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: bioRxiv

Article Title: Zika and Dengue Viruses Differentially Modulate Host mRNA Processing Factors Defining Its Virulence

doi: 10.1101/2024.10.18.619084

Figure Lengend Snippet: (A) Heatmap of normalized RNA-seq Z-score data for mock versus ZIKV-infected (left) and DENV-infected (right) Vero samples at 1-, 3-, 5-DPI, and long-term infected samples. (B) A comparison of normalized RNA-seq reads represented in reads per million for NXT1 and visualized in IGV. Comparisons shown for mock, ZIKV-, and DENV-infected Vero samples at 1-, 3-, 5-DPI, and long-term infected samples. (C) Western blot analysis of anti-NXT1 for mock, ZIKV-, and DENV-infected samples at 1-, 3-, 5-DPI as well as long-term DENV. (D) Immunocytochemistry staining of mock, ZIKV, and DENV at 1- and 5-DPI. Antibodies used were anti-NXT1 (green), anti-ZIKV NS5 (red), and DAPI (blue).

Article Snippet: Primary antibodies used were as follows: Mouse (M) anti-4G2 (Cat# Ab00230-2.0, Absolute Antibody), M anti-DENV NS1 (Type 2/3/4) (Cat# MAB94441-100, R&D Systems), M anti-NXT1 (Cat#67680-1-Ig, Proteintech), M anti-alpha tubulin (Cat#66031-1-Ig, Proteintech), Rabbit (Rb) anti-beta actin (Cat# 026-42210, Li-cor), Rb anti-DENV NS5 (Cat#NBP2-42901, Novus Biologicals), Rb anti-GAPDH (Cat# 5174S, Cell Signaling Technology (CST)), Rb anti-UPF1 (Cat#12040, CST) Rb anti-UPF2 (Cat#11875, CST), Rb anti-ZIKV NS5 (Cat# GTX133312, GeneTex), Rb anti-ZIKV envelope (Cat# GTX133314, GeneTex).

Techniques: RNA Sequencing Assay, Infection, Comparison, Western Blot, Immunocytochemistry, Staining

Journal: bioRxiv

Article Title: Zika and Dengue Viruses Differentially Modulate Host mRNA Processing Factors Defining Its Virulence

doi: 10.1101/2024.10.18.619084

Figure Lengend Snippet: (A) Heatmap of NPC pathway-associated normalized RNA-seq transcripts for mock versus ZIKV-infected SK-N-SH samples at 1-, 3-, and 5-DPI. (B) A comparison of normalized RNA-seq reads represented in reads per million for NXT1 and visualized in IGV. Comparisons shown for mock and ZIKV-infected SK-N-SH samples at 1-, 3-, 5-DPI. (C) Western blot analysis of anti-NXT1 for mock, ZIKV-, and DENV-infected samples at 1-, 3-, 5-DPI as well as long-term DENV-infected sample. (D) Immunocytochemistry staining of mock, ZIKV, and DENV at 1- and 5-DPI. Antibodies used were anti-NXT1 (green), anti-ZIKV NS5 (red), and DAPI (blue).

Article Snippet: Primary antibodies used were as follows: Mouse (M) anti-4G2 (Cat# Ab00230-2.0, Absolute Antibody), M anti-DENV NS1 (Type 2/3/4) (Cat# MAB94441-100, R&D Systems), M anti-NXT1 (Cat#67680-1-Ig, Proteintech), M anti-alpha tubulin (Cat#66031-1-Ig, Proteintech), Rabbit (Rb) anti-beta actin (Cat# 026-42210, Li-cor), Rb anti-DENV NS5 (Cat#NBP2-42901, Novus Biologicals), Rb anti-GAPDH (Cat# 5174S, Cell Signaling Technology (CST)), Rb anti-UPF1 (Cat#12040, CST) Rb anti-UPF2 (Cat#11875, CST), Rb anti-ZIKV NS5 (Cat# GTX133312, GeneTex), Rb anti-ZIKV envelope (Cat# GTX133314, GeneTex).

Techniques: RNA Sequencing Assay, Infection, Comparison, Western Blot, Immunocytochemistry, Staining

Journal: bioRxiv

Article Title: Zika and Dengue Viruses Differentially Modulate Host mRNA Processing Factors Defining Its Virulence

doi: 10.1101/2024.10.18.619084

Figure Lengend Snippet: (A) Top panel-Immunocytochemistry of LMB-treated and untreated Vero samples at 48 hours. Antibodies used were anti-UPF1 (in green), anti-4G2 (in red), and DAPI (in blue). Shown on the right is a graphical representation of the percentage of infected cells at each time point for treated and untreated samples. Bottom panel-Immunocytochemistry of LMB-treated and untreated Vero samples at 48 hours. Antibodies used were anti-NS5 (in green) and DAPI (in blue). Shown on the right is a graphical representation of the percentage of infected cells at each time point for treated and untreated samples. (B) Top panel-Immunocytochemistry of LMB-treated and untreated SK-N-SH samples at 48 hours. Antibodies used were anti-UPF1 (in green), anti-4G2 (in red), and DAPI (in blue). Shown on the right is a graphical representation of the percentage of infected cells at each time point for treated and untreated samples. Bottom panel-Immunocytochemistry of LMB-treated and untreated SK-N-SH samples at 48 hours. Antibodies used were anti-NS5 (in green) and DAPI (in blue). Shown on the right is a graphical representation of the percentage of infected cells at each time point for treated and untreated samples. Two images were counted for each antibody per sample and values were averaged. Error bars represent SEM.

Article Snippet: Primary antibodies used were as follows: Mouse (M) anti-4G2 (Cat# Ab00230-2.0, Absolute Antibody), M anti-DENV NS1 (Type 2/3/4) (Cat# MAB94441-100, R&D Systems), M anti-NXT1 (Cat#67680-1-Ig, Proteintech), M anti-alpha tubulin (Cat#66031-1-Ig, Proteintech), Rabbit (Rb) anti-beta actin (Cat# 026-42210, Li-cor), Rb anti-DENV NS5 (Cat#NBP2-42901, Novus Biologicals), Rb anti-GAPDH (Cat# 5174S, Cell Signaling Technology (CST)), Rb anti-UPF1 (Cat#12040, CST) Rb anti-UPF2 (Cat#11875, CST), Rb anti-ZIKV NS5 (Cat# GTX133312, GeneTex), Rb anti-ZIKV envelope (Cat# GTX133314, GeneTex).

Techniques: Immunocytochemistry, Infection

Journal: Communications Biology

Article Title: Dengue virus-free defective interfering particles have potent and broad anti-dengue virus activity

doi: 10.1038/s42003-021-02064-7

Figure Lengend Snippet: A A lentiviral vector expressing the DENV-2 NS proteins and EGFP. B A lentiviral vectors that expresses the DI-290 RNA as an mRNA that is processed by the HDVr into authentic DI-290 RNA and expresses CFP. C A retroviral vector that expresses DENV-2 S proteins and mCherry. D The transduction of HEK 293T cells can be selected by FACS for triple-positive cells expressing EGFP, mCherry and CFP. E Western blot of HEK-DI-290-ORF cells using antibodies to DENV NS5, NS3, C and E proteins. The relative protein loading in each lane is indicated by a western blot for β-tubulin. F Confocal fluorescence microscopy of HEK 293T, DENV-2 infected HEK 293T, and HEK-DI-290-ORF cells stained with antibodies to DENV E, C, and NS3 (magenta). Cells were counter stained with DAPI (blue). The white bar in the image represents 10 µm. mol. wt. is molecular weight.

Article Snippet: DENV-2 NS3 and DENV-2 NS5 were detected with a rabbit anti-DENV NS3 polyclonal antibody (Sigma Aldrich) and a mouse anti-DENV NS5 monoclonal antibody (GeneTex). dsRNA was detected with a mouse anti-dsRNA monoclonal antibody J2 (SCICONS).

Techniques: Plasmid Preparation, Expressing, Retroviral, Transduction, Western Blot, Fluorescence, Microscopy, Infection, Staining, Molecular Weight

Journal: Communications Biology

Article Title: Dengue virus-free defective interfering particles have potent and broad anti-dengue virus activity

doi: 10.1038/s42003-021-02064-7

Figure Lengend Snippet: Confocal fluorescence microscopy of HEK 293T, HEK-DI-290, HEK-DI-290-ORF, and DENV-2 infected HEK 293T cells that were stained with an anti-double strand RNA antibody (magenta). The cells were counterstained with DAPI to stain nuclei (blue). The white bar in the image represents 10 µm.

Article Snippet: DENV-2 NS3 and DENV-2 NS5 were detected with a rabbit anti-DENV NS3 polyclonal antibody (Sigma Aldrich) and a mouse anti-DENV NS5 monoclonal antibody (GeneTex). dsRNA was detected with a mouse anti-dsRNA monoclonal antibody J2 (SCICONS).

