBV. (D) Raji cells transfected with pCMV3FC-BGLF2 were treated with TPA and NaB to reactivate EBV, then lysed 24 hours later. The cell lysate was then divided in two and IPs performed with IgG- or anti-FLAG-conjugated beads. Western blotting was performed using antibodies against Ago2 and FLAG. 2% of the lysate was run as the input (IN) sample. (E) AGS-BGLF2 cells were treated with Dox for 48 hours to induce BGLF2-FLAG expression or left untreated, then processed as in B. (F) AGS-BGLF2 cells and AGS-EBV-Z cells with WT or BGLF2 KO EBV were treated with Dox for 0, 24 or 48 hours as indicated, then lysed in 9M urea and Western blotted using antibodies against BGLF2, FLAG and vinculin (loading control). The BGLF2 blot detects the FLAG-tagged BGLF2 in the AGS-BGLF2 cells (asterisk) as well as the endogenous BGLF2 produced from the EBV in AGS-EBV-Z cells (arrow). Note that there is a faint background band picked up by the BGLF2 antibody that is similar in size to FLAG-tagged BGLF2 and is seen in all the lanes. (G) AGS-BGLF2 cells were treated with Dox or left untreated as in B, then lysed in 9M urea and Western blotted for Ago2 and actin. Ago2 bands were quantified relative to actin and plotted relative to the uninduced (- Dox) sample. Average values and standard deviation are shown from three independent experiments, where * = 0.01 <P≤0.05. (H) Total RNA was extracted from AGS-BGLF2 cells treated with Dox for 48 hours or left untreated, and Ago2 transcripts were measured relative to actin transcripts and plotted as in D. (I) 293T cells were transfected with plasmids expressing FLAG-tagged BGLF2, KSHV ORF33, HSV-1 UL16, HCMV UL94, or pCMV3FC empty plasmid (EP). Forty-eight hours later, FLAG IPs were performed followed by Western blotting using antibodies against FLAG and Ago2. 2.5% of samples were run as inputs. https://doi.org/10.1371/journal.ppat.1010235.g001 Transfection can result in high levels of protein expression due to the large number of plasmids taken up. Therefore, we also wanted to confirm the BGLF2-RISC interaction (and subsequent effects) with lower BGLF2 expression levels. To this end, we generated an AGS cell line containing a Tet-inducible BGLF2-FLAG (AGS-BGLF2) and showed that FLAG-IPs performed after BGLF2 induction by Dox treatment recovered Ago2 and TNRC6A (Fig 1E). We confirmed that the expression level of Dox-induced BGLF2-FLAG was physiological by comparing to BGLF2 levels in EBV infection. Dox-induced BGLF2 levels were found to be somewhat lower than that seen 24–48 hours after lytic reactivation of AGS-EBV-Z cells (Fig 1F), confirming that the BGLF2-FLAG is not overexpressed. In addition, we noticed that induction of BGLF2 expression resulted in increased Ago2 (and sometimes TNRC6A) protein levels (see Fig 1E, input samples), which might be a consequence of the BGLF2 interaction stabilizing this protein. To further explore this possibility, we quantified Ago2 proteins and Ago2 transcripts from multiple experiments, with and without induced expression of BGLF2-FLAG. We found that BGLF2 expression resulted in a consistent increase in Ago2 proteins without affecting Ago2 mRNA abundance (Fig 1G and 1H), supporting the hypothesis that BGLF2 affects Ago2 at the protein level (possibly due to stabilization by BGLF2 binding) and not the transcriptional level.

RISC interaction is not conserved in BGLF2 homologues in other herpesviruses All herpesviruses contain a homologue of BGLF2, which appear to have conserved functions in virion generation and infectivity [29,51–54]. Whether these proteins have similar roles in modulating cellular processes is unclear. We investigated whether BGLF2 homologues from three human herpesvirus, Kaposi’s sarcoma-associated herpesvirus (KSHV) ORF33, herpes simplex virus 1 (HSV-1) UL16, and human cytomegalovirus (HCMV) UL94, also interacted with RISC. To this end, FLAG-IPs were performed with FLAG-tagged KSHV ORF33, HSV-1 UL16, and HCMV UL94 expressed in 293T cells, followed by Western blotting for endogenous Ago2 (Fig 1I). Unlike BGLF2, none of these BGLF2 homologous were found to interact with Ago2, indicating that this interaction is a unique feature of EBV BGLF2.

