Activity of Recombinant Dengue 2 Virus NS3 Protease in the Presence of a Truncated NS2B Co-factor, Small Peptide Substrates, and Inhibitors*

  title={Activity of Recombinant Dengue 2 Virus NS3 Protease in the Presence of a Truncated NS2B Co-factor, Small Peptide Substrates, and Inhibitors*},
  author={D. Leung and Kate Schroder and H. White and Ning Xia Fang and Martin J. Stoermer and G. Abbenante and J. L. Fern{\'a}ndez Mart{\'i}n and Paul R. Young and David P. Fairlie},
  journal={The Journal of Biological Chemistry},
  pages={45762 - 45771}
Recombinant forms of the dengue 2 virus NS3 protease linked to a 40-residue co-factor, corresponding to part of NS2B, have been expressed in Escherichia coli and shown to be active against para-nitroanilide substrates comprising the P6-P1 residues of four substrate cleavage sequences. The enzyme is inactive alone or after the addition of a putative 13-residue co-factor peptide but is active when fused to the 40-residue co-factor, by either a cleavable or a noncleavable glycine linker. The NS4B… 

Figures and Tables from this paper

Identification of Residues in the Dengue Virus Type 2 NS2B Cofactor That Are Critical for NS3 Protease Activation
Results indicate a pivotal function of conserved residues Trp62, Leu75, and Ile79 in the NS2B cofactor in the structural activation of the dengue virus NS3 serine protease.
Probing the substrate specificity of the dengue virus type 2 NS3 serine protease by using internally quenched fluorescent peptides.
It is shown that the presence of basic amino acids at the P3 and P4 positions is a major specificity-determining feature of the dengue virus NS3 protease.
Yellow fever virus NS3 protease: peptide-inhibition studies.
The findings indicate that the characteristics of YFV protease are very similar to those reported for dengue and West Nile virus proteases, and suggest that pan-flavivirus NS3 protease drugs may be developed for flaviviral diseases.
Functional Characterization of cis and trans Activity of the Flavivirus NS2B-NS3 Protease*
This study describes the first biochemical characterization of flavivirus protease activity using full-length NS3, and confirms that autolytic cleavage of the helicase site was insensitive to protein dilution, confirming thatAutolysis is intramolecular.
Enzymatic Characterization and Homology Model of a Catalytically Active Recombinant West Nile Virus NS3 Protease*
The design, expression, and enzymatic characterization of a catalytically active recombinant WNV protease is described, consisting of a 40-residue component of cofactor NS2B tethered via a noncleavable nonapeptide to the N-terminal 184 residues of NS3.


Purified NS2B/NS3 Serine Protease of Dengue Virus Type 2 Exhibits Cofactor NS2B Dependence for Cleavage of Substrates with Dibasic Amino Acids in Vitro*
It is suggested that heterodimerization of the NS3pro domain with NS2B generates additional specific interactions with the P2 and P3 residues of the substrates.
Activity of purified hepatitis C virus protease NS3 on peptide substrates
Primary structure is an important determinant of the efficiency with which each site is cleaved during polyprotein processing, and slow cleavage of the NS4B-NS5A site in the absence of NS4A is due to low binding affinity of the enzyme for this site.
Evidence that the N-terminal domain of nonstructural protein NS3 from yellow fever virus is a serine protease responsible for site-specific cleavages in the viral polyprotein.
  • T. Chambers, R. Weir, C. Rice
  • Biology
    Proceedings of the National Academy of Sciences of the United States of America
  • 1990
Sequence homology and molecular modeling studies have suggested that the N-terminal one-third of the flavirvirus nonstructural protein NS3 functions as a trypsin-like serine protease. To examine the
Cotranslational Membrane Insertion of the Serine Proteinase Precursor NS2B-NS3(Pro) of Dengue Virus Type 2 Is Required for Efficient in Vitro Processing and Is Mediated through the Hydrophobic Regions of NS2B*
Results showed that cotranslational addition of microsomal membranes to the TnT reaction markedly enhanced the cis cleavage of the 2B/3 site in a dose-dependent manner and the cleavage products, NS2B and NS3(Pro), were membrane-associated.
Homology model of the dengue 2 virus NS3 protease: putative interactions with both substrate and NS2B cofactor.
The model described in this study not only reveals unique features of the flavivirus protease but also provides a structural basis for both cofactor and substrate binding that should prove useful in the early design and development of inhibitors.
Crystal structure of Dengue virus NS3 protease in complex with a Bowman-Birk inhibitor: implications for flaviviral polyprotein processing and drug design.
The crystal structure of a complex of the protease with a Bowman-Birk inhibitor reported here provides a starting point for design of site-specific mutations to probe the mechanism of catalysis by the catalytic domain, its activation by the regulatory domain and by design of specific inhibitors of enzymatic activity.
In vitro processing of dengue virus type 2 nonstructural proteins NS2A, NS2B, and NS3
The data from these expression experiments confirm that NS3 is the viral proteinase responsible for cleavage events generating the amino termini of NS2B and NS3 and presumably for cleavages generating the termine of NS4A and NS5 as well.
The Serine Protease and RNA-Stimulated Nucleoside Triphosphatase and RNA Helicase Functional Domains of Dengue Virus Type 2 NS3 Converge within a Region of 20 Amino Acids
The results reveal that the functional domains required for serine protease and RNA-stimulated NTPase activities map within the region between amino acid residues 160 and 180 of NS3 protein and that a novel motif, the cluster of basic residues 184RKRK, plays an important role for the RNA- Stimulated N TPase activity.
Yellow fever virus NS2B-NS3 protease: charged-to-alanine mutagenesis and deletion analysis define regions important for protease complex formation and function.
The data suggest thatNS2B-NS3 complex formation requires charge interactions involving the N-terminus of the conserved domain of NS2B and 22 N-Terminal residues of NS3, and a role for the putative transmembrane regions of NS 2B in targeting of NS 3 to intracellular membranes is also suggested.
Both nonstructural proteins NS2B and NS3 are required for the proteolytic processing of dengue virus nonstructural proteins
This work constructed recombinant vaccinia viruses expressing various portions of the NS region of the dengue virus type 4 polyprotein and showed that NS2B was needed, apparently in cis, for NS3/NS4A cleavage and for a series of internal cleavages in NS3.