Techniques: Fluorescence, Microscopy, Infection, Staining

Journal: Communications Biology

Article Title: Dengue virus-free defective interfering particles have potent and broad anti-dengue virus activity

doi: 10.1038/s42003-021-02064-7

Figure Lengend Snippet: A Triplicate samples of cell-free culture supernatant of HEK-DI-290 or HEK-DI-290-ORF cells underwent ultracentrifugation. The pelleted material was assayed by RT-qPCR to measure the level of DI-290 RNA. The mean value, SD and calculated a P value is shown. ( B ) Duplicates of the pelleted samples from A were assayed by western blot using and anti- DENV-2 E or anti-DENV-2 C antibodies. The pelleted material of cell-free culture supernatant of DENV-2 infected cells were used as a positive control. C Vero cells were incubated with DENV-2 using an MOI of 0.01 or with culture supernatant from HEK-DI-290-ORF cells. Lysates were made after 7 days and assayed by western blot using an anti-DENV NS3 antibody, or with anti-B-tubulin to monitor the amount of protein loaded in each lane. D HEK-DI-290-ORF cells were grown in a glass spinner flask using serum-free medium. The supernatant was filtered (0.45 µM), nuclease treated and DIPs were pelleted at 100,000 × g . RNA was extracted from the pelleted material and assayed by RT-qPCR to measure the level of DI-290 RNA in the sample. The mean value of DI-290 RNA measured in copies/ mL and SD from four independent cultures is shown. E DENV-2 supernatant (equivalent to 3 × 10 7 NS5 RNA copies) and culture supernatant from HEK-DI-290-ORF cells (equivalent to 3 × 10 7 DI-290 RNA copies) were tenfold serially diluted and incubated with Vero cells. The plates were fixed with acetone/methanol 5 days later and plaques were visualized by staining with an anti-Flavivirus E antibody. F Plaques were counted to determine virus titer 5 days post-infection. Data shown are mean values ± SD from three replicate experiments. mol. wt. is molecular weight.

Article Snippet: DENV-2 NS3 and DENV-2 NS5 were detected with a rabbit anti-DENV NS3 polyclonal antibody (Sigma Aldrich) and a mouse anti-DENV NS5 monoclonal antibody (GeneTex). dsRNA was detected with a mouse anti-dsRNA monoclonal antibody J2 (SCICONS).

Techniques: Quantitative RT-PCR, Western Blot, Infection, Positive Control, Incubation, Staining, Virus, Molecular Weight

Journal: Communications Biology

Article Title: Dengue virus-free defective interfering particles have potent and broad anti-dengue virus activity

doi: 10.1038/s42003-021-02064-7

Figure Lengend Snippet: DIPs purified from HEK-DI-290-ORF cell culture supernatant, purified DENV-2 from infected HEK 293T cells or CHT column purified supernatant from HEK-DI-290 were placed on a 5–50% sucrose gradient and subjected to ultracentrifugation. Gradient fractions were collected from the bottom of the tube and A RNA was extracted from triplicate samples of each fraction that was assayed by RT-qPCR to measure levels of DI-290 RNA. The mean values and SD are shown. Each fraction was assayed by immuno-dot-blot using B anti-DENV-2 E or C anti-DENV-2 C antibodies. A representative of two experiments with similar results is shown.

Article Snippet: DENV-2 NS3 and DENV-2 NS5 were detected with a rabbit anti-DENV NS3 polyclonal antibody (Sigma Aldrich) and a mouse anti-DENV NS5 monoclonal antibody (GeneTex). dsRNA was detected with a mouse anti-dsRNA monoclonal antibody J2 (SCICONS).

Techniques: Purification, Cell Culture, Infection, Quantitative RT-PCR, Dot Blot

Journal: Communications Biology

Article Title: Dengue virus-free defective interfering particles have potent and broad anti-dengue virus activity

doi: 10.1038/s42003-021-02064-7

Figure Lengend Snippet: Vero cells or Vero-ORF expressing DENV-2 S and NS proteins were treated with cell-free culture supernatant of HEK-DI-290 (black bars, a negative control) or DIP supernatant of HEK-DI-290-ORF cells (red bars) for 12 h when the culture medium was replaced. After 48 h, RNA samples were prepared from A cells and B culture supernatant and used in RT-qPCR assays to measure levels of DI-290 RNA. The cellular levels of DI-290 were normalized to GAPDH mRNA in the same sample. The relative level of DI-290 in Vero cells treated with negative control supernatant was set as 1. The error bars indicate the SD. A representative of three experiments with similar results is shown. .

