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Regulated proteolysis of the polyprotein precursor of West Nile virus (WNV) by the essential NS2B-NS3(pro)tease, a promising drug target for WNV inhibitors, is required for the propagation of infectious virions. Structural and drug design studies, however, require pilot-scale quantities of a pure and catalytically active WNV protease that is resistant to self-proteolysis. Autolytic cleavage at the NS2B-NS3 boundary leads to individual, non-covalently associated, NS2B and NS3 domains, together with residual amounts of the intact NS2B-NS3, in the NS2B-NS3pro samples. We modified the cleavage site sequence of the NS2B-NS3 junction region and then developed expression and purification procedures to prepare a covalently linked, single-chain, NS2B-NS3pro K48A mutant construct. This construct exhibits high stability and functional activity and is thus well suited for the follow-up purification and structural and drug design studies.
West Nile virus (WNV), a member of the Flaviviridae family and an emerging pathogen in the US, is transmitted to animals, including humans, by mosquito bites [1, 2]. There are no specific countermeasures against WNV infection in humans. The WNV capsid encloses a single-stranded RNA encoding a 3,400 amino acid residue polypeptide precursor. The precursor is comprised of three structural proteins [C, capsid, M, membrane, and E, envelope] and seven non-structural (NS) proteins arranged in the order C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5 [3, 4]. Proteases from the host (furin and secretase) and from the virus [NS3 serine proteinase (NS3pro)] are required to process the polyprotein precursor into individual functional proteins [5, 6]. NS3pro is responsible for the cleavage of the capsid protein C and at the NS2A/NS2B, NS2B/NS3, NS3/NS4A, NS4A/NS4B and NS4B/NS5 boundaries [7–14].
The full-length NS3 peptide sequence is a multifunctional protein. The N-terminal 184 amino acid residue fragment represents the serine proteinase NS3pro with a conventional His-Asp-Ser catalytic triad. The C-terminal portion of the NS3 protein encodes an RNA helicase. During virus propagation, protease and helicase normally work together in a coordinated fashion . As is the case with a number of flaviviruses, the NS2B protein that is located in the polypeptide precursor upstream of the NS3pro domain functions as a cofactor and promotes the folding and the functional activity of the NS3pro [16–18]. The individual NS3pro domain, lacking the NS2B part, is catalytically inert. The cofactor activity of the 35–48-residue central portion is approximately equivalent to that of the entire NS2B sequence [19, 20]. Inactivating mutations of the NS3pro cleavage sites in the polyprotein precursor abolish viral infectivity . These data suggest that the two-component NS2B-NS3pro is a promising drug target for WNV inhibitors. The sequence of WNV NS2B-NS3pro is 56% identical to that of Dengue virus (DV), thus suggesting a high degree of structural similarity that exists between these two proteinases. A crystal structure of the individual DV NS3pro complexed with mung-bean Bowman-Birk type trypsin inhibitor has been available for several years [21, 22]. This structure, however, represents a virtually inactive protease. When our work was in progress, the high resolution structures of the two-component WNV and DV NS2B-NS3pro in complex with the substrate-based inhibitor benzoyl-norleucine (P4)-lysine (P3)-arginine (P2)-arginine (P1)-aldehyde (Bz-Nle-Lys-Arg-Arg-H) became available .
Structural and drug design studies require the availability of substantial amounts of recombinant NS2B-NS3pro with high proteolytic activity and resistance to self-proteolysis. Normally, because of self-proteolysis of the NS2B-NS3 junction region, individual, non-covalently associated NS2B and NS3 domains, together with residual amounts of intact NS2B-NS3, are present in the NS2B-NS3pro samples . Their presence complicates the isolation and analysis of NS2B-NS3pro. To overcome this difficulty, we modified the cleavage site sequence of the NS2B-NS3 junction region in order to obtain covalently linked, single-chain NS2B-NS3pro that exhibits high stability and functional activity, well suited for follow-up purification and structural and drug design studies.
Reagents were purchased from Sigma-Aldrich (Milwaukee, WI) unless indicated otherwise. Pyroglutamic acid-RTKR-7-amino-4-methylcoumarin (Pyr-RTKR-AMC) was purchased from American Peptide (Sunnyvale, CA).
