Journal: Antiviral research

Article Title: High-throughput screening for the identification of small-molecule inhibitors of the flaviviral protease

doi: 10.1016/j.antiviral.2016.08.014

Figure Lengend Snippet: Active-Site titration of DENV2 protease with BPTI. X-intercept indicates the active concentration of DENV2 protease (25 nM protein was used based on protein concentration).

Article Snippet: BHK-21 cells (ATCC), stably expressing DENV2 replicon RNA ( Alcaraz-Estrada et al., 2013 ; Boonyasuppayakorn et al., 2014 ; Manzano et al., 2011 ) encoding Renilla luciferase reporter (1×10 4 cells/well of a 96-well plate), were treated with varying concentrations (0, 0.5, 1, 2, 5, 10, and 25 μM) of different inhibitors.

Techniques: Titration, Concentration Assay, Protein Concentration

Journal: Antiviral research

Article Title: High-throughput screening for the identification of small-molecule inhibitors of the flaviviral protease

doi: 10.1016/j.antiviral.2016.08.014

Figure Lengend Snippet: Percent Inhibition of DENV2 and WNV proteases by compounds (at 10 and 25 μM) selected from HTS and validation assays.

Article Snippet: BHK-21 cells (ATCC), stably expressing DENV2 replicon RNA ( Alcaraz-Estrada et al., 2013 ; Boonyasuppayakorn et al., 2014 ; Manzano et al., 2011 ) encoding Renilla luciferase reporter (1×10 4 cells/well of a 96-well plate), were treated with varying concentrations (0, 0.5, 1, 2, 5, 10, and 25 μM) of different inhibitors.

Techniques: Inhibition, Biomarker Discovery

Journal: Antiviral research

Article Title: High-throughput screening for the identification of small-molecule inhibitors of the flaviviral protease

doi: 10.1016/j.antiviral.2016.08.014

Figure Lengend Snippet: Ki, EC50, CC50 and SI index of selected compounds.

Article Snippet: BHK-21 cells (ATCC), stably expressing DENV2 replicon RNA ( Alcaraz-Estrada et al., 2013 ; Boonyasuppayakorn et al., 2014 ; Manzano et al., 2011 ) encoding Renilla luciferase reporter (1×10 4 cells/well of a 96-well plate), were treated with varying concentrations (0, 0.5, 1, 2, 5, 10, and 25 μM) of different inhibitors.

Techniques: Inhibition

Journal: Antiviral research

Article Title: High-throughput screening for the identification of small-molecule inhibitors of the flaviviral protease

doi: 10.1016/j.antiviral.2016.08.014

Figure Lengend Snippet: EC50 values for inhibition of DENV2-infected BHK-21 cells determined by plaque assays. Compound C (A), Compound D (B), Compound F (C), Compound G (D), and Compound H (E).

Article Snippet: BHK-21 cells (ATCC), stably expressing DENV2 replicon RNA ( Alcaraz-Estrada et al., 2013 ; Boonyasuppayakorn et al., 2014 ; Manzano et al., 2011 ) encoding Renilla luciferase reporter (1×10 4 cells/well of a 96-well plate), were treated with varying concentrations (0, 0.5, 1, 2, 5, 10, and 25 μM) of different inhibitors.

Techniques: Inhibition, Infection

Journal: Antiviral research

Article Title: High-throughput screening for the identification of small-molecule inhibitors of the flaviviral protease

doi: 10.1016/j.antiviral.2016.08.014

Figure Lengend Snippet: Inhibition of DENV2 replication analyzed by qPCR. BHK-21 cells were infected with DENV2 (MOI 1) and the viral RNA were isolated after 24h and the RNA copy number was determined as described under Materials and Methods. DMSO treated control is taken as 100% and percent reduction of viral RNA by treatment of DENV-2 infected BHK-21 cells with the compounds at the indicated concentrations is plotted. Compounds C, D and H at 10 μM; Compounds F and G at 2 μM concentrations.

Article Snippet: BHK-21 cells (ATCC), stably expressing DENV2 replicon RNA ( Alcaraz-Estrada et al., 2013 ; Boonyasuppayakorn et al., 2014 ; Manzano et al., 2011 ) encoding Renilla luciferase reporter (1×10 4 cells/well of a 96-well plate), were treated with varying concentrations (0, 0.5, 1, 2, 5, 10, and 25 μM) of different inhibitors.

