Journal: Cell reports

Article Title: The ER Membrane Protein Complex Promotes Biogenesis of Dengue and Zika Virus Non-structural Multi-pass Transmembrane Proteins to Support Infection

doi: 10.1016/j.celrep.2019.04.051

Figure Lengend Snippet: (A–D) 293T cell pools stably expressing Cas9 nuclease and an sgRNA targeting EMC6 were subsequently transiently co-transfected with constructs encoding HA-tagged DENV non-structural proteins and NS1-FLAG (A, C, and D) or GFP (B) as a transfection control. (E) ImageJ (NIH) was used to quantify band intensities for western blots of each of the DENV non-structural proteins in EMC6 knockout cells compared with wild-type from (A)–(D). Each dot represents a biological replicate. Bars represent mean ± SD. The Mann-Whitney U test was performed to assess statistical significance (*p < 0.05 and **p < 0.005 compared to NS1). (F–K) HEK293 cells were transfected with siRNAs against EMC1 or a scrambled negative control (scr). Forty-eight hours later, cells were transfected with a replication-defective full-length ZIKV cDNA (F and G) or with plasmids encoding individual S-tagged ZIKV non-structural proteins NS5 (H), NS4A (I), NS4B (J), or NS2B (K). The asterisk in (G) indicates a nonspecific background band. For (A)–(D) and (F)–(K) 24 h post-transfection, cells were lysed and proteins were separated using SDS-PAGE followed by western blotting for the indicated proteins. Each blot is representative of a minimum of two biological replicates. See also .

Article Snippet: Anti-ZIKV NS5 , Genetex , Cat# GTX133312; RRID: AB_2750559.

Techniques: Stable Transfection, Expressing, Transfection, Construct, Control, Western Blot, Knock-Out, MANN-WHITNEY, Negative Control, SDS Page

Journal: Cell reports

Article Title: The ER Membrane Protein Complex Promotes Biogenesis of Dengue and Zika Virus Non-structural Multi-pass Transmembrane Proteins to Support Infection

doi: 10.1016/j.celrep.2019.04.051

Figure Lengend Snippet: (A) The TMHMM version 2.0 algorithm was used to predict the presence of transmembrane helices in 2k-NS4B of DENV (UniProtKB: P29990). This plot depicts the probability of a given residue being located within a transmembrane helix, where greater values reflect greater transmembrane probability. The indicated amino acid positions on the x axis indicate the boundaries of each predicted transmembrane domain with scores above 0.3 for each predicted transmembrane domain. (B) HEK293T cells stably expressing Cas9 and sgRNA targeting EMC6 were co-transfected to express the indicated C-terminally GFP-tagged NS4B mutants and NS1-FLAG as a transfection control. 2k-96 indicates a C-terminal truncation at amino acid 97. 2k-58 indicates a C-terminal truncation at amino acid 59. Δ32–96 indicates a deletion from 32 to 96. Twenty-four hours post-transfection, cells were lysed and proteins were resolved using SDS-PAGE followed by western blotting for the indicated proteins. (C) Band intensitiesforNS4Bwild-type and Δ32–96 in EMC6 knockout cells compared with wild-type cells. Each point represents a biological replicate from cells transfected with NS4B-GFP (circles) or NS4B-HA (triangles). (D) Schematic of NS4B mutations to alter hydrophobicity of the pTM1 and pTM2 helices. Both of these helices are speculated to be membrane associated, with red representing hydrophobic and blue representing charged and polar hydrophilic residues, respectively (middle). Mutagenesis of the hydrophobic residues to lysine (top) is expected to result in decreased membrane association of pTM1 and pTM2. Conversely, mutagenesis of charged or polar residues to leucine (bottom) is expected to result in the insertion of pTM1 and pTM2 into the membrane. (E and F) Wild-type 293T cells or cells stably expressing Cas9 and sgRNA targeting EMC6 were transfected to express the indicated NS4B-HA mutants with substitutions to lysine residues (less hydrophobic) shown in (E) and substitutions to leucine residues (more hydrophobic) shown in (F). Twenty-four hours post-transfection, cells were lysed and proteins were resolved using SDS-PAGE followed by western blotting for the indicated proteins. Blots are representative of a minimum of three independent experiments.(G) Bands were quantitated using ImageJ and represented as a ratio of band intensity in EMC6 knockout versus wild-type cells. Each point represents a biological replicate, with bars representing mean ± SD. Triangles represent quantification from blots of NS4B with PNGase treatment, while circles represent quantification from blots of NS4B without PNGase treatment. Statistical significance was assessed using Dunnett’s test for multiple comparisons (**p < 0.005 and ***p < 0.0005 compared with WT NS4B). (H) Model of EMC interaction with theSec61 translocon and the flavivirus protein. Depicted is a flavivirus genomic RNA (upper left) being translated by a ribosome (blue) at the cytosolic face of the ER. Transmembrane domains are cotranslationally inserted into the ER membrane by the Sec61 translocon (brown). The EMC (yellow) is associated with the translocon and assists in the proper insertion and stabilization of certain multi-pass transmembrane domain proteins at the time of protein translation and/or translocation. The expression of both NS4A and NS4B of DENV and ZIKV is dependent on the EMC. Expression of the next protein in the flavivirus polypeptide, NS5, is also decreased in EMC-deficient cells when NS5 is expressed on the same polyprotein as NS4A and NS4B but not when expressed by itself.

