Systematic identification of hepatitis E virus ORF2 interactome reveals that TMEM134 engages in ORF2-mediated NF-κB pathway

•59 proteins interacting with HEV ORF2 were identified from a human liver cDNA library using an ubiquitin-split yeast two-hybrid approach.•20 ORF2-interacting proteins were located in endoplasmic reticulum (ER).•Eight proteins named ASGR2, BNIP3, C14ORF1, EBP, HMOX2, TMEM134, TMEM14A and TMEM230 were further verified by a pull-down assay.•TMEM134 interacts and co-localizes with ORF2 in the endoplasmic reticulum, negatively regulates ORF2-mediated inhibition of the NF-κB signaling pathway. Hepatitis E virus (HEV) is the causative agent of acute hepatitis E. Open reading frame 2 (ORF2) encodes the capsid protein of HEV, which is the main structural protein and may participate, together with the host factors, in viral entry and egress. However, it is still not clear which host proteins are involved in the interaction with ORF2 and what the functions of these ORF2-interacting proteins are. In this study, we have applied a split-ubiquitin yeast two-hybrid screening approach in combination with the pull-down and coimmunoprecipitation assays, identified and validated multiple interacting partners of ORF2 of genotype 1 and 4, which have diverse biological functions. Among these novel candidates that have not been previously reported, we have found that 20 of them are located in endoplasmic reticulum. TMEM134, which interacts and co-localizes with ORF2 in the endoplasmic reticulum, negatively regulates ORF2-mediated inhibition of the NF-κB signaling pathway. Our study for the first time has systematically mapped the ORF2 interactome in two genotypes of HEV, providing a new insight of understanding the virus-host interaction during the pathogenesis of HEV, and may offer potential therapeutic targets to intervene the HEV life cycle.