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Protein Interactions Network of Hepatitis E Virus RNA and Polymerase With Host Proteins - PubMed

  • ️Tue Jan 01 2019

Protein Interactions Network of Hepatitis E Virus RNA and Polymerase With Host Proteins

Gayatri D Kanade et al. Front Microbiol. 2019.

Abstract

Host-pathogen interactions are crucial for the successful propagation of pathogens inside the host cell. Knowledge of interactions between host proteins and viral proteins or viral RNA may provide clues for developing novel antiviral strategies. Hepatitis E virus (HEV), a water-borne pathogen that causes acute hepatitis in humans, is responsible for epidemics in developing countries. HEV pathology and molecular biology have been poorly explored due to the lack of efficient culture systems. A contemporary approach, to better understand the viral infection cycle at the molecular level, is the use of system biology tools depicting virus-host interactions. To determine the host proteins which participate in the regulation of HEV replication, we indentified liver cell proteins interacting with HEV RNA at its putative promoter region and those interacting with HEV polymerase (RdRp) protein. We employed affinity chromatography followed by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF/MS) to identify the interacting host proteins. Protein-protein interaction networks (PPI) were plotted and analyzed using web-based tools. Topological analysis of the network revealed that the constructed network is potentially significant and relevant for viral replication. Gene ontology and pathway enrichment analysis revealed that HEV RNA promoter- and polymerase-interacting host proteins belong to different cellular pathways such as RNA splicing, RNA metabolism, protein processing in endoplasmic reticulum, unfolded protein response, innate immune pathways, secretory vesicle pathway, and glucose metabolism. We showed that hnRNPK and hnRNPA2B1 interact with both HEV putative promoters and HEV RdRp, which suggest that they may have crucial roles in HEV replication. We demonstrated in vitro binding of hnRNPK and hnRNPA2B1 proteins with the HEV targets in the study, assuring the authenticity of the interactions obtained through mass spectrometry. Thus, our study highlights the ability of viruses, such as HEV, to maneuver host systems to create favorable cellular environments for virus propagation. Studying the host-virus interactions can facilitate the identification of antiviral therapeutic strategies and novel targets.

Keywords: gene ontology analysis; hepatitis E virus; host-protein interactions; protein interactions network; system biology; viral RNA; viral polymerase.

Copyright © 2019 Kanade, Pingale and Karpe.

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Figures

FIGURE 1
FIGURE 1

Identification of HEV interacting host proteins. (A) pcDNA_FLAG-RdRp construct was transfected into Huh7 S10-3 cells. Post 48 h of transfection cells were harvested and checked for expression of RdRp by western blot using anti-FLAG antibody. (B) Mock-transfected Huh 7 S10-3 cells or cells transfected with pcDNA_FLAG_RdRp (expressing FLAG-tagged RdRp) were harvested after 48 h of transfection. Immunoprecipetation was performed by anti-FLAG antibody or an isotype control antibody. RdRp interacting host proteins were eluted with protein G dynabeads and analyzed on SDS PAGE followed by silver staining. (C) HEV sub-genomic promoter (Sg) RNA interacting cellular proteins were pull down by using RNA affinity chromatography. Biotinylated HEV sub-genomic promoter RNA were immobilized on M280 streptavidin dynabeads. RNA immobilized beads were incubated with cell lysate of Huh7 S10-3. Interacting host proteins were eluted and checked on SDS PAGE followed by silver staining for visualization. Non-biotin RNA of the sub-genomic promoter was taken as control. (D) HEV genomic promoter (G) RNA interacting cellular proteins were pull down by using RNA affinity chromatography. HEV genomic promoter RNA interacting host proteins were eluted and checked on SDS PAGE followed by silver staining. For (B–D) lane 1, protein molecular weight ladder; lane 2, negative control pull-down; lane 3, experimental test pull down.

FIGURE 2
FIGURE 2

Construction of HEV-host interaction network. (A) HEV-host interaction network: Interaction map of HEV sub-genomic promoter (Sg), genomic promoter (G) and RdRp with interacting host proteins constructed in Cytoscape 3.6.1. Proteins were classified on the basis of their protein class by Panther gene ontology tool. The corresponding symbols indicating different protein classes have been mentioned on the figure. (B) Venn diagram comparing HEV interacting host proteins with different HEV components. Blue, yellow and green colors indicate proteins interacting with the sub-genomic promoter, genomic promoter, and RdRp, respectively. Common proteins within the data sets have been indicated in the colored intersections. Proteins have been represented as the respective NCBI gene names.

FIGURE 3
FIGURE 3

Construction and analysis of HEV-host PPI network. Interaction map of HEV interacting host proteins further interacting with the other proteins of our data. Black edges represent interactions revealed through mass spectrometry reported in this study. Secondary protein-protein interactions among host proteins revealed through literature mining have been indicated in red colored edges. Proteins have been represented as the respective NCBI gene names.

FIGURE 4
FIGURE 4

Analysis of inter protein interaction network using STRING database. Each edge color indicates a different method of protein-protein interaction prediction as indicated below the figure.

FIGURE 5
FIGURE 5

Gene ontology analysis of HEV- host interactions based on (A) biological process, (B) molecular function, and (C) cellular component category. Y-axis represents the combined enrichment score computed using Enricher.

FIGURE 6
FIGURE 6

Pathway enrichment analysis. (A) Graph shows the enriched pathways targeted by HEV, analyzed by KEGG functional annotation pathway database. Y-axis represents the combined score computed using Enricher. (B) Schematic representation of “protein processing in endoplasmic reticulum” pathway (imported from KEGG: map04141). (C) Schematic representation of the spliceosome pathway targeted by HEV host interacting proteins (imported from KEGG: map03040). Proteins interacting with HEV RNA promoter (G or Sg) or polymerase within the entire pathway are shown in red color.

FIGURE 7
FIGURE 7

Validation of interactions between HNRNPK and HNRNPA2B1 with HEV promoters and RdRp. (A) pTandem_FLAG-RdRp_Myc-HNRNPK plasmid was transfected in Huh 7 S10-3 cells. 48 h post transfection co-IP was performed with anti c-Myc antibody. Interaction of c-Myc tagged HNRNPK with that of FLAG tagged RdRp was checked with western blot by using anti c-Myc antibody. (B) pTandem_FLAG-RdRp_Myc-HNRNPA2B1 plasmid was transfected in Huh 7 S10-3 cells. 48 h post transfection co-IP was performed with anti c-Myc antibody. Interaction of c-Myc tagged HNRNPA2B1 with that of FLAG tagged RdRp was checked with western blot by using anti c-Myc antibody. (C) Huh 7 S10-3 cell lysate was incubated with HEV G and Sg promoter RNA followed by immunoprecipitation with anti HNRNPK or anti HNRNPA2B1 antibody. RT-PCR was performed to detect HNRNP-bound HEV RNAs in the elutes. Figure shows amplified PCR products on 2% agarose gel.

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