HBV infection remains a public health problem worldwide. Because the lack of appropriate cell lines that can support HBV infection efficiently, the cellular and molecular mechanisms of hepadnavirus infection remain incompletely understood. The hepadnavirus animal infection models such as ducks (DHBV) and woodchucks (WHBV) have been used to investigate the viral replication, pathogenesis or hepadnavirus-associated hepatocellular carcinoma. DHBV-PDHs model is a valuable model of hepadnavirus infection with high reproducibility and efficiency . In the present study, global changes in cellular protein expression in DHBV-infected PDHs were explored by 2-DE combined with MS/MS. Among the 75 differentially expressed protein spots, 51 spots have been identified by MS/MS corresponding to 42 proteins, in which 30 spots were matched to orthologous proteins of Gallus gallus or Anas platyrhynchos, 7 spots to other avian species, and 14 spots to non-avian species, while mass spectra of the other 24 protein spots did not match to any proteins in the current databases, possibly due to the incomplete genome sequence of Anas platyrhynchos or low abundance of those protein spots.
In previously studies, Tong performed a proteomic analysis comparing HepG2 with HepG2.2.15 in which HBV genome integrated into cellular chromosome , and Narayan revealed 19 differentially regulated features in HepaRG cells by 2-DE . HepG2.2.15 is a HBV replication cell model but not an infection model, while the human hepatoma HepaRG cells are susceptible to HBV, but 10~20% of cells can be infected regardless of the amount of virus used (MOI > 200) [4, 6]. In previous studies, it has been showed that at MOI of 30, about 50%~60% PDHs can be reproducibly infected with DHBV . Some of differentially expressed proteins identified in the present study, such as alpha-enolase, lamin A, GAPDH and cofilin-2 have not yet been reported in hepadnavirus proteomic analysis.
Viruses depend on host cell metabolism for their replication. Elucidation of the pathways/processes involving in the viral life cycle will help to understand the mechanisms of viral infection. In the 2-DE analysis, the identified differentially expressed proteins were classified into carbohydrate metabolism, amino acid metabolism, cytoskeletal/structural protein, stress response and other functions according to the Gene Ontology criteria. Some of differentially expressed proteins identified in the present study have been reported playing roles in viral infections, as shown in Table 2.
In DHBV infected PDHs, the expression of some carbohydrate metabolic enzymes, such as phosphoglycerate kinase 1, triosephosphate isomerase, phosphoglycerate mutase 1 etc, was up-regulated. The differentially expressed proteins involving in carbohydrate metabolism, suggests perturbed energy metabolism in DHBV infections. Hepatitis C Virus (HCV) infection reprograms the cellular metabolisms to favor glucose fermentation and glycolytic intermediates toward the metabolite synthesis that supports the viral life cycle . In lymphocytic choriomeningitis virus infection, there was a significant increasing in transcripts promoting gluconeogenesis for viral mediate synthesis, and a decreasing in transcripts promoting glycogenolysis in the early stage of infection .
However, GAPDH and alpha-enolase, key enzymes involving in glycolysis and gluconeogenesis, are decreased in DHBV infected PDHs. GAPDH and alpha-enolase have been found associating with the cell membrane and in secreted viral particles of influenza virus, lentiviral vector etc [20, 21]. GAPDH may phosphorylate the HBV core protein, and binds to the preS1 region of the HBV envelope antigen and posttranscriptional regulatory element in regulating expression of surface antigen, suggesting that GAPDH plays an important role in the life-cycle of HBV infection [22–24]. The host cellular carbohydrate metabolism affected by DHBV infection may benefit viral replication.
Alterations of cytoskeleton networks were found in many viral infections [25–27]. Hepadnavirus needs to manipulate and utilize the host cytoskeleton to promote viral infection like many viruses, although the mechanism is still unclear . In DHBV infected PDHs, the microfilament-associated proteins, beta-actin and cofilin-2 were down-regulated, and three microfilament-associated proteins such as transgelin, destrin, and collapsin response mediator protein-2B were up-regulated. Actin plays an active role in maturation of the viruses [29, 30]. Many viruses require actin for viral entry and establishment of infection, including human immunodeficiency virus (HIV), adenovirus, Simian virus 40, and vaccinia virus [31–34]. However, the actin cortex beneath the plasma membrane can also be an obstacle for virus entry or budding . It has been reported that DHBV entry depends on both intact microtubules and their dynamic turnover but not actin cytoskeleton . Therefore the role of actin in DHBV replication is required to further investigation.
