Fig 1.
SeV induces cytoplasmic condensation of EHMT1.
Confocal microscopic images of (A) HDFs infected with EmGFP SeV (green), (B) HDF infected with WT SeV immunolabelled for EHMT1 (red). (C) HEK-293 cells infected with EmGFP SeV (green), immunolabelled for EHMT1 (red). (D) BEAS-2B cells infected with EmGFP SeV (green), immunolabelled for EHMT1 (red) 48 h p.i. Composite images are with DAPI (blue) stained nuclei. Scale bar, 40 μm. Raw confocal microscopic images are deposited on BioImage Archive (Accession id: S-BIAD1362). EHMT1, euchromatic histone methyltransferase 1; HDF, human dermal fibroblast; SeV, Sendai virus; WT, wild-type.
Fig 2.
EHMT1 is recruited to Sendai viral IBs.
Confocal microscopic images of (A) BEAS-2B infected with WT SeV co-immunolabelled with EHMT1 (red) and SeV (green), (B) HEK infected with EmGFP SeV (green) and transfected with mCh_N (grey), immunolabelled with EHMT1 (red), (C) HEK infected with EmGFP SeV (green), transfected with piRFP_P (grey) and immunolabelled with EHMT1 (red). (D, E) HEK co-transfected with mCh_N (red) and piRFP_P (yellow) at a ratio of 1:1 (E) immunolabelled with EHMT1 (green). (F) HEK co-transfected with Egfp_L (green), mCh_N (red), and piRFP_P (grey) in a 3:6:2 ratio. Composites of all images are with DAPI (blue) stained nuclei. Scale bar, 40 μm. Raw confocal microscopic images are deposited on BioImage Archive (Accession id: S-BIAD1362). EHMT1, euchromatic histone methyltransferase 1; IB, inclusion body; SeV, Sendai virus; WT, wild-type.
Fig 3.
A distinct nucleo-cytoplasmic form of EHMT1 associates with SeV IBs.
(A) Western blotting of the whole cell, nuclear and cytoplasmic fractions of Uninfected BEAS-2B cells (UI) and EmGFP SeV infected (SV) at 48 h p.i. Blots probed with EHMT1, LaminB1, and Gapdh. (B) Nuclear and cytoplasmic fractions of BEAS-2B cells resolved on a 4% SDS-PAGE gel, immunoblotted with EHMT1. Confocal microscopic images of HEK transfected with (C) EHMT1B1-3_EGFP (green), (D) EHMT1B1-4_EGFP (green), and (E) EHMT1V09_EGFP (green). Confocal microscopic images of HEK co-transfected with mCh_N (red) and piRFP_P (grey) along with (F) EHMT1B1-3_EGFP (green), (G) EHMT1V09_EGFP (green), and (H) EHMT1B1-4_EGFP (green). Composites of all images are with DAPI (blue) stained nuclei. Scale bar, 40 μm. Source data are provided as S1 Data. Raw confocal microscopic images are deposited on BioImage Archive (Accession id: S-BIAD1362). EHMT1, euchromatic histone methyltransferase 1; IB, inclusion body; SeV, Sendai virus.
Fig 4.
EHMTs enzymatic activity regulates SeV IB formation and viral replication.
(A) Confocal microscopic composite images of BEAS-2B immunolabelled with SeV ab (red), marking the IBs in cells treated with DMSO, 3 μm BIX or 3 μm UNC and infected with WT SeV. (B) Graph plotted for the relative expression of SeV gRNA as assessed by qRT-PCR with the Ct values normalised against GAPDH. (n = 3 replicates, one-way ANOVA, **, p < 0.005, *, p < 0.05) (C) Confocal microscopic images of HEK co-transfected with EGFP_L (green), mCh_N (red), and piRFP_P (grey), treated with DMSO, 3 μm BIX or 3 μm UNC and simultaneously transfected with SeV DI RNA. (D) Graph representing the relative expression of SeV DI RNA in EHMT inhibitor treated conditions, as assessed by qRT-PCR. Ct values were normalised against beta-actin. (n = 4 replicates, one-way ANOVA, ****, p < 0.0001) (E, F) Haemolysis assay performed on WT SeV infected cells treated with DMSO, 3 μm BIX or 3 μm UNC. (E) Representative image of tubes containing supernatant from lysed RBCs, (F) graph depicting percentage haemolysis. (n = 4 replicates, one-way ANOVA, ****, p < 0.0001). Data from (B), (D), and (F) are mean ± SD. Source data are provided as S1 Data. Raw confocal microscopic images are deposited on BioImage Archive (Accession id: S-BIAD1362). EHMT, euchromatic histone methyltransferase; IB, inclusion body; SeV, Sendai virus; WT, wild-type.
Fig 5.