Techniques: Inhibition, Infection, Isolation, Control

Journal: Antiviral research

Article Title: High-throughput screening for the identification of small-molecule inhibitors of the flaviviral protease

doi: 10.1016/j.antiviral.2016.08.014

Figure Lengend Snippet: (A) Absorbance spectra of compounds C, F, G and H. (B) Fluorescence spectra of DENV2 protease (2μM) with various concentrations of compounds, C, F, G and H. (C) Competition of BPTI on the fluorescence quenching in the presence of 50 μM compound and 10 μM BPTI. (D) Kinetic analysis of compounds C, F and G. Lineweaver-Burk (LB) plots, of compounds, C, F, and G.

Article Snippet: BHK-21 cells (ATCC), stably expressing DENV2 replicon RNA ( Alcaraz-Estrada et al., 2013 ; Boonyasuppayakorn et al., 2014 ; Manzano et al., 2011 ) encoding Renilla luciferase reporter (1×10 4 cells/well of a 96-well plate), were treated with varying concentrations (0, 0.5, 1, 2, 5, 10, and 25 μM) of different inhibitors.

Techniques: Fluorescence

Journal: Nature communications

Article Title: Structures of flavivirus RNA promoters suggest two binding modes with NS5 polymerase.

doi: 10.1038/s41467-021-22846-1

Figure Lengend Snippet: Fig. 1 NS5 binding analysis of tRNA-fused stem-loop A (tRNA-SLA). a Interaction between DENV NS5 and SLA. Interactions between NS5 and various SLAs (tRNA-SLADENV and in vitro transcribed SLA70 and SLA80) were examined by electrophoretic mobility shift assay (EMSA). SLA70 and SLA80 contain the first 70 and 80 nucleotides of DENV2 RNA genome, respectively. The tRNA-SLADENV, SLA70 and SLA80 (1 μM) were titrated with increasing concentrations of NS5 from 1 μM to 4 μM. NS5 binds tRNA-SLA, SLA70, and SLA80 similarly. EMSA was performed twice with similar results. b Competition assay between tRNA-SLADENV and other nucleic acids for DENV NS5. The tRNA-SLADENV (1 μM) was titrated with increasing concentrations of DENV NS5 in the presence of tRNAphe, ssDNA (43 nt), and ssRNA (8 nt) (1 μM each). The nucleic acids were stained with ethidium bromide and visualized under UV at 302 nm. The competition assay was repeated twice with similar results. c Interaction between ZIKV NS5 and SLA. Interactions between NS5 and various SLAs (tRNA-SLAZIKV and in vitro transcribed SLA71 and SLA81) were analyzed by EMSA. EMSA was carried out similarly as in a. EMSA was performed twice with similar results. d Competition assay between tRNA-SLAZIKV and other nucleic acids for ZIKV NS5. Competition assay with tRNAphe, ssDNA, and ssRNA was carried out as described above, and repeated twice with similar results. Source data for figures a- d are provided as a Source Data file.

Article Snippet: The DENV2 RNA (∼3 μg) containing either the WT DENV2 SLA, SLA-ZIKV, or SLA-9A was transfected by electroporation (Amaxa Nucleofector II system, Amaxa Biosystems, Cologne, Germany) into BHK-21 cells (American Type Culture Collection, Manassas, VA), as previously described57.

Techniques: Binding Assay, In Vitro, Electrophoretic Mobility Shift Assay, Competitive Binding Assay, Staining

Journal: Nature communications

Article Title: Structures of flavivirus RNA promoters suggest two binding modes with NS5 polymerase.

