Isolation And Identification Of Hepatitis E Virus (HEV) Capsid Interacting Proteins In Host Cells, And Preliminary Study Of The Role Of Grp78/Bip In HEV Infection

Hepatitis E virus (HEV), the pathogen of hepatitis E (HE), is a non-enveloped, enterically transmitted virus, carries a single-strained, positive sense RNA genome with 3 predicted open reading frames (ORFs). It is well known that HE is a major cause of both sporadic and epidemic hepatitis, which is no longer confined to Asia and developing countries but has also become a concern of the developed nations. The mortality of the disease is usually 0.4-2%, but is much higher in pregnant women that would reach to 10-20%. Furthermore, increasing data suggest that HE is zoonotic, and therefore may cause very serious epidemics like SARS or bird flu. However, the mechanism of HEV entry on host cells is still poorly understood due to the absence of a reliable cell-culture system and small animal model. In the present study, we used a recombinant HEV capsid protein - p239 as a bait to screen the HEV capsid interacting proteins from hepatocytes with different origins, and analyzed the potential role of these candidates in HEV capsid entry.The p239 polypeptide locates at aa368-606 of HEV ORF2. It trend to self-assemble into 15-30nm particles in diameter when was renatured from inclusion body expressed in E.coli. p239 had good reactivity with sera of acute HE patients. When used as an immunogen, it protected the rhesus monkeys from HEV infection or hepatitis. In in-vitro cultured hepatocytes, we found that p239 could attach on cellular membrane, and then enter into cytoplasm of HepG2, while the attachment and the entry could be completely blocked by HEV neutralizing mAb 8C11. In addition, pre-incubation of p239 with hepatocytes blocked the infection of wild-type HEV on HepG2 and primary cultured human hepatocytes. These results demonstrate that p239 particles simulate the surface structure of HEV.p239 was then fusion-expressed with calmodulin binding peptide (CBP) tag to generate p239-CBP. The recombinant protein was fixed on the calmodulin Sepharose 4B according to the high affinity between CBP and calmodulin, and then incubated with HepG2 cellular extract or monkey liver tissue extract. The p239 interacting proteins were separated by two dimensional gel electrophoresis, digested with trypsin, and then identified by Matrix-Asisted Laser Desorption/Ionization-Time of Flight-Mass Spectrometry (MALDI-TOF-MS). The screened out proteins included Grp78, HSP90,α-tubulin, p43, ATPase beta subunit, and an unnamed protein. The interaction between p239 and Grp78, HSP90, andα-tubulin, were further proved by co-immunoprecipitation. These results suggest that the platform for p239 interacting protein screening is feasible, and the candidate proteins are potentially functional in HEV infection.Grp78, a member of heat shock protein 70kD families, locates at both cellular plasma and membrane and has been suggested to be the receptor of dengue virus and CAV9. Pull-down and co-IP results showed that p239 directly bound to Grp78 and this binding was sensitive to ATP treatment. Antibody blocking results demonstrated that the interaction was conformation-dependent and the binding site overlapped with the neutralized site 8C11. The localization of Grp78 on plasma membrane was proved by flow cytometry. Meanwhile, a partial co-localization of Grp78 and p239 was observed on plasma membrane by CONFOCAL assay. Further, pre-incubation of purified Grp78 or Grp78-his with p239 significantly blocked the attachment of p239 on HepG2 cells.In general, our results demonstrated: (1) p239 has similar surface structure with wild type HEV, and therefore could be used to study HEV interacting proteins in host cells, and the mechanism of virus entry. (2) A protein-protein interaction screening system was established. By using p239-CBP as bait, we screened out several HEV capsid interacting proteins, which included Grp78, HSP90,α-tubulin, p43, ATPase beta subunit, and an unnamed protein. Partial of the interaction were further proved by co-immunoprecipitation. (3) The interaction between p239 and Grp78 was confirmed by immuno-pull down, antibody blocking and CONFOCAL assay. (4) Recombinant Grp78, Grp78-his or anti-Grp78 sera completely or partially blocked the attachment of p239 on hepatocytes, providing evidences that Grp78 participates in the attachment/entry of HEV capsid protein on host cells.The present study investigates the entry mechanism of HEV on host cells for the first time. Our results may contribute to anti-HEV drug development and vaccine design.