| Abstract|| |
Background: Hepatitis C virus (HCV) represents a serious worldwide healthcare problem. No protective vaccines against HCV have been developed yet due to the fact that HCV is rapidly mutable, allowing the virus to escape from the neutralizing antibodies. Understanding of HCV was initially hampered by the inability to achieve viral replication in cell culture. Given its essential roles in viral polyprotein processing and immune evasion, HCV NS3/4A protease is a prime target for antiviral chemotherapy. We aimed to establish in vivo cell-based assay system for monitoring the activity of NS3/4A protease from HCV genotype 4a, the predominant genotype in Egypt, and the Middle East. Furthermore, the developed system was used to evaluate the inhibitory potency of a series of computer-designed chemically-synthesized compounds against NS3/4A protease from HCV genotype 4a. Materials and Methods: Native as well as mutant cleavage sites to NS3/4A protease were cloned in frame into β-galactosidase gene of TA cloning vector. The target specificity of HCV NS3/4A was evaluated by coexpression of β-galactosidase containing the protease cleavage site with NS3/4A protease construct in bacterial cells. The activity of β-galactosidase was colorimetrically estimated in the cell lysate using orthonitro phenyl β-D-galactopyanoside (ONPG) as a substrate. Results and Conclusions: We successfully developed an efficient cell-based system based on the blue/white selection of bacterial cells that are able to express functional/nonfunctional β-galactosidase enzyme.
Keywords: Genotype 4a; Hepatitis C virus; monitoring; NS3/4A protease
|How to cite this article:|
Naguib MM, Mohamed MR, M. Ali MA, Karim AM. Development of an efficient in vivo cell-based assay system for monitoring hepatitis C virus genotype 4a NS3/4A protease activity. Indian J Pathol Microbiol 2019;62:391-8
|How to cite this URL:|
Naguib MM, Mohamed MR, M. Ali MA, Karim AM. Development of an efficient in vivo cell-based assay system for monitoring hepatitis C virus genotype 4a NS3/4A protease activity. Indian J Pathol Microbiol [serial online] 2019 [cited 2020 Jun 5];62:391-8. Available from: http://www.ijpmonline.org/text.asp?2019/62/3/391/263503
| Introduction|| |
Hepatitis C virus (HCV) infection is a global public health problem. According to the World Health Organization (WHO), there are 130-170 million individuals chronically infected with HCV, corresponding to 2-2.5% of the world's total population. There are considerable regional differences. In some countries, for example, Egypt, the prevalence is as high as 22%. Chronic HCV infection causes normally quiescent hepatocytes to divide repeatedly, leading to fibrosis, cirrhosis, and occasionally progression to hepatocellular carcinoma (HCC).
Comparisons of HCV nucleotide sequences derived from individuals from different geographical regions revealed the presence of at least seven major HCV genotypes. The frequencies of genotype 4 are highest in Central Africa and the Middle East and dominate in Egypt.
The HCV NS3-4A protease plays essential roles in viral polyprotein processing and immune evasion. It comprises NS3, a bi-functional protein with an N-terminal serine protease and a C-terminal NTPase/helicase domain, and NS4A, a 54-amino-acid peptide cofactor that forms a tight complex with NS3 and is essential for optimal activity.,, The contribution of NS4A to NS3 protease activity can be mimicked by a synthetic peptide encompassing amino acid residues 21-34 of NS4. The three-dimensional structure of the NS3 protease domain (residues 1-181) complexed with a synthetic NS4A cofactor (residues 21-34) has shown that the NS4A peptide is an integral component of the NS3 protease structure.
The pegylated interferon alfa (peg-IFNα) and ribavirin (RBV) standard of care has been modified with the recent advent of direct-acting antiviral agents (DAAs), which dramatically improved sustained virological response rates in both treatment-naïve and -experienced patients, thus, improving therapeutic options and treatment outcomes for HCV-infected individuals. Although several viral components have been identified as potential anti-HCV targets, the HCV NS3/4A serine protease remains one of the most attractive targets for antiviral drug development to combat HCV infection due to its crucial role in the viral replication machinery. Numerous HCV NS3/4A protease inhibitors (PIs) have been studied in clinical trials for the treatment of chronic HCV infection. Nevertheless, current NS3/4A PIs typically display variable activities across HCV genotypes, which will likely limit their broad usage against multiple genotypes.
