Proteomics Analysis Of Primary Duck Hepatocytes Infected With DHBV And Preliminary Study On Biological Function Of Duck Annexin A2 In DHBV Infection

Hepatitis B virus (HBV), belonging to the genus Orthohepadnavirus of the Hepadnaviridae family, is a major cause of liver infection in human. Lack of an appropriate in vitro HBV infection system, how hepatocytes responsing to hepadnaviruses infection remains incompletely understood. Duck hepatitis B virus is a member of the Hepadnaviridae family, classified to Avihepadnavirus, sharing similar biological features with HBV including genome organization and structural organization, viral replication process and biological characteristics. The primary duck hepatocytes-duck hepatitis B virus (PDHs-DHBV) system is a valuable hepadnaviruses infection model in vitro with two major advantages, the ready availability of ducks which are the natural host of DHBV, and the high sensitivity to DHBV of primary hepatocytes from ducklings.With highly reproducible and efficient infection, PDHs-DHBV made the infection system for studying the cellular and molecular biology of hepadnavirus infection under control.The development of proteomic methods revolution has enabled us to assess virus-host interactions and the changes of cellular proteins expression at a global scale, to reveal the connections between virus infection and host cellular functions. How viral infection affects the expression of the cellular proteome has attracted attentions. We intend to utilize a natural PDHs-DHBV infection system to explore hepatocytes response to hepadnaviruses infection. Although Anas platyrhynchos genome has not been sequenced, the decoding of the genome of Gallus gallus (chicken) and the advancement of the genomic techniques bring opportunities to investigate on the interaction of DHBV infection and host protein expression. In the present work, we attempted to analyze primary duck hepatocytes with and without duck hepatitis B virus infection by proteomics and explore the molecular mechanisms of hepadnavirus infections. In the present study, we investigated the effect of DHBV infected primary duck hepatocytes by proteomic analysis. A total of 51 differential expressed protein spots were revealed by 2-DE between DHBV infected and uninfected PDHs at 24,72 and 120h postinfection, respectively. Twenty-seven proteins have been identified by MALDI-TOF MS.Differentially expressed proteins included alpha-enolase, destrin, elongation factor-2, lamin A, heat shock protein, and annexin A2 (ANX2), etc. Western blot further confirmed the down-regulated expression of and ANX2 during DHBV infection. However, duck heat shock protein 70 and lamin A were not detected by Western blot analysis with the rabbit anti-human or anti-mouse heat shock protein 70 polyclonal antibodies, and rabbit anti-human lamin A polyclonal antibody.It was revealed that ANX2 was down-regulated in DHBV infected PDHs comparing with uninfected PDHs. And the proteomics analysis showed that ANX2 expression was down-regulated in PDHs losing their susceptibility to DHBV infection compared to PDHs susceptible to DHBV infection when PDHs cultured in the different stages or culture medium. ANX2, belonging to a family of calcium-dependent, phospholipid binding proteins, is involved in many biological processes such as the Ca2+ dependent exocytosis, calcium transport and cell proliferation. It participates in viral infection, including assisting in the assembly of HIV in monocyte-derived macrophages, as a cellular cofactor supporting HIV-1 infection, enhancing cytomegalovirus binding and membrane fusion. This indicated that ANX2 may be involved in DHBV infection.In order to confirm the ANX2 protein change in the proteomics, we prepared polyclone anti ANX2 antibodies. By Western blot we confirmed the result. To explore the role of ANX2 in the DHBV infection, PDHs were treated with ANX2-specific siRNA (A2-701) or with nontargeting control siRNA (N.C.) and infected with DHBV after the treatment four days. Culture medium was collected and cells were lysed at postinfection (p.i.) four days. Supernatant and intracellular DHBV DNA were analyzed by dot blot hybridization and southern blot hybridization. The results have shown that siRNA of ANX2 in PDHs results in decreased DHBV replication.Recombinant plasmids which express DsRed fusion protein with duck ANX2 and EGFP fusion protein with DHBpreS/S were co-transfected with EGFP-DHBpreS/S fusion protein recombinant plasmid into 293T cell line.The results showed that duck ANX2 co-localized in cytoplasm with DHBVpreS/S by confocal analysis This indicated that ANX2 may interact with DHBVpreS/S. In order to confirm the interaction between them, co-immunoprecipitate and pull down were used. Anti-ANX2 and anti DHBVpreS Western blot showed that DHBVpreS/S coprecipitates with ANX2 from PDHs. Purified ANX2-His protein precipitated on a nickel matrix after incubation with lysates from DHBV infected PDHs and coprecipitated DHBV preS/S. These results supported that ANX2 interacted with DHBV preS/S in PDHs and cell-free systems.In conclusion, this study explored hepatocytes responses to hepadnaviruses infection by cellular proteome analysis, using a natural PDHs-DHBV infection system. The data obtained may lead to better understanding of interactions between hepadnaviruses and hepatocytes and molecular mechanisms of hepadnavirus infection.