Article Snippet: DENV-2 NS3 and DENV-2 NS5 were detected with a rabbit anti-DENV NS3 polyclonal antibody (Sigma Aldrich) and a mouse anti-DENV NS5 monoclonal antibody (GeneTex). dsRNA was detected with a mouse anti-dsRNA monoclonal antibody J2 (SCICONS).

Techniques: Expressing, Negative Control, Quantitative RT-PCR

Journal: Communications Biology

Article Title: Dengue virus-free defective interfering particles have potent and broad anti-dengue virus activity

doi: 10.1038/s42003-021-02064-7

Figure Lengend Snippet: A Huh7 cells in triplicate wells were infected with DENV-2 at an MOI of 1 for 3 h. The virus was removed and the cells were treated with either cell-free supernatant from HEK 293T cells, HEK-DI-290 cells or DIPs from HEK-DI-290-ORF cells. For the latter two treatments, DI-290 RNA was used at five copies of DI-290 RNA/cell. Culture supernatant from each well was collected at the time points indicated (d.p.i.), and the level of DENV-2 RNA in each sample was measured by RT-qPCR using primers to detect the NS5 region. Infection in the presence of HEK 293T supernatant was set at 100%. Error bars indicate the SD. A P value comparing negative control treatments to DIPs is shown. B As in A , DENV-2 infected Huh7 cells were treated with The DIP fraction (DIP frac.), DIP in culture supernatant (DIP sup), the CHT flow-through fraction (CHT F.T. frac.) or HEK 293T supernatant (HEK 293T sup.) as a negative control. DI-290 RNA levels for the first three treatments were normalized to ten copies of DI-290 RNA/cell. At the time points indicated, the level of DENV-2 RNA in culture supernatant was measured by RT-qPCR using primers for the DENV-2 NS5 region. The P values shown were calculated with a two-tailed Student’s t test. A representative of three experiments with similar results is shown.

Article Snippet: DENV-2 NS3 and DENV-2 NS5 were detected with a rabbit anti-DENV NS3 polyclonal antibody (Sigma Aldrich) and a mouse anti-DENV NS5 monoclonal antibody (GeneTex). dsRNA was detected with a mouse anti-dsRNA monoclonal antibody J2 (SCICONS).

Techniques: Infection, Virus, Cell Culture, Quantitative RT-PCR, Negative Control, Two Tailed Test

Journal: Communications Biology

Article Title: Dengue virus-free defective interfering particles have potent and broad anti-dengue virus activity

doi: 10.1038/s42003-021-02064-7

Figure Lengend Snippet: A Triplicate wells of DENV-2 infected HuH7 cells (MOI 1.0 for 3 h) were treated with serially diluted DIPs as shown. After 2 d.p.i., the level of viral RNA in culture supernatant was measures by RT-qPCR using primers to the DENV-2 NS5 region. Mean values are shown with error bars indicating the SD. Using triplicate wells, DENV-2 replication in infected cells (DENV-2 only) was compared to cells treated with purified DIPs (DENV-2 + inact. DIPs) or with DIPs treated with UV light irradiation (DENV-2 + inact. DIPs). In B , the level of viral RNA in culture supernatant was measure by RT-qPCR as previously described. In C , viral titers in culture supernatant were measure using an immuno-plaque assay. B , C Mean values are shown and error bars show the SD. The P values shown were calculated with a two-tailed Student’s t test. D In triplicate wells, Huh7 cells were infected with prototype strains of all DENV serotypes at an MOI of 1 for 3 h. The infected cells were treated with purified DIPs equivalent to five copies of DI-290 RNA/cell. The levels of DENV-2 RNA in culture supernatant were measured by RT-qPCR as previously described. All measurements show mean values and error bars indicate the SD. The P values shown were calculated with a two-tailed Student’s t test. The data show representative outcome of two identical experiments with similar results.

Article Snippet: DENV-2 NS3 and DENV-2 NS5 were detected with a rabbit anti-DENV NS3 polyclonal antibody (Sigma Aldrich) and a mouse anti-DENV NS5 monoclonal antibody (GeneTex). dsRNA was detected with a mouse anti-dsRNA monoclonal antibody J2 (SCICONS).

Techniques: Infection, Quantitative RT-PCR, Purification, Irradiation, Plaque Assay, Two Tailed Test

Journal: PLoS ONE

Article Title: Glutathionylation of dengue and Zika NS5 proteins affects guanylyltransferase and RNA dependent RNA polymerase activities

doi: 10.1371/journal.pone.0193133

Figure Lengend Snippet: The second method was to immunoprecipitate the dengue protein in cell lysate with specific dengue protein antibody and protein A/G beads and determine that the dengue protein was glutathionylated as shown by western blot detection with an anti-GSH antibody. The quantity of NS5 is not detectable in whole lysate. Lane 1 is DENV-infected cell lysate. Lane 2 is IP sample of NS5.