Cloning of the DNA sequence encoding the wild-type two-component NS2B-NS3pro from WNV was described earlier . The 48 amino acid residue central portion of NS2B (residues 1393–1440 of the WNV polyprotein precursor) and the NS3 (residues 1476–1687 of the WNV polyprotein precursor) sequences were linked by a flexible GGGGSGGGG linker. The WNV autolytic site-deficient NS2B-NS3pro(K48A) construct was prepared with 5’-CCAGGAGCACCTTGGGCGGGCGGGGGAGGT-3’ and 5’-ACCTCCCCCGCCCGCCCAAGGTGCTCCTGG-3’ forward and reverse primers, respectively (mutant nucleotides are underlined) using a QuickChange mutagenesis kit (Stratagene, San Diego, CA). After confirming their authenticity by sequencing, the constructs were re-cloned into the pET101 expression vector. Competent E.coli BL21 (DE3) Codon Plus cells (Stratagene, San Diego, CA) were transformed with the recombinant pET101 vectors. Transformed cells were grown at 30°C in a Luria–Bertani broth containing ampicillin (0.1 mg/ml). Cultures were induced with 0.6 mM isopropyl β-D-thiogalactoside for 16 h at 18°C. E.coli cells (6 g/L of E.coli culture) were then collected by centrifugation (5,000xg; 15 min), resuspended in 20 ml PBS containing 1 M NaCl, 1 mM phenylmethylsulfonyl fluoride and lyzozyme (5 mg/ml), and disrupted by sonication (30 sec pulse, 30 sec interval; 8 pulses) on ice. The debris were then removed by centrifugation at 20,000xg, 30 min.
The NS2B-NS3pro WT and K48A constructs, C-terminally tagged with a 6xHis tag, were each purified from the soluble fraction of E.coli cell extract using affinity chromatography on a 1.6x10 cm Co2+-chelating Sepharose Fast Flow column (Amersham, Piscataway, NJ) equilibrated with PBS - 1 M NaCl - 1 mM phenylmethylsulfonyl fluoride. NS2B-NS3pro was eluted with 100 ml of 10–500 mM gradient of imidazole in PBS - 1 M NaCl - 1 mM phenylmethylsulfonyl fluoride buffer (elution rate 0.5 ml/min). The fractions (1.5 ml each; 10 fractions total) containing NS2B-NS3pro were collected and diluted 10-fold with PBS and the protease samples were then re-chromatographed under the same conditions on a Co2+-chelating Sepharose Fast Flow column. The presence of NS2B-NS3pro in the fractions was confirmed by 4–20% gradient gel electrophoresis and also by the activity assay. In the activity assays 1 μl fraction aliquots and a Pyr-RTKR-AMC substrate were used. The NS2B-NS3pro K48A protein fractions were concentrated with a 5 kDa-cutoff concentrator (Millipore, Billerica, MA) and dialyzed against 10 mM Tris-HCl, pH 8.0, containing 0.005% Brij35. Typical yields of the purified NS2B-NS3pro K48A were 10 mg/L of E. coli culture.
The assay for NS3pro cleavage activity was performed in 10 mM Tris-HCl buffer, pH 8.0, containing 20% glycerol and 0.005% Brij 35. The Pyr-RTKR-AMC substrate and enzyme concentrations, unless indicated otherwise, were 24 μM and 10 nM, respectively. The total assay volume was 0.1 ml. Initial reaction velocities were monitored continuously at λex 360 nm and λem 460 nm on a Spectramax Gemini EM fluorescence spectrophotometer (Molecular Devices, Sunnyvale, CA). All assays were performed in triplicate in a 96 well plate. The values of Km and kcat were derived from a double reciprocal plot of 1/V0 vs. 1/[S], using the Lineweaver-Burk transformation: 1/V0 = Km/Vmax x 1/[S] + 1/Vmax, where V0 is the initial velocity of the substrate hydrolysis and [S] is the substrate concentration, Vmax is the maximum rate of hydrolysis, and Km is the Michaelis-Menten constant. The concentration of the catalytically active proteinase was measured using a fluorescence assay by titration against a standard aprotinin solution of a known concentration.
CD spectra (190–250 nm) of the NS2B-NS3pro and NS2B-NS3pro K48A purified samples (both at 1.4 mg/ml in 15 mM potassium phosphate buffer, pH 7.8 ) were collected in a 1 mm pathlength quartz cuvette at ambient temperature using a 62ADC spectrometer (AVIV Instruments, Lakewood, NJ). Prior to the CD spectra tests, the NS2B-NS3pro sample was incubated for 16 h at 24°C to complete its autolytic conversion into the individual NS3pro enzyme and the NS2B domain. The resulting samples were re-checked by SDS gel-electrophoresis to confirm that autolytic conversion had been completed. The results are expressed as the [Θ]MRW (Mean Residue Weight) ellipticity.
Size-exclusion chromatography of the NS2B-NS3pro and NS2B-NS3pro K48A purified samples was performed on a Superdex 200HR 10/30 pre-packed column (Amersham) on an FPLC System. The column was equilibrated with 50 mM Tris-HCl buffer, pH 7.5, containing 2 mM DTT and 0.2 M NaCl at a constant flow rate of 0.5 ml/min.
To determine the N-terminal sequence of the NS3pro domain and, consequently, the autolytic cleavage site, following SDS gel-electrophoresis the purified NS2B-NS3pro sample (4 μg) was transferred onto an Immobilon membrane support. The 30 kDa NS3pro band was subjected to N-terminal sequencing at ProSeq (Boxford, MA).
