Febri Gunawan Sugiokto

Epstein-Barr virus (EBV) establishes lifelong persistent infection in over 90% of the world’s population. The virus persists as an episome in the host cells during latency and periodically reactivates through transcriptional activation of the immediate-early (IE) genes. While epigenetic regulation is central to maintaining viral latency, the host factors that enforce repression at these promoters remain incompletely defined. Here, we employed a novel Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-based en gineered DNA-binding molecule-mediated ch romatin i mmuno p recipitation coupled with m ass s pectrometry (enChIP-MS) approach to identify proteins associated with the promoter of EBV IE gene ZTA . This approach revealed an enrichment of multiple heterogeneous nuclear ribonucleoproteins and identified HNRNPA2B1 as a potential regulator of EBV ZTA gene expression. Functional analyses across multiple EBV+ cancer cell models demonstrated that HNRNPA2B1 acts as a restriction factor for EBV lytic reactivation. Depletion of HNRNPA2B1 led to increased expression of IE and downstream lytic genes, enhanced RNA polymerase II recruitment to the ZTA and RTA promoters, and elevated the proportion of cells entering the lytic cycle. Conversely, enforced expression of HNRNPA2B1 suppressed EBV lytic reactivation. Mechanistically, HNRNPA2B1 enhances repressive viral chromatin states by facilitating recruitment of the histone demethylase LSD1 to EBV IE gene promoters, thereby limiting the activating histone H3 lysine 4 trimethylation. Together, these findings identify HNRNPA2B1 as a key epigenetic regulator of EBV latency and link RNA-binding proteins to epigenetic control of viral reactivation. IMPORTANCE: This study identifies HNRNPA2B1 as a previously unrecognized host factor that promotes Epstein-Barr virus (EBV) latency through direct regulation of viral chromatin at immediate-early gene promoters. By integrating locus-specific chromatin proteomics with functional and mechanistic analyses, our work reveals how an RNA-binding protein HNRNPA2B1 recruits a histone-modifying enzyme to control EBV reactivation. These findings provide new insights into host-virus interactions that control EBV latency and reactivation and highlight the role of RNA-binding proteins in chromatin regulation that may be broadly relevant to other latent DNA viruses.

Epstein-Barr virus nuclear antigen 1 (EBNA1) is essential for the replication and stable maintenance of the viral episome in infected cells. Here, we identify the SUMO E3 ligase PIAS1 as a key regulator of EBNA1 through site-specific SUMOylation. Our chromatin immunoprecipitation sequencing analysis revealed that PIAS1 is enriched at the viral origin of plasmid replication ( oriP ), where it physically associates with EBNA1 and catalyzes its SUMOylation. Using mutational analysis, we identified three lysine residues on EBNA1 (K17, K75, and K241) as major SUMOylation sites. Disruption of these sites compromises EBNA1’s ability to restrict Epstein-Barr virus (EBV) lytic replication. In addition, both PIAS1 depletion and the disruption of EBNA1 SUMOylation lead to reduced retention of EBNA1- OriP -based EBV mini-replicon, indicating the importance of EBNA1 SUMOylation in viral episome maintenance. Together, these results uncover a conserved post-translational mechanism by which PIAS1-mediated SUMOylation modulates EBNA1 function and EBV episome maintenance, suggesting a broader role for SUMOylation in viral latency, lytic replication, and persistence. IMPORTANCE: Epstein-Barr virus (EBV) persists in infected cells by maintaining its episome through the viral protein EBNA1. We discovered that PIAS1 SUMOylates EBNA1 at specific sites, a process essential for EBNA1 to retain the viral episome and suppress reactivation. When SUMOylation is disrupted, the EBV-based replicon becomes less stable, and EBV is more likely to reactivate. These findings reveal a new layer of host control of EBV latency and reactivation and highlight PIAS1-mediated EBNA1 SUMOylation as a key mechanism regulating viral persistence.

Synovial Sarcoma (SS) is driven by the SS18::SSX fusion oncoprotein and is ultimately refractory to therapeutic approaches. SS18::SSX alters ATP-dependent chromatin remodeling BAF (mammalian SWI/SNF) complexes, leading to the degradation of canonical (cBAF) complexes and amplified expression of SS18::SSX-containing non-canonical BAF (ncBAF or GBAF) complexes that drive an SS-specific transcription program and tumorigenesis. We demonstrate that SS18::SSX activates the SUMOylation program. The small molecule SUMOylation inhibitor, TAK-981, de-SUMOylates the cBAF/PBAF component, SMARCE1, stabilizing and restoring cBAF on chromatin, shifting SS models away from SS18::SSX-driven transcription. The result is DNA damage, cell death and tumor inhibition across both human and mouse SS tumor models. TAK-981 synergizes with cytotoxic chemotherapy through increased DNA damage, leading to tumor regression. Targeting the SUMOylation pathway in SS restores cBAF complexes and blocks the SS18::SSX transcriptome, identifying an unappreciated role of SUMOylation in SS and a subsequent therapeutic vulnerability.

