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: 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: Primary antibodies used for WB are rabbit anti-UBA1 (Invitrogen, MA5-35786), mouse anti-HA epitope (BioLegend, reference 901501), rabbit anti-FLAG epitope (Proteintech, reference 20543–1-AP) and rabbit anti-ZIKV NS5 (GeneTex, GTX133328).

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: Primary antibodies used for WB are rabbit anti-UBA1 (Invitrogen, MA5-35786), mouse anti-HA epitope (BioLegend, reference 901501), rabbit anti-FLAG epitope (Proteintech, reference 20543–1-AP) and rabbit anti-ZIKV NS5 (GeneTex, GTX133328).

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: Primary antibodies used for WB are rabbit anti-UBA1 (Invitrogen, MA5-35786), mouse anti-HA epitope (BioLegend, reference 901501), rabbit anti-FLAG epitope (Proteintech, reference 20543–1-AP) and rabbit anti-ZIKV NS5 (GeneTex, GTX133328).

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

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: 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: 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: 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: 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: 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: 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: 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: 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: 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: Virus, Infection, Membrane

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: ( 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: 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