PPP2R5B regulates ANPEP expression and TGEV entry via dephosphorylation of HSF1 at Ser304/Ser308

Transmissible gastroenteritis virus (TGEV) is an enteric coronavirus responsible for severe gastrointestinal disease in swine; however, the host determinants and mechanisms underlying viral entry remain poorly defined. This study systematically examines the role of the serine/threonine protein Phosphatase 2A (PP2A) regulatory subunit B'β (PPP2R5B) during TGEV Infection. Genetic ablation of PPP2R5B in PK-15 cells significantly reduced TGEV infectivity. Stage-specific analyses revealed that loss of PPP2R5B selectively impaired viral entry, while viral replication and release were unaffected. Transcriptomic profiling and functional validation identified the TGEV receptor Aminopeptidase N (ANPEP) as a critical downstream effector of PPP2R5B. PPP2R5B-mediated regulation of ANPEP expression was independent of the AMPK, PI3K/Akt, ERK, and Wnt/β-catenin signaling pathways. Instead, PPP2R5B directly interacted with heat shock factor 1 (HSF1) and promoted dephosphorylation of HSF1 at Ser304 and Ser308, thereby reducing HSF1-dependent transcriptional activation of ANPEP. These findings reveal a previously unrecognized PPP2R5B-HSF1-ANPEP regulatory axis that governs TGEV cellular entry and suggest that host dephosphorylation-dependent regulation may represent a potential target for Antiviral intervention.IMPORTANCECoronavirus entry is a critical determinant of viral tropism and pathogenicity. This study identifies PPP2R5B, a regulatory subunit of the PP2A Phosphatase complex, as a host factor that facilitates TGEV entry by controlling the expression of the cellular receptor ANPEP. We demonstrate that PPP2R5B regulates ANPEP transcription through modulation of HSF1 phosphorylation, independent of several canonical signaling pathways. These findings uncover a host phosphatase-dependent regulatory mechanism underlying coronavirus entry and highlight host signaling components as potential targets for Antiviral intervention.