Reactivation of Epstein-Barr Virus by HIF-1α Requires p53

Abstract

<jats:p>We previously reported that the cellular transcription factor, HIF-1α, binds a hypoxia-response element (HRE) located within the promoter of Epstein-Barr virus's (EBV's) latent-lytic switch <jats:italic>BZLF1</jats:italic> gene, Zp, inducing viral reactivation. Here, EBV-infected cell lines derived from gastric cancers and Burkitt lymphomas were incubated with HIF-1α-stabilizing drugs: iron chelator [Desferal® (DFO,)]; neddylation inhibitor [Pevonedistat® (MLN-4924)]; and prolyl hydroxylase inhibitor [Roxadustat® (FG-4592)]. DFO and MLN-4924, but not FG-4592, induced accumulation of both lytic EBV proteins and phosphorylated p53 in cell lines that contain a wild-type p53 gene. FG-4592 also failed to activate transcription from Zp in a reporter assay despite inducing accumulation of HIF-1α and transcription from another HRE-containing promoter. Unexpectedly, DFO failed to induce EBV reactivation in cell lines that express mutant or no p53 or when p53 expression was knocked down with shRNAs. Likewise, HIF-1α failed to activate transcription from Zp when p53 was knocked out by CRISPR-Cas9. Importantly, DFO induced binding of p53 as well as HIF-1α to Zp in ChIP assays, but only when the HRE was present. Nutlin-3, a drug known to induce accumulation of phosphorylated p53, synergized with DFO and MLN-4924 in inducing EBV reactivation. Conversely, KU-55933, a drug that inhibits ataxia telangiectasia mutated thereby preventing p53 phosphorylation, inhibited DFO-induced EBV reactivation. Lastly, activation of Zp transcription by DFO and MLN-4924 mapped to its HRE. Thus, we conclude that induction of <jats:italic>BZLF1</jats:italic> gene expression by HIF-1α requires phosphorylated, wild-type p53 as a co-activator, with HIF-1α binding recruiting p53 to Zp.</jats:p> <jats:p><jats:bold>IMPORTANCE</jats:bold> EBV, a human herpesvirus, is latently present in most nasopharyngeal carcinomas, Burkitt lymphomas, and some gastric cancers. To develop a lytic-induction therapy for treating patients with EBV-associated cancers, we need a way to efficiently reactivate EBV into lytic replication. EBV's <jats:italic>BZLF1</jats:italic> gene product, Zta, usually controls this reactivation switch. We previously showed that HIF-1α binds the <jats:italic>BZLF1</jats:italic> gene promoter, inducing Zta synthesis, and HIF-1α-stabilizing drugs can induce EBV reactivation. Here, we determined which EBV-positive cell lines are reactivated by classes of HIF-1α-stabilizing drugs. We found, unexpectedly, that HIF-1α-stabilizing drugs only induce reactivation when they also induce accumulation of phosphorylated, wild-type p53. Fortunately, p53 phosphorylation can also be provided by drugs such as Nutlin-3, leading to synergistic reactivation of EBV. These findings indicate that some HIF-1α-stabilizing drugs may be helpful as part of a lytic-induction therapy for treating patients with EBV-positive malignancies that contain wild-type p53.</jats:p>

Age-dependent effects of the recombinant spike protein/SARS-CoV-2 on the M–CSF– and IL-34-differentiated macrophages in vitro

“…Similarly, p53 and its downstream gene p21 are upregulated in fibrotic lung disease and are known to be activated in response to DNA damage [ 16 , 17 ]. Furthermore, p53 intervenes in viral activation and has been identified in the bronchial lavage fluid of COVID-19 patients [ 18 , 19 ].…”

ΔNp63α promotes Epstein-Barr virus latency in undifferentiated epithelial cells

“…Furthermore, p53 and ΔNp63ɑ bind to some of the same promoters, and ΔNp63ɑ can repress p53 activation at these promoters [ 51 , 55 , 69 ]. Since p53 was previously reported to be a positive regulator of EBV lytic reactivation and binds to the Z promoter via the Sp1 transcription factor and HIF-1α [ 70 – 73 ], we determined if ΔNp63ɑ’s repression of EBV lytic reactivation is due to competition with p53. For these experiments, ΔNp63ɑ expression was inhibited by siRNAs in a NOKs-Akata cell line that had CRISPR-Cas9 mediated knockout of p53 ( Figs 5A and S6 ).…”

“… A) The CRISPR/CAS9 technique was used to knock out p53 expression in NOKs-Akata cells, as described previously [ 73 ], and expression of the p53 protein and tubulin loading control was examined by immunoblot to confirm knock-out of p53 expression. B) NOKs-Akata cells in which the p53 gene was knocked-out were transfected with siRNAs against ΔNp63ɑ or a control sequence.…”

“…Here we confirmed that cis-platinum treatment of EBV-infected epithelial cells does, indeed, result in loss of intact ΔNp63ɑ expression and showed that restoration of ΔNp63ɑ expression in cis-platinum treated cells reduces the amount of lytic EBV reactivation ( Fig 12A and 12B ). Thus, the use of chemotherapy drugs such as cis-platinum that can not only induce DNA damage and p53/ATM activation (known activators of lytic EBV reactivation [ 71 , 73 ]), but can simultaneously degrade ΔNp63ɑ, is predicted to be the most effective approach for achieving efficient lytic induction therapy in EBV-positive NPC tumors.…”

Isoforms of the p53 Family and Gastric Cancer: A Ménage à Trois for an Unfinished Affair

“…These mechanisms may explain an increase in the p53 protein level despite a reduction of TP53 mRNA in some EBV-infected GC [ 311 ]. In addition, the functional interaction between the EBV and p53 is reciprocal, as p53/ATM [ 312 ] and the hypoxia inducible factor HIF1A [ 313 ] participate in the reactivation of the viral lytic phase through their binding to a hypoxia response element (HRE) located within the promoter of the EBV latent-lytic switch BZLF1 gene Zp.…”