# A Prion‐Like Domain in EBV EBNA1 Promotes Phase Separation and Enables SRRM1 Splicing

## TL;DR

This study shows that the EBV protein EBNA1 acts like a prion, forming protein aggregates that help tumors grow by altering gene splicing.

## Contribution

The first evidence that EBNA1 is a prion-like protein, linking its phase separation to tumor progression.

## Key findings

- EBNA1 contains a prion-like domain that drives liquid–liquid phase separation.
- EBNA1 interacts with SRSF1 to regulate SRRM1 splicing and promote tumor growth.
- Deleting the prion-like domain inhibits tumor cell proliferation and protein aggregation.

## Abstract

Epstein‐Barr virus (EBV) nuclear antigen 1 (EBNA1) is necessary to maintain stability of EBV episomes, EBV replication, and causes host genomic instability and promotes tumor cells survival. Recent studies have shown that viruses utilize liquid–liquid phase separation (LLPS) within host cells to form sub‐cellular compartments known as “virus factories”. Prion‐like domains (PrLDs), which resemble structural domains of low complexity, are shown to drive LLPS in vivo. In the current study, a PrLD is identified in EBNA1 and aggregation of EBNA1 proteins is observed in EBV‐positive tumors. EBNA1 condensate interacting molecules are examined and are found that EBNA1 interacts with the splicing factor SRSF1 to regulate alternative splicing of SRRM1 and promote tumor progression. Deleting the EBNA1 PrLD results in defects in protein aggregation, LLPS, alternative splicing regulation, and nasopharyngeal carcinoma cells proliferation. Targeting the PrLD of EBNA1 inhibits the formation of protein aggregation, promotes alternative splicing of SRRM1, and inhibits the progression of nasopharyngeal carcinoma. Here, we report for the first time that EBNA1, a protein from the human oncogenic virus EBV, is a prion‐like protein, combining algorithm prediction and experimental validation. That implies a possible molecular pathogenic mechanism of EBNA1 in neurodegenerative diseases.

This study discoveries that EBV EBNA1 behaves as a prion‐like protein, verified using cell‐based assays and the Saccharomyces cerevisiae Sup35p prion identification system. The prion‐like domain of EBNA1 drives liquid–liquid phase separation. EBNA1 interacts with the splicing factor SRSF1 to regulate the expression of the SRRM1 splicing isoforms, thereby promoting EBV‐associated tumor development.