Study Of Proteins Related With The Different Susceptibility Of Primary Duck Hepatocytes To Duck Hepatitis B Virus Infection

Hepatitis B virus (HBV) is a major pathogen of acute and chronic hepatitis. HBV infection may result in acute or chronic hepatitis, and may eventually develop to cirrhosis and hepatocelluar carcinomar. HBV belongs to hepadnaviruses family. HBV is primarily hepatotropic virus and only infects human and chimpanzee, but not non-primate laboratory animals. Since lacking of an in vitro infection system, the molecular mechanism of the early steps of HBV life cycle is unclear till now, especially the host proteins related with viral infection as well as effects of viral infections on protein expression of host cell.All members of hepadnaviruses share unique genomic and virion structure features and replicate by using the same strategy through reverse transcription of viral RNA into DNA as an essential step of viral genome replication. The infection models of duck hepatitis B virus (DHBV) and woodchuck hepatitis virus (WHV) are used as alternative system for studying pathogenesis and replication of HBV.It has been reported that the susceptibility of primary duck hepatocytes (PDHs) to DHBV decreased along with the prolonging culture time and PDHs can be maintain its susceptibility to DHBV when culture medium is substituted with fetal bovin serum-free L-15 medium contained 1.5% DMSO. This phenomenon indicating there may be some changes of host protein expression when the PDHs cultured in the different culture conditions, which may affect the susceptibility to DHBV. Since the transient and dynamic property of viral entry, it is difficult to study the early events of viral infection.Nowadays, the proteomic approach is considered to be the key technology in the global analysis of protein expression and in the understanding of gene function in the post-genomic era. By studying global patterns of protein content and activity andhow these change during development or in response to disease, proteomics research has boosted our understanding of systems-level cellular behavior and mechanism of disease. In the present work, we attempted to make comprehensive and comparative analysis of primary duck hepatocytes with different susceptibility to duck hepatitis B virus infection. The differential proteins of PDHs with different susceptibility were identified by using 2-DE and MALDI-TOF-MS/MS to DHBV and the proteins' functions predicted by using bioinformatics method. We choose some candidate differential protein, amplified their cDNA and cloned into recombinant expression plasmids. Interactions between certain fusion host protein and DHBV surface antigen DHBpreS/S were studied by colocalization in 293T cell line and coimmunoprecipitation.Part I Establishment and modification of duck hepatocytes separation methodAccording to the published reports, we established and modified method of isolating the duck hepatocytes from duckline's liver to culture primary duck hepatocytes. Three different liver perfusion methods were applied, namely liver was perfused via the cardiac atrium, the thoracic cavity artery, and the portal vein respectively. By comparing these three methods, modified perfusion method via portal vein was adopted. Compared with the other methods, portal vein perfusion method can perfuse the duck liver thoroughly in less time, use less perfusion liquid and decrease the opportunity of bacterial contamination. The isolated duck hepatocytes attached to bottom of cell culture dish in 4 to 6 hours post plating and full extend at the next day.The primary duck hepatocytes could be infected with DHBV naturally. DHBV DNA in the supernatant could be detected by Dot hybridization and intracellular DHBV DNA from DHBV infected PDHs detected by Southern hybridization.Part II Proteomics analysis of primary duck hepatocytes with different susceptibility to DHBVThe susceptibility to DHBV of PDHs growth in different culture medium and at different culture times were studied by using southern hybridization analysis and immunohistochemistry analysis after DHBV infection. The results showed that PDHs may lose their susceptibility to DHBV infection gradually and changes in culture condition may also lead to the susceptibility decrease.To determine whether PDHs' susceptibility to DHBV infection alters during prolonged culture time, we infected PDHs with DHBV serum at 2 4, 6 8 and 10 days post plating. After incubation overnight with infectious DHBV, PDHs were cultured in L-15 medium with 5% fetal bovin serum (FBS), harvested at day 5 after infection, and total DHBV DNA from PDHs was detected by Southern Blot hybridization with DHBV DNA probe. The highest level of DHBV DNA was detected in infected PDHs at day 2 after plating and intracellular DHBV DNA decreased along with the cultured days, suggesting that PDHs were losing their susceptibility to DHBV infection gradually, that indicated that in 5% FBS culture medium PDHs in the early cultured stage were more susceptibility to DHBV than PDHs cultured for 8 days.In FBS-free L-15 culture medium containing 1.5% DMSO for day 2, 4, 8 after cell plating PDHs showed the same susceptibility to DHBV, but by day 10's PDHs susceptibility to DHBV decreased. These results suggest that the FBS-free cell culture medium containing 1.5% DMSO maintained the PDHs' susceptibility to a large extent. The results confirmed by DHBV surface antigen detection of DHBV infected PDHs by immunohistochemistry and immunofluorescence assay.We performed 2D-electrophoresis with proteins of PDHs that have different susceptibility to DHBV infection with differences in culture time and medium. With mass spectra, we found 132 proteins were expressed differently in PDHs at different culture time and 123 in that with different culture medium. Among the 31 proteins shared by the two comparison groups, 26 had consistent correlation with the change of susceptibility. The results indicated these 26 proteins most probably be susceptibility-related to some extent.Part III Functional study of host proteins related with the differential susceptibility of PDHs to DHBV infectionIn order to analyze the functions of host proteins related with the differential susceptibility of PDHs to DHBV infection, total duck liver RNA was extracted and be reverse transcripted into cDNA. Duck Ctm4, annexin A2, 14-3-3 tau and HPPD cDNA eukaryotic expression plasmids were constructed respectively. Since the pivotal role of DHBpreS/S in the process of DHBV infection, we observed the interaction between host proteins and DHBpreS/S using colocalization and coimmunoprecipitation . We constructed recombinant plasmids which express DsRed fusion protein with duck Ctm4, annexin A2, 14-3-3 tau or HPPD and EGFP fusion DHBpreS/S protein. Recombinant plasmids which expressed the duck host fusion proteins were co-transfected with EGFP-DHBpreS/S fusion protein recombinant plasmid into 293T cell line. By Confocal analysis the results showed that duck annexin A2 and HPPD could co-localized in cell plasma with DHBpreS/S. In order to confirm the interaction between host protein and DHBpreS/S, recombinant plasmids expressed the duck host proteins with myc tag were co-transfected with DHBpreS/S expression plasmid. With co-immunoprecipitation analysis, it showed that duck annexin A2 and HPPD could be co-immunoprecipitated with DHBpreS/S, suggesting the both duck proteins can interact with DHBpreS/S and may play the roles in DHBV infection.Part IV Proteomics analysis of primary duck hepatocytes infected and uninfected with DHBVAs no cell model can support HBV natural infection successfully currently, the changes of structure and function of the host cells caused by HBV infection are still unclear. In this part, we infected PDHs with DHBV naturally, then analyzed the protein alteration with proteomics method. After 2-DE and MALDI-TOF-MS/MS,total 127 proteins were identified, which 55 were up-expressed in PDHs infected with DHBV and 72 were down-expressed.Preliminary function prediction showed that 13% of the 127 differentially expressed proteins are involved in amino acid transport and metabolism; 3% are involved in carbohydrate transport and metabolism; 17% are involved in energy production and conversion; 13% are involved in signal transduction mechanisms. Subcell location showed that most of the distinct proteins are in the cytoplasma, the others are mitochondrial proteins and nuclear proteins. We found that anti-HIV infection factor APOBEC3G was up-expressed in PDHs infected with DHBV, which indicated this factor may also serve anti-viral effective on hepadnaviruses infection process.