Identification of residues in the dengue virus type 2 NS2B cofactor that are critical for NS3 protease activation

Abstract

Proteolytic processing of the dengue virus polyprotein is mediated by host cell proteases and the virus-encoded NS2B-NS3 two-component protease. The NS3 protease represents an attractive target for the development of antiviral inhibitors. The three-dimensional structure of the NS3 protease domain has been determined, but the structural determinants necessary for activation of the enzyme by the NS2B cofactor have been characterized only to a limited extent. To test a possible functional role of the recently proposed Phix(3)Phi motif in NS3 protease activation, we targeted six residues within the NS2B cofactor by site-specific mutagenesis. Residues Trp62, Ser71, Leu75, Ile77, Thr78, and Ile79 in NS2B were replaced with alanine, and in addition, an L75A/I79A double mutant was generated. The effects of these mutations on the activity of the NS2B(H)-NS3pro protease were analyzed in vitro by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of autoproteolytic cleavage at the NS2B/NS3 site and by assay of the enzyme with the fluorogenic peptide substrate GRR-AMC. Compared to the wild type, the L75A, I77A, and I79A mutants demonstrated inefficient autoproteolysis, whereas in the W62A and the L75A/I79A mutants self-cleavage appeared to be almost completely abolished. With exception of the S71A mutant, which had a k(cat)/K(m) value for the GRR-AMC peptide similar to that of the wild type, all other mutants exhibited drastically reduced k(cat) values. These 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.

FIG. 1.

Organization of the dengue virus polyprotein and the NS2B(H)-NS3(pro) construct. (A) Sites on the dengue virus polyprotein cleaved by host-encoded proteases (▿) and the virus-encoded two-component protease NS2B-NS3 (▾). The NS3 protease domain (NS3pro) is shaded, and the 40-residue minimum cofactor region which supports catalytic activity of the NS3 protease is indicated by a bar. (B) Structural organization of the expression construct NS2B(H)-NS3pro as generated by SOE-PCR. The construct contains the N-terminal polyhistidine tag for purification purposes, the central activation sequence of NS2B from residues 48 to 95 followed by residues 121 to 130, and 180 N-terminal residues of NS3 representing the protease domain. The residues of the catalytic triad, His51, Asp75, and Ser135, are indicated, and the sequence of the native NS2B/NS3 polyprotein cleavage junction is shown.

FIG. 2.

Sequence alignment of the conserved central domain within the NS2B cofactors of members of the Flaviviridae family. Numbers on the left indicate positions of amino acid residues in the polyprotein sequence. Residues which are identical in all sequences are shaded in dark grey, and conserved residues are shaded in light grey. The location of the Φx₃Φ motif is indicated above the alignment, and residues within the dengue virus NS2B cofactor that were changed to alanine are labeled by dots. Abbreviations: DEN, dengue virus; JEV, Japanese encephalitis virus; KUNJIN, Kunjin virus; MVE, Murray valley encephalitis virus; TBE, tick-borne encephalitis virus; WNV, West Nile virus; YFV, yellow fever virus.

FIG. 3.

Kinetics of proteolytic autoprocessing of the NS2B(H)-NS3(pro) mutant derivatives. (A) Samples of wild-type NS2B(H)-NS3pro and the mutant proteins were refolded by successive dialysis, incubated at 37°C, and analyzed on Coomassie blue-stained Tricine-SDS-PAGE gels as described in the text. Lane M, protein molecular size markers. Numbers above the lanes indicate incubation times ranging from 0 to 24 h. (B) Band intensities of wild-type NS2B(H)-NS3pro and the mutant polypeptides as observed on Tricine-SDS-PAGE gels were quantitated by densitometric analysis with the ONE-D scan program, and the fraction of cleaved NS2B(H)-NS3(pro) precursor was plotted as a function of incubation time.

FIG. 4.

Steady-state cleavage kinetics of the GRR-AMC substrate by NS2B(H)-NS3pro derivatives in vitro. Reactions were performed in triplicate at a 0.15 μM protein concentration over a range of substrate concentrations from 10 to 250 μM. Assays were performed with 60-min incubation periods, and kinetic parameters were obtained by nonlinear regression analysis of initial velocities prior to 10% substrate depletion. The L75A/I79A double mutant and the W62A mutant had no measurable activity at a 10-fold-higher enzyme concentration (1.5 μM). Data were plotted in a double reciprocal format.

FIG. 5.

Molecular model of the interaction between the NS2B core segment and the NS3 protease. The model was generated by Deepview Swiss-Pdbviewer. (A) NS2B(H) (residues 56 to 93) mapped onto the X-ray crystal structure of NS3. The surface of NS3pro is shown in blue, and NS2B(H) is shown as a ribbon in yellow. The location of Trp62 at the N terminus of NS2B(H) is shown. (B) Interaction of residues Leu75, Ile77, and Ile79 with the hydrophobic pocket of NS3. The surface of NS3 is colored by the residue type (yellow, polar; blue, basic; red, acidic; white, nonpolar); the NS2B segment is shown as a yellow stick with the side chains of Leu75, Ile77, and Ile79 in grey. (C) Association of the Trp62 residue (red) of NS2B(H) (yellow ribbon) with the N-terminal proline cluster of NS3 (side chains in green and NS3 in blue).