Previous studies showed that the presence of the 48 residue central NS2B domain linked to the N-terminus of NS3 significantly enhanced the accumulation of soluble recombinant NS3pro in E.coli [18, 20, 23]. We used a similar approach to express the WNV NS2B-NS3pro. The 48 residue central portion of the NS2B sequence was linked with the NS3pro domain via a GGGGSGGGG linker. To facilitate its isolation, the NS2B-NS3pro construct was C-terminally tagged with a (His)x6 tag and then expressed in E.coli. After induction with isopropyl-β-D-thiogalactoside, large amounts of NS2B-NS3pro were produced as a soluble protein. The soluble fraction was loaded onto a Co2+-agarose affinity column. The NS2B-NS3pro protein was then eluted with a 10–500 mM gradient of imidazole concentrations (Fig. 1). The pooled, partially purified, NS2B-NS3pro fractions were then re-chromatographed on a Co2+-agarose column to acquire the homogenous NS2-NS3pro samples. During purification, we observed an incomplete autolytic conversion of the 36 kDa NS2B-NS3pro construct into the individual 30 kDa NS3pro and the NS2B sequence (5–6 kDa).
To complete the autolytic conversion, the purified samples (0.02–0.2 mg/ml) were incubated overnight at pH 8 and 24°C yielding the individual NS3pro domain and the NS2B (the latter is not visible in Fig. 2 because of its low molecular mass). The resulting NS3 samples retained the C-terminal His tag since the NS3 moiety was readily recognized by a His-tag antibody. By using N-terminal microsequencing, we determined that in the course of autolysis the NS3pro activity cleaved the linker sequence and that the N-terminal sequence of the individual NS3pro was GGGSGG suggesting cleavage at the K48G↓GGGSGGGG site (the linker sequence is underlined). Consistent with our data, Erbel et al.  also determined that the WNV NS2B-NS3pro construct undergoes self-cleavage during expression and purification at the K48G↓G site of the glycine linker. We believe that the cleavage at this unconventional K48G↓G site occurs because of disordered loop structure of the linker region. The structure of the WNV constructs and the relative positions of the mutations are shown in Fig. 2. Inhibitors of the NS3pro activity, including aprotinin (Ki = 25 nM), nona-D-Arg-amide (Ki = 6 nM) and decanoyl-Arg-Val-Lys-Arg-chloromethylketone (Ki = 400 nM) completely inhibited autolysis of the NS2B-NS3pro construct (data not shown). Size-exclusion chromatography confirmed that the NS2B sequence remained non-covalently bound with the NS3pro domain following autocatalytic cleavage of the NS2B-NS3pro construct (Fig. 3).
Based on these sequence data, we constructed the autolysis-resistant K48A mutant. Metal-chelating chromatography followed by re-chromatography of the pooled fractions was sufficient for isolation of the homogenous NS2B-NS3pro K48A construct with a high (45%) yield (Fig. 1 and and2;2; Table 1). The data from gel-electrophoresis and size-exclusion chromatography confirmed that the K48A mutant represented a single-chain protein and that it was fully resistant to autolysis at the linker region (Fig. 2 and and3).3). We conclude that the K48A mutation of the C-terminal amino acid residue of the NS2B sequence inactivated the autolytic cleavage site in the NS2B-NS3pro construct. The K48A mutation, however, did not have any effect on the catalytic activity of the NS2B-NS3pro construct, and the K48A construct was highly efficient in cleaving Pyr-RTKR-AMC (Table 2). Circular dichroism studies also confirmed structural integrity of the wild-type and the K48A mutant constructs (Fig. 4).
Overall, we conclude that the NS2B-NS3pro K48A mutant, which exhibits high stability and functional activity and which can be purified in pilot-scale quantities from E.coli cells as a soluble recombinant protein, is well suited for follow-up structural and drug design studies. Consistent with this suggestion, we have already accomplished the crystallization and X-ray structural analysis of the NS2B-NS3pro K48A construct with an 1.8 resolution, while our previous attempts with the wild-type NS2B-NS3pro were unsuccessful. Because the structural analysis was performed using the K48A construct complexed with aprotinin rather than with the substrate-based inhibitor, a highly valuable information about the P1’–P4’ subsites of NS2B-NS3pro has been acquired in the course of our work (A. Aleshin, S. Shiryaev, A. Strongin, R. Liddington, Structural evidence for multiple levels of regulation of flaviviral protease activity, submitted for publication).
The structural organization of the two-component NS2B-NS3pro is similar in many flavivirus types, including WNV, Dengue types 1–4, Yellow fever, Japanese and Murray valley encephalitis and Kunjin viruses. Accordingly, we predict that similar modifications of the NS2B-NS3pro boundary (which may involve residues distinct from K48 because this lysine residue is conserved only in WNV and Japanese encephalitis viruses) will be sufficient to inactivate the autolytic cleavage site, facilitate isolation and improve the stability and presentation of many flaviviral NS2B-NS3 constructs. These mutant, autolysis-resistant flaviviral protease constructs can then be efficiently used in drug design and structural studies.
This work was supported by NIH Grants AI056385, AI61139 and U54 RR020843 to AYS.
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