scholarly journals Transient kinetic analysis of the elongation mode of Dengue Virus RNA polymerase domain

2010 ◽  
Vol 24 (S1) ◽  
Author(s):  
Zhinan Jin ◽  
Jerome Deval ◽  
Kenneth A. Johnson ◽  
David C. Swinney
Keyword(s):  
Dengue Virus ◽  
Rna Polymerase ◽  
Kinetic Analysis ◽  
Polymerase Domain ◽  
Virus Rna

scholarly journals Pre-Steady-State Kinetic Analysis of the Elongation Mode of Dengue Virus RNA Polymerase Domain

2010 ◽  
Vol 98 (3) ◽  
pp. 451a-452a
Author(s):  
Zhinan Jin ◽  
Jerome Deval ◽  
Kenneth A. Johnson ◽  
David C. Swinney
Keyword(s):  
Steady State ◽  
Dengue Virus ◽  
Rna Polymerase ◽  
Kinetic Analysis ◽  
Polymerase Domain ◽  
Virus Rna ◽  
Steady State Kinetic ◽  
State Kinetic

scholarly journals Full-length Dengue Virus RNA-dependent RNA Polymerase-RNA/DNA Complexes

2011 ◽  
Vol 286 (38) ◽  
pp. 33095-33108 ◽  
Author(s):  
Michal R. Szymanski ◽  
Maria J. Jezewska ◽  
Paul J. Bujalowski ◽  
Cecile Bussetta ◽  
Mengyi Ye ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Rna Polymerase ◽  
Full Length ◽  
Rna Dependent Rna Polymerase ◽  
Dna Complexes ◽  
Virus Rna

scholarly journals Conformational Flexibility of the Dengue Virus RNA-Dependent RNA Polymerase Revealed by a Complex with an Inhibitor

2013 ◽  
Vol 87 (9) ◽  
pp. 5291-5295 ◽  
Author(s):  
C. G. Noble ◽  
S. P. Lim ◽  
Y.-L. Chen ◽  
C. W. Liew ◽  
L. Yap ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Rna Polymerase ◽  
Conformational Flexibility ◽  
Rna Dependent Rna Polymerase ◽  
Virus Rna

scholarly journals High Fidelity of Yellow Fever Virus RNA Polymerase

2004 ◽  
Vol 78 (2) ◽  
pp. 1032-1038 ◽  
Author(s):  
Konstantin V. Pugachev ◽  
Farshad Guirakhoo ◽  
Simeon W. Ocran ◽  
Fred Mitchell ◽  
Megan Parsons ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Rna Polymerase ◽  
Yellow Fever ◽  
Error Rate ◽  
Yellow Fever Virus ◽  
Protein A ◽  
Fever Virus ◽  
Genome Replication ◽  
Virus Rna

ABSTRACT Three consecutive plaque purifications of four chimeric yellow fever virus-dengue virus (ChimeriVax-DEN) vaccine candidates against dengue virus types 1 to 4 were performed. The genome of each candidate was sequenced by the consensus approach after plaque purification and additional passages in cell culture. Our data suggest that the nucleotide sequence error rate for SP6 RNA polymerase used in the in vitro transcription step to initiate virus replication was as high as 1.34 × 10−4 per copied nucleotide and that the error rate of the yellow fever virus RNA polymerase employed by the chimeras for genome replication in infected cells was as low as 1.9 × 10−7 to 2.3 × 10−7. Clustering of beneficial mutations that accumulated after multiple virus passages suggests that the N-terminal part of the prM protein, a specific site in the middle of the E protein, and the NS4B protein may be essential for nucleocapsid-envelope interaction during flavivirus assembly.

scholarly journals De Novo Synthesis of Negative-Strand RNA by Dengue Virus RNA-Dependent RNA Polymerase In Vitro: Nucleotide, Primer, and Template Parameters

2003 ◽  
Vol 77 (16) ◽  
pp. 8831-8842 ◽  
Author(s):  
Masako Nomaguchi ◽  
Matt Ackermann ◽  
Changsuek Yon ◽  
Shihyun You ◽  
R. Padmanbhan
Keyword(s):  
Dengue Virus ◽  
Rna Polymerase ◽  
De Novo ◽  
Mutational Analysis ◽  
Synthesis Product ◽  
De Novo Synthesis ◽  
High Concentration ◽  
Rna Dependent Rna Polymerase ◽  
Terminal Nucleotide ◽  
Virus Rna

