Journal: The Journal of General Virology

Article Title: Ubiquitin-like modifier-activating enzyme 1 interacts with Zika virus NS5 and promotes viral replication in the infected cell

doi: 10.1099/jgv.0.002063

Figure Lengend Snippet: UBA1 interacts with orthoflaviviral NS5 proteins. ( a ) UBA1 is co-immunoprecipitated with ZIKV, USUV and WNV NS5 proteins. 293 T cells were transfected with constructs expressing HA-tagged versions of each NS5 (ZIKV, USUV and WNV), EYFP (pEYFP-N1, shown as pEYFP) or an empty vector (pcDNA3.1, shown as pcDNA). NS5 proteins were immunoprecipitated with anti-HA agarose beads. ( b ) UBA1 coprecipitated with recombinant His-tagged ZIKV NS5 in assays using Ni-NTA agarose resin (Invitrogen). For the assay, 3 mg of whole-cell protein, extracted from 293 T cells, was incubated in the presence of either 90 µg of recombinant BSA or His-tagged ZIKV NS5.

Article Snippet: The plates were then processed following the immunocytochemistry protocols previously described in our laboratory [ ], using mouse anti-UBA1 (Santa Cruz, sc-53555), rabbit anti-UBA1 (Invitrogen, MA5-35786), rabbit anti-ZIKV NS5 (GeneTex, GTX133328) and mouse anti-dsRNA (MUbio, reference 10010200) at a 1:500 dilution.

Techniques: Immunoprecipitation, Transfection, Construct, Expressing, Plasmid Preparation, Recombinant, Incubation

Journal: The Journal of General Virology

Article Title: Ubiquitin-like modifier-activating enzyme 1 interacts with Zika virus NS5 and promotes viral replication in the infected cell

doi: 10.1099/jgv.0.002063

Figure Lengend Snippet: ZIKV NS5 colocalizes with UBA1 in the infected cell. Fluorescence signals emitted by UBA1 and NS5 overlap in nuclear and cytoplasmic regions of the infected cells at 36 h post-infection (merge). To detect NS5 (green) and UBA1 (red), specific rabbit anti-NS5 and mouse anti-UBA1 antibodies were used. Nuclei were stained with DAPI (blue). All signals combined are represented (merge). ( a, c ) UBA1 and NS5 are detected in the nuclei of ZIKV-infected cells. ( b, d ) Close-up view of the areas where colocalization between UBA1 and NS5 was observed. An amplification of the image selected in the dotted square area in ( a ) and (c) is provided in (b) and ( d ), respectively. ( e ) Colocalization of NS5 and UBA1 in the cytoplasmic region of A549 cells. ( f ) An amplification of the image selected in the dotted square area in ( e ) is provided. Arrows indicate those regions where colocalization is detected.

Article Snippet: The plates were then processed following the immunocytochemistry protocols previously described in our laboratory [ ], using mouse anti-UBA1 (Santa Cruz, sc-53555), rabbit anti-UBA1 (Invitrogen, MA5-35786), rabbit anti-ZIKV NS5 (GeneTex, GTX133328) and mouse anti-dsRNA (MUbio, reference 10010200) at a 1:500 dilution.

Techniques: Infection, Fluorescence, Staining, Amplification

Journal: The Journal of General Virology

Article Title: Ubiquitin-like modifier-activating enzyme 1 interacts with Zika virus NS5 and promotes viral replication in the infected cell

doi: 10.1099/jgv.0.002063

Figure Lengend Snippet: Enhanced ZIKV replication in cells overexpressing UBA1. ( a, b ) UBA1 is overexpressed in 293 T cells transfected with either pUBA1 ( a ) or pFLAG-UBA1 ( b ) when compared to untransfected cultures (mock) or cells transfected with an empty construct (pcDNA3.1, namely, pcDNA). ( a ) Larger levels of UBA1 detected in cells transfected with pUBA1; tubulin was used as a reference loading control. ( b ) Larger amounts of UBA1 detected in cells transfected with pFLAG-UBA1 when using a specific anti-UBA1 antibody. A positive band of the expected size for UBA1 was detected with an anti-FLAG antibody. Tubulin was used as a reference loading control. Representative images were included, which were obtained from two independent biological samples (two different WBs for each sample). ( c ) Virus titres obtained in cells previously transfected with pUBA1, pFLAG-UBA1 or an empty vector (pcDNA). Cells were infected with ZIKV at an m.o.i. of 1 TCID 50 /cell, and samples collected at different time points were titrated (four independent biological replicas). Significant differences with respect to mock-transfected cells (pcDNA) are indicated (two-way ANOVA, Dunnett’s multiple comparisons test; * P <0.05). ( d ) Representative WB of ZIKV NS5 and UBA1 protein levels in cells previously transfected (36 h earlier) with pUBA1, pFLAG-UBA1 or an empty vector (pcDNA); as a loading reference control, an anti-tubulin antibody was used (four independent biological replicas). ( e ) Ratio of NS5 and UBA1 protein levels relative to mock-transfected cells (empty vector) at 36 h post-infection. Tubulin was used as a reference control (four biological replicas).

Article Snippet: The plates were then processed following the immunocytochemistry protocols previously described in our laboratory [ ], using mouse anti-UBA1 (Santa Cruz, sc-53555), rabbit anti-UBA1 (Invitrogen, MA5-35786), rabbit anti-ZIKV NS5 (GeneTex, GTX133328) and mouse anti-dsRNA (MUbio, reference 10010200) at a 1:500 dilution.

