Epstein‐Barr virus‐encoded EBNA‐5 forms trimolecular protein complexes with MDM2 and p53 and inhibits the transactivating function of p53

Abstract

We report that MDM2, a negative regulator of p53, can bind to EBNA-5. Using GST pull-down assay, immunoprecipitation, surface plasmon resonance and immunostaining of lymphoblastoid cells, we found that trimolecular complexes are formed between EBNA-5, MDM2 and p53, where MDM2 serves as a bridge. The EBNA-5 binding to MDM2 counteracted destabilizing effect of the latter on the p53. In ubiquitination and degradation assays in vitro, EBNA-5 inhibited p53 polyubiquitination (but not monoubiquitination) in a concentration-dependent manner. This resembles the effect of p14ARF on p53. Moreover, EBNA-5 was found to inhibit the degradation of p53 in vitro. High levels of p53 expression were maintained in LCLs. The binding of EBNA-5 to MDM2 also could impair the functional activity of p53. The p53-dependent genes P21 and VDR were not induced in EBV-infected, in contrast to mitogen-activated cells. This may explain the tolerance of established LCLs to high levels of p53 without undergoing apoptosis.

Epstein-Barr Virus Immortalization of Human B-Cells Leads to Stabilization of Hypoxia-Induced Factor 1 Alpha, Congruent with the Warburg Effect

“…The MCF7 breast carcinoma cell line and LCLs (described in [2], [6]) were cultured at 37°C in Iscove's medium containing 10% fetal bovine serum and appropriate antibiotics. Periodic staining with Hoechst 33258 (Sigma-Aldrich, St. Louis, MO) confirmed the absence of mycoplasma.…”

“…EBNA-1 is essential for the maintenance of viral episomes and regulates viral promoter usage; EBNA-2 activates the viral LMP-1 promoter and a number of cellular genes, such as CD23, C-MYC, cyclin D1 and HES . EBNA-5 enhances EBNA-2-dependent transcription and inactivates p53 by forming a trimolecular complex with MDM2 and p53 [2]. EBNA-6 is involved in chromatin remodeling and the regulation of the RB pathway (for a review, see [3]).…”

Why is F19Ap53 unable to bind MDM2? Simulations suggest crack propagation modulates binding

“…It is possible that organisms such as viruses exploit this mechanism and degrade p53 very efficiently. 42 It will be interesting to see if the replacement of Y104 in MDM2 by R (R103 is the equivalent residue in MDM4) will attenuate the signal transmission.…”

Inhibition of Germinal Centre Apoptotic Programmes by Epstein-Barr Virus

“…There is evidence that EBNA-3C can act as a deubiquitinase which leads to stabilisation of the p53-negative regulator MDM2. The levels of p53 in EBNA-3C over-expressing cells are consequently reduced through p53 degradation [86], although in LCLs, this effect may be diminished by binding of EBNA-LP (EBNA-5) to MDM2 which reportedly blocks its ability to target p53 for degradation [87]. The formation of EBNA-LP/MDM2/p53 complexes has been proposed to block p53-mediated transcription (of p21) and provide an explanation as to how rapidly proliferating LCLs tolerate high levels of wild-type p53 without succumbing to p53-induced cell cycle arrest [87].…”

“…The levels of p53 in EBNA-3C over-expressing cells are consequently reduced through p53 degradation [86], although in LCLs, this effect may be diminished by binding of EBNA-LP (EBNA-5) to MDM2 which reportedly blocks its ability to target p53 for degradation [87]. The formation of EBNA-LP/MDM2/p53 complexes has been proposed to block p53-mediated transcription (of p21) and provide an explanation as to how rapidly proliferating LCLs tolerate high levels of wild-type p53 without succumbing to p53-induced cell cycle arrest [87]. During the lytic cycle, BZLF-1 mediates p53 degradation independently of MDM2 function thereby blocking the potential for p53-mediated gene transcription during productive viral infection [88].…”