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. 2018 Jan 23;3(1):e00135-17.
doi: 10.1128/mSystems.00135-17. eCollection Jan-Feb 2018.

Host-Virus Protein Interaction Network Reveals the Involvement of Multiple Host Processes in the Life Cycle of Hepatitis E Virus

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Free PMC article

Host-Virus Protein Interaction Network Reveals the Involvement of Multiple Host Processes in the Life Cycle of Hepatitis E Virus

Chandru Subramani et al. mSystems. .
Free PMC article

Abstract

Comprehensive knowledge of host-pathogen interactions is central to understand the life cycle of a pathogen and devise specific therapeutic strategies. Protein-protein interactions (PPIs) are key mediators of host-pathogen interactions. Hepatitis E virus (HEV) is a major cause of viral hepatitis in humans. Recent reports also demonstrate its extrahepatic manifestations in the brain. Toward understanding the molecular details of HEV life cycle, we screened human liver and fetal brain cDNA libraries to identify the host interaction partners of proteins encoded by genotype 1 HEV and constructed the virus-host PPI network. Analysis of the network indicated a role of HEV proteins in modulating multiple host biological processes such as stress and immune responses, the ubiquitin-proteasome system, energy and iron metabolism, and protein translation. Further investigations revealed the presence of multiple host translation regulatory factors in the viral translation/replication complex. Depletion of host translation factors such as eIF4A2, eIF3A, and RACK1 significantly reduced the viral replication, whereas eIF2AK4 depletion had no effect. These findings highlight the ingenuity of the pathogen in manipulating the host machinery to its own benefit, a clear understanding of which is essential for the identification of strategic targets and development of specific antivirals against HEV. IMPORTANCE Hepatitis E virus (HEV) is a pathogen that is transmitted by the fecal-oral route. Owing to the lack of an efficient laboratory model, the life cycle of the virus is poorly understood. During the course of infection, interactions between the viral and host proteins play essential roles, a clear understanding of which is essential to decode the life cycle of the virus. In this study, we identified the direct host interaction partners of all HEV proteins and generated a PPI network. Our functional analysis of the HEV-human PPI network reveals a role of HEV proteins in modulating multiple host biological processes such as stress and immune responses, the ubiquitin-proteasome system, energy and iron metabolism, and protein translation. Further investigations revealed an essential role of several host factors in HEV replication. Collectively, the results from our study provide a vast resource of PPI data from HEV and its human host and identify the molecular components of the viral translation/replication machinery.

Keywords: hepatitis E virus; host-pathogen interactions; protein-protein interactions.

