Identification of a New Phosphorylated Host Interactor of the Epstein–Barr Virus (EBV) Kinase BGLF4 Suggests Key Points for EBV-Specific Antiviral Drug Targeting

Epstein-Barr virus (EBV) is a human pathogenic and oncogenic herpesvirus, with worldwide importance, at times associated with serious to life-threatening symptoms, especially in immunocompromised hosts. The available preventive options against EBV disease are limited to medically elaborate and cost-intensive measures of cell-based immunotherapy. The development of novel options of anti-EBV drug targeting is currently a matter of intense international efforts. A putative target of the antiviral therapy approach is the EBV-encoded protein kinase BGLF4, which fulfills a multifaceted role in productive viral replication. So far, viral BGLF4 interactor proteins and phosphorylated substrates have occasionally been reported, but in particular cellular interactors await further characterization concerning both, their relevance for BGLF4 functionality and their accessibility to antiviral drugs. In this study, we have analyzed host cell-BGLF4 interaction, BGLF4 kinase properties, and BGLF4-directed small molecules. The main results are as follows: <b>(i)</b> a mass spectrometry-based interactomic study was performed with EBV-producing Akata-BX1 cells, thereby identifying the human pyruvate dehydrogenase (PDH) as a relevant BGLF4 interactor; <b>(ii)</b> BGLF4-PDH interaction was confirmed by protein coimmunoprecipitation, subcellular cofractionation, and confocal imaging; <b>(iii)</b> the BGLF4-mediated phosphorylation of PDH was demonstrated by an in vitro kinase assay (IVKA); <b>(iv)</b> a reduction in PDH phosphorylation was shown for selected kinase inhibitors, which also exerted BGLF4-directed inhibitory potential in a quantitative qSox-IVKA, and <b>(v)</b> these hit compounds showed anti-EBV activity in lytically induced P3HR-1 cells using qPCR measurement, as well as PDH-inhibitory activity using standardized PDH assays. These data lead to an improved understanding of EBV-host interaction that may open novel anti-EBV preventive opportunities. Combined, the findings point to PDH as a new cellular interactor of the EBV kinase BGLF4. Also, notably, the data on pharmacological intervention with kinase activity or substrate phosphorylation may possibly provide as yet untapped options of antiviral drug targeting.