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

Abstract

NS3 protein of dengue virus type 2 has a serine protease domain within the N-terminal 180 residues. NS2B is required for NS3 to form an active protease involved in processing of the viral polyprotein precursor. The region carboxy terminal to the protease domain has conserved motifs present in several viral RNA-stimulated nucleoside triphosphatase (NTPase)/RNA helicases. To define the functional domains of protease and NTPase/RNA helicase activities of NS3, full-length and amino-terminal deletion mutants of NS3 were expressed in Escherichia coli and purified. Deletion of 160 N-terminal residues of NS3 (as in NS3del.2) had no detrimental effect on the basal and RNA-stimulated NTPase as well as RNA helicase activities. However, mutagenesis of the conserved P-loop motif of the RNA helicase domain (K199E) resulted in loss of ATPase activity. The RNA-stimulated NTPase activity was significantly affected by deletion of 20 amino acid residues from the N terminus or by substitutions of the cluster of basic residues, 184RKRK-->QNGN, of NS3del.2, although both mutant proteins retained the conserved RNA helicase motifs. Furthermore, the minimal NS3 protease domain, required for cleavage of the 2B-3 site, was precisely defined to be 167 residues, using the in vitro processing of NS2B-NS3 precursors. Our 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 NTPase activity.

FIG. 1

DEN2 NS3 expression constructs. The full-length and N-terminal deletion constructs of NS3 were cloned into the pET-PFH or pET-PH vector as described in Materials and Methods. These constructs encode recombinant NS3 proteins with a Met residue at the N terminus and His tags (PFH or PH) at the C terminus. The filled boxes in pET-NS3mt.1 and -2 refer to the substitution mutants of NS3 in which the P-loop motif ¹⁹⁹GKT is changed to GET and the stretch of basic residues ¹⁸⁴RKRK is changed to QNGN, respectively. Conserved boxes 3 and 4 of 10 flavivirus NS3 sequences, indicated as 3 & 4, are from a previous report (36) based on the model of Bazan and Fletterick (3). The asterisk indicates the endpoint of the minimal protease domain.

FIG. 2

Expression of NS3 polypeptides in E. coli. (A) Recombinant DEN2 NS3 proteins were expressed and purified from E. coli BL21(DE3) cells by using Ni-NTA affinity column chromatography as described in Materials and Methods. Left panel, Coomassie blue-stained gel of Ni ²⁺-NTA-purified proteins separated by SDS-PAGE. Right panel, Western blot prepared with anti-DEN2 NS3 polyclonal antibodies. Lanes: M, protein molecular weight marker; 1, NS3-PFH; 2, NS3del.1-PH; 3, NS3del.2-PH; 4, NS3AC-PFH; 5, NS3del.2-PH; 6, NS3mt.2-PH; 7, NS3mt.1-PH. (B) NS3del.2 protein was further purified by Sephadex G-75 column chromatography under denaturing conditions, and eluates were refolded described in Materials and Methods. The refolded fractions were analyzed by SDS-PAGE and stained with Coomassie blue. Lanes: M, molecular weight standards; 1 to 5, pooled fractions 21 to 26, 27, 28, 29, and 30, respectively. (C) SDS-PAGE followed by Coomassie blue staining (left panel) and Western blot analysis (right panel) of the refolded NS3AC polypeptide.

FIG. 3

NTPase assays in the absence or presence of polyribonucleotides. (A) ATPase assays were carried out in the absence or presence of different homopolymers at 0.5 mM (concentration measured as mononucleotides). The specific activities of the NS3del.2 ATPase under each reaction condition were calculated based on the protein used (1.45 μg or 27 pmol/assay) and measured ATP hydrolysis rates. Specific activity is defined as moles of ADP generated per mole of protein/per second. Each point on the plot represents the mean value of triplicate assays, and each error bar represents the standard deviation. (B) ATPase assays of the purified NS3del.2 protein were carried out under standard reaction conditions with or without 0.5 mM poly(A). The initial velocity, ΔA₃₄₀, was proportional to the amount of protein (18.62 pmol/μg of protein) used in the assay. Each point is the mean value of two measurements.

FIG. 4

Biochemical and kinetic analysis of ATPase activity of NS3. (A) Effect of divalent cation. The effects of various Mg²⁺ or Mn²⁺ concentrations on the ATPase were determined in 50 mM HEPES-K⁺ (pH 7.5)–16 mM (NH₄)₂SO₄–0.5 mM ATP with or without 0.5 mM poly(A), as indicated. (B) Effect of ionic strength. The reactions were carried out in 50 mM HEPES-K⁺ (pH 7.5)–2.5 mM MgCl₂–0.5 mM ATP and increasing concentration of KCl in the absence or presence of 0.5 mM poly(A). The increased ionic strength [I = 1/2 · (Ci · Zi²)] is indicated. (C) Effect of ATP concentration. The ATPase activity of NS3del.2 was measured at increasing concentrations of ATP but constant Mg²⁺ concentration (2.5 mM), with the rest of the components the same. The ATP concentrations used in the assays are 0.03, 0.05, 0.1, 0.2, 0.3, 0.5, and 0.75 mM. K_m and K_cat constants for NS3del.2 protein in the absence (○) or presence (□) of 0.5 mM poly(A) were determined from Lineweaver-Burk plots.

FIG. 5

Enzyme-coupled NTPase assays. The NTPase assays were carried out under standard conditions as described in Materials and Methods in the absence or presence of 0.5 mM poly(A) and with 0.5 mM each NTP (ATP, GTP, CTP, and UTP) as substrates. Relative activities in triplicates were plotted in a bar graph; each error bar represents the standard deviation.

FIG. 6

ATPase activities of NS3 mutants. The standard enzyme-coupled ATPase assays were carried out in triplicate in the presence or absence of 0.5 mM poly(A) and 0.5 mM ATP. Relative activity denotes the specific activity units as defined for Fig. 3.

FIG. 7

Mapping the boundary of the NS3 protease domain. NS2B-NS3 precursor constructs containing successive C-terminal deletions of the protease domain were expressed in TNT system in the presence of canine microsomal membranes as described in Materials and Methods. The lysates were centrifuged to isolate the microsomal membrane fraction, and the processing reactions were analyzed by SDS-PAGE and autoradiography. Lanes: 1, NS2B-NS3(183aa); 2, NS2B-NS3(176aa); 3, NS2B-NS3(170aa); 4, NS2B-NS3(164aa); 5 to 9: NS2B-NS3(169aa) to NS2B-NS3(165aa).

FIG. 8

RNA helicase assay of NS3del.2 protein. RNA helicase assays were performed as described in Materials and Methods. The double-stranded RNA substrate used for helicase assay is shown. Lanes: 1, RNA substrate heated at 90°C before loading; 2, no protein added (negative control); 3, standard RNA helicase reaction mixture omitting ATP but containing 54 pmol of NS3del.2; 4, standard RNA helicase reaction mixture except containing 3 mM EDTA and 54 pmol of NS3del.2; 5 to 9, standard RNA helicase reaction mixtures containing 54, 27, 14, 7, and 3.5 pmol of NS3del.2, respectively. Autoradiography of the polyacrylamide gel is shown.