Fig 1.
Identifications of the interaction partners of HEV replication complex in ORF1 trans-complementation and replicon systems by proteomics approaches.
(A) Schematic representation of how SILAC and mass spectrometry identify host proteins interacting with ORF1 specifically. Cells expressing GFP-Flag or ORF1-Flag are cultured in “Heavy” or “Light” culture medium. Then, cells are lysed, immunoprecipitated with Flag antibody and analyzed by mass spectrometry. Proteins specifically appeared in ORF1 co-immunoprecipitated sample at high abundance were components of replication complex. (B) Interaction network of host factors associated with ORF1 (ORF1/GFP fold change>3 and score>5) was analyzed by STRING [60], and was drawn with Cytoscape. (C) Gene ontology analysis of host factors interacting with ORF1 was performed with the database for annotation, visualization and integrated Discovery (DAVID) [61]. (D) Schematic representation of Kernow C1/p6 ORF1HA-Flag BSR2AGluc replicon in which ORF1 is tagged with HA-Flag. (E) Workflow of purification and characterization of ORF1 replication complex with HEK293T Kernow C1/p6 ORF1HA-Flag BSR2AGluc replicon cells. (F) Interaction network of host proteins in ORF1 replication complex was analyzed by STRING, and was drawn with Cytoscape. (G) Comparison of proteins interacting with ORF1 in ORF1 trans-complementation and Kernow C1/p6 ORF1HA-Flag BSR2AGluc replicon systems.
Fig 2.
PRMT5/WDR77 complex interacts with ORF1.
(A) Co-IP assay of ORF1-Flag, WDR77-HA and PRMT5-HA. HEK293T cells were co-transfected with ORF1-Flag or EGFP-Flag and WDR77-HA, PRMT5-HA constructs. Cell lysates were incubated with Flag antibody. Immunoprecipitated samples were blotted with Flag and HA antibody. One star indicates WDR77 and two stars indicate PRMT5. (B) Co-IP assay of ORF1-Flag and endogenous PRMT5, WDR77. HEK293T cells was transfected with ORF1-Flag or EGFP-Flag plasmid, and cell lysates were incubated with Flag antibody. Immunoprecipitated sample were blotted with Flag, PRMT5 and WDR77 antibodies. (C) Western blot analysis of ORF1 replication complex purified from HEK293T Kernow C1/p6 ORF1HA-Flag BSR2AGluc replicon cells. Replication complex was immunoprecipitated with Flag antibody and blotted with Flag, PRMT5 and WDR77 antibodies. HEK293T cells expressing EGFP-Flag were included as the negative control. (D) Immunofluorescence of ORF1 and WDR77 in HEK293T Kernow C1/p6 ORF1HA-Flag BSR2AGluc replicon cells. Cells were stained with HA (green) and WDR77 (red) antibodies prior to analysis by confocal microscopy. Perinuclear puncta-like structures are shown (white arrowheads). The cell nuclei were stained with DAPI (blue). Line profiles corresponding to the white lines show colocalization. (E-F) Co-IP assay of ORF1 full-length, truncations and PRMT5, WDR77 or HA-WDR77. Flag tagged ORF1 full-length, its truncations or EGFP-Flag was expressed in HEK293T cells and immunoprecipitated by Flag antibody. Immunoprecipitated sample were blotted with Flag, PRMT5, WDR77 and HA antibodies. All data are representative of three independent experiments.
Fig 3.
PRMT5/WDR77 complex inhibits HEV infection.
(A) Western blot analysis of lysates from HepG2C3A cells transduced with PRMT5, WDR77 or non-targeting control plentiCRISPRv2 sgRNA lentivirus. Tubulin was used as the loading control. (B) HepG2C3A cells were infected with HEV Kernow C1/p6 virus. Immunofluorescence of ORF2 was performed at day 3 post infection with ORF2 monoclonal antibody (2G8) [20]. (C) Flow cytometry analysis of HEV infection by ORF2 staining using 2G8 antibody was performed at day 3 post infection. (D) RT-qPCR analysis of HEV genomic RNAs at day 3 post infection. (E) Intracellular viral titer of Kernow C1/p6 was determined at day 7 post transfection in S10-3 cells. Values are means plus standard deviations (SD) (error bars) (n = 3). *, P < 0.05; ***, P < 0.001 by one-way ANOVA. All data are representative of three independent experiments.
Fig 4.
PRMT5/WDR77 complex could not restrict HCV and SARS-CoV-2 infection.
(A) Western blot analysis of lysates from S10-3 cells transduced with PRMT5, WDR77 or non-targeting control sgRNA lentivirus. Tubulin was used as the loading control. (B) Flow cytometry analysis of EGFP expression at day 4 post HCV-NS5A-EGFP infection at MOI of 0.1. (C) Western blot analysis of lysates from Caco-2-N cells transduced with PRMT5, WDR77 or non-targeting control sgRNA lentivirus. Actin was used as the loading control. (D) Flow cytometry analysis of GFP expression at 24 hours post SARS-CoV-2 GFP/ΔN trVLP infection at MOI of 0.05. Data in all panels were representative of three independent experiments.
Fig 5.
PRMT5/WDR77 complex restricts HEV genome replication.
(A) Western blot analysis of lysates from S10-3 cells transduced with PRMT5, WDR77 or non-targeting control plentiCRISPRv2 sgRNA lentivirus. Actin was used as the loading control. (B-D) S10-3 cells were transfected with HEV replicon RNAs of Kernow C1/p6 Gluc replicon (B), Sar55 Gluc replicon (C) and pSHEV3 Gluc replicon (D). Supernatants were collected and Gluc activity was measured at day 2 post transfection. Data are normalized with non-targeting control sgRNA. Values are means plus standard deviations (SD) (error bars) (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001 by one-way ANOVA. All data are representative of three independent experiments.
Fig 6.
PRMT5/WDR77 complex catalyzes ORF1 R458 methylation to inhibit HEV replication in a methyltransferase activity dependent manner.
(A) Western blot analysis of lysates from S10-3 PRMT5 knockdown cells transduced with cDNA constructs of PRMT5 WT or E435Q/E444Q mutant. (B) PRMT5 KO cells rescued with PRMT5 WT or E435Q/E444Q mutant were transfected with Kernow C1/p6 Gluc replicon RNAs. Supernatant was collected and Gluc activity was measured at day 2 post transfection. Gluc activity was normalized with non-targeting sgRNA expressing vector control. (C) Schematic representation of arginine methylation identified by mass spectrometry in ORF1. (D) Replicon RNAs of Kernow C1/p6 Gluc WT and mutants were transfected into WT S10-3 or PRMT5 knockdown cells, and luciferase activity was measured at day 2 post transfection. (E) S10-3 cells were transfected with Kernow C1/p6 WT or R458K RNAs and RT-qPCR was performed at day 7 post transfection. (F) Relative abundance of methylation on R458 in HEK293T WT and PRMT5 knockdown cells. The precursor abundance ratio at R458 was calculated as the abundance of methylated R458 divided by the total abundance of R458. The relative abundance of methylation in WT cells was set as 100%. (G) WB analysis of S10-3 cells overexpressing PRMT5 and WDR77. (H) S10-3 cells overexpressing PRMT5 and WDR77 were transfected with Kernow C1/p6 Gluc WT or R458K mutant replicon RNAs and Gluc activity was measured at day 2 post transfection. Values are means plus standard deviations (SD) (error bars) (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significantly different by one-way ANOVA or Student’s t test. All data are representative of three independent experiments.