Journal: PLOS Pathogens

Article Title: Paramyxovirus matrix protein redirects METTL3 for dual regulation of viral replication and immune evasion

doi: 10.1371/journal.ppat.1013755

Figure Lengend Snippet: HeLa cells were transfected with FLAG-METTL3 expression plasmid, and at 24 h post-transfection (hpt), the cells were infected with rBPIV3-EGFP at an MOI of 1. At 48 h post-infection (hpi), the cells were fixed and costained with anti-FLAG antibody (Ab) for METTL3 and anti-BPIV3-N Ab (A). Arrowheads indicate regions where N and METTL3 fluorescence signals colocalize within the cells. The bar graph shows the distribution of METTL3 subcellular localization. For classification-based quantification, more than 30 METTL3 single- or METTL3/N double-positive cells per condition were randomly selected and classified into two localization patterns (“nucleus only” or “cytoplasmic or nucleus”). The number of cells displaying each localization pattern was counted for each condition from three independent experiments, and the results are presented as the percentage of total cells analyzed (B). HeLa cells were transfected with FLAG-METTL3 plasmid and, 24 hpt, infected with rBPIV3-EGFP at an MOI of 1. At 48 hpi, cells were fixed and co-stained with antibodies against dsRNA and FLAG-METTL3 (C) or dsRNA and endogenous METTL3 (endo METTL3) (D). Representative images from three independent experiments are shown. Manders’ colocalization coefficient between dsRNA and FLAG-METTL3 (E) or dsRNA and endogenous METTL3 (F) fluorescence signals in panels C and D was calculated using the Coloc 2 plugin in Fiji/ImageJ software. The coefficient was determined for each cell, and results represent analyses of 6–7 individual cells per condition from three independent experiments. Bars indicate the mean ± SD. White boxes in panels C and D indicate regions shown as magnified views in the corresponding lower-right insets. Data are representative of three independent experiments (n = 3). Asterisks indicate significance (* p < 0.05); ns, not significant.

Article Snippet: The following antibodies were used as primary Abs: mouse anti-MeV-M monoclonal Ab (mAb) (clone E388) kindly provided by T. A. Sato, mouse anti-FLAG Ab (proteintech, Cat. #66008–7-Ig), rabbit-FLAG Ab (proteintech, Cat. #66008–4-Ig), mouse anti-HA monoclonal Ab (mAb) (proteintech, Cat. #66006–2-Ig), rabbit anti-HA tag pAb (MBL, Cat. #561), rabbit anti-CRM1 (exportin-1) pAb (proteintech, Cat. #27917–1-AP), mouse anti-double-stranded RNA Ab clone J2 (Nordic MUbio, Cat. #10010200), mouse anti-m6A mAb (Sigma, Cat. #MS1124), rabbit anti-METTL3 pAb (GeneTex, Cat. #GTX105037), rabbit anti-METTL14 pAb (Sigma-Aldrich, Cat. #HPA038002), and HRP-conjugated alpha tubulin mAb (proteintech, Cat. #HRP-66031).

Techniques: Transfection, Expressing, Plasmid Preparation, Infection, Fluorescence, Staining, Software

Journal: PLOS Pathogens

Article Title: Paramyxovirus matrix protein redirects METTL3 for dual regulation of viral replication and immune evasion

doi: 10.1371/journal.ppat.1013755

Figure Lengend Snippet: HeLa cells were cotransfected with METTL3 and M expression plasmid or an empty vector. At 48 h post-transfection (hpt), the cells were fixed and costained with anti-BPIV3-M antibody (Ab) and anti-FLAG Ab for METTL3 (A). HeLa cells were transfected with METTL3 expression plasmid, and at 24 hpt, the cells were infected with rBPIV3-EGFP at an MOI of 1. At 48 h post-infection (hpi), the cells were fixed and costained with anti-FLAG antibody (Ab) for METTL3 and anti-BPIV3-M Ab (B). Cell nuclei were stained with DAPI. Insets represent an enlargement of the areas indicated by a small square. Arrowheads indicate cytoplasmic colocalization of M with METTL3. Representative images from three independent experiments are shown. For classification-based quantification, more than 20 METTL3 single-positive or METTL3/M double-positive cells per condition were randomly selected and categorized into two subcellular localization patterns (“nucleus only” or “cytoplasmic or nucleus”). The number of cells in each category was counted, and the data are presented as the percentage of total cells analyzed from n = 3 independent experiments. (C and D). Asterisks indicate statistically significant differences (* p < 0.05); ns, not significant.