Over the past 10 years, the growth in scientific understanding of the HCV lifecycle and new technologies to measure HCV replication played a critical role in the development of a number of models to study the HCV lifecycle and screen for potential HCV inhibitors in vitro. These models include cell-free enzyme assays for the HCV NS3-4A protease and RNA-dependent RNA polymerase, hepatoma cell lines harboring subgenomic and genomic replicons (nucleic acids capable of autonomous replication), an infectious cell culture system, and humanized mouse models infectable by HCV. Nevertheless, the cell culture HCV replication system based on a subgenomic HCV was restricted to replicons derived from genotype 1 and 2a isolates,,,, thus limiting the ability to evaluate potential inhibitors across a spectrum of clinically relevant genotypes. Furthermore, there is not yet a cell culture system for the most prevalent genotypes in Egypt and the Middle East.
| Materials and Methods|| |
Cloning of HCV NS3/4A protease sequence into the pGEX-4T-1 expression vector
The HCV NS32-181 segment was amplified by polymerase chain reaction (PCR) from its pUC119 plasmid containing HCV genome (residues 29-5523) and oligonucleotide primers, +3301 (sense; residues 3301-3320), and –4200 (sense; residues 4182-4200) oligonucleotides that span the HCV genome sequences plus BamH I and Sal I (Promega, Madison, WI, USA) cut sites for forward and reverse primers, respectively. Nested PCR was then performed with a 5'oligonucleotide (HCVproL) encoding an BamH I site, residues 21-34 of NS4, and a dipeptide linker, Gly-Gly, along with residues 2-8 of NS3 (residues 3361-3381). The 3'oligonucleotide (HCV proR) is complementary to residues 175-181 of NS3 (residues 3880-3901) and encoded an in-frame stop codon flanked by an Sal I site. The sequences of the oligonucleotide primers that were used are represented in [Table 1]. PCR amplification product of the first PCR reaction was analyzed on 1% agarose gel and purified with rapid elution using GenJet gel extraction kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) to be used as a template for the nested PCR. The amplified product of the nested PCR was analyzed by electrophoresis on a 1% agarose gel and purified using the GenJet gel extraction kit. The purified PCR product was initially cloned in the TA vector and the ligation product was then used to transform One Shot TOP10 Chemically Competent E. coli (Invitrogen by Life Technologies, Carlsbad, CA, USA). Plasmids from positive clones that contain HCV NS32-181/421-34 protease construct were extracted and digested by BamH I and Sal I. pGEX-4T-1 vector (General Electric Healthcare, Little Chalfont, Buckinghamshire, UK) was also digested by BamH I and Sal I. The digested pGEX-4T-1 was dephosphorylated by calf-intestinal alkaline phosphatase (CIAP, Promega, Madison, WI, USA) to reduce the probability of self-ligation. The dephosphorylated vector was separated on 1% agarose gel and purified with rapid elution using GenJet gel extraction kit. The purified insert and purified dephosphorylated vector were separated on 1% agarose gel to determine their molar ratio for ligation. Two ligation reactions were performed, one for cloning the insert in pGEX-4T-1 vector and the other was self-ligation of digested pGEX-4T-1 as a control to check the presence of background during the main transformation reaction. Both ligation products were used to transform Top 10 competent cells. The transformed TOP 10 cells were allowed to grow on an ampicillin agar medium, selected randomly from that plate, and the presence of recombinant pGEX-4T-1 was checked by isolating plasmid DNA from the selected colonies, following set up of master plates, then performing colony PCR reaction using G27 and G28 universal primers on pGEX-4T-1 vector in order to verify the presence of the insert (HCV NS32-181/421-34 protease construct of about 750 bp in length (620 bp of insert + about 130 bp of vector).
