Journal: mBio

Article Title: Whole-blood model reveals granulocytes as key sites of dengue virus propagation, expanding understanding of disease pathogenesis

doi: 10.1128/mbio.01505-24

Figure Lengend Snippet: Hirudin compatibility with a whole-blood DENV infection model. ( A ) Hirudin preserves complement function. Plasma was separated from hirudinized whole blood or EDTA-treated hirudinized blood after 2 h, then incubated with yeast cells. C3b deposition on yeast was detected by flow cytometry. ( B ) Diagram of the in vitro whole-blood DENV infection model (18 h post-infection). At 2 and 18 h post-infection, each cell population was analyzed based on their size, granularity, and CD markers for cell viability and dengue antigens. ( C ) Hirudin does not interfere with DENV infectivity. Hirudinized blood from DENV-naïve donors was incubated with mock or DENV (10⁷ or 10⁸ genome copies/mL). Infectious DENV titers in plasma were determined by focus-forming unit (FFU) assay at 2 h (white bars) and 18 h (gray bars) post-infection. ( D ) Representative gating strategy for analyzing DENV-infected whole blood. Cells were gated based on forward scatter (FSC), side scatter (SSC), and established CD markers to distinguish platelets (CD41a + ), granulocytes (CD66 + ), monocytes (CD14 + ), B (CD19 + ) and T (CD3 + ) lymphocytes, and NK cells (CD56 + ). ( E, F ) Hirudin maintains leukocyte viability in a whole-blood infection model. Cell viability in each white blood cell (WBC) population was determined by live/dead staining and flow cytometry after incubation with mock (white bars), DENV 10⁷ genome copies/mL (gray bars), or 10⁸ genome copies/mL (black bars) at 2 h ( E ) and 18 h ( F ) post-infection. ( G–I ) DENV infection in each cell population in a whole-blood infection model. Percentages of DENV-positive cells, as determined by surface DENV envelope ( E ), protein ( G ), intracellular NS1 ( H ), and intracellular NS3 ( I ) in each WBC cell population were determined at 2 h (white circles) and 18 h (gray circles) post-infection. Data are presented as individual dot plots from 8 to 10 independent experiments. Student’s t -test was used to compare expression levels of DENV antigens among each time-point and each condition in each cell population. Asterisks (*, **, and ***) indicate statistical significance ( P < 0.05, P < 0.01, and P < 0.005, respectively).

Article Snippet: Anti-DENV NS5 polyclonal antibody (GTX103350) was purchased from GeneTex (CA, USA).

Techniques: Infection, Clinical Proteomics, Incubation, Flow Cytometry, In Vitro, Staining, Expressing

Journal: mBio

Article Title: Whole-blood model reveals granulocytes as key sites of dengue virus propagation, expanding understanding of disease pathogenesis

doi: 10.1128/mbio.01505-24

Figure Lengend Snippet: DENV infection and replication efficiency in different blood cell populations. ( A ) Schematic diagram of the experimental setup for analyzing DENV infection and replication in sorted blood cell population 18 h post-infection with DENV at 10⁸ genome copies/mL. ( B ) Purity assessment of each sorted WBC population from mock-infected (white bars) or DENV-infected (black bars) whole blood. ( C ) Confocal microscopy images of DENV-infected WBCs. Intracellular DENV NS1 and NS5 proteins are shown in green, and nuclei are stained with Hoechst dye (blue). ( D ) Quantification of cell-associated DENV RNA in each sorted cell population by qRT-PCR. ( E ) Infectious DENV titers (FFU/10³ cells) in supernatants from co-cultures of sorted blood cell populations with permissive BHK cells for 2 days. ( F ) DENV genome copies (per 10³ cells) in supernatants from co-cultures as in panel E . ( G ) Specific infectivity ( si ) of DENV in each cell population, calculated as the ratio of infectious DENV titers (FFU) to viral genome copies. Data are representative of three independent experiments. N.D. indicates "not detected."

Article Snippet: Anti-DENV NS5 polyclonal antibody (GTX103350) was purchased from GeneTex (CA, USA).

Techniques: Infection, Confocal Microscopy, Staining, Quantitative RT-PCR

Journal: mBio

Article Title: Whole-blood model reveals granulocytes as key sites of dengue virus propagation, expanding understanding of disease pathogenesis

doi: 10.1128/mbio.01505-24

Figure Lengend Snippet: DENV antigen detection in whole blood from adult and pediatric patients. ( A ) Representative gating strategy for flow cytometry analysis of DENV-infected patient whole blood. Cell populations were identified based on forward scatter (FSC), side scatter (SSC), and the following CD markers: platelets (CD41a + ), granulocytes (CD66 + ), monocytes (CD14 + ), B cells (CD19 + ) and T cells (CD3 + ), and NK cells (CD56 + ). ( B–D ) Percentages of cells positive for surface DENV envelope ( E ), protein ( B ), intracellular NS1 ( C ), or intracellular NS3 ( D ) in the indicated cell populations from adult DENV-infected patients ( n = 24). Whole blood was collected at the following two time points: acute (1–6 days before defervescence) and convalescent (7–24 days after defervescence). Each symbol represents an individual patient. The X / X number in each plot indicates the number of patients with detectable positive cells out of the total number of analyzed for that cell population. ( E ) Kinetics of NS1-positive cells in the indicated cell populations from pediatric DENV-infected patients ( n = 8; four with dengue fever [DF], four with dengue hemorrhagic fever [DHF]). Whole blood was collected daily during the acute phase, with one convalescent sample. Lines represent individual patients.

Article Snippet: Anti-DENV NS5 polyclonal antibody (GTX103350) was purchased from GeneTex (CA, USA).

Techniques: Flow Cytometry, Infection

Journal: mBio

Article Title: Whole-blood model reveals granulocytes as key sites of dengue virus propagation, expanding understanding of disease pathogenesis

doi: 10.1128/mbio.01505-24

Figure Lengend Snippet: Demographic of dengue patients

Article Snippet: Anti-DENV NS5 polyclonal antibody (GTX103350) was purchased from GeneTex (CA, USA).

Techniques:

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-DENV NS5 (Rabbit polyclonal) , Genetex , Cat#: GTX124253 RRID: AB_11169932 , WB (1:1000).

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-DENV NS5 (Rabbit polyclonal) , Genetex , Cat#: GTX124253 RRID: AB_11169932 , WB (1:1000).

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-DENV NS5 (Rabbit polyclonal) , Genetex , Cat#: GTX124253 RRID: AB_11169932 , WB (1:1000).

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-DENV NS5 (Rabbit polyclonal) , Genetex , Cat#: GTX124253 RRID: AB_11169932 , WB (1:1000).

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-DENV NS5 (Rabbit polyclonal) , Genetex , Cat#: GTX124253 RRID: AB_11169932 , WB (1:1000).

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-DENV NS5 (Rabbit polyclonal) , Genetex , Cat#: GTX124253 RRID: AB_11169932 , WB (1:1000).

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: ( 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