Transferrin Receptor 1 is Required for Efficient Hepatitis E Virus Production

Hepatitis E virus (HEV) infection is a major cause of acute viral hepatitis worldwide. HEV is a positive-strand RNA virus encoding three open reading frames (ORFs). The ORF3 protein is a small membrane-associated protein essential for viral particle secretion; however, its precise role in the viral life cycle remains incompletely understood. Here, we performed immunoprecipitation followed by mass spectrometry to identify host proteins interacting with the HEV ORF3 protein. Candidate interactors were validated by co-immunoprecipitation, confirming physical interactions between ORF3 and cysteine-rich and transmembrane domain-containing protein 1 (CYSTM1), Ras-related protein Rab24, and transferrin receptor 1 (TfR1). Confocal microscopy demonstrated colocalization of all three host factors with ORF3 protein. Gene silencing and knockout revealed that each protein contributes to virus production, with TfR1 depletion producing the most pronounced effect. In cells harboring replicating HEV, TfR1 colocalized with the ORF2 (capsid) and ORF3 proteins at Rab11A-positive recycling endosomes. Silencing of TfR1 in primary human hepatocytes, followed by HEV RNA transfection or infection, confirmed its role in virus production, particularly in the assembly of infectious particles, consistent with its colocalization with the HEV ORF2 protein. Collectively, our proteomics-based analysis identifies TfR1, along with CYSTM1 and Rab24, as novel ORF3-interacting host factors required for efficient production of infectious HEV. These findings provide new insights into the role of ORF3 protein in viral assembly and highlight TfR1 as a key host factor in the HEV life cycle.