BGLF2 induces the loss of p-bodies We also investigated the association of BGLF2 with p-bodies, which are present in all cells, using Dcp1a as a marker. Although we did not see any obvious enrichment of BGLF2 at p-bodies in HeLa cells, we noticed that the number of these bodies was reduced in the presence of BGLF2 (Fig 5C and 5D). This effect was verified by counting the number of p-bodies per cell in cells expressing or not expressing BGLF2, showing a 4-fold reduction in p-bodies in the presence of BGLF2. A similar effect on p-bodies was seen when BGLF2 was expressed in HONE-1 cells (S1B Fig). We also investigated this effect in the AGS cells with Tet-inducible BGLF2, which have low levels of BGLF2 expression. Dox-induction of BGLF2 for 24 hours led to 3-fold reduction in p-bodies (Fig 5E). We conclude that BGLF2 consistently induces the loss of p-bodies, which would be expected to disrupt miRNA function, and that formation of stress granules is not a requirement for this p-body disruption.

BGLF2 interferes with let-7 miRNA function The ability of BGLF2 to interact with RISC and disrupt p-bodies suggests that BGLF2 might affect the ability of RISC to mediate miRNA function. We tested this hypothesis by examining effects of BGLF2 on the well-studied let-7 family of miRNAs. To this end, we used a Renilla luciferase reporter containing two tandem let-7a binding sites (2xLet7a). We first confirmed that this reporter was regulated by let-7 miRNAs by transfecting it with and without a let-7 sponge construct that ursurps let-7 miRNAs, thereby reducing targeting of the luciferase reporter [57]. As expected, this sponge resulted in an increase in the Renilla luciferase activity (Fig 6A). The Renilla luciferase activity of the 2xLet7a reporter was then compared with and without BGLF2 expression and normalized to effects on a firefly luciferase construct lacking let-7a sites (an internal control). BGLF2 consistently increased Renilla luciferase expression to a similar extent as the let-7 sponge (Fig 6A) and dependent on the presence of the let-7 sites. This suggested that BGLF2 interferes with let-7a miRNA function. PPT PowerPoint slide

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TIFF original image Download: Fig 6. BGLF2 inhibits let-7a function in reporter assays. (A) HONE-1 were co-transfected with a Renilla luciferase reporter with or without let-7 binding sites (+ or– 2xLet7a), a firefly luciferase control plasmid and either mSCV-based let-7 sponge plasmids, mSCV empty plasmid, pCMV3FC-BGLF2 or pCMV3FC empty plasmid as indicated. Renilla and firefly luciferase were quantified and Renilla values were normalized to firefly values. (B) The same experiment as in A except that a firefly luciferase reporter containing the Dicer 3’UTR (+ Dicer 3’UTR) or no UTR (- Dicer 3’UTR) was used and compared to a Renilla luciferase negative control plasmid. (C) AGS-BGLF2 cells were treated with Dox (+) for 48 hours to induce BGLF2-FLAG expression or left untreated (-), then lysed in 9M urea and Western blotted for Dicer, FLAG and actin (Top). Dicer bands were quantified relative to actin and induced samples (+Dox) were plotted relative to the uninduced (-Dox) sample (Bottom). Average values and standard deviation are shown from three independent experiments. (D) HONE-1 cells were co-transfected with a firefly luciferase reporter containing SUMO1 3’UTR, SUMO2 3’UTR or no 3’UTR (pGL4.10; as indicted), a Renilla luciferase control plasmid, and either pCMV3FC-BGLF2 or pCMV3FC (empty plasmid). Firefly luciferase activity was normalized to Renilla luciferase activity for each sample. For each luciferase assay, average values from 3 independent experiments (with standard deviation) are shown relative to the value for the empty plasmid. * = 0.01<P≤0.05, ** = 0.001<P≤0.01, *** = 0.0001<P≤0.001, **** = P≤0.0001. https://doi.org/10.1371/journal.ppat.1010235.g006 We further examined the effect of BGLF2 on let-7 miRNAs by using a firefly luciferase reporter with and without a 3’UTR from the Dicer gene, which contains multiple let-7 miRNA binding sites and has been shown to be regulated by let-7 miRNAs [58,59]. We confirmed regulation of this reporter by let-7 miRNAs using the let-7 sponge and then tested the effect of co-expression with BGLF2 (using a Renilla luciferase reporter lacking the Dicer 3’UTR as an internal control; Fig 6B). Consistent with the 2xLet7a reporter results, BGLF2 significantly increased firefly luciferase levels in the reporter containing the Dicer 3’UTR, but not in reporters lacking this sequence, providing further evidence that BGLF2 disrupts let-7 miRNAs activity. To determine whether this up-regulation of Dicer by BGLF2 also occurred in endogenous Dicer, we compared Dicer protein levels in AGS cells with Tet-inducible BGLF2, before and after BGLF2 expression. Consistent with the reporter results, Dicer levels increased after BGLF2 expression (Fig 6C).