Epstein-Barr virus nuclear antigen 1 (EBNA1) is essential for the replication and stable maintenance of the viral episome in infected cells. Here, we identify the SUMO E3 ligase PIAS1 as a key regulator of EBNA1 through site-specific SUMOylation. Our Chromatin-Immunoprecipitation Sequencing (ChIP-seq) analysis revealed that PIAS1 is enriched at the viral origin of plasmid replication ( oriP ), where it physically associates with EBNA1 and catalyzes its SUMOylation. Using mutational analysis, we identified three lysine residues on EBNA1 (K17, K75, and K241) as major SUMOylation sites. Disruption of these sites compromises EBNA1’s ability to restrict EBV lytic replication. In addition, both PIAS1 depletion and the disruption of EBNA1 SUMOylation lead to reduced retention of EBNA1- OriP -based EBV mini-replicon, indicating the importance of EBNA1 SUMOylation in viral episome maintenance. Together, these results uncover a conserved post-translational mechanism by which PIAS1-mediated SUMOylation modulates EBNA1 function and EBV episome maintenance and suggests a broader role for SUMOylation in viral latency, lytic replication and persistence. IMPORTANCE: Epstein-Barr virus (EBV) persists in infected cells by maintaining its episome through the viral protein EBNA1. We discovered that PIAS1 SUMOylates EBNA1 at specific sites, a process essential for EBNA1 to retain viral episome and suppress reactivation. When SUMOylation is disrupted, EBV-based replicon becomes less stable and EBV is more likely to reactivate. These findings reveal a new layer of host control of EBV latency and reactivation, and highlight PIAS1-mediated EBNA1 SUMOylation as a key mechanism regulating viral persistence.

As a ubiquitous human pathogen, the Epstein–Barr virus (EBV) has established lifelong persistent infection in about 95% of the adult population. The EBV infection is associated with approximately 200,000 human cancer cases and 140,000 deaths per year. The presence of EBV in tumor cells provides a unique advantage in targeting the viral genome (also known as episome), to develop anti-cancer therapeutics. In this review, we summarize current strategies targeting the viral episome in cancer cells. We also highlight emerging technologies, such as clustered regularly interspersed short palindromic repeat (CRISPR)-based gene editing or activation, which offer promising avenues for selective targeting of the EBV episome for anti-cancer therapy. We discuss the challenges, limitations, and future perspectives associated with these strategies, including potential off-target effects, anti-cancer efficacy and safety.

Epstein-Barr virus (EBV) is a ubiquitous human tumor virus that establishes lifelong, persistent infections in B cells. The presence of EBV in cancer cells presents an opportunity to target these cells by reactivating the virus from latency. In this study, we developed a novel approach for EBV reactivation termed clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9-mediated EBV reactivation (CMER) strategy. Using modified CRISPR-associated protein 9 (dCas9) fused with VP64, we designed 10 single guide RNAs (sgRNAs) to target and activate the EBV immediate-early gene promoter. In Akata Burkitt lymphoma cells, 9 out of 10 CMER sgRNAs effectively reactivated EBV. Among these, CMER sgRNA-5 triggered robust reactivation across various cell types, including lymphoma, gastric cancer, and nasopharyngeal carcinoma cells. Importantly, the combination of CMER and ganciclovir selectively eliminated EBV-positive cells, regardless of their cell origin. These findings indicate that targeted virus reactivation by CMER, combined with nucleoside analog therapy, holds promise for EBV-associated cancer treatment. IMPORTANCE: This study explores a novel strategy called clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9-mediated Epstein-Barr virus (EBV) reactivation (CMER) to reactivate the Epstein-Barr virus in cancer cells. EBV is associated with various cancers, and reactivating EBV from latency offers a potential therapeutic strategy. We utilized an enzymatically inactive CRISPR-associated protein 9 (dCas9) fused with VP64 and designed 10 single guide RNAs to target the EBV immediate-early gene promoter. Nine of these sgRNAs effectively reactivated EBV in Burkitt lymphoma cells, with CMER sgRNA-5 demonstrating strong reactivation across different cancer cell types. Combining CMER with ganciclovir selectively eliminated EBV-positive cells, showing promise for EBV-associated cancer treatment.