Article Snippet: The following antibodies were used as primary Abs: mouse anti-MeV-M monoclonal Ab (mAb) (clone E388) kindly provided by T. A. Sato, mouse anti-FLAG Ab (proteintech, Cat. #66008–7-Ig), rabbit-FLAG Ab (proteintech, Cat. #66008–4-Ig), mouse anti-HA monoclonal Ab (mAb) (proteintech, Cat. #66006–2-Ig), rabbit anti-HA tag pAb (MBL, Cat. #561), rabbit anti-CRM1 (exportin-1) pAb (proteintech, Cat. #27917–1-AP), mouse anti-double-stranded RNA Ab clone J2 (Nordic MUbio, Cat. #10010200), mouse anti-m6A mAb (Sigma, Cat. #MS1124), rabbit anti-METTL3 pAb (GeneTex, Cat. #GTX105037), rabbit anti-METTL14 pAb (Sigma-Aldrich, Cat. #HPA038002), and HRP-conjugated alpha tubulin mAb (proteintech, Cat. #HRP-66031).

Techniques: Expressing, Plasmid Preparation, Transfection, Infection, Staining

Journal: PLOS Pathogens

Article Title: Paramyxovirus matrix protein redirects METTL3 for dual regulation of viral replication and immune evasion

doi: 10.1371/journal.ppat.1013755

Figure Lengend Snippet: (A) Schematic representation of BPIV3-M point mutants. BPIV3-M contains a nuclear export signal (NES) at L106 and L107 and a ubiquitination site (Ubi site) at K258. L106 and L107, or K258 in BPIV3-M, were substituted with alanine (M-L106A/L107A) or arginine (M-K258R) residues as indicated, respectively, and the substituted residue is shown as a red letter. (B) HeLa cells were cotransfected with expression plasmids of wt-BPIV3-M, M-L106A/L107A, and K258R along with FLAG-METTL3 expression plasmid. At 48 h post-transfection (hpt), the cells were costained with anti-M antibody (Ab) and anti-FLAG Ab for METTL3. (C) HeLa cells were transfected with the METTL3 expression plasmid. At 24 hpt, the cells were infected with rBPIV3-EGFP at an MOI of 1. At 4 h post-infection (hpi), DMSO or 5 ng/mL Leptomycin B (LMB) was added, and the cells were incubated for 48 h in the presence of LMB. Then, the cells were fixed, and METTL3 and BPIV3-N were costained. (D) HeLa cells were transfected with siRNA targeting Exportin-1 (si-Exportin-1) or control siRNA (si-Control). At 48 hpt, cells were lysed and subjected to immunoblotting with anti–Exportin-1 antibody to confirm knockdown efficiency. (E) HeLa cells were cotransfected with siRNA for Exportin-1 and METTL3 expression plasmid. At 24 hpt, the cells were infected with rBPIV3-EGFP at an MOI of 1. The cells were fixed at 48 hpi and costained for BPIV3-N and METTL3. Representative images from three independent experiments are shown. For classification-based quantification, more than 20 METTL3/M double-positive cells (F), METTL3/N double-positive cells (G) , or METTL3/N double-positive cells under siRNA conditions (H) per condition were randomly selected and categorized into two subcellular localization patterns (“nucleus only” or “cytoplasmic or nucleus”). The number of cells in each category was counted, and the data are presented as the percentage of total cells analyzed from n = 3 independent experiments. Asterisks indicate statistically significant differences (* p < 0.05).

