YM-53601 Suppresses Hepatitis E Virus Infection in Cell Culture and Supports the Role of FDFT1-Dependent Cholesterol Homeostasis in the HEV Life Cycle

Hepatitis E virus (HEV) is a major cause of acute hepatitis worldwide and can cause chronic infection in immunocompromised individuals. Ribavirin is used off-label for chronic hepatitis E, but its limitations highlight the need for alternative antiviral therapies. We screened 327 compounds from an anti-hepatitis C virus (anti-HCV) compound library using three distinct HEV reporter systems and identified YM-53601, an inhibitor of farnesyl-diphosphate farnesyltransferase 1 (FDFT1; squalene synthase), as a compound with marked anti-HEV activity. YM-53601 showed marked suppression of HEV propagation in cell culture models of acute and chronic infection. Combination with ribavirin showed an additive antiviral interaction and was associated with sustained viral suppression during prolonged culture. Time-of-addition analysis indicated that YM-53601 acts predominantly at an early post-attachment step associated with HEV internalization. TAK-475, another FDFT1 inhibitor, reproduced this early-step antiviral phenotype, whereas small interfering RNA (siRNA)-mediated FDFT1 depletion suppressed HEV propagation and additionally impaired HEV RNA replication. YM-53601 and TAK-475 markedly reduced the methyl-β-cyclodextrin (MβCD)-extractable free cholesterol fraction, used here as an operational measure of readily accessible cellular cholesterol, whereas FDFT1 depletion produced a greater reduction in residual intracellular free cholesterol. Furthermore, cholesterol supplementation partially restored early cell-associated HEV RNA levels following YM-53601 or TAK-475 treatment and HEV RNA replication-associated reporter activity following FDFT1 knockdown. Together, these findings suggest that FDFT1-dependent cholesterol homeostasis contributes to multiple stages of HEV infection. Pharmacological modulation of this pathway therefore warrants further investigation as a potential host-directed therapeutic strategy against HEV infection.