To Study The Interaction Of HEV ORF2 Protein With The Liver Cell Proteins

Hepatitis E virus(HEV) is the causative agent of hepatitis E， mainlytransmitted via the faecal-oral route. It is a nonenveloped virus with a7.2kilobases positive-stranded RNA genome, containing three partiallyoverlapping open reading frames called ORF1, ORF2and ORF3. The ORF2proteinis the viral capsid protein, which was proposed to encapsidate the viralRNA genome. It is suggested that in the HEV replication cycle, the ORF2protein binds a specific yet uncharacterized receptor on the surface oftarget cells, starting the process of viral propagation. The virus has fourgenotypes, which shares appropriately80%nucleotides identity. Genotypes1and2exclusively infect humans, whereas genotypes3and4also infectpigs and several other mammalian species, with pigs serving as reservoirsfor HEV.In this study, yeast two-hybrid system was used to screen a pig livercDNA library for identify proteins interacting with HEV. The functions ofthese pig proteins in the HEV propagation were analyzed. Furthermore, toinvestigate the molecular mechanism of host range restriction of differentHEV genotypes, the differences between pig proteins interacting withgenetype1HEV and pig proteins interacting with genotype4HEV wereanalyzed.The screening for pig proteins that interact with ORF2protein derivedfrom genotype1HEV strain and genotype4strain were performed three timesusing split-ubiquitin yeast two-hybrid. After yeast co-transform experimentremoving non-specific interactions, we obtain9pig proteins interact withgenotype1HEV ORF2and17proteins interact with genotype4HEV ORF2. Fromthe result, we found that there are eight pig proteins named YIPF5, SEC61B,SERP1, ATL2, CTB5A, CMTM6, MTCH2, NNT that only interact with genotype4HEV ORF2. Subcellular locations of these eight proteins include membrane(CMTM6)，mitochondrion (MTCH2,NNT), endoplasmicreticulum(YIPF5, SEC61B,SERP1, ATL2, CYB5A).The9proteins interact with genotype1HEV ORF2and17proteins interactwith genotype4HEV ORF2obtained after yeast co-transform experiment wereconformed by Chemiluminescent co-immunoprecipitation assay. Meanwhile,Chemiluminescent co-immunoprecipitation assay was used as a quantitative method to compare the binding capacities of ORF2proteins from genotype1and4HEV with all the positive proteins in the yeast co-transform experiment.Protein YIPF5and SEC61B show significant binding difference interactingwith genotype1and4HEV ORF2, the binding capacities with genotype4HEVORF2is stronger than genotype1. Thus, the two proteins may be related tohost range restriction of HEV genotype1and4.The result provide us a clue to further study of replication and hostrange restriction of HEV.