BGLF2 increases SUMO proteins through miRNA effects BGLF2 was previously shown to globally increase SUMOylation of cellular proteins in multiple cell lines [23]. One mechanism by which SUMOylation can be globally increased is by increasing the level of free SUMO [60,61]. Interestingly, SUMO1, SUMO2, and SUMO3 transcripts all contain multiple let-7 recognition sites and have been shown to be regulated by let-7 miRNAs [61]. Since BGLF2 can interfere with let-7 function, we investigated the possibility that this interference results in increased free SUMO levels which may drive SUMOylation. We first confirmed that BGLF2 induced SUMOylation in the Tet-inducible AGS-BGLF2 cells by preparing whole cell lysates with and without Dox treatment and Western blotting using antibodies against SUMO1 (Fig 7A) or SUMO2/3 (Fig 7B). Consistent with previous studies [23], BGLF2 expression led to an increase in SUMOylated cellular proteins, which was more obvious for SUMO2/3 than for SUMO1. As a control to confirm the requirement of BGLF2, parental AGS cells lacking the BGLF2 cassette were also treated with Dox and processed in the same manner, but as expected, this treatment did not lead to increased SUMOylation. The lysates from the AGS-BGLF2 cells were then reanalysed (on a higher percentage gel) for free SUMO. BGLF2 was found to consistently increase the levels of free SUMO1 (Fig 7C) and SUMO2/3 (Fig 7D), with a larger effect on SUMO2/3. This mirrors the effect on SUMOylation and suggests that the increase in SUMOylation caused by BGLF2 may be due to increased SUMO protein levels. PPT PowerPoint slide

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TIFF original image Download: Fig 7. BGLF2 induces SUMOylation and increases free SUMO post-transcriptionally. (A) and (B) AGS or AGS-BGLF2 cells were treated with Dox for 48 hours or left untreated. Whole cell lysates were then analysed by Western blotting using antibodies against FLAG, actin and either SUMO1 (A) or SUMO2/3 (B). The bracket marks the position of cellular proteins with increased SUMOylation with BGLF2. (C) and (D) Lysates from A and B were analysed for free SUMO by Western blotting using a higher percentage gel and antibodies against FLAG, actin and either SUMO1 (C) or SUMO2/3 (D) Free SUMO bands were quantified from 3 independent experiments and average values for the induced (+Dox) samples were plotted relative to the uninduced (-Dox) samples which were set to one. * = 0.01<P≤0.05, **** = P≤0.0001. (E) Samples generated as in A and B were analysed for SUMO1, SUMO2, or SUMO3 transcripts by qRT-PCR. Transcripts were normalized to actin transcript levels and average values for induced (+Dox) samples from 3 independent experiments are shown relative to uninduced (-Dox) samples which were set to one. https://doi.org/10.1371/journal.ppat.1010235.g007 We then investigated whether the increase in free SUMO could be due to BGLF2 effects on miRNA. We first examined whether BGLF2 expression affected the level of SUMO transcripts by collecting total RNA from the same cells used for Western blotting and performing RT-qPCR for SUMO1, SUMO2, SUMO3, and actin transcripts, the later of which was used as a normalization control. Unlike the effect of BGLF2 on SUMO proteins, SUMO transcripts were unaffected by BGLF2 (Fig 7E), indicating that the regulation of SUMO levels occurred post-transcriptionally. We next investigated whether BGLF2 regulates SUMO transcripts through their 3’UTRs, which are known to be regulated by multiple let-7 miRNAs [61]. To this end, we generated firefly luciferase reporters containing fragments of the SUMO1 or SUMO2 3’UTRs and examined effects of co-expression with BGLF2, using a Renilla luciferase reporter lacking 3’-UTRs as a normalization control. BGLF2 was found to consistently increase firefly luciferase reporter activity (Fig 6D), suggesting that BGLF2 regulates SUMO transcripts through their 3’UTRs. Since these sequences contain let-7 recognition motifs, the results are consistent with BGLF2 increasing SUMO expression by interfering with let-7 miRNAs.

BGLF2 inhibits let-7 interaction with Ago2 We next further investigated the mechanism by which BGLF2 inhibits let-7 function. Our model was that BGLF2 sequesters RISC, making it less available to interact with cellular miRNAs. We tested this model by determining how BGLF2 expression affects the association of let-7 miRNAs with Ago2. To this end, Ago2-IPs were performed in AGS-BGLF2 cells with and without induction of BGLF2 (compared to an IgG control) and recovered miRNAs were quantified by RT-qPCR, using primers specific to let-7a-5p and let-7g-5p miRNAs, both of which regulate SUMO1, SUMO2 and SUMO3 3’UTRs [61]. We then compared the amount of let-7a-5p and let-7g-5p recovered with Ago2 as compared to the IgG control, in the presence and absence of BGLF2. These values were also normalized to Ago2 recovery, as determined by Western blotting of the same samples (S2A Fig). As shown in Fig 8A, the degree of recovery of both let-7 miRNAs with Ago2 decreased about 3-fold in the presence of BGLF2. We also checked the total level of these let-7 miRNAs in the lysates prior to Ago2-IP, to determine if the decreased recovery with Ago2 could be due to a decrease in the cellular levels of these miRNAs. However, the cellular levels of let-7a-5p and let-7g-5p were not decreased by BGLF2 but were somewhat increased (although not statistically significant; Fig 8B). The results support a model in which BGLF2 inhibits let-7 miRNA incorporation into Ago2. PPT PowerPoint slide

