Stabilization of dengue virus polymerase in de novo initiation assay provides advantages for compound screening

Pornwaratt Niyomrattanakit, Kah Fei Wan, Ka Yan Chung, Siti Nurdiana Abas, Cheah Chen Seh, Hongping Dong, Chin Chin Lim, Alexander Theodore Chao, Chang Bok Lee, Shahul Nilar, Julien Lescar, Pei-Yong Shi, David Beer, Siew Pheng Lim

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Dengue virus (DENV) NS5 protein comprises an N-terminal methyltransferase domain and a C-terminal RNA-dependent RNA polymerase domain (RdRp). DENV RdRp is responsible for viral RNA synthesis via a de novo initiation mechanism and represents an attractive target for anti-viral therapy. Herein we describe the characterization of its de novo initiation activities by PAGE analyses and the knowledge gained was used to develop a fluorescent-based assay. A highly processive and robust assay was achieved by addition of cysteine in the assay buffer. This stabilized the apo-enzyme, and rendered optimal de novo initiation activity while balancing its intrinsic terminal transferase activity. Steady-state kinetic parameters of the NTP and RNA substrates under these optimal conditions were determined for DENV1-4 FL NS5. Heavy metal ions such as Zn++ and Co++ as well as high levels of monovalent salts, suppressed DENV polymerase de novo initiation activities. This assay was validated with nucleotide chain terminators and used to screen two diverse small library sets. The screen data obtained was further compared with concurrent screens performed with a DENV polymerase elongation fluorescent assay utilizing pre-complexed enzyme-RNA. A higher hit-rate was obtained for the de novo initiation assay compared to the elongation assay (∼2% versus ∼0.1%). All the hits from the latter assay are also identified in the de novo initiation assay, indicating that the de novo initiation assay performed with the stabilized apo-enzyme has the advantage of providing additional chemical starting entities for inhibiting this enzyme.

Original languageEnglish (US)
Pages (from-to)36-46
Number of pages11
JournalAntiviral Research
Volume119
DOIs
StatePublished - Jul 1 2015
Externally publishedYes

Fingerprint

Dengue Virus
RNA Replicase
Enzymes
RNA
Heavy Ions
Viral RNA
Methyltransferases
Transferases
Heavy Metals
Libraries
Cysteine
Buffers
Nucleotides
Salts
Therapeutics

Keywords

  • Activity
  • Assay
  • Compounds
  • De novo initiation
  • Dengue virus NS5 protein
  • Elongation
  • Inhibition
  • Keywords Flavivirus
  • Kinetics
  • RNA dependent
  • RNA polymerase

ASJC Scopus subject areas

  • Virology
  • Pharmacology

Cite this

Niyomrattanakit, P., Wan, K. F., Chung, K. Y., Abas, S. N., Seh, C. C., Dong, H., ... Lim, S. P. (2015). Stabilization of dengue virus polymerase in de novo initiation assay provides advantages for compound screening. Antiviral Research, 119, 36-46. https://doi.org/10.1016/j.antiviral.2015.04.007

Stabilization of dengue virus polymerase in de novo initiation assay provides advantages for compound screening. / Niyomrattanakit, Pornwaratt; Wan, Kah Fei; Chung, Ka Yan; Abas, Siti Nurdiana; Seh, Cheah Chen; Dong, Hongping; Lim, Chin Chin; Chao, Alexander Theodore; Lee, Chang Bok; Nilar, Shahul; Lescar, Julien; Shi, Pei-Yong; Beer, David; Lim, Siew Pheng.

In: Antiviral Research, Vol. 119, 01.07.2015, p. 36-46.

Research output: Contribution to journalArticle

Niyomrattanakit, P, Wan, KF, Chung, KY, Abas, SN, Seh, CC, Dong, H, Lim, CC, Chao, AT, Lee, CB, Nilar, S, Lescar, J, Shi, P-Y, Beer, D & Lim, SP 2015, 'Stabilization of dengue virus polymerase in de novo initiation assay provides advantages for compound screening', Antiviral Research, vol. 119, pp. 36-46. https://doi.org/10.1016/j.antiviral.2015.04.007
Niyomrattanakit, Pornwaratt ; Wan, Kah Fei ; Chung, Ka Yan ; Abas, Siti Nurdiana ; Seh, Cheah Chen ; Dong, Hongping ; Lim, Chin Chin ; Chao, Alexander Theodore ; Lee, Chang Bok ; Nilar, Shahul ; Lescar, Julien ; Shi, Pei-Yong ; Beer, David ; Lim, Siew Pheng. / Stabilization of dengue virus polymerase in de novo initiation assay provides advantages for compound screening. In: Antiviral Research. 2015 ; Vol. 119. pp. 36-46.
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AU - Seh, Cheah Chen

AU - Dong, Hongping

AU - Lim, Chin Chin

AU - Chao, Alexander Theodore

AU - Lee, Chang Bok

AU - Nilar, Shahul

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AU - Shi, Pei-Yong

AU - Beer, David

AU - Lim, Siew Pheng

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