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Literature summary for 2.7.7.48 extracted from

  • Lu, G.; Bluemling, G.R.; Collop, P.; Hager, M.; Kuiper, D.; Gurale, B.P.; Painter, G.R.; De La Rosa, A.; Kolykhalov, A.A.
    Analysis of ribonucleotide 5-triphosphate analogs as potential inhibitors of Zika Virus RNA-dependent RNA polymerase by using nonradioactive polymerase assays (2017), Antimicrob. Agents Chemother., 61, e01967-16 .
    View publication on PubMedView publication on EuropePMC

Activating Compound

Activating Compound Comment Organism Structure
dithiothreitol 10 mM, required to achieve maximal polymerase activity Zika virus

Cloned(Commentary)

Cloned (Comment) Organism
a codon-optimized cDNA sequence of ZIKV NS5 (amino acids 2522 to 3423; strain H/PF/2013) is synthesized de novo. The ZIKV NS5 polymerase portion is PCR amplified and cloned into a pMal-c5X vector under tac promoter control. Expression in Escherichia coli Zika virus
expression in Escherichia coli Dengue virus

Protein Variants

Protein Variants Comment Organism
additional information active-site mutation, GDD -> GAA, in amino acid positions 663 to 665 of NS5 causes the complete loss of NS5 polymerase activity Zika virus

Inhibitors

Inhibitors Comment Organism Structure
3'-dATP incorporation of the 3'-deoxynucleotide triphosphate fully prevents extension of a product in the presence of the next correct ribonucleotide Zika virus
3'-dCTP incorporation of the 3'-deoxynucleotide triphosphate fully prevents extension of a product in the presence of the next correct ribonucleotide Zika virus
3'-dGTP incorporation of the 3'-deoxynucleotide triphosphate fully prevents extension of a product in the presence of the next correct ribonucleotide Zika virus
3'-dUTP incorporation of the 3'-deoxynucleotide triphosphate fully preventes extension of a product in the presence of the next correct ribonucleotide Dengue virus
3'-dUTP incorporation of the 3'-deoxynucleotide triphosphate fully prevents extension of a product in the presence of the next correct ribonucleotide Zika virus
4'-C-azidocytidine-5'-triphosphate causes only partial chain termination Zika virus
beta-D-2'-alpha-deoxy-2'-beta-fluoro-2'-C-methyluridine-5'-triphosphate causes chain termination after incorporation into nascent RNA Dengue virus
beta-D-2'-alpha-deoxy-2'-beta-fluoro-2'-C-methyluridine-5'-triphosphate causes chain termination after incorporation into nascent RNA Zika virus
beta-D-2'-beta-C-ethynyl-7-deaza-adenosine-5'-triphosphate causes chain termination after incorporation into nascent RNA Zika virus
beta-D-2'-beta-C-ethynyluridine-5'-triphosphate causes chain termination after incorporation into nascent RNA Dengue virus
beta-D-2'-beta-C-ethynyluridine-5'-triphosphate causes chain termination after incorporation into nascent RNA Zika virus
beta-D-2'-beta-C-methyladenosine-5'-triphosphate causes chain termination after incorporation into nascent RNA Zika virus
beta-D-2'-beta-C-methylcytidine-5'-triphosphate causes chain termination after incorporation into nascent RNA Zika virus
beta-D-2'-beta-C-methylguanosine-5'-triphosphate causes chain termination after incorporation into nascent RNA Zika virus
beta-D-2'-beta-C-methyluridine-5'-triphosphate causes chain termination after incorporation into nascent RNA Dengue virus
beta-D-2'-beta-C-methyluridine-5'-triphosphate causes chain termination after incorporation into nascent RNA Zika virus
beta-D-2'-deoxy-2'-alpha-fluoro-2'-beta-C-methylcytidine-5'-triphosphate causes chain termination after incorporation into nascent RNA Zika virus
beta-D-2'-deoxy-2'-alpha-fluoro-2'-beta-C-methylguanosine-5'-triphosphate causes chain termination after incorporation into nascent RNA Zika virus
imidazole tolerable at low concentrations, but inhibits at concentrations higher than 25 mM Zika virus
NaCl tolerable at low concentrations, but inhibits at concentrations higher than 25 mM Zika virus

Metals/Ions

Metals/Ions Comment Organism Structure
Mn2+ Mn2+x02is required for enzymatic activity, while Mg2+x02 is not Zika virus

Organism

Organism UniProt Comment Textmining
Dengue virus
-
-
-
Zika virus A0A024B7W1 genome polyprotein
-

Purification (Commentary)

Purification (Comment) Organism
-
Dengue virus
-
Zika virus

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
nucleoside triphosphate + RNAn
-
Dengue virus diphosphate + RNAn+1
-
?
nucleoside triphosphate + RNAn DNA can serve as a template for ZIKV NS5 with an efficiency similar to that of an RNA template. The enzyme (NS5) can utilize single-stranded DNA but not double-stranded DNA as a template or a primer to synthesize RNA. Both full-length NS5 and a truncated NS5 containing the polymerase domain can carry out in vitro RNA-dependent RNA synthesis Zika virus diphosphate + RNAn+1
-
?

Synonyms

Synonyms Comment Organism
NS5
-
Dengue virus
NS5
-
Zika virus
RNA-dependent RNA polymerase
-
Dengue virus
RNA-dependent RNA polymerase
-
Zika virus

IC50 Value

IC50 Value IC50 Value Maximum Comment Organism Inhibitor Structure
0.00033
-
pH 7.5, 35°C Dengue virus beta-D-2'-beta-C-ethynyluridine-5'-triphosphate
0.00046
-
pH 7.5, 35°C, measured in the presence of 0.001 mM competing UTP, RNA template Zika virus beta-D-2'-beta-C-ethynyluridine-5'-triphosphate
0.00067
-
pH 7.5, 35°C, measured in the presence of 0.001 mM competing UTP, RNA template Zika virus 3'-dUTP
0.00068
-
pH 7.5, 35°C Dengue virus 3'-dUTP
0.00494
-
pH 7.5, 35°C Dengue virus beta-D-2'-beta-C-methyluridine-5'-triphosphate
0.00578
-
pH 7.5, 35°C, measured in the presence of 0.001 mM competing UTP, RNA template Zika virus beta-D-2'-beta-C-methyluridine-5'-triphosphate
0.05513
-
pH 7.5, 35°C Dengue virus beta-D-2'-alpha-deoxy-2'-beta-fluoro-2'-C-methyluridine-5'-triphosphate
0.09076
-
pH 7.5, 35°C, measured in the presence of 0.001 mM competing UTP, RNA template Zika virus beta-D-2'-alpha-deoxy-2'-beta-fluoro-2'-C-methyluridine-5'-triphosphate