Virus host protein interaction network analysis reveals that the HEV ORF3 protein may interrupt the blood coagulation process

Abstract

Hepatitis E virus (HEV) is endemic worldwide and a major cause of acute liver disease in developing countries. However, the molecular mechanisms of liver pathology and clinical disease are not well understood for HEV infection. Open reading frame 3 (ORF3) of HEV encodes a small phosphoprotein, which is assumed to be involved in liver pathology and clinical disease. In this study, the interactions between the HEV ORF3 protein and human proteins were investigated using a stringent, high-throughput yeast two-hybrid (Y2H) analysis. Thirty two proteins were shown to interact with genotype 1 ORF3, 28 of which have not been reported previously. These novel interactions were evaluated by coimmunoprecipitation of protein complexes from transfected cells. We found also that the ORF3 proteins of genotype 4 and rabbit HEV interacted with all of the human proteins identified by the genotype 1 ORF3 protein. However, the putative ORF3 protein derived from avian HEV did not interact with the majority of these human proteins. The identified proteins were used to infer an overall interaction map linking the ORF3 protein with components of the host cellular networks. Analysis of this interaction map, based on functional annotation with the Gene Ontology features and KEGG pathways, revealed an enrichment of host proteins involved in complement coagulation, cellular iron ion homeostasis and oxidative stress. Additional canonical pathway analysis highlighted the enriched biological pathways relevant to blood coagulation and hemostasis. Consideration of the clinical manifestations of hepatitis E reported previously and the results of biological analysis from this study suggests that the ORF3 protein is likely to lead to an imbalance of coagulation and fibrinolysis by interacting with host proteins and triggering the corresponding pathological processes. These results suggest critical approaches to further study of the pathogenesis of the HEV ORF3 protein.

Figure 1. Chemiluminescent detection of protein-protein interactions by co-immuno precipitation.

On the x-axis, “NC1 and NC2” represent 293 cell lysate and negative control; “1∼28” represent FTL, HP, APCS, APOH, NR0B2, AMBP, VTN, TF, MT2A, COX2, CD151, HPX, IGFBP4, FXN, FGA, ALDOB, HSPA8, CYP2E1, SERPINC1, CD63, RGS19, FGB, NAT1, DCN, TSG101,CXCL10, HGD and MAT1A. In the co-immunoprecipitation, the interacting protein complexes were precipitated by both Anti-HEV ORF3 polyclonal antibody(indicated by light blue squares) and GFP1 polyclonal antibody (indicated by magenta squares)., The negative controls were precipitated by Anti-AcGFP antibody.

Figure 2. A visualized map of HEV ORF3 protein-human protein interactions.

Yellow nodes: HEV ORF3 protein; green nodes: host proteins identified as interacting partners of the HEV ORF3 protein using the Y2HGold system; tiny nodes: secondary interactors of the host proteins interacting with the ORF3 protein.

Figure 3. Enriched canonical pathways in the HEV ORF3 protein and human protein interaction network (p≦0.05).

The canonical pathways are mapped to the x-axisand the y-axis represents the % of genes mapped to a given pathway within the network and in human genome.

Figure 4. Network illustration of interactions between HEV ORF3 interacting proteins and host proteins associated to “Hemostasis”.