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Title: Establishment of a robust dengue virus NS3-NS5 binding assay for identification of protein-protein interaction inhibitors
Authors: Takahashi, H.
Takahashi, C.
Moreland, N.J.
Chang, Y.-T. 
Sawasaki, T.
Ryo, A.
Vasudevan, S.G. 
Suzuki, Y.
Yamamoto, N.
Keywords: AlphaScreen technology
Dengue virus
Protein-protein interaction inhibitors
Wheat-germ cell-free protein synthesis
Issue Date: Dec-2012
Citation: Takahashi, H., Takahashi, C., Moreland, N.J., Chang, Y.-T., Sawasaki, T., Ryo, A., Vasudevan, S.G., Suzuki, Y., Yamamoto, N. (2012-12). Establishment of a robust dengue virus NS3-NS5 binding assay for identification of protein-protein interaction inhibitors. Antiviral Research 96 (3) : 305-314. ScholarBank@NUS Repository.
Abstract: Whereas the dengue virus (DENV) non-structural (NS) proteins NS3 and NS5 have been shown to interact in vitro and in vivo, the biological relevance of this interaction in viral replication has not been fully clarified. Here, we first applied a simple and robust in vitro assay based on AlphaScreen technology in combination with the wheat-germ cell-free protein production system to detect the DENV-2 NS3-NS5 interaction in a 384-well plate. The cell-free-synthesized NS3 and NS5 recombinant proteins were soluble and in possession of their respective enzymatic activities in vitro. In addition, AlphaScreen assays using the recombinant proteins detected a specific interaction between NS3 and NS5 with a robust Z' factor of 0.71. By employing the AlphaScreen assay, we found that both the N-terminal protease and C-terminal helicase domains of NS3 are required for its association with NS5. Furthermore, a competition assay revealed that the binding of full-length NS3 to NS5 was significantly inhibited by the addition of an excess of NS3 protease or helicase domains. Our results demonstrate that the AlphaScreen assay can be used to discover novel antiviral agents targeting the interactions between DENV NS proteins. © 2012 Elsevier B.V.
Source Title: Antiviral Research
ISSN: 01663542
DOI: 10.1016/j.antiviral.2012.09.023
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