Abstract
The chemical probe C60 efficiently triggers Epstein-Barr Virus (EBV) reactivation from latency through an unknown mechanism. Here, we identify the Cullin exchange factor CAND1 as a biochemical target of C60. We also identified CAND1 in an shRNA library screen for EBV lytic reactivation. Gene expression profiling revealed that C60 activates the p53 pathway and protein analysis revealed a strong stabilization and S15 phosphorylation of p53. C60 reduced Cullin1 association with CAND1 and led to a global accumulation of ubiquitylated substrates. C60 also stabilized the EBV immediate early protein ZTA through a Cullin-CAND1-interaction motif in the ZTA transcription activation domain. We propose that C60 perturbs the normal interaction and function of CAND1 with Cullins to promote the stabilization of substrates like ZTA and p53, leading to EBV reactivation from latency. Understanding the mechanism of action of C60 may provide new approaches for treatment of EBV associated tumors, as well as new tools to stabilize p53.
Highlights
We identified several molecules with a common tetrahydrocarboline core that stimulated ZTA and EA-D expression in multiple different cell lines latently infected with Epstein-Barr Virus (EBV), including those derived from Burkitt lymphoma, nasopharyngeal carcinoma, and lymphoblastoid cell lines immortalized in vitro [28]
Strategies to reactivate EBV from latency are of current clinical interest for precision treatment of cancers latently infected with EBV [5, 7, 12, 33]
By genetic shRNA library screening, we demonstrated that Cullin-associated and neddylation-dissociated 1 (CAND1) acts as a restriction factor for EBV reactivation from latency (Fig 2)
Summary
Epstein-Barr Virus (EBV) is a human gammaherpesvirus that establishes latent infection in Blymphocytes in over 90% of adults worldwide [1]. While the pathways that activate the BZLF1 promoter have been investigated extensively, relatively less is known about the mechanisms that regulate ZTA protein function and stability, and whether this can be modulated to control the reactivation process. ZTA can interact with p53 through its b-ZIP domain [21] and can target p53 for degradation through a mechanism that is dependent on the Zta-Cullin interaction [20] In this context, ZTA has been shown to function as an adaptor in the Elongin B/C-Cul2/5-SOCS (ECS) ubiquitin ligase complex. BPLF1 promotes viral DNA replication through inhibiting multiple Cullin-Ring-ubiquitin ligase (CRL), including those that ubiquitylate cellular S phase licensing factor CDT1 [24]. We provide evidence that C60 works through a novel mechanism that stabilizes p53 and ZTA by altering CAND1-dependent Cullin-ubiquitin ligase substrate exchange and selection
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