Purified NS2B/NS3 Serine Protease of Dengue Virus Type 2 Exhibits Cofactor NS2B Dependence for Cleavage of Substrates with Dibasic Amino Acids in Vitro*

@article{Yusof2000PurifiedNS,
  title={Purified NS2B/NS3 Serine Protease of Dengue Virus Type 2 Exhibits Cofactor NS2B Dependence for Cleavage of Substrates with Dibasic Amino Acids in Vitro*},
  author={Rohana Yusof and Stephen R Clum and Mary G. Wetzel and H. M. Krishna Murthy and Raji Padmanabhan},
  journal={The Journal of Biological Chemistry},
  year={2000},
  volume={275},
  pages={9963 - 9969}
}
Dengue virus type 2 NS3, a multifunctional protein, has a serine protease domain (NS3pro) that requires the conserved hydrophilic domain of NS2B for protease activity in cleavage of the polyprotein precursor at sites following two basic amino acids. In this study, we report the expression of the NS2B-NS3pro precursor inEscherichia coli as a fusion protein with a histidine tag at the N terminus. The precursor was purified from insoluble inclusion bodies by Ni2+ affinity and gel filtration… 

Figures and Tables from this paper

Activity of Recombinant Dengue 2 Virus NS3 Protease in the Presence of a Truncated NS2B Co-factor, Small Peptide Substrates, and Inhibitors*
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
In vitro determination of dengue virus type 2 NS2B-NS3 protease activity with fluorescent peptide substrates.
TLDR
The 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.
Identification of Residues in the Dengue Virus Type 2 NS2B Cofactor That Are Critical for NS3 Protease Activation
TLDR
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.
Functional Characterization of cis and trans Activity of the Flavivirus NS2B-NS3 Protease*
TLDR
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.
Probing the substrate specificity of the dengue virus type 2 NS3 serine protease by using internally quenched fluorescent peptides.
TLDR
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.
...
...

References

SHOWING 1-10 OF 57 REFERENCES
Serine protease of hepatitis C virus expressed in insect cells as the NS3/4A complex.
TLDR
This work expressed the full-length NS3 and NS4A in insect cells as a soluble fusion protein with an N-terminal polyhistidine tag and purified the two proteins to homogeneity, finding that the monodisperse, soluble form of the NS3/4A complex is associated with the highest protease activity.
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*
TLDR
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.
Processing of Japanese encephalitis virus non-structural proteins: NS2B-NS3 complex and heterologous proteases.
TLDR
Both protease components are associated as a complex, presumably representing the active JE virus protease, despite the considerable sequence conservation of NS2B and NS3 between the two viruses.
Dengue 2 virus NS2B and NS3 form a stable complex that can cleave NS3 within the helicase domain.
TLDR
An NS3-specific antiserum is used, under nondenaturing conditions, to demonstrate that NS2B and NS3 form a complex both in vitro and in vivo, and a potential conserved cleavage site that resembles other sites cleaved by the NS3/NS2B proteinase is revealed.
Mutagenesis of the NS3 Protease of Dengue Virus Type 2
TLDR
A mutational analysis of the region of NS3 which contains the catalytic serine, five putative substrate binding residues, and several residues that are highly conserved among flavivirus proteases and among all serine proteases indicates that D129 is not a major determinant of substrate binding and that its interaction with the substrate, if it occurs at all, is not essential.
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
TLDR
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.
Both nonstructural proteins NS2B and NS3 are required for the proteolytic processing of dengue virus nonstructural proteins
TLDR
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.
NS2B-3 proteinase-mediated processing in the yellow fever virus structural region: in vitro and in vivo studies
TLDR
Expressing a YF polyprotein extending from C through the N-terminal one-third of NS3 revealed that C-prM processing, but not translocation, was dependent on an active NS2B-3 proteinase, suggesting that signalase-mediated cleavage in the lumen of the endoplasmic reticulum may be dependent on prior cleavage at the anchC dibasic site.
In vitro processing of dengue virus type 2 nonstructural proteins NS2A, NS2B, and NS3
TLDR
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.
...
...