Techniques: Transfection, Construct, Control, Virus, Plasmid Preparation, Infection

Journal: bioRxiv

Article Title: PTBP1 is upregulated by Zika virus infection via HIF-1α signal and hijacks NS1 protein to induce NS1 degradation to restrain viral replication

doi: 10.1101/2024.11.19.624259

Figure Lengend Snippet: (A-C) A549 cells were infected with ZIKV at an MOI of 1 for different durations (0, 12, 24, and 48 h). The cells were collected for ZIKV RNA level detected by qRT-PCR (A), PTBP1 mRNA (B), and protein (C) levels detected by qRT-PCR and Western blot, respectively. (D-F) A549 cells were infected with ZIKV at MOIs of 0, 0.25, 0.5, and 1.0 for 24 h. ZIKV RNA level was detected by qRT-PCR (A), while PTBP1 mRNA (B) and protein (C) levels were detected by qRT-PCR and Western blot, respectively. Graphs were expressed as Mean±SD, n = 3. ns, not-significant; **, P < 0.01; ***, P < 0.001.

Article Snippet: Rabbit polyclonal antibodies against ZIKV NS5 (Cat: GTX133312) and NS1 (Cat: GTX634159) were obtained from Genetex Inc. (Irvine, CA, USA).

Techniques: Infection, Quantitative RT-PCR, Western Blot

Journal: bioRxiv

Article Title: PTBP1 is upregulated by Zika virus infection via HIF-1α signal and hijacks NS1 protein to induce NS1 degradation to restrain viral replication

doi: 10.1101/2024.11.19.624259

Figure Lengend Snippet: (A) A549 cells were infected with ZIKV at an MOI of 1 at different time points (0, 12, 24, and 48 h). Cells were collected at the corresponding time points for Western blot analysis. (B and C) A549 cells were stimulated with different doses of IOX2 (0, 20, and 40 μM) for 12 h. Cells were collected for qRT-PCR (B) and Western blot (C) analysis, respectively. (D and E) A549 cells were stimulated with IOX2 (40 μM) for 12 h, followed by infection with ZIKV (MOI = 1) for an additional 24 h. Cells were collected for qRT-PCR (D) and Western blot (E) analysis, respectively. (F and G) A549 cells were stimulated with a HIF-1α inhibitor YC-1 (20 μM) for 16 hours, followed by infection with ZIKV (MOI = 1) for another 24 h. Cells were collected for qRT-PCR (F) and Western blot (G) analysis, respectively. Graphs were expressed as Mean±SD, n = 3. ns, not-significant; **, P < 0.01; ***, P < 0.001.

Article Snippet: Rabbit polyclonal antibodies against ZIKV NS5 (Cat: GTX133312) and NS1 (Cat: GTX634159) were obtained from Genetex Inc. (Irvine, CA, USA).

Techniques: Infection, Western Blot, Quantitative RT-PCR

Journal: bioRxiv

Article Title: PTBP1 is upregulated by Zika virus infection via HIF-1α signal and hijacks NS1 protein to induce NS1 degradation to restrain viral replication

doi: 10.1101/2024.11.19.624259

Figure Lengend Snippet: (A) LV-PTBP1 and LV-Vector control cells were subjected to Western blot analysis to detect the expression of FLAG-tagged protein. (B-D) LV-PTBP1 and LV-Vector cells were infected with ZIKV (MOI = 1) at different time points (0, 24, and 48 h). Cells were collected at the corresponding time points for qRT-PCR (B) and Western blot (C) analysis, respectively. Supernatants was subjected to TCID50, and the virus titer was calculated (D). (E) The stable expressing shPTBP1 and shNC control cells were subjected to Western blot analysis. (F-H) shPTBP1 and shNC cells were infected with ZIKV (MOI = 1) at different time points (0, 24, and 48 h). Cells were collected at the corresponding time points for qRT-PCR (F) and Western blot (G) analysis, respectively. Supernatants were subjected to TCID50, and the virus titer was calculated (H). Graphs were expressed as Mean±SD, n = 3. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Rabbit polyclonal antibodies against ZIKV NS5 (Cat: GTX133312) and NS1 (Cat: GTX634159) were obtained from Genetex Inc. (Irvine, CA, USA).

Techniques: Plasmid Preparation, Control, Western Blot, Expressing, Infection, Quantitative RT-PCR, Virus

Journal: bioRxiv

Article Title: PTBP1 is upregulated by Zika virus infection via HIF-1α signal and hijacks NS1 protein to induce NS1 degradation to restrain viral replication

doi: 10.1101/2024.11.19.624259

Figure Lengend Snippet: (A-C) shPTBP1 cells and shNC control cells were treated with different concentrations of IOX2 (0, 30, 40, 50 μM) for 12 hours and then infected with ZIKV (MOI = 1). Cells were collected 24 hours post-infection for qRT-PCR (A) and Western blot (B) analysis, respectively. Supernatants were subjected to TCID50, and the virus titer was calculated (C). Graphs were expressed as Mean±SD, n = 3. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Rabbit polyclonal antibodies against ZIKV NS5 (Cat: GTX133312) and NS1 (Cat: GTX634159) were obtained from Genetex Inc. (Irvine, CA, USA).