Article Snippet: The following antibodies were used as primary Abs: mouse anti-MeV-M monoclonal Ab (mAb) (clone E388) kindly provided by T. A. Sato, mouse anti-FLAG Ab (proteintech, Cat. #66008–7-Ig), rabbit-FLAG Ab (proteintech, Cat. #66008–4-Ig), mouse anti-HA monoclonal Ab (mAb) (proteintech, Cat. #66006–2-Ig), rabbit anti-HA tag pAb (MBL, Cat. #561), rabbit anti-CRM1 (exportin-1) pAb (proteintech, Cat. #27917–1-AP), mouse anti-double-stranded RNA Ab clone J2 (Nordic MUbio, Cat. #10010200), mouse anti-m6A mAb (Sigma, Cat. #MS1124), rabbit anti-METTL3 pAb (GeneTex, Cat. #GTX105037), rabbit anti-METTL14 pAb (Sigma-Aldrich, Cat. #HPA038002), and HRP-conjugated alpha tubulin mAb (proteintech, Cat. #HRP-66031).

Techniques: Ubiquitin Proteomics, Residue, Expressing, Plasmid Preparation, Transfection, Infection, Incubation, Control, Western Blot, Knockdown

Journal: PLOS Pathogens

Article Title: Paramyxovirus matrix protein redirects METTL3 for dual regulation of viral replication and immune evasion

doi: 10.1371/journal.ppat.1013755

Figure Lengend Snippet: (A) 293T cells were cotransfected with M expression plasmid together with FLAG-METTL3 expression plasmid. For infected cells, the cells were transfected with FLAG-METTL3 expression plasmid and infected with rBPIV3-EGFP at an MOI of 1 at 24 h post-transfection (hpt). At 72 hpt, the cells were harvested and subjected to coimmunoprecipitation with anti-FLAG antibody (Ab). The precipitates were analyzed by western blotting using anti-BPIV3-M Ab. (B) Schematic representation of human METTL3. ZF1 and ZF2: two zinc finger domains, MTD: methyltransferase domain, DPPW: DPPW motif. The 1-400, 1-380, 1-200, and 161-580 deletion mutants lacking each domain are shown. 293T cells were cotransfected with the deletion mutants along with BPIV3-M. At 48 hpt, the cells were harvested and subjected to coimmunoprecipitation with anti-FLAG Ab as described in the legend of . (C) Schematic representation of FLAG- and GST-tagged deletion mutants of the BPIV3 M protein used to map the METTL3 interaction domain. Cells were transfected with a FLAG-METTL3 expression plasmid, and cells were harvested at 48–72 h post-transfection. Deletion mutants of the M protein fused with FLAG and GST tags were synthesized using a wheat germ cell-free expression system. Each FLAG-GST-tagged M deletion mutant was incubated with glutathione–sepharose beads at 4°C, followed by the addition of lysates containing FLAG-METTL3. After incubation, the beads were extensively washed, and bound proteins were analyzed by western blotting. Both METTL3 and M deletion mutants were detected using an anti-FLAG antibody. Each experiment was performed at least twice.

Article Snippet: The following antibodies were used as primary Abs: mouse anti-MeV-M monoclonal Ab (mAb) (clone E388) kindly provided by T. A. Sato, mouse anti-FLAG Ab (proteintech, Cat. #66008–7-Ig), rabbit-FLAG Ab (proteintech, Cat. #66008–4-Ig), mouse anti-HA monoclonal Ab (mAb) (proteintech, Cat. #66006–2-Ig), rabbit anti-HA tag pAb (MBL, Cat. #561), rabbit anti-CRM1 (exportin-1) pAb (proteintech, Cat. #27917–1-AP), mouse anti-double-stranded RNA Ab clone J2 (Nordic MUbio, Cat. #10010200), mouse anti-m6A mAb (Sigma, Cat. #MS1124), rabbit anti-METTL3 pAb (GeneTex, Cat. #GTX105037), rabbit anti-METTL14 pAb (Sigma-Aldrich, Cat. #HPA038002), and HRP-conjugated alpha tubulin mAb (proteintech, Cat. #HRP-66031).

