Monoclonal antibodies against dengue NS2B and NS3 proteins for the study of protein interactions in the flaviviral replication complex

► The NS3–NS5 interaction is critical for dengue replication. ► Monoclonal antibodies to NS2B and NS3 have been generated to study dengue replication. ► The 3F8 IgG (anti-NS3) can pull-down NS3 and NS5 from dengue infected cells. ► The 3F10 IgG (anti-NS2B) detects an interaction between NS3 and NS5...

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Bibliographic Details
Published in:	Journal of virological methods 2012, Vol.179 (1), p.97-103
Main Authors:	Moreland, Nicole J., Tay, Moon Y.F., Lim, Elfin, Rathore, Abhay P.S., Lim, Angeline P.C., Hanson, Brendon J., Vasudevan, Subhash G.
Format:	Article
Language:	eng
Subjects:	Animals Antibodies, Monoclonal - immunology Antibodies, Monoclonal - isolation & purification Antibodies, Viral - immunology Antibodies, Viral - isolation & purification Biological and medical sciences Dengue Dengue virus Dengue Virus - physiology enzyme activity enzyme-linked immunosorbent assay Enzyme-Linked Immunosorbent Assay - methods epitopes Flavivirus fluorescent antibody technique Fundamental and applied biological sciences. Psychology Humans hydrophilicity immunoglobulin G Immunoglobulin G - immunology Immunoglobulin G - isolation & purification Immunohistochemistry - methods Immunoprecipitation - methods Mice Microbiology monoclonal antibodies Monoclonal antibody NS2B NS3 Protein Binding Protein Interaction Mapping proteinases Reproducibility of Results RNA RNA Helicases - immunology RNA Helicases - metabolism RNA-directed RNA polymerase Sensitivity and Specificity Serine Endopeptidases - immunology Serine Endopeptidases - metabolism serotypes Techniques used in virology viral nonstructural proteins Viral Nonstructural Proteins - immunology Viral Nonstructural Proteins - metabolism Virology Western blotting Yellow fever virus
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Summary:	► The NS3–NS5 interaction is critical for dengue replication. ► Monoclonal antibodies to NS2B and NS3 have been generated to study dengue replication. ► The 3F8 IgG (anti-NS3) can pull-down NS3 and NS5 from dengue infected cells. ► The 3F10 IgG (anti-NS2B) detects an interaction between NS3 and NS5 in vitro. ► Protein interaction assays incorporating the IgG will be valuable for drug discovery research. The replication of dengue virus (DENV) RNA requires at least two viral non-structural (NS) proteins, NS3 and NS5. To facilitate the study of the DENV replication complex, human monoclonal IgG that are specific for NS proteins have been generated and characterised. The anti-NS3 IgG, 3F8, binds a conserved epitope (aa526-531) in the NS3 helicase domain, and cross-reacts with NS3 from all four DENV serotypes and the related yellow fever virus. The anti-NS2B IgG, 3F10, binds aa49-66 of NS2B (CF18), which forms part of the 47 aa hydrophilic cofactor region required for NS3 protease activity. The specificity of the IgG for their respective non-structural proteins has been demonstrated by immunofluorescence of cells infected with DENV and Western blotting. 3F8 is able to co-immunoprecipitate NS3 and NS5 from BHK-21 cells infected with DENV2, and 3F10 is able to detect an interaction between recombinant NS2B CF18NS3 full-length protein and the NS5 RNA-dependent RNA polymerase (RdRp) domain in an ELISA-based binding assay. The assay is specific and highly reproducible, with a clear binding curve seen when RdRp is incubated with increasing amounts of full-length NS3, but not the NS3 protease domain. The NS3 helicase domain competes with NS3 full-length for NS5 RdRp binding, with a K d. of 2.5 μM. Since NS3 and NS5 are required for DENV replication, this fascile assay could be used to screen for non-nucleoside, allosteric inhibitors that disrupt the interaction between the two proteins.
ISSN:	0166-0934 1879-0984