A straightforward experimental approach to expression, purification, refolding, and enzymatic analysis of recombinant dengue virus NS2B(H)-NS3pro protease.

Dengue virus threatens around 2.5 billion people worldwide; about 50 million become infected every year, and yet no vaccine or drug is available for prevention and/or treatment. The flaviviral NS2B-NS3pro complex is indispensable for flaviviral replication and is considered to be an important drug target. The aim of this study was to develop a simple and generally applicable experimental strategy to construct, purify, and assay a highly active recombinant NS2B(H)-NS3pro complex that would be useful for high-throughput screening of potential inhibitors. The sequence of NS2B(H)-NS3pro was generated by overlap extension PCR (SOE-PCR) and cloned into the pTrcHisA vector. Hexahistidine-tagged NS2B(H)-NS3pro complex was expressed in E. coli predominantly as insoluble protein and purified to >95% purity by single-step immobilized metal affinity chromatography. SDS-PAGE followed by immunoblotting of the purified enzyme demonstrated the presence of the NS2B(H)-NS3pro precursor and its autocleavage products, NS3pro and NS2B(H), as 37, 21, and 10 kDa bands, respectively. Kinetic parameters, Km, kcat, and kcat/Km for the fluorophore-linked protease model substrate Ac-nKRR-amc were obtained using inner-filter effect correction. The kinetic parameters Km, kcat, and kcat/Km for Ac-nKRR-amc substrate were 100 µM, 0.112 s(-1), and 1120 M(-1)·s(-1), respectively. A simplified procedure for the cloning, overexpression, and purification of the NS2B(H)-NS3pro complex was applied, and a highly active recombinant NS2B(H)-NS3pro complex was obtained that could be useful for the design of high-throughput assays aimed at flaviviral inhibitor discovery.

Modulation of enzymatic activity of dengue virus nonstructural protein NS3 nucleoside triphosphatase/helicase by poly(U).

The nonstructural protein 3 (NS3) appears to be the most promising target for anti-flavivirus therapy because of its multiple enzymatic activities that are indispensable for virus replication. NS3 of dengue virus type 2 (DEN2) is composed of two domains, a serine protease in the N-terminal domain (NS3pro) and RNA-stimulated nucleoside triphosphatase (NTPase)/RNA helicase at the C-terminus (NS3h). NS3 plays an important role in viral replication and the coordinated regulation of all the catalytic activities in the full-length NS3 protein. In this study, a plasmid harboring the NS3 helicase domain (NS3h) was constructed by PCR. The 56.5 kDa NS3h protein was purified by metal-chelate affinity chromatography followed by renaturation, mediated by artificial chaperone-assisted refolding, which yielded the active helicase. NTPase activity was assayed with Malachite Green. The NTPase activity in the presence of poly(U) showed a higher turnover number (kcat) and a lower Km value than without poly(U). The activity increased approximately fourfold in the presence of polynucleotides. This indicates that NTPase activity of dengue NS3 can be stimulated by polynucleotides. A helicase assay based on internal fluorescence quenching was conducted using short internally quenched DNA oligonucleotides as substrates. Significant fluorescence signaling increase was observed in the absence of polynucleotides such as poly(U). No unwinding activity was observed with addition of poly(U). The approach we describe here is useful for the further characterization of substrate specificity and for the design of high-throughput assays aimed at discovery of inhibitors against NS3 NTPase/helicase activities.

Effects on larvicidal activity of single proline substitutions in alpha3 or alpha4 of the Bacillus thuringiensis Cry4B toxin.