Techniques: Control, Infection, Quantitative RT-PCR, Western Blot, Virus

Journal: bioRxiv

Article Title: PTBP1 is upregulated by Zika virus infection via HIF-1α signal and hijacks NS1 protein to induce NS1 degradation to restrain viral replication

doi: 10.1101/2024.11.19.624259

Figure Lengend Snippet: (A) HEK293T cells were transfected with the HA-Vector (2.0, 1.75, 1.5, 1.25, and 0.75 μg) and FLAG-Vector (1.0, 0, 0, 0, 0 μg) or FLAG-NS1 (0, 0, 0, 0, 0.5 μg), or FLAG-NS5 (0, 0, 0, 0, 0.5 μg) or HA-PTBP1 (0, 0, 0.25, 0.5, 1.0) along with eGFP-C1 (0, 0.25, 0.25, 0.25, 0.25 μg) plasmids. After 30 h, the cells were collected and subjected to Western blot analysis. (B) The scheme of two main models of protein degradation: UPS (ubiquitin-proteasome system) and ALP (autophagy-lysosome pathway). (C and D) HEK293T cells (C or A549 cells (D) were transfected with 1.5 μg of each plasmid: HA-Vector, FLAG-NS1, and HA-PTBP1, along with FLAG-NS1. At 18 h post-transfection, the cells were treated with DMSO, 3-MA (1.0 mM), MG132 (2.5 μM), or NH 4 Cl (5 mM) for another 12 h. The protein levels were analyzed by Western blot. (E) A549 cells were transfected with 1.0 μg of each plasmid: HA-Vector or HA-PTBP1 along with FLAG-NS1. At 24 h post-transfection, the cells were labeled with anti-FLAG and anti-HA primary antibodies followed by corresponding fluorescent secondary antibodies. The lysosomes were stained with Lyso-Tracker, while nucleus was stained with DAPI. The images displaying NS1 (red), PTBP1 (green), Lysosome (white), and DAPI (blue) were captured under a confocal fluorescence microscope. Scale bar = 20 μm.

Article Snippet: Rabbit polyclonal antibodies against ZIKV NS5 (Cat: GTX133312) and NS1 (Cat: GTX634159) were obtained from Genetex Inc. (Irvine, CA, USA).

Techniques: Transfection, Plasmid Preparation, Western Blot, Ubiquitin Proteomics, Labeling, Staining, Fluorescence, Microscopy

Journal: bioRxiv

Article Title: PTBP1 is upregulated by Zika virus infection via HIF-1α signal and hijacks NS1 protein to induce NS1 degradation to restrain viral replication

doi: 10.1101/2024.11.19.624259

Figure Lengend Snippet: Upon ZIKV infection, HIF-1α signal is activated to promote PTBP1 expression. Meanwhile, ZIKV viral RNA is translated into structural and nonstructural proteins, which participate in viral replication. The HIF-1α-induced PTBP1 interacts with ZIKV NS1 and then promotes the degradation of NS1 protein in a lysosomal pathway, and finally disrupts ZIKV replication.

Article Snippet: Rabbit polyclonal antibodies against ZIKV NS5 (Cat: GTX133312) and NS1 (Cat: GTX634159) were obtained from Genetex Inc. (Irvine, CA, USA).

Techniques: Infection, Expressing

Journal: eLife

Article Title: Zika virus causes placental pyroptosis and associated adverse fetal outcomes by activating GSDME

doi: 10.7554/eLife.73792

Figure Lengend Snippet: JEG-3 or GSDME-KO JEG-3 cells were infected with ZIKV at a multiplicity of infection (MOI) of 1. At indicated time post-infection, cells were subjected to microscopy, cytotoxicity, and Western blot analyses. ( A ) JEG-3 cells were infected with ZIKV for 24 hr. Representative cell morphology was shown. Scale bar, 50 μm. ( B ) LDH levels in supernatant and cell viability were measured at indicated time post-infection (n=3). ( C ) Immunoblot analyses of GSDME-FL, GSDME-N, and GSDMD-FL in ZIKV-infected JEG-3 cells at indicated time post-infection. (D–F) JEG-3 and GSDME-KO JEG-3 cells were infected with ZIKV for 24 hr. Immunoblot analyses of GSDME-FL and GSDME-N by Western blot ( D ). Representative cell morphology was shown. Scale bar, 50 μm ( E ). LDH levels in supernatant and cell viability were measured (n=3) ( F ). LDH release (B, F) is presented as the mean ± SEM of three independent experiments, and the two-tailed unpaired Student’s t-test was used to calculate significance. ***, p<0,001; ****, p<0.0001; ns, no significance. Figure 1—source data 1. Raw data for .

Article Snippet: Mouse monoclonal anti-ZIKV NS5 antibody was generated in our lab. Rabbit monoclonal anti-GSDME (ab215191) antibody was purchased from Abcam.

Techniques: Infection, Microscopy, Western Blot, Two Tailed Test

Journal: eLife

Article Title: Zika virus causes placental pyroptosis and associated adverse fetal outcomes by activating GSDME

doi: 10.7554/eLife.73792

Figure Lengend Snippet: Negative control (NC), GSDME KO, GSDME-overexpressed and GFP-overexpressed JEG-3 cells were infected with ZIKV at a multiplicity of infection (MOI) of 0.1. The supernatants of ZIKV-infected cells were harvested at 12–72 hr post-infection for plaque assay, and the titration was performed on Vero cells (n=3). Figure 1—figure supplement 1—source data 1. Raw data for .

Article Snippet: Mouse monoclonal anti-ZIKV NS5 antibody was generated in our lab. Rabbit monoclonal anti-GSDME (ab215191) antibody was purchased from Abcam.

Techniques: Negative Control, Infection, Plaque Assay, Titration

Journal: eLife

Article Title: Zika virus causes placental pyroptosis and associated adverse fetal outcomes by activating GSDME

doi: 10.7554/eLife.73792

Figure Lengend Snippet: (A–C) Relevant cells were infected with ZIKV at a multiplicity of infection (MOI) of 1. At indicated time post-infection, cells were subjected to microscopy, cytotoxicity, and Western blot analyses. Phase-contrast images of ZIKV-induced pyroptotic cell death in HeLa, HEK 293T, Huh-7, A549, and SH-SY5Y cells at 72 hr post-infection. Scale bar, 50 μm ( A ). Comparison of ATP cell viability, LDH release-based cell death (n=3) ( B ), and GSDME cleavage ( C ) in ZIKV-infected relevant cells. ( D ) Table summarizing results shown in ( A–C ), and from ZIKV infection experiments with relevant cell lines. The abundance of GSDME, the replication and infection levels of ZIKV in cells, and the ZIKV-induced LDH release were quantified. The cells showing the highest abundance of GSDME (SHSY-5Y), susceptibility to ZIKV (JEG-3), and degree of pyroptosis (JEG-3) were considered as references. Those cells are defined as high (severe) if their corresponding index is higher than the 75% of reference, medium (moderate) if the index is between 75% and 25%, and low (none) when the index is less than the 25% of the reference. ( E and F ) Analyses of ATP cell viability, LDH release-based cell death (n=3) ( E ), and GSDME cleavage ( F ) in JEG-3 cells at 36 hr post-infection. Unpaired t-test versus mock. LDH release (B, E) is presented as the mean ± SEM of three independent experiments, and the two-tailed unpaired Student’s t-test was used to calculate significance. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; ns, no significance. Figure 2—source data 1. Raw data for .

Article Snippet: Mouse monoclonal anti-ZIKV NS5 antibody was generated in our lab. Rabbit monoclonal anti-GSDME (ab215191) antibody was purchased from Abcam.

Techniques: Infection, Microscopy, Western Blot, Comparison, Two Tailed Test

Journal: eLife

Article Title: Zika virus causes placental pyroptosis and associated adverse fetal outcomes by activating GSDME

doi: 10.7554/eLife.73792

Figure Lengend Snippet: ( A ) Immunoblot analyses of GSDME-FL in indicated cell lines. (B–C) Relevant cells were infected with ZIKV at a multiplicity of infection (MOI) of 0.1. At indicated time post-infection, cells were subjected to immunofluorescence analysis (scale bar, 400 μm) ( B ). The supernatants of ZIKV-infected cells were harvested at 12–72 hr post-infection, and the plaque assay was performed on Vero cells (n=3) ( C ). Figure 2—figure supplement 1—source data 1. Raw data for .

Article Snippet: Mouse monoclonal anti-ZIKV NS5 antibody was generated in our lab. Rabbit monoclonal anti-GSDME (ab215191) antibody was purchased from Abcam.

Techniques: Western Blot, Infection, Immunofluorescence, Plaque Assay

Journal: eLife

Article Title: Zika virus causes placental pyroptosis and associated adverse fetal outcomes by activating GSDME

doi: 10.7554/eLife.73792

Figure Lengend Snippet: ( A ) Immunoblot analyses of GSDME-FL, GSDME-N, and casepase-3 in ZIKV-infected JEG-3 cells. ( B ) The grayscale analysis of . ( C ) Immunoblot analyses of GSDME-FL, GSDME-N, caspase-9, and casepase-8 in ZIKV-infected JEG-3 cells. ( D ) The grayscale analysis of . ( E ) The grayscale analysis of . ( F ) The grayscale analysis of . All data are presented as the mean ± SEM of three independent experiments, and the two-tailed unpaired Student’s t-test was used to calculate significance. *, p<0.5; **, p<0.01; ***, p<0.001; ns, no significance. Figure 3—figure supplement 1—source data 1. Raw data for .

Article Snippet: Mouse monoclonal anti-ZIKV NS5 antibody was generated in our lab. Rabbit monoclonal anti-GSDME (ab215191) antibody was purchased from Abcam.

Techniques: Western Blot, Infection, Two Tailed Test

Journal: eLife

Article Title: Zika virus causes placental pyroptosis and associated adverse fetal outcomes by activating GSDME

doi: 10.7554/eLife.73792

Figure Lengend Snippet: (A–C) JEG-3 cells were infected with ZIKV at a multiplicity of infection (MOI) of 1 followed by incubated with either 10 μM VX-765, 25 μM Z-DEVD-FMK, 25 μM Z-VAD-FMK, or 10 μM GSK872. At 24 hr post-infection, the cells were subjected to microscopy ( A ), cytotoxicity (n=3) ( B ), and Western blot analyses ( C ). Scale bar, 50 μm. (D–F) JEG-3 cells were infected with ZIKV at an MOI of 1 followed by incubated with either 25 μM Z-IETD-FMK or 25 μM Z-LEHD-FMK. At 24 hr post-infection, the cells were subjected to microscopy ( D ), cytotoxicity (n=3) ( E ), and Western blot analyses ( F ). Scale bar, 50 μm. ( G–H ) Caspase-1, caspase-3, and caspase-8 KO JEG-3 cells or wild-type (WT) cells were infected with ZIKV at an MOI of 1. At 24 hr post infection, LDH levels in supernatant, cell viability (n=3) ( G ) and cleavage of GSDME, caspase-1, caspase-3, and caspase-8 were measured in ZIKV-infected JEG-3 cells. ( I–J ) JEG-3 cells were mock-infected or infected with ZIKV at an MOI of 1. At 24 hr post-infection, the mRNA level of TNF-α ( I ) and concentration of TNF-α in the culture supernatant of JEG-3 cells ( J ) (n=3) were determined by RT-qPCR and enzyme-linked immunosorbent assay (ELISA), respectively. (K–L) JEG-3 cells were infected with ZIKV at an MOI of 1 followed by incubated with 2 μM R-7050. At 24 hr post-infection, the cells were subjected to cytotoxicity (n=3) ( K ) and Western blot analyses ( L ). Unpaired t-test versus mock. All data are presented as the mean ± SEM of three independent experiments, and the two-tailed unpaired Student’s t-test was used to calculate significance. **, p<0.01; ***, p<0.001; ****, p<0.0001; ns, no significance. Figure 3—source data 1. Raw data for .

Article Snippet: Mouse monoclonal anti-ZIKV NS5 antibody was generated in our lab. Rabbit monoclonal anti-GSDME (ab215191) antibody was purchased from Abcam.