Article Snippet: 1 mg of cell lysates were immunoprecipitated with 1 mg/ml of an anti-DENV 2 NS5 protein monoclonal antibody (GeneTex).

Techniques: Western Blot, Infection

Journal: PLoS ONE

Article Title: Glutathionylation of dengue and Zika NS5 proteins affects guanylyltransferase and RNA dependent RNA polymerase activities

doi: 10.1371/journal.pone.0193133

Figure Lengend Snippet: 2A Shown are the two domains of NS5 with respective activity and residue length underneath . The 14 relative cysteine positions are shown with the identified glutathionylated cysteines shown in bold red. 2B Shown is a ribbon representation of full length dengue NS5 (PDB ID 4V0R) with the identified glutathionylated cysteines shown in red and highlighted in green the GTP in the methyltransferase/guanylyltransferase domain.

Article Snippet: 1 mg of cell lysates were immunoprecipitated with 1 mg/ml of an anti-DENV 2 NS5 protein monoclonal antibody (GeneTex).

Techniques: Activity Assay, Residue

Journal: PLoS ONE

Article Title: Glutathionylation of dengue and Zika NS5 proteins affects guanylyltransferase and RNA dependent RNA polymerase activities

doi: 10.1371/journal.pone.0193133

Figure Lengend Snippet: HEK293T/17 cells were mock infected and infected with DENV 2 for 2 h. The infected cells were harvested at 0, 2 and 6 hour post infection (hpi). The immunoprecipitates with DENV NS5 protein antibody were run on non-reduced SDS gel and western blot was performed using anti-GSH antibody.

Article Snippet: 1 mg of cell lysates were immunoprecipitated with 1 mg/ml of an anti-DENV 2 NS5 protein monoclonal antibody (GeneTex).

Techniques: Infection, SDS-Gel, Western Blot

Journal: PLoS ONE

Article Title: Glutathionylation of dengue and Zika NS5 proteins affects guanylyltransferase and RNA dependent RNA polymerase activities

doi: 10.1371/journal.pone.0193133

Figure Lengend Snippet: A) DENV and Zika NS5 protein were not treated and pre-treated with 5 mM GSSG for 10 min at RT. These proteins then were used to perform the guanylyltransferase activity assay. The samples were resolved on 10% SDS-PAGE. The extent of enzyme guanylylation activity was measured by GTP-Cy5 signal tracking using Azure ™ cSeries. Gels were then stained with Coomassie Blue to normalize for protein loading. The bottom panel shows equivalent aliquots of the same samples used in a parallel gel that was transferred for Western blot and detection by anti-GSH antibody. B) The ratio of quantified guanylyltransferase bands to protein load of panel A is shown as a bar graph.

Article Snippet: 1 mg of cell lysates were immunoprecipitated with 1 mg/ml of an anti-DENV 2 NS5 protein monoclonal antibody (GeneTex).

Techniques: Activity Assay, SDS Page, Staining, Western Blot

Journal: PLoS ONE

Article Title: Glutathionylation of dengue and Zika NS5 proteins affects guanylyltransferase and RNA dependent RNA polymerase activities

doi: 10.1371/journal.pone.0193133

Figure Lengend Snippet: The sequences are nonstructural protein NS5 from Dengue virus 2 NP_739590.2, Dengue virus 1 NP_722465.1, Dengue virus 3 YP_001531176.2, Dengue virus 4 NP_740325.1, Zika virus AMR39834.1, Japanese encephalitis virus NP_775674.1, West Nile virus YP_001527887.1, Tick-borne encephalitis virus NP_775511.1, Yellow fever virus NP_776009.1, Murray Valley encephalitis virus NP_722539.1, Langat virus NP_740302.1. Cysteines are highlighted in yellow with the positions of the 3 glutathionylated cysteines identified in this study shown in green highlight in the alignment. Blue bars illustrate the residues of the finger subdomains, the red bars show the residues of the palm domain and the purple bars show the residues of the thumb domain . Conservation of amino acid sequence is shown by 100 percent conserved as white on black, between 80 to 100 percent conserved as white on dark grey, between 60 to 80 percent conserved as black on light grey, and less than 60 percent conserved as black on white.

Article Snippet: 1 mg of cell lysates were immunoprecipitated with 1 mg/ml of an anti-DENV 2 NS5 protein monoclonal antibody (GeneTex).

Techniques: Virus, Sequencing