Article Snippet: Anti-ZIKV NS5 , Genetex , Cat# GTX133312; RRID: AB_2750559.

Techniques: Residue, Stable Transfection, Expressing, Transfection, Control, SDS Page, Western Blot, Knock-Out, Membrane, Mutagenesis, Translocation Assay

Journal: Cell reports

Article Title: The ER Membrane Protein Complex Promotes Biogenesis of Dengue and Zika Virus Non-structural Multi-pass Transmembrane Proteins to Support Infection

doi: 10.1016/j.celrep.2019.04.051

Figure Lengend Snippet:

Article Snippet: Anti-ZIKV NS5 , Genetex , Cat# GTX133312; RRID: AB_2750559.

Techniques: Recombinant, Protease Inhibitor, Membrane, Luciferase, Transfection, Virus, Software

Journal: bioRxiv

Article Title: Inhibition of the DENV2 and ZIKV RNA polymerases by Galidesivir triphosphate measured using a continuous fluorescence assay

doi: 10.1101/2022.12.20.521302

Figure Lengend Snippet: Optimisation of the SYTO 9 fluorescence-based RdRp assay using DENV2 NS5. Unless otherwise stated reactions contained 200 nM DENV2 NS5, 2.5 mM MnCl 2 , 40 μg/mL poly(U), 2.5 μM SYTO 9 and were initiated by addition of 0.5 mM ATP. (Panel A) Time-dependent fluorescence (excitation 485/emission 528 nm) from a DENV2 NS5 catalyzed reaction, and a matched negative control without ATP. (Panels B to F) Dependence of measured rate on concentrations of: SYTO 9 dye (Panel B), poly(U) (Panels C and D*), Mn 2+ (Panel E) and DENV2 NS5 (Panel F). Measured rates in fluorescence units per second are given as a percentage relative to the maximum rate for that experiment. In panels B, C, E and F the hollow circles show the results of individual technical replicates, while the solid lines are drawn through the median of each set of technical replicates. * Panel C shows measured rate as a function of poly(U) concentration while panel D shows the biphasic reaction progress curves characteristically observed with poly(U) concentrations under 10 μg/mL.

Article Snippet: Genes encoding the full length NS5 proteins from DENV2 (GenBank accession no. NC001474 , UniProt accession no. P29990 ), and ZIKV (GenBank accession no. NC012532 , UniProt accession no. Q32ZE1 ) were commercially synthesized and cloned into plasmid pUC57 by GenScript Biotech.

Techniques: Fluorescence, Negative Control, Concentration Assay

Journal: bioRxiv

Article Title: Inhibition of the DENV2 and ZIKV RNA polymerases by Galidesivir triphosphate measured using a continuous fluorescence assay

doi: 10.1101/2022.12.20.521302

Figure Lengend Snippet: Enhancement of NS5 DENV2 activity by the addition of short poly(A) primers. Reactions contained 200 nM DENV2 NS5, 1.5 mM MnCl 2 , 40 μg/mL poly(U), 3 μM SYTO 9 and were initiated with 10 μM ATP. In addition, reactions contained either no primer ( de novo reaction) or 10 μM of a non-5’-phosphorylated poly(A) primer. The primers tested were A2 (ApA), A3 (ApApA), A4 (ApApApA) and A5 (ApApApApA). Grey bars represent the mean relative rate of three technical replicates, and hollow circles indicate the individual replicate values.