Techniques: Infection, Incubation, Microscopy, Western Blot, Concentration Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: eLife

Article Title: Zika virus causes placental pyroptosis and associated adverse fetal outcomes by activating GSDME

doi: 10.7554/eLife.73792

Figure Lengend Snippet: (A–B) JEG-3 cells were infected with ZIKV or UV-inactivated ZIKV at a multiplicity of infection (MOI) of 1. At 24 hr post-infection, the cleavage of gasdermin E (GSDME) ( A ), LDH release, and cell viability (n=3) ( B ) were analyzed. (C–D) JEG-3 cells were seeded in six-well plates followed by transfection with 2 μg of indicated plasmids. At 24 hr post-transfection, the cleavage of GSDME ( C ), LDH release, and cell viability (n=3) ( D ) were analyzed. Unpaired t-test versus control. Asterisks indicate specific bands. All data are presented as the mean ± SEM of three independent experiments, and the two-tailed unpaired Student’s t-test was used to calculate significance. ****, p<0.0001; ns, no significance. Figure 4—figure supplement 1—source data 1. Raw data for .

Article Snippet: Mouse monoclonal anti-ZIKV NS5 antibody was generated in our lab. Rabbit monoclonal anti-GSDME (ab215191) antibody was purchased from Abcam.

Techniques: Infection, Transfection, Control, Two Tailed Test

Journal: eLife

Article Title: Zika virus causes placental pyroptosis and associated adverse fetal outcomes by activating GSDME

doi: 10.7554/eLife.73792

Figure Lengend Snippet: (A–C) JEG-3 cells were seeded in six-well plates and were transfected with 1 μg ZIKV 5’ untranslated region (UTR) or 3’ UTR. At 24 hr post-transfection, cells were subjected to microscopy ( A ), cytotoxicity (n=3) ( B ), and Western blot analyses ( C ). Scale bar, 50 μm. (D–F) RIG-I, TLR7, or TLR8 knockdown JEG-3 cells or wild-type (WT) JEG-3 cells were infected with ZIKV at a multiplicity of infection (MOI) of 1. At 24 hr post-infection, cells were subjected to microscopy ( D ), cytotoxicity (n=3) ( E ), and immunoblot analyses ( F ). Scale bar, 50 μm. (G–H) ZIKV-infected JEG-3 cells were infected with ZIKV. At 24 hr post-infection, the cells were subjected to cytotoxicity (n=3) ( G ) and Western blot analyses ( H ). (I–J) JEG-3 cells and RIG-I KO JEG-3 cells were seeded in six-well plates and were transfected with 1 μg ZIKV 5’ UTR. At 24 hr post-transfection, cells were subjected to cytotoxicity (n=3) ( I ) and Western blot analyses ( J ). (K–L) JEG-3 or RIG-I KO JEG-3 cells were mock-infected or infected with ZIKV at an MOI of 1. The mRNA level ( K ) and concentration of TNF-α in the culture supernatant ( L ) (n=3) was determined by RT-qPCR and enzyme-linked immunosorbent assay (ELISA), respectively. All data are presented as the mean ± SEM of three independent experiments, and the two-tailed unpaired Student’s t-test was used to calculate significance. **, p<0.01; ***, p<0.001; ****, p<0.0001; ns, no significance. RIG-I, retinoic acid-inducible gene I; TLR, Toll-like receptor. Figure 4—source data 1. Raw data for .

Article Snippet: Mouse monoclonal anti-ZIKV NS5 antibody was generated in our lab. Rabbit monoclonal anti-GSDME (ab215191) antibody was purchased from Abcam.

Techniques: Transfection, Microscopy, Western Blot, Knockdown, Infection, Concentration Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: eLife

Article Title: Zika virus causes placental pyroptosis and associated adverse fetal outcomes by activating GSDME

doi: 10.7554/eLife.73792

Figure Lengend Snippet: ( A ) Primary MTCs were isolated from mouse placentas, and incubated at 37°C in a 5% CO 2 incubator. After 72 hr, immunofluorescence analysis was conducted by using antibody against trophoblast-specific marker CK7 (scale bar, 400 μm). (B–C) MTCs were infected with ZIKV at a multiplicity of infection (MOI) of 5. At 48 hr post-infection, LDH release, cell viability (n=3) ( B ), and the cleavage of GSDME ( C ) were analyzed. All data are presented as the mean ± SEM of three independent experiments, and the two-tailed unpaired Student’s t-test was used to calculate significance. *, p<0.05; **, p<0.01; ***, p<0.001; ns, no significance. Figure 5—figure supplement 1—source data 1. Raw data for .

Article Snippet: Mouse monoclonal anti-ZIKV NS5 antibody was generated in our lab. Rabbit monoclonal anti-GSDME (ab215191) antibody was purchased from Abcam.

Techniques: Isolation, Incubation, Immunofluorescence, Marker, Infection, Two Tailed Test