The possible role of alpha-helices 3 and 4 in toxicity of the dipteran-active Bacillus thuringiensis Cry4B delta-endotoxin was investigated by employing proline substitutions via site-directed mutagenesis. Similar to the wild-type Cry4B, the mutant toxins were over-expressed in Escherichia coli as cytoplasmic inclusions and were structurally stable upon solubilization and trypsin activation. The substitution of glutamine 149 by proline in the center of helix 4 (Q149P) resulted in a nearly complete loss of toxicity against Aedes aegypti mosquito-larvae. However, single proline replacements near the center of helix 3 (V119P) and at the N-terminus of helix 4 (Q140P) did not decrease larvicidal activity. The toxicity of E. coli cells expressing the wild-type toxin was significantly reduced by two-hour preincubation with the non-toxic mutant (Q149P), thus indicating that the primary binding step was not affected by the proline substitution in helix 4. The results therefore reveal a crucial role for helix 4 of the Cry4B toxin in toxicity, possibly in membrane insertion and pore formation rather than in receptor recognition.

Effects on toxicity of eliminating a cleavage site in a predicted interhelical loop in Bacillus thuringiensis CryIVB delta-endotoxin.

When activated by treatment with mosquito (Aedes aegypti) gut extract, the Bacillus thuringiensis CryIVB delta-endotoxin lysed A. aegypti cells in vitro. SDS-PAGE and N-terminal sequence determination showed that in addition to removal of the C-terminal half of the molecule, the activated toxin had undergone proteolytic cleavage at two internal regions producing 47-48-kDa and 16-18-kDa polypeptides. Aligning the CryIVB protein sequence with the crystallographic structure of the CryIIIA toxin suggested that one set of cleavages occurred in a region before the start of the N-terminal helical bundle and the second cleavage site occurred in a predicted loop between helices 5 and 6 in the bundle at arginine-203. To investigate the suggestion by Li et al. that interhelical proteolysis is important in the cytolytic mechanism of these toxins, arginine-203 was substituted by alanine. The mutated toxin now resisted proteolysis at this position and showed a marked decrease in cytolysis in vitro but an increase in larvicidal activity.

Comparison of Bacillus thuringiensis subsp. israelensis CryIVA and CryIVB cloned toxins reveals synergism in vivo.

When the gene for the mosquitocidal protein CryIVA was expressed in two strains of Bacillus thuringiensis (Bt) cured of their resident delta-endotoxin genes, the protein accumulated as large inclusions. The inclusions produced in the Bt subsp. kurstaki recipient strain were twice as soluble at alkaline pH as the inclusions produced in Bt subsp. israelensis. Solubilized protoxins were activated by treatment with mosquito gut extracts or trypsin for varying lengths of time and tested for in vitro cytotoxicity on cell lines of three genera of mosquito. CryIVA treated with any of the mosquito gut extracts for 6 h showed significant toxicity against Anopheles gambiae cells and slight activity on Culex quinquefasciatus cells. For CryIVB, the only significant cytotoxicity observed was against Aedes aegypti cells after treatment with Aedes gut extract. In in vivo bioassays, both CryIVA, purified from either of the Bt recipient strains, and CryIVB inclusions were similarly toxic to A. aegypti and A. gambiae larvae but CryIVA was 25-fold more toxic to C. quinquefasciatus. Synergism in vivo between the two toxins was revealed when results from assaying single toxins and mixtures were compared. Mixtures of CryIVA and CryIVB proved to be 5-fold more toxic to Culex than either toxin used singly and showed a reduced but similar synergism when tested against Aedes and Anopheles larvae. The synergism was not duplicated in vitro using cell lines from these three insects.

Cytotoxicity of a cloned Bacillus thuringiensis subsp. israelensis CryIVB toxin to an Aedes aegypti cell line.

A cloned CryIVB toxin was purified from a cured strain of Bacillus thuringiensis (BT) containing the cryIVB gene on the recombinant plasmid Cam135. Solubilized protoxin was treated with Aedes gut extract or trypsin for varying times and tested for toxicity in vitro on three dipteran and one lepidopteran cell line. Treatment with the Aedes extract but not trypsin, produced an active toxin which lysed only Aedes aegypti cells out of those tested. This activation was time-dependent reaching a maximum after 6 h. Both the Aedes extract-treated and trypsin-treated toxin killed A. aegypti larvae, but this toxicity declined rapidly with increasing time of exposure to the proteolytic preparations.

