Functional analysis of EBNA1 protein by using a recombinant EBV with gene replacement

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EBNA1 protein, a viral protein encoded by Epstein-Barr virus (EBY), is a multi-functional protein. It is well known to be essential for the replication and segregation of EBV episomes in latently-infected cells, and it also regulates viral gene expression as a transcriptional activator. We generated a recombinant EBV, designated HMG1-EBNA1 knock-in EBV, in which the EBNA1 gene is replaced with a HMGI-EBNA1 chimeilc gene. The HMG1-EBNA1 chimeric gene encodes an EBNA1 mutant with greatly-impaired transcriptional activity, while keeping the ability of EBNA1 to episomally riiaintain EBV based plasmids. Akata cells harboring wild-type EBV and HMG1-EBNA1 knock-in virus were established. The mixture of wild-type EBV and HMGI-EBNA1 knock-in EBV Was used to infect: EBV-negative Akata cells and cord blood lymphocytes. We found that HMG1-EBNA1 knock-in virus was maintained as episomes in Akata cells. By contrast, the analyses of BACmids that had been rescued from the established lymphoblastoid cell lines revealed that the HMG1-EBNA1. chimeric gene was frequently replaced with the wild-type EBNA1 gene, presumably due to the recombination between HMGI-EBNA1 knock-in EBY and wild-type EBV. These results strongly suggest that the regulation of viral gene expression by EBNA1 as a transactivator is critical, for EBV-induced B cell growth transformation.