|Table 1: The sequences of forward and reverse primers used for PCR amplification of HCV NS3/4A protease cDNA fragments|
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Expression of the recombinant pGEX-4T-1 containing HCV NS3/4A protease sequence
The selected positive colony was allowed to grow overnight, and then the overnight culture was diluted and incubated at 37°C in incubator shaker for additional 2 h till the OD at 600 nm reached 0.4-0.6, then IPTG was added at a final concentration of 1 mM to induce the transcription and consequently the translation of the glutathione S-transferase (GST)-fusion protein. At 0, 1, 2, 3, 4, and 5 h post-induction, the whole culture was centrifuged in a Sorvall GSA rotor at 5,000 rpm at 4°C for 10 min. The cells pelleted from the culture were washed with PBS, resuspended in fusion protein extraction buffer (50 mM Tris-HCl, pH 8.0; 0.15 M NaCl; 5 mM EDTA, pH 8.0; 1% NP-40, and 1 mM PMSF), incubated for 15-20 min on ice, disrupted with 50 strokes of a tight fitting pestle in a Dounce homogenizer, and centrifuged in a Sorvall SS34 rotor at 12,000 rpm at 4°C for 10 min to remove cell debris. The clear lysate supernatant was further clarified by filtration through a 0.45 μm filter. The recombinant protein was purified by affinity chromatography where the filtered supernatant was loaded onto a polypropylene column (Poly-Prep Chromatography Columns, Bio-Rad Laboratories, Hercules, CA, USA) packed with glutathione cellulose resin (Carl Roth GmbH + Co. KG, Schoemperlenstraße, Karlsruhe, Germany) according to the manufacturer's instructions. The protein concentration was determined using the Pierce bicinchoninic acid (BCA) Protein Assay Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) following the manufacturer's instructions. Expression of the fusion protein was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting.
Cloning of HCV5A/5B cleavage site into pGEM-T vector
Synthetic oligonucleotides encoding the HCV proteolytic NS5A-5B cleavage site (EDVVCCSMSYTWTG) and mutant NS5A-5B cleavage site (EDVVGGSMSYTWTG) with 3'A-overhang nucleotide were used in TA cloning to generate β-gal.HCV5A5B and β-gal.HCVmt5A5B plasmids, respectively. The recombinant vectors were then used to transform E. coli Top10 competent cells. Blue colonies were collected and the presence of the cloned cleavage sites was verified by colony PCR using M13F and M13R universal primers on pGEM-T vector, with an expected size of about 260 bp. The PCR product was separated on 2% agarose gel.
Co-transformation of the cells containing NS protease with β-gal HCV5A5B and β-gal HCV mt5A5B plasmids
Cells containing NS protease was separately co-transformed with β-gal HCV5A5B and β-gal HCV mt5A5B plasmids. Recombinant pGEX-4T-1 from blue bacterial colonies was prepared and the prepared plasmids were then used to co-transform cells containing NS protease. The competent cells were separately transformed with β-gal HCV5A5B and β-gal HCV mt5A5B plasmids. Plasmid DNA was extracted from blue colonies to verify the presence of both plasmids (NS protease and cleavage site plasmids), and separated on 1% agarose gel.
Assessment of the β-galactosidase activity in both β-gal.HCV5A5B and β-gal.HCVmt5A5B constructs
The selected positive colony was allowed to grow overnight, and then the overnight culture was diluted and incubated at 37°C in incubator shaker for additional 2 h till the OD at 600 nm reached 0.4-0.6, then IPTG was added at a final concentration of 1 mM to induce the transcription and consequently, the translation of the glutathione S-transferase (GST)-fusion protein. The O.D of the 1, 2, 3, 4, and 5 h post-induction aliquots at 600 nm was adjusted to be similar to that of the zero aliquot. The cells from each aliquot were harvested by centrifugation, lysed by 100 μl lysis buffer (50 mM Tris-HCl pH 7.4, 300 mM NaCl, 10% glycerol, 0.5% NP40, and 2 mM β-Mercaptoethanol), incubated for 30 min. at 37°C, and centrifuged for 10 min. The activity of β-galactosidase was assessed using β-Gal assay kit (Invitrogen by Life Technologies, Carlsbad, CA, USA). The specific activity of β-galactosidase was calculated from the following equation: Specific activity = nmoles of ortho-nitro phenyl β-D-galactopyanoside (ONPG) hydrolyzed/t/mg protein. nmoles of ONPG hydrolyzed = (OD420) (8 × 105 nL)/(4500 nL/nmole-cm)(1 cm), where 4,500 is the extinction coefficient, t = the time of incubation in min at 37°C (i.e., 30 min), and mg protein is the amount of protein assayed.