Journal: eLife

Article Title: Zika virus causes placental pyroptosis and associated adverse fetal outcomes by activating GSDME

doi: 10.7554/eLife.73792

Figure Lengend Snippet: ( A ) Scheme of infection and the follow-up analyses. The WT or Gsdme -/- female mice were mated with male mice of their respective genotypes, and the pregnant mice were infected intravenously with 1×10 6 PFU of ZIKV H/PF/2013 strain or with an equal volume of Vero cell culture supernatant at embryonic day 9.5 (E9.5) of pregnancy. The mice were sacrificed at E16.5 and the placentas and individual fetuses were collected for follow-up experiments. ( B ) Representative images of fetuses from mock- and ZIKV-infected WT and Gsdme -/- dams at E16.5 are shown. ( C ) Impact of ZIKV infection on fetuses from WT and Gsdme -/- dams at E16.5. The percentage of fetuses that were affected (i.e., had undergone resorption, or exhibited any sign of growth restriction, or malformation) is shown. Numbers on bars indicate normal fetuses (top) or affected fetuses (bottom). ( D ) Representative hematoxylin and eosin staining showed pathological features of placentas at E16.5. Arrows indicate necrotic trophoblast cells. Arrowheads indicate thrombi. Scale bar, 50 μm. (E–F) Propidium iodide (PI) was intravenously injected into the mice before scarification. Representative placenta section images are shown ( E ) and PI-positive cells were quantified (n=3) ( F ). Scale bar, 400 μm. ( G ) ZIKV RNA levels of maternal spleens, serum, and brains of WT and Gsdme -/- dams infected with ZIKV. ( H ) ZIKV RNA levels of all placentas and fetal heads carried by WT and Gsdme -/- dams infected with ZIKV. The number of samples in each group is listed. Data for all panels are pooled from three to five independent experiments. For ( C ), significance was determined by Fisher’s exact test. For ( F ), significance was determined by a two-tailed Student’s t-test. For ( G ) and ( H ), the Mann-Whitney test was used to calculate significance. Data shown are median with interquartile range, and the dotted line depicts the limit of detection. *, p<0.05; **, p<0.01; ****, p<0.0001; ns, no significance. Figure 5—source data 1. Raw data for .

Article Snippet: Mouse monoclonal anti-ZIKV NS5 antibody was generated in our lab. Rabbit monoclonal anti-GSDME (ab215191) antibody was purchased from Abcam.

Techniques: Infection, Cell Culture, Staining, Injection, Two Tailed Test, MANN-WHITNEY

Journal: eLife

Article Title: Zika virus causes placental pyroptosis and associated adverse fetal outcomes by activating GSDME

doi: 10.7554/eLife.73792

Figure Lengend Snippet: Pregnant wild-type (WT) and Gsdme -/- dams were infected with ZIKV or Vero cell culture supernatant (Mock) as described in . ( A ) Measurements of daily weight of dams after ZIKV infection are shown as the percentage of maternal starting weight for ZIKV-infected WT mice (n=8) and Gsdme -/- (n=9) mice. Significance was determined by one-way ANOVA corrected using Bonferroni’s test for multiple comparisons. ( B ) The percentage of dams showing abnormal pregnancy (at least one fetus showed morphological abnormality or suffered demise/resorption). Numbers on bars indicate normal pregnancy (top) or abnormal pregnancy (bottom). Significance was determined by Fisher’s exact test. *, p<0.05. ( C ) The weight of fetuses (WT Mock, n=65; WT ZIKV, n=109; Gsdme -/- Mock, n=58; Gsdme -/- ZIKV, n=76). Data shown are median with interquartile range, and Mann-Whitney test was used to calculate significance. *, p<0.05; ****, p<0.0001. Data are pooled from three to five independent experiments. Figure 5—figure supplement 2—source data 1. Raw data for .

Article Snippet: Mouse monoclonal anti-ZIKV NS5 antibody was generated in our lab. Rabbit monoclonal anti-GSDME (ab215191) antibody was purchased from Abcam.

Techniques: Infection, Cell Culture, MANN-WHITNEY

Journal: eLife

Article Title: Zika virus causes placental pyroptosis and associated adverse fetal outcomes by activating GSDME

doi: 10.7554/eLife.73792

Figure Lengend Snippet: (A–B) The pregnant C57BL/6N mice were mock-infected or intravenously infected with 1×10 6 PFU of ZIKV H/PF/2013 strain at embryonic day 9.5 (E9.5). At E16.5, placentas were collected and the mRNA level and concentration of TNF-α in mouse placentas were determined by RT-qPCR ( A ) and enzyme-linked immunosorbent assay (ELISA) ( B ), respectively (n=6). (C–G) The pregnant C57BL/6N mice were intravenously infected with 1×10 6 PFU of ZIKV H/PF/2013 strain at E9.5, followed by treatment with R-7050 (7 mg/kg, i.p.) or DMSO every other day. At E16.5, mice were sacrificed and the placentas and individual fetuses were collected. Representative images of fetuses from mock- and ZIKV-infected dams treated with R-7050 or vehicle at E16.5 are shown. Scale bar, 1 cm ( C ). The percentages of fetuses that were affected (i.e., had undergone resorption, or exhibited any sign of growth restriction, or malformation) were calculated. Numbers on bars indicate normal fetuses (top) or affected fetuses (bottom) ( D ). Representative hematoxylin and eosin staining was performed to show the pathological features of placentas at E16.5. The asterisks indicate abnormal spheroid structure. Arrows indicate necrotic trophoblast cells. Arrowheads indicate thrombi. Scale bar, 50 μm ( E ). Propidium iodide (PI) was intravenously injected into the mice before scarification. Representative placenta section images are shown ( F ) and PI-positive cells were quantified (n=3) ( G ). Scale bar, 400 μm. For ( A ), ( B ), and ( G ) significance was determined by a two-tailed unpaired Student’s t-test. For ( D ), significance was determined by Fisher’s exact test. **, p<0.01; ***, p<0.001; ****, p<0.0001. Figure 6—source data 1. Raw data for .

Article Snippet: Mouse monoclonal anti-ZIKV NS5 antibody was generated in our lab. Rabbit monoclonal anti-GSDME (ab215191) antibody was purchased from Abcam.

Techniques: Infection, Concentration Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Staining, Injection, Two Tailed Test

Journal: eLife

Article Title: Zika virus causes placental pyroptosis and associated adverse fetal outcomes by activating GSDME

doi: 10.7554/eLife.73792

Figure Lengend Snippet: The weight of fetuses from mock- and ZIKV-infected dams treated with R-7050 or vehicle at E16.5 (vehicle, n=41; vehicle + ZIKV, n=39; R-7050 + ZIKV, n=47). Data shown are median with interquartile range, and Mann-Whitney test was used to calculate significance. ****, p<0.0001. Figure 6—figure supplement 1—source data 1. Raw data for .

