Screening And Confirmation Of Liver Proteins Interacting With HEV ORF2

The open reading frame2(ORF2) of Hepatitis E virus (HEV) encodes thecapsid protein. It plays an important role in the HEV infection and host range. HEVhas mainly four genotypes. Genotype1and2exclusively infect human, whilegenotype3and4can infect both human and swine. Both genotype3and4arezoonotic and swine is the main reservoirs. So far, the mechanism of infection and hostrange limitation remains unclear. This study aims at screening and validatingORF2-binding partners. It will provide the clues for the research of HEV infectionand cross-species transmission.To study the mechanism of HEV infection and find the candidate receptor, theinteracting proteins in human liver with ORF2were screened and verified in thepresent study. Firstly, split-ubiquitin yeast two-hybrid system was used to screenproteins from a human liver cDNA library three times with ORF2of HEV genotype4(aa112-660) as the bait protein. False positive proteins were ruled out by yeastco-transformation assay.43potential proteins that may interact with ORF2wereeventually acquired. Secondly, these proteins interacting with genotype1and4wasverified by pull-down. Eight ORF2-binding partners were verified, which is ASGR2,BNIP3, C14orf1, EBP, HMOX2, TMEM134, TMEM14A and TMEM230. Finally, theinteraction of TMEM134and ORF2was further confirmed byco-immunoprecipitation.In order to study the mechanism of HEV host range limitation, the present studyalso analyzed the interaction between ORF2of different genotypes and differentspecies.(1) All the swine specific proteins that only interacted with ORF2of genotype4in the yeast two-hybrid screens were verified by pull-down. The results showed thatfour of them, named NNT, MTCH2, STX12and SEC61B, interacted with ORF2ofboth genotype1and4and one protein named ATL2can only interact with genotype4but not genotype1.(2) ATL2cloned from human and swine liver interacted withORF2of genotype4but not genotype1.(3) By cloning swine and mouse ASGR2genes, we compared their difference interacting with ORF2. It shows that bothASGR2cloned from human and swine liver could interact with ORF2.In summary,43potential proteins interacted with ORF2were acquired, and8ofthese proteins were confirmed as ORF2-binding partners. TMEM134was furthervalidated, suggesting it may be related with the infection of HEV. ATL2interactedwith only genotype4but not genotype1. These findings provide the clues anddirections for studying the mechanism of HEV infection and cross-species limitation.