ABSTRACT By using a purified dengue virus RNA-dependent RNA polymerase and a subgenomic 770-nucleotide RNA template, it was shown previously that the ratio of the de novo synthesis product to hairpin product formed was inversely proportional to increments of assay temperatures (20 to 40°C). In this study, the components of the de novo preinitiation complex are defined as ATP, a high concentration of GTP (500 μM), the polymerase, and the template RNA. Even when the 3′-terminal sequence of template RNA was mutated from -GGUUCU-3′ to -GGUUUU-3′, a high GTP concentration was required for de novo initiation, suggesting that high GTP concentration plays a conformational role. Furthermore, utilization of synthetic primers by the polymerase indicated that AGAA is the optimal primer whereas AG, AGA, and AGAACC were inefficient primers. Moreover, mutational analysis of the highly conserved 3′-terminal dinucleotide CU of the template RNA indicated that change of the 3′-terminal nucleotide from U to C reduced the efficiency about fivefold. The order of preference for the 3′-terminal nucleotide, from highest to lowest, is U, A∼G, and C. However, change of the penultimate nucleotide from C to U did not affect the template activity. A model consistent with these results is that the active site of the polymerase switches from a “closed” form, catalyzing de novo initiation through synthesis of short primers, to an “open” form for elongation of a double-stranded template-primer.

scholarly journals A Fluorescence-Based Alkaline Phosphatase–Coupled Polymerase Assay for Identification of Inhibitors of Dengue Virus RNA-Dependent RNA Polymerase

2011 ◽  
Vol 16 (2) ◽  
pp. 201-210 ◽  
Author(s):  
Pornwaratt Niyomrattanakit ◽  
Siti Nurdiana Abas ◽  
Chin Chin Lim ◽  
David Beer ◽  
Pei-Yong Shi ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Dengue Virus ◽  
Rna Polymerase ◽  
High Throughput Screening ◽  
Drug Target ◽  
Rna Dependent Rna Polymerase ◽  
Signal Stability ◽  
Nucleotide Analogue ◽  
Virus Rna ◽  
Adenosine Nucleotide

The flaviviral RNA-dependent RNA polymerase (RdRp) is an attractive drug target. To discover new inhibitors of dengue virus RdRp, the authors have developed a fluorescence-based alkaline phosphatase–coupled polymerase assay (FAPA) for high-throughput screening (HTS). A modified nucleotide analogue (2′-[2-benzothiazoyl]-6′-hydroxybenzothiazole) conjugated adenosine triphosphate (BBT-ATP) and 3′UTR-U30 RNA were used as substrates. After the polymerase reaction, treatment with alkaline phosphatase liberates the BBT fluorophore from the polymerase reaction by-product, BBTPPi, which can be detected at excitation and emission wavelengths of 422 and 566 nm, respectively. The assay was evaluated by examining the time dependency, assay reagent effects, reaction kinetics, and signal stability and was validated with 3′dATP and an adenosine-nucleotide triphosphate inhibitor, giving IC50 values of 0.13 µM and 0.01 µM, respectively. A pilot screen of a diverse compound library of 40,572 compounds at 20 µM demonstrated good performance with an average Z factor of 0.81. The versatility and robustness of FAPA were evaluated with another substrate system, BBT-GTP paired with 3′UTR-C30 RNA. The FAPA method presented here can be readily adapted for other nucleotide-dependent enzymes that generate PPi.

Biochemical and kinetic analysis of the influenza virus RNA polymerase purified from insect cells

2010 ◽  
Vol 391 (1) ◽  
pp. 570-574 ◽  
Author(s):  
Shijian Zhang ◽  
Leiyun Weng ◽  
Liqing Geng ◽  
Jinlan Wang ◽  
Jingling Zhou ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Kinetic Analysis ◽  
Insect Cells ◽  
Virus Rna

Biochemical characterization of the inhibition of the dengue virus RNA polymerase by beta-d-2′-ethynyl-7-deaza-adenosine triphosphate

2010 ◽  
Vol 87 (2) ◽  
pp. 213-222 ◽  
Author(s):  
Derek R. Latour ◽  
Andreas Jekle ◽  
Hassan Javanbakht ◽  
Robert Henningsen ◽  
Peter Gee ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Rna Polymerase ◽  
Adenosine Triphosphate ◽  
Biochemical Characterization ◽  
Virus Rna

scholarly journals Characterization of the Elongation Complex of Dengue Virus RNA Polymerase

2010 ◽  
Vol 286 (3) ◽  
pp. 2067-2077 ◽  
Author(s):  
Zhinan Jin ◽  
Jerome Deval ◽  
Kenneth A. Johnson ◽  
David C. Swinney
Keyword(s):  
Dengue Virus ◽  
Rna Polymerase ◽  
Elongation Complex ◽  
Virus Rna
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