B Cell-Specific Transcription Activator PAX5 Recruits p300 To Support EBNA1-Driven Transcription

ABSTRACT

The binding of Epstein-Barr Virus (EBV) nuclear antigen 1 (EBNA1) to the latent replication origin (oriP) triggers multiple downstream events to support virus-induced pathogenesis and tumorigenesis. Although EBV is widely recognized as a B-lymphotropic infectious agent, little is known about how tissue-specific factors are involved in the establishment of latency. Here, we showed that EBNA1 binds B cell activator PAX5 to promote EBNA1/oriP-dependent binding and transcription. In addition to showing that short hairpin RNA (shRNA)-mediated PAX5 knockdown substantially abrogated the above EBNA1-dependent functions, two mini-EBV reporter plasmids were used to perform nonlytic nano-luciferase (nLuc) activity and chromatin immunoprecipitation (ChIP) assays to show how EBNA1 cooperates with PAX5 to activate the transcription at the oriP site. The expression plasmids of two PAX5 mutants, V₂₆G (EBNA1 binding mutant) and P₈₀R (which remained EBNA1 associated), were used to assess their capability to restore the defects caused by PAX5 depletion in EBNA1/oriP-mediated binding, transcription, and maintenance of the genome copy number of the mini-EBV episome reporter in BJAB cells stably expressing EBNA1 or that of the EBV genome in EBV-infected BJAB cells. Since p300 is known to be associated with PAX5, we showed that the loss of function of the P₈₀R mutant in support of EBNA1/oriP-mediated transcription under PAX5 depletion conditions was linked to its defective binding to p300. ChIP-quantitative PCR (qPCR) confirmed that P₈₀R indeed failed to recruit p300 to the oriP DNA. Our discovery suggests that EBV has evolved an exquisite strategy to take advantage of tissue-specific factors to enable the establishment of viral latency.

IMPORTANCE Although B cells are known to be the primary target for EBV infection, there is limited knowledge regarding the mechanism that determines this preferable tissue tropism. An in-depth understanding of the potential link of tissue-specific factors with the viral genes and their functioning is key to deciphering how EBV induces persistent infection in the distinct types of host cells. In this study, a substantial protein-protein interaction mediated by the B cell-specific activator PAX5 and EBNA1 was identified as the general requirement for the binding of EBNA1 to the latent replication origin and for downstream events. Of importance, the EBNA1-PAX5-p300 network is directly linked to EBNA1-dependent transcription. These findings suggest that targeting the viral gene-associated tissue-specific factors may lead to new therapeutic strategies for EBV-associated malignancies.