EHMTN/C influences SeV IB formation and viral replication via non-epigenetic mechanisms.
(A, B) HEK transfected with empty Cas9 plasmid or EH_132 gRNA (GFP reporter), infected with WT SeV. (A) Confocal microscopic composite images of the cells immunolabelled with SeV ab (red). (B) Graph plotted for the relative expression of SeV gRNA normalised with GAPDH as assessed by qRT-PCR (n = 5 replicates, unpaired t test, ****, p < 0.0001). (C–E) HEK transfected with SpCas9/EH_132, mCh_EH1_FL (mEL), followed by infection with WT SeV. (C) Schematic representation of the experimental procedure. (D) Confocal microscopic composite images of HEK expressing SpCas9/EH_132 (green), mCh_EH1_FL (grey), and immunolabelled with SeV ab (red). (E) Graph plotted for the relative expression of SeV gRNA normalised with GAPDH as assessed by qRT-PCR. (n = 3 replicates, unpaired t test, p < 0.0001 (****)) (F, G) HEK were transfected with SpCas9 or EH_132, infected with WT SeV and simultaneously treated with BX795. (F) Confocal microscopic composite images of HEK expressing SpCas9/EH_132 (green), immunolabelled for SeV (red). Nuclei of all images are counterstained with DAPI (blue). Scale bar, 40 μm. (G) Graph plotted for the relative expression of SeV gRNA normalised with GAPDH as assessed by qRT-PCR. (n = 3 replicates, one-way ANOVA, *, p < 0.05). Data from (B), (E), and (G) are mean ± SD. Source data are provided as S1 Data. Raw confocal microscopic images are deposited on BioImage Archive (Accession id: S-BIAD1362). EHMT, euchromatic histone methyltransferase; IB, inclusion body; SeV, Sendai virus; WT, wild-type.
Fig 6.
EHMT1N/C interacts with and methylates the SeV nucleoprotein upon infection.
(A, B) GFP IP from the cytoplasmic fraction of cells triple transfected with mCh_N, piRFP_P and (A) EHMT1B1-3_EGFP or (B) EHMT1V09_EGFP; elute western blotted and probed for GFP (top) and mCherry (bottom). (C, D) mCherry IP from cells triple transfected with mCh_N, piRFP_P and (C) EHMT1B1-3_EGFP or (D) EHMT1V09_EGFP, elute western blotted and probed for GFP (top) and mCherry (red). (E) EHMT1 IP from cells transfected either with mCh_N or co-transfected with mCh_N + piRFP_P, elute western blotted and probed with EHMT1, mCherry, and SeV. (F) EHMT1 RNA-IP graph representing fold enrichment of beta-actin and SeV gRNA, normalised with IgG. Data are mean ± SD. (n = 3, Ratio paired t test; *, p < 0.05) SeV gRNA demonstrated about 1,000-fold higher enrichment in comparison with beta-actin. (G, H) EHMT1 IP from the cytoplasmic fraction of cells infected with SeV, elute western blotted and probed with (G) SeV and (H) meK. (I) BEAS-2B cells were treated with BIX/UNC and simultaneously infected with WT SeV; cells were fractionated 24 h post infection. The cytoplasmic fraction was western blotted and probed with EHMT1, SeV, meK, and GAPDH. Source data are provided as S1 Data. EHMT1, euchromatic histone methyltransferase 1; SeV, Sendai virus; WT, wild-type.
Fig 7.
Incorporation of EHMT1N/C into SeV IBs correlates with the formation of large IB platforms and enhanced SeV replication.
(A) Live cell confocal microscopic images of HEK transfected with mCh_N (red) and infected with EmGFP SeV (green) at 48 h p.i.; arrows represent fusion and fission of SeV IBs. (B) Confocal microscopic images of BEAS-2B cells infected with WT SeV, fixed and co-immunolabelled for EHMT1 (red) and SeV (green) at indicated time points. (C–E) BEAS-2B cells infected with WT or EmGFP SeV were fixed and immunolabelled with SeV at various time points postinfection. Confocal microscopic images were analysed by ImageJ. (C, E) Graph representing the ±SEM of total number of IBs formed per cell for WT SeV and EmGFP SeV infected cells, respectively. (D, F) Graph representing the area ±SEM of IBs >10 μm2 per cell for WT SeV and EmGFP SeV infected cells, respectively [n > 25 cells, Brown–Forsythe and Welch ANOVA tests]. (G, H) Replication of SeV genomic RNA at indicated time points postinfection. Graphs representing the Ct values for SeV genomic RNA normalised with GAPDH for (G) WT SeV and (H) EmGFP SeV. Data from G and H are mean ± SD. (n = 4 replicates, repeated measures (RM) one-way ANOVA) (I) BEAS-2B cells were infected with WT SeV, fixed and co-immunolabelled with EHMT1 and SeV at indicated time points postinfection. Graph representing the percentage colocalization of EHMT1 with SeV in the 3 subpopulations of IBs, analysed by the Volocity Image Analysis software. Data are ±SEM. (n > 25 cells, ordinary one-way ANOVA) p-value: 0.1234 (ns), 0.0332 (*), 0.0021 (**), 0.0002 (***), <0.0001 (****). (J) Western blotting of the cytoplasmic fraction of SeV infected cells at indicated time points postinfection, immunoprobed for SeV, meK, and GAPDH. Source data are provided as S1 Data. Raw confocal microscopic images are deposited on BioImage Archive (Accession id: S-BIAD1362). EHMT1, euchromatic histone methyltransferase 1; IB, inclusion body; SeV, Sendai virus; WT, wild-type.