Article Snippet: Genes encoding the full length NS5 proteins from DENV2 (GenBank accession no. NC001474 , UniProt accession no. P29990 ), and ZIKV (GenBank accession no. NC012532 , UniProt accession no. Q32ZE1 ) were commercially synthesized and cloned into plasmid pUC57 by GenScript Biotech.

Techniques: Activity Assay, Relative Rate

Journal: bioRxiv

Article Title: Inhibition of the DENV2 and ZIKV RNA polymerases by Galidesivir triphosphate measured using a continuous fluorescence assay

doi: 10.1101/2022.12.20.521302

Figure Lengend Snippet: ATP dependence of DENV2 NS5 activity in the absence and presence of an A2 primer. The apparent K M for ATP was determined in the absence of primer ( de novo , Panel A), and in the presence of A2 (ApA-primer at concentrations of 10, 20 and 40 μM (Panels B, C and D respectively). Rates are reported as nucleotides incorporated per molecule of DENV2 NS5 per minute. Reactions contained 200 nM DENV2 NS5, 0 to 40 μM A2 primer, 40 μg/mL poly(U), 2.5 mM MnCl 2 , 3 μM SYTO 9, and were initiated with ATP at concentrations ranging from 0 to 1500 μM. Hollow circles show the experimental measurements, while solid lines show the fit of the data to the Michaelis-Menten equation.

Article Snippet: Genes encoding the full length NS5 proteins from DENV2 (GenBank accession no. NC001474 , UniProt accession no. P29990 ), and ZIKV (GenBank accession no. NC012532 , UniProt accession no. Q32ZE1 ) were commercially synthesized and cloned into plasmid pUC57 by GenScript Biotech.

Techniques: Activity Assay

Journal: bioRxiv

Article Title: Inhibition of the DENV2 and ZIKV RNA polymerases by Galidesivir triphosphate measured using a continuous fluorescence assay

doi: 10.1101/2022.12.20.521302

Figure Lengend Snippet: Inhibition of DENV2 (Panel A) and ZIKV (Panel B) NS5 RdRp activity by Gal-TP in A2 primed reactions. Dose-response curves for Gal-TP against NS5 RdRp activity were determined in three independent experiments as shown. Inhibition assay components are given in . Rates are given as a percentage relative to the uninhibited rate in the absence of Gal-TP. Data (hollow circles) were fit to a four-parameter logistic model (solid lines) to determine IC 50 values.

Article Snippet: Genes encoding the full length NS5 proteins from DENV2 (GenBank accession no. NC001474 , UniProt accession no. P29990 ), and ZIKV (GenBank accession no. NC012532 , UniProt accession no. Q32ZE1 ) were commercially synthesized and cloned into plasmid pUC57 by GenScript Biotech.

Techniques: Inhibition, Activity Assay

Journal: bioRxiv

Article Title: Inhibition of the DENV2 and ZIKV RNA polymerases by Galidesivir triphosphate measured using a continuous fluorescence assay

doi: 10.1101/2022.12.20.521302

Figure Lengend Snippet: Inhibition of DENV2 (Panel A) and ZIKV (Panel B) NS5 RdRp activity by 3’-dATP in A2 primed reactions. Dose-response curves for 3’-dATP against NS5 RdRp activity were determined in three independent experiments as shown. Inhibition assay components are given in . Rates are given as a percentage relative to the uninhibited rate in the absence of 3’-dATP. Data (hollow circles) were fit to a four-parameter logistic model (solid lines) to determine IC 50 values.

Article Snippet: Genes encoding the full length NS5 proteins from DENV2 (GenBank accession no. NC001474 , UniProt accession no. P29990 ), and ZIKV (GenBank accession no. NC012532 , UniProt accession no. Q32ZE1 ) were commercially synthesized and cloned into plasmid pUC57 by GenScript Biotech.

Techniques: Inhibition, Activity Assay