Biosynthesis of 130-kilodalton mosquito larvicide in the cyanobacterium Agmenellum quadruplicatum PR-6.

The 130-kilodalton mosquito larvicidal gene, cloned from Bacillus thuringiensis var. israelensis, was introduced into the cyanobacterium Agmenellum quadruplicatum PR-6 by plasmid transformation. Transformed cells synthesized 130-kilodalton delta-endotoxin protein and showed mosquito larvicidal activity. Results demonstrate a potential use of a cyanobacterium for biological control of mosquitoes.

The mosquito larvicidal activity of 130 kDa delta-endotoxin of Bacillus thuringiensis var. israelensis resides in the 72 kDa amino-terminal fragment.

Bacillus thuringiensis var. israelensis produces 130 kDa delta-endotoxin which is highly toxic to mosquito-larvae. The mosquito-larvicidal activity was delineated by sequential deletions from ends of the 1136 amino acids delta-endotoxin. A maximum of 459 amino acids could be removed from the carboxy-terminal of the toxin without a significant loss of the larvicidal activity. However, no more than 38 amino acids could be deleted from the amino-terminal without losing the toxicity. The truncated peptide of 72 kDa exhibited similar toxicity to the 130 kDa toxin and was between 39th and 677th amino acids.

Common features of Bacillus thuringiensis toxins specific for Diptera and Lepidoptera.

The complete nucleotide sequence of a cloned gene encoding a 130-kDa crystal protein of Bacillus thuringiensis (B.t.) subspecies israelensis has been determined. The recombinant protein (Bt8) was purified and shown to be a mosquito-specific toxin with a LC50 value of 43 ng/ml to third-instar larvae of Aedes aegypti. Bt8 is processed by proteases or midgut extracts of mosquito larvae into toxic fragments of 68-78 kDa. Deletion mapping indicated that the active fragment of Bt8 is localized in the N-terminal half of the protoxin molecule. The deduced amino acid sequence of Bt8 has been compared with that of Bt2, a Lepidoptera-specific toxin, previously cloned from Bacillus thuringiensis berliner. Highly homologous amino acid stretches are present in the C-terminal half of the proteins. The N-terminal parts show much less sequence homology but they display a strikingly similar distribution of hydrophilic and hydrophobic amino acids. In addition, Bt8 and Bt2 show a significant immunological cross-reaction. The data indicate that although these B.t. delta endotoxins exhibit a different insect-host specificity, they are structurally related and might use a similar mechanism to interact with insect cell membranes.

Cloning and expression of 130-kd mosquito-larvicidal delta-endotoxin gene of Bacillus thuringiensis var. Israelensis in Escherichia coli.

Five recombinant E. coli clones exhibiting toxicity to Aedes aegypti larvae were obtained from a library of 800 clones containing XbaI DNA fragments of 110 kb plasmid from B. thuringiensis var. israelensis. All the five clones (pMU 14/258/303/388/679) had the same 3.8-kb insert and encoded a major protein of 130 kDa which was highly toxic to A. aegypti larvae. Three clones (pMU 258/303/388) transcribed the 130 kD a gene in the same direction as that of lac Z promoter of pUC12 vector whereas the transcription of the other two (pMU 14/679) was in the opposite direction. A 1.9-kb fragment of the 3.8 kb insert coded for a protein of 65 kDa. Partial DNA sequence of the 3.8 kb insert, corresponding to the 5'-terminal of the 130 kDa gene, revealed a continuous reading frame, a Shine-Dalgarno sequence and a tentative 5'-regulatory region. These results demonstrated that the 3.8 kb insert is a minimal DNA fragment containing a regulatory region plus the coding sequence of the 130 kDa protein that is highly toxic to mosquito larvae.