Proteasomal inhibition triggers viral oncoprotein degradation via autophagy-lysosomal pathway.

Chandrima Gain,Arijit Ghosh,Shaoni Bhattacharjee,Erle S Robertson,Samaresh Malik,Benu Brata Das,Abhik Saha

doi:10.1371/journal.ppat.1008105

Abstract

Epstein-Barr virus (EBV) nuclear oncoprotein EBNA3C is essential for B-cell transformation and development of several B-cell lymphomas particularly those are generated in an immuno-compromised background. EBNA3C recruits ubiquitin-proteasome machinery for deregulating multiple cellular oncoproteins and tumor suppressor proteins. Although EBNA3C is found to be ubiquitinated at its N-terminal region and interacts with 20S proteasome, the viral protein is surprisingly stable in growing B-lymphocytes. EBNA3C can also circumvent autophagy-lysosomal mediated protein degradation and subsequent antigen presentation for T-cell recognition. Recently, we have shown that EBNA3C enhances autophagy, which serve as a prerequisite for B-cell survival particularly under growth deprivation conditions. We now demonstrate that proteasomal inhibition by MG132 induces EBNA3C degradation both in EBV transformed B-lymphocytes and ectopic-expression systems. Interestingly, MG132 treatment promotes degradation of two EBNA3 family oncoproteins–EBNA3A and EBNA3C, but not the viral tumor suppressor protein EBNA3B. EBNA3C degradation induced by proteasomal inhibition is partially blocked when autophagy-lysosomal pathway is inhibited. In response to proteasomal inhibition, EBNA3C is predominantly K63-linked polyubiquitinated, colocalized with the autophagy-lysosomal fraction in the cytoplasm and participated within p62-LC3B complex, which facilitates autophagy-mediated degradation. We further show that the degradation signal is present at the first 50 residues of the N-terminal region of EBNA3C. Proteasomal inhibition reduces the colony formation ability of this important viral oncoprotein, induces apoptotic cell death and increases transcriptional activation of both latent and lytic gene expression which further promotes viral reactivation from EBV transformed B-lymphocytes. Altogether, this study offers rationale to use proteasome inhibitors as potential therapeutic strategy against multiple EBV associated B-cell lymphomas, where EBNA3C is expressed.

Highlights

Epstein-Barr virus (EBV) is a large DNA virus that belongs to the gammaherpesvirus subfamily and persistently infects majority of the worldwide human population [1,2,3]
EBV encoded EBNA3 family of nuclear latent antigens comprising of EBNA3A, EBNA3B, and EBNA3C are unique to immunoblastic lymphomas
We demonstrate that proteasomal inhibition leads to degradation of oncogenic EBNA3A and EBNA3C proteins, whereas EBNA3B remains unaffected

Summary

Introduction

Epstein-Barr virus (EBV) is a large DNA virus that belongs to the gammaherpesvirus subfamily and persistently infects majority of the worldwide human population [1,2,3]. EBV is detected as the etiologic factor of majority of infectious mononucleosis (IM), a lymphoproliferative disorder attributable to a striking increase in CD8+ T-cell activation and subsequent expansion of B-lymphocytes upon viral infection [5]. This clinical manifestation of primary EBV infection indicates its competence to embark on latent infection in B-lymphocytes and to impel B-cell proliferation in vivo. In vitro EBV transforms nascent B-lymphocytes into continuously proliferating lymphoblastoid cell lines (LCLs) with phenotypic resemblance of activated B-blasts [6,7] These LCLs express similar gene sets that have been detected in EBV associated lymphoid malignancies in humans, such as immunoblasic Bcell lymphomas [1,2]. Five viral antigens including EBNA2, EBNA3A, EBNA3C, EBNA-LP and LMP1 act in concert to transform naïve B-

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