M6A RNA modification pathway plays important roles in diverse cellular processes and viral life cycle. Here, we investigated the relationship between PIAS1 and the m6A reader protein YTHDF2, which is involved in regulating RNA stability by binding to m6A-modified RNA. We found that both the N-terminal and C-terminal regions of YTHDF2 interact with PIAS1. We showed that PIAS1 promotes the SUMOylation of YTHDF2 at three specific lysine residues. We also demonstrated that PIAS1 enhances the anti-EBV activity of YTHDF2. We further revealed that PIAS1 mediates the SUMOylation of other YTHDF family members, namely, YTHDF1 and YTHDF3, to limit EBV replication. These findings together illuminate an important regulatory mechanism of YTHDF proteins in controlling viral RNA decay and EBV replication through PIAS1-mediated SUMOylation.

YTHDF2 is a member of the YTH protein family that binds to N6-methyladenosine (m6A)-modified RNA, regulating RNA stability and restricting viral replication, including Epstein-Barr virus (EBV). PIAS1 is an E3 SUMO ligase known as an EBV restriction factor, but its role in YTHDF2 SUMOylation remains unclear. In this study, we investigated the functional regulation of YTHDF2 by PIAS1. We found that PIAS1 promotes the SUMOylation of YTHDF2 at three specific lysine residues (K281, K571, and K572). Importantly, PIAS1 enhances the antiviral activity of YTHDF2, and SUMOylation-deficient YTHDF2 shows reduced anti-EBV activity. Mechanistically, YTHDF2 lacking SUMOylation exhibits reduced binding to EBV transcripts, leading to increased viral mRNA stability. Furthermore, PIAS1 mediates SUMOylation of YTHDF2’s paralogs, YTHDF1 and YTHDF3. These results collectively uncover a unique mechanism whereby YTHDF2 controls EBV replication through PIAS1-mediated SUMOylation, highlighting the significance of SUMOylation in regulating viral mRNA stability and EBV replication. Importance N6-methyladenosine (m6A) RNA modification pathway plays important roles in diverse cellular processes and viral life cycle. Here, we investigated the relationship between PIAS1 and the m6A reader protein YTHDF2, which is involved in regulating RNA stability by binding to m6A-modified RNA. We found that both the N-terminal and C-terminal regions of YTHDF2 interact with PIAS1. We showed that PIAS1 promotes the SUMOylation of YTHDF2 at three specific lysine residues. We also demonstrated that PIAS1 enhances the anti-Epstein-Barr virus (EBV) activity of YTHDF2. We further revealed that PIAS1 mediates the SUMOylation of other YTHDF family members, namely YTHDF1 and YTHDF3, due to their similarities with YTHDF2. These findings together illuminate an important regulatory mechanism of YTHDF2 in controlling viral RNA decay and EBV replication through PIAS1-mediated SUMOylation.

The discovery of an N ⁶ -methyladenosine (m ⁶ A) RNA modification pathway has fundamentally altered our understanding of the central dogma of molecular biology. This pathway is controlled by methyltransferases (writers), demethylases (erasers), and specific m ⁶ A binding proteins (readers).

Biofilm formation is one of the main causes of increased antibiotic resistance in Acinetobacter baumannii infections. Bacteriophages and their derivatives, such as tail proteins with depolymerase activity, have shown considerable potential as antibacterial or antivirulence agents against bacterial infections. Here, we gained insights into the activity of a capsular polysaccharide (CPS) depolymerase, derived from the tailspike protein (TSP) of φAB6 phage, to degrade A. baumannii biofilm in vitro. Recombinant TSP showed enzymatic activity and was able to significantly inhibit biofilm formation and degrade formed biofilms; as low as 0.78 ng, the inhibition zone can still be formed on the bacterial lawn. Additionally, TSP inhibited the colonization of A. baumannii on the surface of Foley catheter sections, indicating that it can be used to prevent the adhesion of A. baumannii to medical device surfaces. Transmission and scanning electron microscopy demonstrated membrane leakage of bacterial cells treated with TSP, resulting in cell death. The therapeutic effect of TSP in zebrafish was also evaluated and the results showed that the survival rate was significantly improved (80%) compared with that of the untreated control group (10%). Altogether, we show that TSP derived from φAB6 is expected to become a new antibiotic against multi-drug resistant A. baumannii and a biocontrol agent that prevents the formation of biofilms on medical devices.