In vitro determination of dengue virus type 2 NS2B-NS3 protease activity with fluorescent peptide substrates.
The NS2B-NS3(pro) polyprotein segment from the dengue virus serotype 2 strain 16681 was purified from overexpressing E. coli by metal chelate affinity chromatography and gel filtration. Enzymatic activity of the refolded NS2B-NS3(pro) protease complex was determined in vitro with dansyl-labeled peptide substrates, based upon native dengue virus type 2 cleavage sites. The 12mer substrate peptides and the cleavage products could be separated by reversed-phase HPLC, and were identified by UV and fluorescence detection. All of the peptide substrates (representing the DEN polyprotein junction sequences at the NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 sites) were cleaved by the recombinant protease NS2B-NS3(pro). No cleavage was observed with an enzymatically inactive S135A mutant of the NS3 protein, or with a modified substrate peptide of the NS3/NS4A polyprotein site that contained a K2093A substitution. Enzymatic activity was dependent on the salt concentration. A 50% decrease of activity was observed in the presence of 0.1 M sodium chloride. Our results show that the NS3 protease activity of the refolded NS2BNS3(pro) protein can be assayed in vitro with high specificity by using cleavage-junction derived peptide substrates.
Khumthong
Rabuesak
R
Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Phutthamonthon No. 4 Rd., Nakornpathom 73170, Thailand.
Angsuthanasombat
Chanan
C
Panyim
Sakol
S
Katzenmeier
Gerd
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D004271:DNA, Fungal / Q000235:genetics; D003716:Dengue Virus / Q000201:enzymology* / Q000235:genetics; D010450:Endopeptidases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000737:chemistry; D020365:RNA Helicases; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases; D013379:Substrate Specificity; D017361:Viral Nonstructural Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D004271:DNA, Fungal; D010450:Endopeptidases; D005456:Fluorescent Dyes; D009842:Oligopeptides; D020365:RNA Helicases; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C080102:NS2B protein, flavivirus; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases