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

Abstract

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 in Escherichia coli as a fusion protein with a histidine tag at the N terminus. The precursor was purified from insoluble inclusion bodies by Ni 2؉ affinity and gel filtration chromatography under denaturing conditions. The denatured precursor was refolded to yield a purified active protease complex. Biochemical analysis of the protease revealed that its activity toward either a natural substrate, NS4B-NS5 precursor, or the fluorogenic peptide substrates containing two basic residues at P1 and P2, was dependent on the presence of the NS2B domain. The peptide with a highly conserved Gly residue at P3 position was 3-fold more active as a substrate than a Gln residue at this position. The cleavage of a chromogenic substrate with a single Arg residue at P1 was NS2B-independent. These results suggest that heterodimerization of the NS3pro domain with NS2B generates additional specific interactions with the P2 and P3 residues of the substrates.Dengue viruses, members of the family Flaviviridae, are transmitted by mosquitos. There are four serotypes (types 1 to 4) that cause widespread human diseases such as dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. About 40% of the world population living in tropical and subtropical regions of the world is at risk for infection. Of the 1 million cases of dengue hemorrhagic fever cases per year, about 5% are fatal. There is currently no effective vaccine or antiviral drug to protect against dengue diseases (1, 2). The virus has a single strand RNA genome of positive polarity. The viral RNA has a type 1 cap at the 5Ј-end and is devoid of poly(A) at the 3Ј-end. The RNA genome codes for a single polyprotein precursor (3,391 amino acid residues for DEN2-New Guinea C strain) (3) arranged in the order C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5.1 Processing of the N-terminal region by the host signal peptidase associated with the endoplasmic reticulum yields three structural proteins that are assembled into the virion (C, prM, and E) (4 -6). Processing of C-prM by signal peptidase occurs via a post-translational mechanism in which cleavage of the cytoplasmic domain of C in the C-prM precursor by the viral protease precedes the signal peptidase cleavage (7-9). prM undergoes further cleavage to M at a late step during virion morphogenesis mediated by a cellular protease in the post-Golgi acidic compartment (10). In addition, an endoplasmic reticulum membrane-bound host protease with the characteristics of the signalase mediates cleavages at the NS1-NS2A (11) and NS4A-NS4B junctions (12)(13)(14).A trypsin-like serine protease domain within the N-terminal 180 amino acid residues of NS3 was identified by sequence comparisons (15,16). Analysis of polyprotein proces...

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

“…The flanking hydrophobic residues of NS2B are likely to function by promoting association between the protease complex and infected cell membranes (17). The essential nature of the central hydrophilic domain for proteolytic activity has been recently confirmed in vitro with Escherichia coli expressed and purified recombinant NS3pro complexed with this NS2B co-factor domain (18).…”

Policresulen, a novel NS2B/NS3 protease inhibitor, effectively inhibits the replication of DENV2 virus in BHK-21 cells

“…Previous studies have shown that NS3 contains two functional domains: a serine protease at the N-terminus and an RNA helicase at the C-terminus [12][13][14] . NS2B is an essential element that acts as a co-factor to greatly enhance the activity of NS3 by 3300-to 6600-fold [15,16] . As a viral protease complex, NS2B/ NS3 is mainly in charge of cleaving the viral poly-protein precursor to generate structural proteins and nonstructural proteins, and the disruption of NS2B/NS3 protease function distinctly inhibits viral replication [17] .…”

Mechanisms of activation and inhibition of Zika virus NS2B-NS3 protease

“…In the past, successful structure-based drug development against NS3pro of the hepatitis C virus (HCV) has generated a series of United States Food and Drug Administration (FDA)-approved inhibitors with potent efficacy for viral eradication [6]. Flavivirus NS3pro exists predominantly in an inactive state; however, upon association with the NS2B viral protein, its enzymatic activity increases by 3 300-6 600-fold [7]. The recent structure of ZIKV NS2B-NS3pro in complex with a boronate inhibitor has provided some important insights for drug design [8].…”