Lamin A is key structural components of the nuclear lamina and lamins, involving in DNA replication and gene expression, as well as presenting a natural barrier against most DNA viruses such as human cytomegalovirus (HCMV), Kaposi's sarcoma-associated herpesvirus, herpes simplex virus (HSV) 1 and Epstein-Barr virus . Lamin A/C is phosphorylated in HSV-infected cells supporting a role in regulating virus capsid nuclear egress [37, 38]. Infection of Epstein-Barr virus induced disassembly of the nuclear lamina and redistribution of nuclear lamin for the nuclear egress . The expression of lamin A with different isoforms, were up-regulated in DHBV infected PDHs, suggesting that lamin A may play a role in DHBV replication.
In DHBV infected PDHs, up-regulated expressions of amino acid metabolism enzymes, catalyzing interconversion of glutamate, histidine, and proline (Glutamate dehydrogenase 1, Urocanate hydratase, Delta-1-pyrroline-5-carboxylate dehydrogenase, the orthologs in human referred to protein 16, 19 and 20, 22 in Table 1), indicate that glutamine metabolism is enhanced. Switching the anaplerotic substrate from glucose to glutamine to accommodate the biosynthetic and energetic needs of the viral infection and to allow glucose to be used biosynthetically was reported in HCMV infection . HCV-infected cells exhibit increased levels of the enzymes catalyzing glutamine flux to replenish metabolic intermediates through the latter half of the citric acid cycle providing substrates for ATP production . Thus similar mechanism of glutamine metabolism may be at work in DHBV infection.
Stress response associated proteins including endoplasmic reticulum stress associated proteins such as Hsp70, and chaperonin containing t-complex polypeptide 1 (TCP1) and oxidative stress associated proteins such as antioxidant enzymes Mn superoxide dismutase and peroxiredoxin-3 (similar to antioxidant protein isoform 2) were found to be up-regulated post DHBV infection. Hsp70 assists folding of many newly synthesized polypeptides, and refolding of the proteins misfolded [41, 42]. Hsp70 can enhance flock house virus replication [43, 44]. Hsp70 and Hsp90 participate in dengue virus entry as a receptor complex . Moreover, HBV P protein activation in vitro is fundamentally dependent on heat shock protein 70 family Hsc70/Hsp40 . In HBV-replicating HepAD38 cell, expressions of heat shock proteins (Hsp70 and Hsp90) and Mn superoxide dismutase increase, after HBV replication induced by tetracycline . In humanized transgenic mice, inhibition of HBV replication results in suppression of Mn superoxide dismutase expression in hepatocytes [47, 48]. It suggests that oxidative stress can be induced by hepadnavirus replication as Epstein-Barr virus .
Annexin A2, belongs 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  and supporting the replication of influenza viruses by mediating activation of plasminogen . It has been reported that HBV polymerase activity was inhibited by interacted with S100A10, a protein binding to annexin A2 . In HepG2.2.15 compared with HepG2, annexin A2 was revealed down-regulated , which was consistent with our observation in DHBV-PDHs model and confirmed by Western blot analysis. It indicated that annexin A2 may involve in hepadnavirus infection and warrants further investigation.
Beta-actin and GAPDH are usually referred as the internal standards for detections of RNA transcription and protein expression of genes. However, those proteins were found to be down-regulated post DHBV infection by 2-DE analysis. Recently, accumulated evidence showed that in HBV-related hepatocellular carcinoma or viral infections, beta-actin and GAPDH are unsuitable controls in quantitative mRNA expression or Western blot analysis due to variations in expression [56–60], though there are controversial observations . These findings therefore highlight the importance of re-evaluating the housekeeping genes whose expressions may be affected by hepadnavirus infection.