Techniques: Expressing, Plasmid Preparation, Infection, Transfection, Western Blot, Synthesized, Mutagenesis, Incubation

Journal: PLOS Pathogens

Article Title: Paramyxovirus matrix protein redirects METTL3 for dual regulation of viral replication and immune evasion

doi: 10.1371/journal.ppat.1013755

Figure Lengend Snippet: (A) A schematic diagram of the RNA immunoprecipitation (RIP) assay used to detect METTL3-associated N mRNA. (B) 293T cells were transfected with METTL3 expression plasmids. At 24 h post-transfection (hpt), the cells were infected with rBPIV3-EGFP at an MOI of 1 for 48 h. The infected cells were lysed, and the extracts were subjected to RNA immunoprecipitation assay with anti-FLAG antibody (Ab). After immunoprecipitation, cDNA of BPIV3-N mRNA captured by METTL3 was synthesized and the amount of N mRNA was quantified by qPCR. Followed by normalization to input RNA values, relative enrichment values of N mRNA were expressed relative to control IgG values using the ΔΔCt method. (C) SRAMP was used to predict the m6A site of the N gene derived from the BPIV3 genome. The vertical axis shows the m6A score and the horizontal axis shows the position number of the base of the N gene where N6-methyladenosine is present within the m6A motif. A yellow bar indicates low-scoring m6A site, N327, and red bars indicate high-scoring m6A sites, N872, N1146, N1372, N1427, and N1443. (D) Schematic representation of the MeRIP (m6A RNA immunoprecipitation) assay. (i) The m6A-containing RNA fragments were bound by an m6A-specific Ab. (ii) Viral N mRNA containing m6A modifications was fragmented. (iii) Antibody–RNA complexes were captured using protein affinity beads. (iv) RNA was eluted, reverse transcribed into cDNA, and amplified by qPCR using primers specific for the indicated N gene regions (N327, N872, N1146, and N1372–1443). (E) 293T cells were infected with rBPIV3-EGFP at an MOI of 1. At 72 h post-infection, the infected cells were harvested, and only mRNA from total RNA was purified. The m6A-modified mRNA was immunoprecipitated with m6A-specific Ab and magnetic beads. Followed by cleavage of the captured mRNA with the cleavage enzyme, the m6A-specific Ab-captured mRNA was eluted from magnetic beads and purified. cDNA was synthesized by reverse transcription reaction, and the m6A sites in N mRNA were then identified by qPCR. The relative value of each to the mRNA value of input RNA was calculated using the ΔΔCt method. Data represent the mean ± SD from n = 3 independent experiments. Asterisks represent statistically significant differences (* p < 0.05). ns; not significant.

Article Snippet: The following antibodies were used as primary Abs: mouse anti-MeV-M monoclonal Ab (mAb) (clone E388) kindly provided by T. A. Sato, mouse anti-FLAG Ab (proteintech, Cat. #66008–7-Ig), rabbit-FLAG Ab (proteintech, Cat. #66008–4-Ig), mouse anti-HA monoclonal Ab (mAb) (proteintech, Cat. #66006–2-Ig), rabbit anti-HA tag pAb (MBL, Cat. #561), rabbit anti-CRM1 (exportin-1) pAb (proteintech, Cat. #27917–1-AP), mouse anti-double-stranded RNA Ab clone J2 (Nordic MUbio, Cat. #10010200), mouse anti-m6A mAb (Sigma, Cat. #MS1124), rabbit anti-METTL3 pAb (GeneTex, Cat. #GTX105037), rabbit anti-METTL14 pAb (Sigma-Aldrich, Cat. #HPA038002), and HRP-conjugated alpha tubulin mAb (proteintech, Cat. #HRP-66031).

Techniques: RNA Immunoprecipitation, Transfection, Expressing, Infection, Immunoprecipitation, Synthesized, Control, Derivative Assay, Reverse Transcription, Amplification, Purification, Modification, Magnetic Beads