Figures

FIG 1
FIG 1
Identification of the host interaction partners of HEV proteins in the human liver and fetal brain cDNA libraries. (A) Schematic of the g-1 HEV genome and of the proteins encoded by it. UTR, untranscribed region. (B) (Upper panel) Western blot showing GAPDH protein level in Huh7 cell extract expressing the indicated Flag-tagged viral proteins. (Lower left panel) Coimmunoprecipitation of Flag-tagged viral proteins expressed in Huh7 cells using Flag-agarose beads and Western blotting using anti-Flag antibody. The right panel shows a higher exposure of the same blot (cropped) to reveal the signal of Met-Flag protein. The crosshatch symbol (#) denotes that PCP and Hel and V and ORF4 migrate at similar sizes. Mol Wt, molecular weight. (C) (Upper panel) Western blot showing GAPDH protein level in Huh7 cell extract expressing the indicated HA-tagged viral proteins. (Lower panel) Coimmunoprecipitation of HA-tagged viral proteins expressed in Huh7 cells using HA-agarose beads and Western blotting using anti-HA antibody. (D) Aliquots of CoIP samples (shown in panels B and C) were immunoblotted using the indicated antibodies.
FIG 2
FIG 2
Construction and analysis of the HEV-host PPI network. (A) Venn diagram comparing the data from the primary interaction partners (HHEV) with the available proteomic data sets. Red, yellow, green, and pink indicate HHEV and differentially regulated proteins in g-1 HEV-infected humans, g-3 HEV-infected swine, and a g-4 HEV-infected A549 cell line, respectively. (B) Schematic of the network analysis. Black, red, and blue nodes indicate viral protein and primary and secondary interaction partners, respectively. Red edge, virus-human (primary interaction partners) protein interaction (Virus-HHEV). Blue edge, all human-human (H-H) protein-protein interactions, including interactions among primary interaction partners (HHEV-HHEV) and experimentally validated interactions among all human proteins (H-H). (C) Virus-(HHEV-HHEV) PPI network. Yellow node, validated by CoIP. Yellow node with green border, virus-HHEV interactions validated by CoIP, published data. (D) Analysis of the HHEV-H data set using STRING database. (E) The translation factor pathway, imported from WikiPathways (ID: WP107). HEV primary and secondary interaction partners are shown in red and blue, respectively. eIF2S1, the target of eIF2AK4, is indicated in yellow.
FIG 3
FIG 3
Several host translation factors are present in the protein complex consisting of the viral RdRp, X, helicase, methyltransferase, PCP, V domain, and ORF4. (A) Coomassie (left) and Western blot (using ORF4 antibody; right) analysis of GST-ORF4 protein, purified from E. coli. (B) Coomassie (left) and Western blot (using X antibody; right) analysis of the X protein, purified from E. coli. (C) Silver stain (left) and Western blot (using myc antibody, right) analysis of RdRp-myc protein, purified by myc-agarose beads, from Huh7 cells overexpressing the protein. (D) Western blot (using Flag antibody) analysis of RdRp, helicase, PCP, and V proteins, purified by Flag-agarose beads, from Huh7 cells overexpressing them. (E) Western blot analysis (using HA antibody) of Y purified by HA-agarose beads, from Huh7 cells overexpressing them. (F) Myc-agarose pulldown assay using RdRp-Myc protein, followed by Western blot analysis of viral proteins and host factors using the indicated antibodies. (G) GST pulldown assay using GST-ORF4 protein, followed by Western blot analysis of virus and host factors using the indicated antibodies.
FIG 4
FIG 4
ORF1 polypeptide associates with host translation factors and assembles a multiprotein complex. (A) Western blot analysis of pGBKT7 and pGBKT7 ORF1 expressing Y2H gold whole-cell extract using anti-Myc antibody (upper panel). A shorter-exposure image is shown to clarify the band at the 75-kDa position (lower panel). Single asterisks (*) and double asterisks (**) denote processed ORF1 fragments. (B) Western blot analysis of pGBKT7 and pGBKT7 ORF1 expressing Y2H gold whole-cell extract using anti-Flag antibody. Single crosshatch symbols (#) and double crosshatch symbols (##) denote processed ORF1 fragments. (C) Coimmunoprecipitation of ORF1-Flag-associated proteins in Huh7 cells using Flag agarose beads (pUNO and pUNO ORF1, lanes 1 and 2 from left). GST-ORF4 was added to the lysate prior to addition of Flag agarose beads. Aliquots of IP samples were subjected to Western blotting using the indicated antibodies. Data represent results of a Flag-agarose pulldown assay of a mixture of in vitro-translated ORF1-Flag, GST-ORF4, and Huh7 whole-cell extracts (pGBKT7 and pGBKT7 ORF1, lanes 3 and 4 from left). Aliquots of eluates were subjected to Western blotting using the indicated antibodies. An uppercase Greek phi (Φ) denotes a processed ORF1 fragment. (D) Western blot analysis of pGBKT7 and pGBKT7 ORF1 synthesized by TNT using anti-Flag antibody. An uppercase Greek phi (Φ) denotes the processed ORF1 fragment.
FIG 5
FIG 5
Essential role of host translation regulatory factors in the assembly of viral translation/replication complex. (A) QRT-PCR of sense and antisense RNA of mock, WT HEV, and GAA HEV infection in Huh7 cells expressing various siRNAs/shRNA, as indicated. Relative RNA levels (normalized to that of GAPDH) are represented as means ± SEM. (B) QRT-PCR of sense and antisense RNA of g-1 HEV-infected ORF4-Huh7 cells expressing various siRNAs/shRNA, as indicated. Relative RNA levels (normalized to that of GAPDH) are represented as means ± SEM. (C) (Upper panel) HEV RdRp assay using the indicated siRNA/shRNAs and RdRp-Flag-transfected cells. (Lower panel) Western blot analysis of aliquots of RdRp protein used in the assay, performed using Flag antibody. (D) (Upper panel) HEV RdRp assay using affinity purified RdRp-Flag and the indicated viral proteins from Huh7 cells. (Lower panel) Western blot analysis of aliquots of RdRp protein used in the assay, performed using Flag antibody. (E) Myc-agarose pulldown assay of the indicated siRNAs/shRNA and RdRp-myc-transfected Huh7 cells, followed by Western blot analysis of various viral and host factors, performed using the indicated antibodies.

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