Article Snippet: Mouse monoclonal anti-ZIKV NS5 antibody was generated in our lab. Rabbit monoclonal anti-GSDME (ab215191) antibody was purchased from Abcam.

Techniques: Infection, MANN-WHITNEY

Journal: eLife

Article Title: Zika virus remodels and hijacks IGF2BP2 ribonucleoprotein complex to promote viral replication organelle biogenesis

doi: 10.7554/eLife.94347

Figure Lengend Snippet: ( A ) Huh 7.5 cells were infected with ZIKV H/PF/2013 at an MOI of 5. Cells were collected at 48 and 72 hr post-infection (hpi). Cell extracts were prepared and analyzed by western blotting using the indicated antibodies. Actin-normalized protein signals are shown. ( B ) Huh7.5 cells were infected with ZIKV H/PF/2013 with an MOI of 10 or left uninfected. Two days post-infection, cells were fixed, immunolabeled for the indicated factors, and imaged by confocal microscopy. Scale bar = 10 µm. The Manders’ coefficient (mean ± SEM) representing the fraction of dsRNA (cyan) and NS3 (red) signals overlapping with IGF2BP2 signal is shown (n=number of cells). ( C ) Co-immunoprecipitation assays using HA antibodies were performed with extracts from Huh7.5 cells stably expressing IGF2BP2-HA (+) or control-transduced cells (-) which were infected with ZIKV at an MOI of 10 for 2 days. Purified complexes were analyzed for their protein content by western blotting. ( D ) Means of quantified NS5 signals from (C) (normalized to actin [extracts] or IGF2BP2 [IP]) ± SEM are shown based on nine independent experiments. ****: p<0.0001; ns: not significant (unpaired t-test). Figure 4—source data 1. Data points to generate the bar graphs of and quantify mean Manders’ coefficients of . Figure 4—source data 2. PDF file containing original western blots for , indicating the relevant bands and conditions. Figure 4—source data 3. Original files for western blot analysis displayed in .

Article Snippet: Antibody , Anti-ZIKV NS5 (Rabbit polyclonal) , Genetex , Cat#: GTX133312 RRID: AB_2750559 , WB (1:5000).

Techniques: Infection, Western Blot, Immunolabeling, Confocal Microscopy, Immunoprecipitation, Stable Transfection, Expressing, Control, Purification

Journal: eLife

Article Title: Zika virus remodels and hijacks IGF2BP2 ribonucleoprotein complex to promote viral replication organelle biogenesis

doi: 10.7554/eLife.94347

Figure Lengend Snippet: ( A ) Schematic representation of reporter ZIKV H/PF/2013 sub-genomic replicons (sgR2A) and replication-deficient genomes because of mutations in NS5 RNA-dependent RNA polymerase (RdRp) sequence (sgR2A GAA). ( B–C ) Huh7.5 were transduced with short-hairpin RNA (shRNA)-expressing lentiviruses and subjected to electroporation with in vitro -transcribed sgR2A or sgR2A GAA RNAs 2 days later. In-cell bioluminescence was measured ( B ) 48 or ( C ) 4 hr post-electroporation and normalized to the non-target shRNA (shNT) control condition. In (C), the luciferase activity was normalized to the transfection efficiency, i.e., the Renilla luciferase (Rluc) activity at 4 hr post-electroporation. Means ± SEM are shown based on four independent experiments. ***: p<0.001; NS: not significant (unpaired t-test). Figure 6—source data 1. Data points to generate all bar graphs of .

Article Snippet: Antibody , Anti-ZIKV NS5 (Rabbit polyclonal) , Genetex , Cat#: GTX133312 RRID: AB_2750559 , WB (1:5000).

Techniques: Sequencing, Transduction, shRNA, Expressing, Electroporation, In Vitro, Control, Luciferase, Activity Assay, Transfection

Journal: eLife

Article Title: Zika virus remodels and hijacks IGF2BP2 ribonucleoprotein complex to promote viral replication organelle biogenesis

doi: 10.7554/eLife.94347

Figure Lengend Snippet: ( A ) Huh7.5 cells stably expressing IGF2BP2-HA (+) and control cells (-) were infected with ZIKV H/PF/2013 at an MOI of 10 or left uninfected. Two days later, cell extracts were prepared and subjected to RNase A treatment (+) or not (-) before anti-HA immunoprecipitations. The resulting complexes were analyzed by western blotting for their abundance in the indicated proteins. ( B ) The RNA content in cell extracts was analyzed on an agarose gel for controlling the efficiency of the RNase A treatment. ( C ) ZIKV NS5 levels in the IP samples were quantified and means of protein signals (normalized to IGF2BP2) ± SEM based on three independent experiments are shown. ***: p<0.001 (unpaired t-test). Figure 8—figure supplement 3—source data 1. Data points to generate the bar graphs in . Figure 8—figure supplement 3—source data 2. PDF file containing original western blots for panel A, indicating the relevant bands and conditions. Figure 8—figure supplement 3—source data 3. Original files for western blot analysis displayed in panel A.

Article Snippet: Antibody , Anti-ZIKV NS5 (Rabbit polyclonal) , Genetex , Cat#: GTX133312 RRID: AB_2750559 , WB (1:5000).

