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Small molecule perturbation of the CAND1-Cullin1-ubiquitin cycle stabilizes p53 and triggers Epstein-Barr virus reactivation.

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.

SUBMITTER: Tikhmyanova N

PROVIDER: S-EPMC5531659 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Small molecule perturbation of the CAND1-Cullin1-ubiquitin cycle stabilizes p53 and triggers Epstein-Barr virus reactivation.

Tikhmyanova Nadezhda N Tutton Steve S Martin Kayla A KA Lu Fang F Kossenkov Andrew V AV Paparoidamis Nicholas N Kenney Shannon S Salvino Joseph M JM Lieberman Paul M PM

PLoS pathogens 20170717 7

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 g ...[more]

PMID: 28715492

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Project description:Epstein-Barr virus (EBV) reactivation involves the ordered induction of approximately 90 viral genes that participate in the generation of infectious virions. Using strand-specific RNA-seq to assess the EBV transcriptome during reactivation, we found extensive bidirectional transcription extending across nearly the entire genome. In contrast, only 4% of the EBV genome is currently bidirectionally annotated. Most of the newly identified transcribed regions show little evidence of coding potential, supporting noncoding roles for most of these RNAs. Based on previous cellular long noncoding RNA size calculations, we estimate that there are likely hundreds more EBV genes expressed during reactivation than was previously known. Limited 5' and 3' rapid amplification of cDNA ends (RACE) experiments and findings of novel splicing events by RNA-seq suggest that the complexity of the viral genome during reactivation may be even greater. Further analysis of antisense transcripts at some of the EBV latency gene loci showed that they are "late" genes, they are nuclear, and they tend to localize in areas of the nucleus where others find newly synthesized viral genomes. This raises the possibility that these transcripts perform functions such as new genome processing, stabilization, organization, etc. The finding of a significantly more complex EBV transcriptome during reactivation changes our view of the viral production process from one that is facilitated and regulated almost entirely by previously identified viral proteins to a process that also involves the contribution of a wide array of virus encoded noncoding RNAs. Epstein-Barr virus (EBV) is a herpesvirus that infects the majority of the world's population, in rare cases causing serious disease such as lymphoma and gastric carcinoma. Using strand-specific RNA-seq, we have studied viral gene expression during EBV reactivation and have discovered hundreds more viral transcripts than were previously known. The finding of alternative splicing and the prevalence of overlapping transcripts indicate additional complexity. Most newly identified transcribed regions do not encode proteins but instead likely function as noncoding RNA molecules which could participate in regulating gene expression, gene splicing or even activities such as viral genome processing. These findings broaden the scope of what we need to consider to understand the viral manufacturing process. As more detailed studies are undertaken they will likely change the way we view this process as a whole.

Project description:Epstein-Barr virus (EBV) maintains a life-long infection due to the ability to alternate between latent and lytic modes of replication. Lytic reactivation starts with derepression of the Zp promoter controlling BZLF1 gene expression, which binds and is activated by the c-Jun transcriptional activator. Here, we identified the cellular Arkadia-like 1 (ARKL1) protein as a negative regulator of Zp and EBV reactivation. Silencing of ARKL1 in the context of EBV-positive gastric carcinoma (AGS) cells, nasopharyngeal carcinoma (NPC43) cells, and B (M81) cells led to increased lytic protein expression, whereas overexpression inhibited BZLF1 expression. Similar effects of ARKL1 modulation were seen on BZLF1 transcripts as well as on Zp activity in Zp reporter assays, showing that ARKL1 repressed Zp. Proteomic profiling of ARKL1-host interactions identified c-Jun as an ARKL1 interactor, and reporter assays for Jun transcriptional activity showed that ARKL1 inhibited Jun activity. The ARKL1-Jun interaction required ARKL1 sequences that we previously showed mediated binding to the CK2 kinase regulatory subunit CK2?, suggesting that CK2? might mediate the ARKL1-Jun interaction. This model was supported by the findings that silencing of CK2?, but not the CK2? catalytic subunit, abrogated the ARKL1-Jun interaction and phenocopied ARKL1 silencing in promoting EBV reactivation. Additionally, ARKL1 was associated with Zp in reporter assays and this was increased by additional CK2?. Together, the data indicate that ARKL1 is a negative regulator of Zp and EBV reactivation that acts by inhibiting Jun activity through a CK2?-mediated interaction.IMPORTANCE Epstein-Barr virus (EBV) maintains a life-long infection due to the ability to alternate between latent and lytic modes of replication and is associated with several types of cancer. We have identified a cellular protein (ARKL1) that acts to repress the reactivation of EBV from the latent to the lytic cycle. We show that ARKL1 acts to repress transcription of the EBV lytic switch protein by inhibiting the activity of the cellular transcription factor c-Jun. This not only provides a new mechanism of regulating EBV reactivation but also identifies a novel cellular function of ARKL1 as an inhibitor of Jun-mediated transcription.

Dataset Information

Small molecule perturbation of the CAND1-Cullin1-ubiquitin cycle stabilizes p53 and triggers Epstein-Barr virus reactivation.

Publications

Small molecule perturbation of the CAND1-Cullin1-ubiquitin cycle stabilizes p53 and triggers Epstein-Barr virus reactivation.

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