doi: 10.1038/s41467-021-22846-1

Figure Lengend Snippet: Fig. 3 Comparison of DENV and ZIKV stem-loop A (SLA) structures. a Sequence alignment of flavivirus SLAs. The SLA sequences from DENV1 (NCBI accession number, NC_001477), DENV2 (NC_001474), DENV3 (NC_001475), DENV4 (NC_002640), WNV (NC_001563), JEV (NC_001437) and ZIKV (KU527068) are aligned. The overall sequence identity among the seven listed SLAs is ~25%. Conserved nucleotides are shaded in pink. The top stem- loop, side loop, and the bottom stem are indicated for DENV2 (top) and ZIKV SLA (bottom). The AG motif and the U-bulge are boxed, and the side loop regions in DENV and ZIKV SLAs involved in the kissing-loop interaction are underlined. b Superposition of the DENV and ZIKV SLA structures. The DENV and ZIKV SLA structures were overlaid by the conserved bottom stem. DENV SLA is colored as in Fig. 2a and ZIKV SLA in light blue. The nucleotides in the AG motif and the U-bulge are indicated in DENV (black letters) and ZIKV (blue letters). The top stem-loops of DENV and ZIKV SLAs are related by ~180° rotation. c Kissing-loop interactions in the DENV SLA dimer. One tRNA-SLADENV molecule is shown as in Fig. 2a, and the other molecule is shown in yellow. The self-complementary, side loop sequence that forms kissing-loop interactions is shown below. d Kissing-loop interactions in the ZIKV SLA dimer. The tRNA-SLAZIKV dimer is colored as in Fig. 2c. The palindromic sequence that forms kissing loop interactions between the side loops is shown below.

Article Snippet: The DENV2 RNA (∼3 μg) containing either the WT DENV2 SLA, SLA-ZIKV, or SLA-9A was transfected by electroporation (Amaxa Nucleofector II system, Amaxa Biosystems, Cologne, Germany) into BHK-21 cells (American Type Culture Collection, Manassas, VA), as previously described57.

Techniques: Comparison, Sequencing

Journal: Nature communications

Article Title: Structures of flavivirus RNA promoters suggest two binding modes with NS5 polymerase.

doi: 10.1038/s41467-021-22846-1

Figure Lengend Snippet: Fig. 4 Self-complementarity of the side loop is required for viral replication. a The size exclusion chromatography of tRNA-SLADENV. The RNA elutes in two peaks, the early peak likely corresponding to a dimer (D, 91 kDa), and the second peak corresponding to the monomer (M, 45.5 kDa). Protein molecular weight standards are shown on top of the elution profile of tRNA-SLADENV. The size exclusion chromatography was performed twice with similar results. b Side loop in SLA mediates RNA-RNA interactions in solution. Interaction between tRNA-SLADENV and fluorescein-labeled RNAs (F-RNA) was examined by EMSA. See Supplementary Fig. 5 for the RNA sequences. The tRNA-SLADENV interacts with SLA, SLA(-), and the ssRNA complementary to the side loop sequence (Side-M). The tRNA-SLADENV does not bind 8-mer random sequence (negative control), the ssRNA complementary to the top stem (Top), or the self-complementary side-loop RNA (Side). Thus, side loop in SLA mediates RNA-RNA interactions. Binding experiments repeated twice with similar results. Source data are provided as a Source Data file. c Replication of DENV containing SLA mutations. DENV2 SLA was replaced with either ZIKV (SLA-ZIKV) or 9 A (SLA-9A) in the side-loop, and replication of WT and mutant viruses were monitored by immunofluorescence staining using monoclonal anti-NS1 antibody (green fluorescence color) and nuclear staining with 4’,6-diamidino-2-phenylindole (DAPI, blue fluorescent color). Scale bar represents 50 μm. Replication experiments were repeated three times with similar results. d RNA-seq analysis of recovered SLA-9A virus. The numbers and percentage of each haplotypes were calculated from 8,489 total reads. The cutoff of inclusion is >4% abundance. Altered nucleotides in 8 haplotypes are shown in red. Potential intermolecular side-loop interactions resulting from the mutations are shown on the right.

Article Snippet: The DENV2 RNA (∼3 μg) containing either the WT DENV2 SLA, SLA-ZIKV, or SLA-9A was transfected by electroporation (Amaxa Nucleofector II system, Amaxa Biosystems, Cologne, Germany) into BHK-21 cells (American Type Culture Collection, Manassas, VA), as previously described57.

Techniques: Size-exclusion Chromatography, Molecular Weight, Labeling, Sequencing, Negative Control, Binding Assay, Mutagenesis, Staining, RNA Sequencing, Virus