Fig 8.
Methyltransferase activity of EHMT1N/C catalyses the growth of IBs to form larger structures.
(A–M) Graphs representing the number and area of IBs analysed from confocal microscopic images by ImageJ analysis tool. (A–C) BEAS-2B cells were infected with WT SeV and treated with 3 μm of BIX/UNC. The cells were fixed and immunolabelled with SeV antibody 16 h p.i. Graphs representing (A) total number of IBs per cell, (B) distribution of area of the whole IB population, (C) area of IBs >10 μm2. (N = 3 replicates, n > 100 cells, ordinary one-way ANOVA) (D–F) HEK were co-transfected with mCh_N, piRFP_P, and EGFP_L in a 3:6:2 ratio, followed by transfection with SeV DI RNA 12 h post transfection of the plasmids and simultaneously treated with 3 μm of BIX/UNC. The cells were fixed and processed for confocal microscopy 12 h post transfection of RNA. Graphs representing (D) total number of IBs, (E) overall area of the whole IB population, and (F) area of IBs >10 μm2. (N = 3 replicates, n > 100 cells, ordinary one-way ANOVA) (G–I) HEK were transfected with empty SpCas9 or EH_132 plasmid to deplete the levels of EHMT1 and infected with WT SeV; cells were immunolabelled with SeV 16 h p.i. Graphs representing (G) total number of IBs per cell, (H) distribution of area of the whole IB population, (I) area of IBs >10 μm2. (N = 3 replicates, n > 100 cells, unpaired t test), p-value: 0.1234 (ns), 0.0332 (*), 0.0021 (**), 0.0002 (***), <0.0001 (****). (J–M) HEK were transfected with SpCas9 or EH_132, infected with WT SeV and either untreated or simultaneously treated with BX795. Graphs representing (J) total number of IBs per cell, (K) distribution of area of the whole IB population, (L) area of IBs >10 μm2 and (M) number of IBs of the 0.1–1 μm2 category. (N = 3 replicates, n > 100 cells, unpaired t test between the indicated groups). Data from A to M are ±SEM. Source data are provided as S1 Data. EHMT1, euchromatic histone methyltransferase 1; IB, inclusion body; SeV, Sendai virus; WT, wild-type.
Fig 9.
Cytoplasmic condensation of EHMT1 is conserved among other members of the single-strand RNA virus family.
Confocal microscopic images of (A) MEF uninfected or infected with Chandipura virus (ChpV) at 6 h and 8 h p.i., immunolabelled with EHMT1 (yellow) and ChpV_L (red). Arrows indicate cytoplasmic condensates of EHMT1 colocalizing with ChpV IBs marked by the L protein. (B, C) HepG2 cells uninfected or infected with Dengue virus, immunolabelled with (B) Nsp1 (red) or (C) EHMT1 (red) at 24 h and 48 h p.i. Composite of all images are with DAPI (blue) stained nuclei. Scale bar, 40 μm. Raw confocal microscopic images are deposited on BioImage Archive (Accession id: S-BIAD1362). EHMT1, euchromatic histone methyltransferase 1; IB, inclusion body; MEF, mouse embryonic fibroblast.
Fig 10.
Model demonstrating distinct Nucleo-cytoplasmic form of EHMT1, recruited by the virus to IBs, facilitates IB coalescence by methylation of the Nucleoprotien.
Upon SeV infection, the cytoplasmic form of EHMT1, EHMT1N/C is recruited to SeV IBs by the N and P proteins in a time-dependent manner as infection progresses. EHMT1N/C plays an enzymatically active role in coalescence of IBs by methylating N, leading to the formation of large IBs and efficient viral replication. Inhibition of EHMTs activity or depletion of EHMT1N/C leads to impairment in coalescence of IBs. EHMT, euchromatic histone methyltransferase; IB, inclusion body; SeV, Sendai virus.
Table 1.
Primers used for cloning.
Table 2.
Primers used for qRT-PCR.