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TIFF original image Download: Fig 8. BGLF2 inhibits let-7 miRNA incorporation into Ago2. (A) Ago2 was immunoprecipitated from AGS-BGLF2 cells treated (+Dox) or untreated (-Dox) with Dox using anti-Ago2 antibody or IgG negative control antibody. Recovered let-7a-5p and let-7g-5p miRNAs were quantified by RT-qPCR, normalized to spike in UniSp6 synthetic RNA control and calculated as fold enrichment in Ago2-IP over IgG control IP. Values were also normalized to recovery of Ago2 protein in the IP. Average values for +Dox samples from 3 independent experiments are shown relative to -Dox samples, which were set to 1. (B) let-7a-5p and let-7g-5p miRNAs from cell lysates in A (prior to immunoprecipitations) were quantified by RT-qPCR, normalized to U6 RNA and plotted as in A. (C) AGS-EBV-Z cell pools containing a non-targeting (WT) or BGLF2-targeting (BGLF2 KO) CRISPR/Cas9 were reactivated by Dox treatment for 48 hours followed by Western blotting using antibodies against the EBV proteins BGLF2, BZLF1, vcap18, and BGLF4, as well as FLAG (to detect FLAG-tagged Cas9), Ago2, and actin. Two BZLF1 bands are detected from the endogenous virus and Tet-inducible expression cassette (asterisk). (D) Ago2 immunoprecipitations were performed as in A using lysates generated as in C. Let-7a-5p and let-7g-5p miRNAs were quantified in lysates (input) and Ago2 IP samples and normalized to spike in UniSp6 synthetic RNA. Let-7a-5p and let-7g-5p values in Ago2-IP samples were also normalized to Ago2 protein recovered in each IP. Average values for 3 independent experiments are shown as percentage of input let-7a-5p and let-7g-5p miRNA. (E) Ago2 immunoprecipitations were performed as in D. and transcripts for SUMO1, SUMO2 and SUMO3 were quantified by RT-qPCR and normalized to Ago2 protein abundance in each IP. Average values for 4 independent experiments are shown as percentage of SUMO transcripts in the input. * = 0.01<P≤0.05, ** = 0.001<P≤0.01. https://doi.org/10.1371/journal.ppat.1010235.g008 We further investigated this model in the context of EBV lytic infection by comparing WT EBV to EBV with a BGLF2 knockout (KO). The BGLF2 KO was generated by lentivirus-delivered CRISPR/Cas9 targeting in the context of AGS-EBV-Z cells, which are AGS-EBV cells with a Tet-inducible BZLF1 cassette that enables efficient reactivation to the lytic cycle by Dox treatment [22]. These cells were compared to AGS-EBV-Z cells transduced with a negative control lentivirus expressing CRISPR/Cas9 but lacking a guide RNA. In both cases cell pools were used, and not individual clones, to ensure that a particular integration site of the lentivirus was not responsible for the effect. Reactivation of WT and BGLF2 KO virus with Dox confirmed that the KO virus produced little BGLF2 (Figs 1F and 8C), while other viral lytic proteins remained unaffected (Fig 8C). Comparison of the lysates also showed that Ago2 levels were unaffected by the BGLF2 KO. Ago2-IPs were performed with these lysates and recovered amounts of let-7a-5p and let-7g-5p miRNAs were determined as above and expressed as a percentage of the let-7a-5p and let-7g-5p miRNAs in the lysates. As shown in Fig 8D, the BGLF2 KO resulted in a consistent increase in let-7a-5p and let-7g-5p associated with Ago2. We further investigated this effect by asking if this increase in let-7 miRNAs incorporated into Ago2 resulted in the expected increase in associated SUMO transcripts. Although there was a lot of variability in recovery of SUMO transcripts, the recovery of SUMO2 and SUMO3 transcripts with Ago2 was significantly increased ~3-fold in the BGLF2 KO lysates (as compared to WT EBV; Fig 8E). This is consistent with the greater effect of BGLF2 on SUMO2/3 than SUMO1. These results further support a model in which BGLF2 inhibits let-7 miRNA function by interfering with the incorporation of these miRNAs into Ago2 complexes.

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[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1010235

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