Publication: Modulation of enzymatic activity of dengue virus nonstructural protein NS3 nucleoside triphosphatase/helicase by poly(U)
Issued Date
2013-08-01
Resource Type
ISSN
16083040
00062979
00062979
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2-s2.0-84882637281
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Mahidol University
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SCOPUS
Bibliographic Citation
Biochemistry (Moscow). Vol.78, No.8 (2013), 925-932
Suggested Citation
M. Junaid, C. Angsuthanasombat, J. E S Wikberg, N. Ali, G. Katzenmeier Modulation of enzymatic activity of dengue virus nonstructural protein NS3 nucleoside triphosphatase/helicase by poly(U). Biochemistry (Moscow). Vol.78, No.8 (2013), 925-932. doi:10.1134/S0006297913080105 Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/31268
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Title
Modulation of enzymatic activity of dengue virus nonstructural protein NS3 nucleoside triphosphatase/helicase by poly(U)
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Abstract
The nonstructural protein 3 (NS3) appears to be the most promising target for anti-flavivirus therapy because of its multiple enzymatic activities that are indispensable for virus replication. NS3 of dengue virus type 2 (DEN2) is composed of two domains, a serine protease in the N-terminal domain (NS3pro) and RNA-stimulated nucleoside triphosphatase (NTPase)/RNA helicase at the C-terminus (NS3h). NS3 plays an important role in viral replication and the coordinated regulation of all the catalytic activities in the full-length NS3 protein. In this study, a plasmid harboring the NS3 helicase domain (NS3h) was constructed by PCR. The 56.5 kDa NS3h protein was purified by metal-chelate affinity chromatography followed by renaturation, mediated by artificial chaperone-assisted refolding, which yielded the active helicase. NTPase activity was assayed with Malachite Green. The NTPase activity in the presence of poly(U) showed a higher turnover number (kcat) and a lower Kmvalue than without poly(U). The activity increased approximately fourfold in the presence of polynucleotides. This indicates that NTPase activity of dengue NS3 can be stimulated by polynucleotides. A helicase assay based on internal fluorescence quenching was conducted using short internally quenched DNA oligonucleotides as substrates. Significant fluorescence signaling increase was observed in the absence of polynucleotides such as poly(U). No unwinding activity was observed with addition of poly(U). The approach we describe here is useful for the further characterization of substrate specificity and for the design of high-throughput assays aimed at discovery of inhibitors against NS3 NTPase/helicase activities. © 2013 Pleiades Publishing, Ltd.