Establishment of a robust dengue virus NS3-NS5 binding assay for identification of protein-protein interaction inhibitors.
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.
Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Center for Translational Medicine, 14 Medical Drive, #15-02, Level 15, Singapore 117599, Singapore. micht@nus.edu.sg
Hirotaka
H
Takahashi
Chikako
C
Takahashi
Nicole J
NJ
Moreland
Young-Tae
YT
Chang
Tatsuya
T
Sawasaki
Akihide
A
Ryo
Subhash G
SG
Vasudevan
Youichi
Y
Suzuki
Naoki
N
Yamamoto
D019943:Amino Acid Substitution; D002474:Cell-Free System; D003716:Dengue Virus / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods*; D004791:Enzyme Inhibitors / Q000378:metabolism*; D005822:Genetic Vectors; D010957:Plasmids; D014176:Protein Biosynthesis; D025941:Protein Interaction Mapping / Q000379:methods*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014158:Transcription, Genetic; D014908:Triticum; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D000085242:Viral Proteases
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D014764:Viral Proteins; C504449:NS3 protease, dengue virus; D000085242:Viral Proteases