Proteomic screening identifies HNRNPA2B1 as a novel factor that controls Epstein-Barr virus reactivation through chromatin regulation

Epstein-Barr virus (EBV) establishes lifelong persistent infection in most human populations. The virus persists as an episome in the host cells during latency and periodically reactivates through transcriptional activation of the immediate-early 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 engineered DNA-binding molecule-mediated chromatin immunoprecipitation coupled with mass spectrometry (enChIP-MS) approach to identify proteins associated with the promoter of EBV immediate-early (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+ 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 KDM1A to EBV IE gene promoters, thereby limiting the activating histone H3K4 trimethylation. Together, these findings identify HNRNPA2B1 as a key epigenetic regulator of EBV latency and link RNA-binding proteins to chromatin control of viral reactivation.