Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase-7

<p><b>Copyright information:</b></p><p>Taken from "Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase"</p><p>http://www.biomedcentral.com/1471-2199/8/59</p><p>BMC Molecular Biology 2007;8():59-59.</p><p>Published online 11 Jul 2007</p><p>PMCID:PMC1934914.</p><p></p>hown below the structures predicted by the Mfold program. Partial structures representing the lower stem-region are shown. The deleted and added nucleotides are indicated by ρ and boxed, respectively. (B) RNA synthesis directed by the wild-type 83-nt RNA and its derivatives. RdRp assays were performed with the RNA templates indicated above the autoradiogram, and products were resolved a 5% polyacrylamide sequencing gel (20 × 40 cm) containing 8 M urea. Arrowhead indicates the 81-nt internally initiated RNA product. Representative data from three independent experiments are shown

Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase-8

<p><b>Copyright information:</b></p><p>Taken from "Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase"</p><p>http://www.biomedcentral.com/1471-2199/8/59</p><p>BMC Molecular Biology 2007;8():59-59.</p><p>Published online 11 Jul 2007</p><p>PMCID:PMC1934914.</p><p></p>eactions were performed in the presence of cold UTP and [α-P] UTP (lane 3), or in the presence of single [α-P] UTP (lane 4). An RNA product from the standard RdRp reaction mixture is shown as a control (lane 2). Lane 1, 5'-end labeled 83-nt RNA size marker

Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase-5

<p><b>Copyright information:</b></p><p>Taken from "Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase"</p><p>http://www.biomedcentral.com/1471-2199/8/59</p><p>BMC Molecular Biology 2007;8():59-59.</p><p>Published online 11 Jul 2007</p><p>PMCID:PMC1934914.</p><p></p>the 3'-terminal region of JEV genome, were left untreated (-) or digested with nuclease S1 (S1), and resolved on a denaturing polyacrylamide gel. Nuclease S1 treatments were performed in 50 mM NaCl (L; low salt) or 500 mM NaCl (H; high salt). Arrowhead indicates the position of the 83-nt RNA template

Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase-0

<p><b>Copyright information:</b></p><p>Taken from "Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase"</p><p>http://www.biomedcentral.com/1471-2199/8/59</p><p>BMC Molecular Biology 2007;8():59-59.</p><p>Published online 11 Jul 2007</p><p>PMCID:PMC1934914.</p><p></p>and ion-exchange chromatography using an SP-Sepharose column. (A) Imidazole elution profile of JEV NS5 from Ni-NTA resin. (B) Gel filtration chromatography elution profile of JEV NS5. (C) NaCl-elution profile of JEV NS5 from an SP-Sepharose column. (A-C) Fractions from each purification step were resolved by SDS-12% PAGE and stained with Coomassie brilliant blue. (D) JEV NS5 and its mutant NS5from a peak fraction eluted from an SP-Sepharose column were resolved by SDS-12% PAGE and visualized by silver staining. (E) Western blot analysis of the purified JEV NS5 and NS5using an anti-Hisantibody. The sizes of protein markers are indicated in kilodaltons. Closed and open arrowheads indicate the full-length JEV NS5, and its major cleaved form identified by MALDI-TOF analysis, respectively

Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase-6

<p><b>Copyright information:</b></p><p>Taken from "Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase"</p><p>http://www.biomedcentral.com/1471-2199/8/59</p><p>BMC Molecular Biology 2007;8():59-59.</p><p>Published online 11 Jul 2007</p><p>PMCID:PMC1934914.</p><p></p>oduct synthesized by JEV NS5 using the 83-nt RNA template. Products were resolved on a 5% polyacrylamide sequencing gel (20 × 40 cm) containing 8 M urea. The RNA size markers, 5'-end labeled RNA template (End), and a set of labeled RNA fragments generated by alkaline hydrolysis of the 5'-end labeled RNA template (End/OH), were resolved on the same gel. Arrowhead indicates the internally initiated 81-nt RNA product. (B) The close-up autoradiogram of the same gel shown in (A). (C) Secondary structure of the 83-nt RNA template predicted by the Mfold program. Bent arrow denotes the predicted RNA synthesis initiation site

Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase-1

<p><b>Copyright information:</b></p><p>Taken from "Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase"</p><p>http://www.biomedcentral.com/1471-2199/8/59</p><p>BMC Molecular Biology 2007;8():59-59.</p><p>Published online 11 Jul 2007</p><p>PMCID:PMC1934914.</p><p></p>) RNA template in the presence (+) or absence (-) of the primer oligo(U). (B) RdRp assays were performed with the purified wild-type NS5 (GDD) and its mutant NS5(GAD) in the presence (+) or absence (-) of a poly(A) RNA template. Relative RdRp activities (%), which were obtained by comparing the P-UMP incorporation measured by liquid scintillation counting with that obtained for the reaction with the template and primer, 3.0 × 10cpm, are presented

Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase-3

<p><b>Copyright information:</b></p><p>Taken from "Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase"</p><p>http://www.biomedcentral.com/1471-2199/8/59</p><p>BMC Molecular Biology 2007;8():59-59.</p><p>Published online 11 Jul 2007</p><p>PMCID:PMC1934914.</p><p></p>nce of increasing concentrations of MgClor MnCl(lanes 2–6 and 7–11; 0.5, 1.0, 2.5, 5.0, and 10 mM of MgCland MnCl, respectively). (B) RdRp assays were performed with the 83-nt RNA representing the 3' end of the plus-strand JEV genome, in the absence of metal ions (lane 1) or in the presence of 2.5 mM of the divalent metal ion indicated above the autoradiogram (lanes 2 and 3). RdRp products were denatured and resolved on a medium size (20 × 20 cm) denaturing 5% polyacrylamide gel, and subjected to autoradiography

Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase-4

<p><b>Copyright information:</b></p><p>Taken from "Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase"</p><p>http://www.biomedcentral.com/1471-2199/8/59</p><p>BMC Molecular Biology 2007;8():59-59.</p><p>Published online 11 Jul 2007</p><p>PMCID:PMC1934914.</p><p></p>or absence (-) of JEV 3'(+)UTR RNA (A and C), 3' (-)UTR RNA (B and C), and the 83-nt RNA (D) template. RdRp products were analyzed as in Figure 4 by autoradiography. Arrowheads indicate the template positions visualized by ethidium-bromide staining of the gels

Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase-2

<p><b>Copyright information:</b></p><p>Taken from "Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase"</p><p>http://www.biomedcentral.com/1471-2199/8/59</p><p>BMC Molecular Biology 2007;8():59-59.</p><p>Published online 11 Jul 2007</p><p>PMCID:PMC1934914.</p><p></p>rmed with the poly(A)/(U)template under the indicated conditions. The RdRp activity was measured as in Figure 2 and is presented as the percentage of that observed under each optimal condition. Shown is the mean and standard error from three independent experiments

Interaction of the HCV core protein with GLD-2 in the cytoplasm is required for miR-122 expression regulation.

<p>(A) Huh7 cells were transfected with an empty vector (Ctrl) or an expression vector encoding Flag-tagged core (Core) or NS5B protein. Lysates were immunoprecipitated with anti-Flag (top) or anti-GLD-2 (bottom) antibody, followed by immunoblotting for the indicated proteins. (B) Indirect double-immunofluorescence staining of the core protein and GLD-2 in Huh7 cells transiently expressing the Flag-tagged core protein. Nuclei were visualized by DAPI staining. (C and D) Subcellular localization of GFP-fused full-length HCV core protein and its truncated derivatives transiently expressed in Huh7 cells for 48 h was analyzed by immunofluorescence microscopy (C). Nucl, nucleus; Cyto, cytoplasm. (D) represents the levels of miR-122 and GFP-fused core proteins measured by northern blot (NB) analysis and immunoblotting (IB). The numbers below the northern blot indicate the miR-122 level compared with the GFP-transfected control, quantified as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005714#ppat.1005714.g001" target="_blank">Fig 1C</a>. Total cell lysates resolved by SDS-PAGE were analyzed by immunoblotting using anti-GFP antibody (bottom). (E) RT-PCR quantification of miR-122 levels in Huh7 cells transiently expressing GFP or the indicated GFP-fused core proteins. (F) Analysis of interaction between GLD-2 and the indicated GFP-fused core proteins by co-immunoprecipitation experiments. (G) Quantitative analysis of colocalization between GLD-2 and the indicated GFP-fused core proteins in Huh7 cells. Huh7/HCV RNA, Huh7 cells transfected with HCV (JFH-1) RNA. Scale bar, 10 μm.</p