Tacrolimus inhibits CVB3-targeted regulation of TFEB by PPP3/calcineurin

Recent evidence indicates that Coxsackievirus B3 (CVB3) infection, a common cause of viral myocarditis, triggers the nuclear translocation of transcription factor EB (TFEB) through a mechanism dependent on the serine/threonine phosphatase Protein Phosphatase 3 (PPP3)/calcineurin, independent of its cleavage. Tacrolimus (TAC), a calcineurin inhibitor widely used in immunosuppressive therapy for cardiovascular conditions such as myocarditis and post-transplant vasculopathy, may modulate TFEB activity in this context. This study investigated the effect of TAC on TFEB regulation during CVB3 infection in HeLa cells. Our results demonstrate that TAC significantly suppresses both the nuclear accumulation and transcriptional activity of TFEB. Conversely, knockdown of Protein Phosphatase 3 Catalytic Subunit (PPP3C) enhances TFEB protein expression and its nuclear localization, indicating that TAC calcineurin-dependent mechanism beyond simple enzymatic inhibition. Moreover, TAC similarly inhibits the nuclear expression and transcriptional activity of both Δ60-TFEB (a cleavage fragment lacking the first 60 amino acids) and TFEBQS60LP (cleavage-resistant mutant). Knockdown of PPP3C leads to increased nuclear distribution of these TFEB variants, confirming that TAC targets PPP3/calcineurin to regulate TFEB and its modified forms. These findings suggest that TAC interferes with the CVB3-induced activation of TFEB, thereby influencing cellular autophagy and viral replication. Importantly, TAC treatment attenuates CVB3-induced autophagic response and reduces viral protein expression and RNA levels in infected cells. Collectively, this study reveals a novel role for TAC in modulating TFEB subcellular localization and activity in the context of CVB3 infection, with potential implications for the pharmacological management of viral myocarditis and associated cardiovascular pathologies.