Techniques: Stable Transfection, Expressing, Control, Infection, Western Blot, Agarose Gel Electrophoresis

Journal: eLife

Article Title: Zika virus remodels and hijacks IGF2BP2 ribonucleoprotein complex to promote viral replication organelle biogenesis

doi: 10.7554/eLife.94347

Figure Lengend Snippet: ( A ) Schematic representation of the pIRO system. Upon transfection in cells expressing the T7 RNA polymerase, this plasmid allows the cytoplasmic transcription of NS1-NS5 polyprotein under the control of T7 promoter, in a ZIKV replication-independent manner. NS1-5 polyprotein synthesis is under the control of ECMV IRES. The presence of both ZIKV 3’ NTR and 5’ cyclization sequence (5’ CS) is required for efficient vesicle packet (VP) induction. Finally, the activity of HDV ribozyme ensures that the 3’ terminus of the RNA is similar to that of viral RNA (vRNA) genome. Huh7-Lunet-T7 were transduced with short-hairpin RNA (shRNA)-expressing lentiviruses at an MOI of 5–10. Two days later, transduced cells were transfected with pIRO-Z plasmid. Sixteen hours later, cells were analyzed for ( B ) IGF2BP2 mRNA levels by RT-qPCR to measure knockdown efficiency, ( C–D ) transfection efficiency by confocal imaging of NS3-labeled cells, and ( E ) for VP content by transmission electron microscopy. Electron micrographs were used to measure ( F ) the percentage of cells with VPs and ( G ) the diameter of VPs in each condition. ***: p<0.001; NS: not significant (unpaired t-test). Figure 10—source data 1. Data points to generate all graphs of .

Article Snippet: Antibody , Anti-ZIKV NS5 (Rabbit polyclonal) , Genetex , Cat#: GTX133312 RRID: AB_2750559 , WB (1:5000).

Techniques: Transfection, Expressing, Plasmid Preparation, Control, Sequencing, Activity Assay, Transduction, shRNA, Quantitative RT-PCR, Knockdown, Imaging, Labeling, Transmission Assay, Electron Microscopy

Journal: eLife

Article Title: Zika virus remodels and hijacks IGF2BP2 ribonucleoprotein complex to promote viral replication organelle biogenesis

doi: 10.7554/eLife.94347

Figure Lengend Snippet: Step 1: After NS protein synthesis early after virus entry, IGF2BP2 associates with NS5 and vRNA, thus excluding PUM2 and TNRC6A mRNA from the ribonucleprotein (RNP). Step 2: The infection-induced association between IGF2BP2 RNP and ATL2 allows the targeting of vRNA/NS5 to the endoplasmic reticulum (ER). Step 3: Viral factors and ATL2 induce the bending of the ER membrane and the formation of vesicle packets (VPs) allowing highly processive vRNA synthesis. Step 4: IGF2BP2 might be involved in the packaging of vRNA into assembling viruses by targeting the genome to the VP pore. The recruitment of IGF2BP2 to the replication compartment might be dependent on its mTOR complex 1 (mTORC1)-dependent phosphorylation status.

Article Snippet: Antibody , Anti-ZIKV NS5 (Rabbit polyclonal) , Genetex , Cat#: GTX133312 RRID: AB_2750559 , WB (1:5000).

Techniques: Virus, Infection, Membrane, Phospho-proteomics

Journal: eLife

Article Title: Zika virus remodels and hijacks IGF2BP2 ribonucleoprotein complex to promote viral replication organelle biogenesis

doi: 10.7554/eLife.94347

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-ZIKV NS5 (Rabbit polyclonal) , Genetex , Cat#: GTX133312 RRID: AB_2750559 , WB (1:5000).

Techniques: Plasmid Preparation, Amplification, Virus, Cloning, Derivative Assay, Transfection, Construct, Control, shRNA, Immunofluorescence, Staining, Recombinant, In Vitro, Sequencing, SYBR Green Assay, ISH Cell Assay, Software

Journal: eLife

Article Title: Zika virus remodels and hijacks IGF2BP2 ribonucleoprotein complex to promote viral replication organelle biogenesis

doi: 10.7554/eLife.94347

Figure Lengend Snippet:

Article Snippet: Rabbit anti-DENV NS4B (GTX124250; cross-reactive for ZIKV), rabbit anti-ZIKV NS4B (GTX133311), rabbit anti-ZIKV NS3 (GTX133309), rabbit anti-ZIKV NS5 (GTX133312), rabbit anti-ZIKV NS4A (GTX133704), rabbit anti-DENV NS5 (GTX124253), and mouse monoclonal anti-DENV-NS3 (GTX629477; cross-reactive for ZIKV) were obtained from Genetex (Irvine, CA, USA).

Techniques: Plasmid Preparation, Amplification, Virus, Clone Assay, Derivative Assay, Transfection, Construct, Control, shRNA, Immunofluorescence, Staining, Recombinant, In Vitro, Sequencing, SYBR Green Assay, ISH Cell Assay, Software

Journal: eLife

Article Title: Zika virus remodels and hijacks IGF2BP2 ribonucleoprotein complex to promote viral replication organelle biogenesis

doi: 10.7554/eLife.94347

Figure Lengend Snippet:

Article Snippet: Rabbit anti-DENV NS4B (GTX124250; cross-reactive for ZIKV), rabbit anti-ZIKV NS4B (GTX133311), rabbit anti-ZIKV NS3 (GTX133309), rabbit anti-ZIKV NS5 (GTX133312), rabbit anti-ZIKV NS4A (GTX133704), rabbit anti-DENV NS5 (GTX124253), and mouse monoclonal anti-DENV-NS3 (GTX629477; cross-reactive for ZIKV) were obtained from Genetex (Irvine, CA, USA).

Techniques: Plasmid Preparation, Amplification, Virus, Clone Assay, Derivative Assay, Transfection, Construct, Control, shRNA, Immunofluorescence, Staining, Recombinant, In Vitro, Sequencing, SYBR Green Assay, ISH Cell Assay, Software