EBNA3C facilitates RASSF1A downregulation through ubiquitin-mediated degradation and promoter hypermethylation to drive B-cell proliferation.

EBV latent antigen 3C (EBNA3C) is essential for EBV-induced primary B-cell transformation. Infection by EBV induces hypermethylation of a number of tumor suppressor genes, which contributes to the development of human cancers. The Ras association domain family isoform 1A (RASSF1A) is a cellular tumor suppressor, which regulates a broad range of cellular functions, including apoptosis, cell-cycle arrest, mitotic arrest, and migration. However, the expression of RASSF1A is lost in many human cancers by epigenetic silencing. In the present study, we showed that EBNA3C promoted B-cell transformation by specifically suppressing the expression of RASSF1A. EBNA3C directly interacted with RASSF1A and induced RASSF1A degradation via the ubiquitin-proteasome-dependent pathway. SCFSkp2, an E3-ubiquitin ligase, was recruited by EBNA3C to enhance RASSF1A degradation. Moreover, EBNA3C decreased the transcriptional activity of RASSF1A promoter by enhancing its methylation through EBNA3C-mediated modulation of DNMTs expression. EBNA3C also inhibited RASSF1A-mediated cell apoptosis, disrupted RASSF1A-mediated microtubule and chromosomal stability, and promoted cell proliferation by upregulating Cyclin D1 and Cyclin E expression. Our data provides new details, which sheds light on additional mechanisms by which EBNA3C can induce B-cell transformation. This will also facilitate the development of novel therapeutic approaches through targeting of the RASSF1A pathway. Author summary Epstein-Barr virus (EBV) which is associated with multiple lymphoid and epithelial malignancies was the first recognized oncogenic virus in humans. EBNA3C, an essential latent antigen encoded by EBV interacts with numerous host transcription factors and plays an important role in the transformation of primary B-cells. RASSF1A, a tumor suppressor plays a vital role in regulating apoptosis, cell-cycle arrest, and mitotic arrest and is implicated in the development of a number of different cancers. We now demonstrate that EBNA3C can physically interact with RASSF1A and induce RASSF1A degradation through the ubiquitin-proteasome-dependent pathway. Further, the E3 ubiquitin ligase SCFSkp2 was recruited by EBNA3C to mediate RASSF1A degradation. Moreover, RASSF1A mRNA expression was also suppressed by EBNA3C. EBNA3C repressed the transcriptional activity of the RASSF1A promoter through induction of its methylation by enhancing DNMT3a expression. EBNA3C regulation of RASSF1A promoted cell proliferation, inhibited RASSF1A-mediated apoptosis and disrupted RASSF1A-mediated microtubule and chromosomal stability. Overall, our results add to our understanding of the many strategies employed by EBNA3C to induce B-cell transformation, which will contribute to new therapeutics for interventions targeting EBV association malignancies.