Assessment of the potential inhibitory activity of the test compounds on the activity of HCV NS3/4A protease
A set of computer designed chemically-synthesized test compounds (7a [BE113], 7b [BE114], 8a [BE115], and 8b [BE116], supporting information) were dissolved as 10 mM stocks in 100% dimethyl sulfoxide (DMSO) and diluted to 50 μM final concentration. The inhibitory efficacy of the test compounds against the HCV NS3/4A protease was evaluated by determining the activity of the β-galactosidase enzyme, which represents the substrate of the NS3/4A protease that escapes the cleavage by the NS3/4A protease, thus, an increased β-galactosidase activity indicates a higher inhibitory potency of the test compound against NS3/4A protease. A cell containing NS protease, β-gal HCV5A5B and β-gal HCV mt5A5B constructs was lysed in 100 μl lysis buffer and 100 μl of the test compound was added and the mixture was incubated at 37°C. Phenylmethane sulfonyl fluoride (PMSF), a nonspecific serine protease inhibitor, at a concentration of 1 M was used as a positive control, whereas DMSO was used as a negative control. A volume of 20 μl of the reaction mixture was transferred at different time intervals (zero, 15 min, 30 min, 45 min, 1 h, and 1.5 h) for estimating the β-galactosidase activity. For conformation, a volume of 50 μl of the cell suspension containing NS3 protease, protease substrate, and the test compound were mixed and the mixture was incubated at 37°C. A volume of 20 μl of the reaction mixture at different time points (zero, 15 min, 30 min, 45 min, 1 h, and 1.5 h) was transferred for estimating the β-galactosidase activity using β-gal assay kit.
An alternative assay for measuring the β-galactosidase activity to evaluate the inhibitory potency of the test compounds against HCV NS3/4A protease
Following a 5 hr-induction by IPTG at a final concentration of 0.5 mM, the protease enzyme and substrate were purified by harvest and lysis of the cells expressing HCV NS3/4A protease and cells expressing NS3 protease cleavage site, respectively. Equal volumes (50 μl) of the protease enzyme and protease substrate in the presence of the test compound at a concentration of 10 mM, were mixed and the mixture was incubated at 37°C. A volume of 10 μl of the reaction mixture at different time points (zero, 15, 30, 45, 60, and 90 minutes) was transferred for estimating the β-galactosidase activity using the β-gal assay kit, with 1 M PMSF and DMSO were used as a positive control and negative control, respectively.
| Results|| |
Cloning of HCV NS3/4A protease sequence into the pGEX-4T-1 expression vector
PCR amplification of HCV NS32-181 segment by nested PCR reaction using pUC119 plasmid containing HCV genome (residues 29 to 5523) as a template and oligonucleotide primers whose positions relative to HCV genome is described in [Figure 1] and [Figure 2].
|Figure 1: The positions of the oligonucleotide primers used for PCR amplification|
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|Figure 2: Agarose gel analysis for PCR amplification of HCV NS32-181segment. (a) The PCR product of the first PCR reaction (900 bp); (b) The nested PCR product (630 bp); M is 1Kb DNA marker|
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Expression of the recombinant pGEX-4T-1 containing HCV NS3/4A protease sequence
Western blotting analysis of the induced GST fusion NS3/4A protease against anti-GST antibody at different time intervals post-induction is shown in [Figure 3].
|Figure 3: Western blotting analysis of the induced GST fusion NS3/4A protease against anti-GST antibody at different time intervals post-induction. M, high molecular weight protein marker; Lane 1, nonrecombinant GST; Lanes 2, 3, 4, and 5, recombinant protein fused to GST at zero, 1, 3 and 5 hours post-induction, respectively. The molecular weight of GST alone is 25 KDa, whereas that of the recombinant protein is about 54 KDa|
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Assessment of the activity of β-galactosidase enzyme encoded by recombinant pGEM-T vector containing either the native substrate for NS3/4A protease enzyme or the mutant form of the NS3/4A protease substrate
The results showed that HCV3/4A protease enzyme had a proteolytic activity towards its native substrate encoded by β-gal HCV 5A5B construct, whereas had no remarkable proteolytic activity towards its mutant substrate encoded by β-gal mt HCV 5A5B construct [Figure 4].
|Figure 4: Assessment of the activity of β-galactosidase enzyme encoded by recombinant pGEM-T vector containing either the native substrate for NS3/4A protease enzyme (β-gal HCV 5A5B construct) or the mutant form of the NS3/4A protease substrate (β-gal mt. HCV 5A5B construct) |
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Evaluating the inhibitory potency of the test compounds on the activity of HCV NS3/4A protease
The results showed that compounds BE114 (7b) and BE115 (8a) had moderate inhibitory efficacy against HCV NS3/4A protease, whereas compounds BE113 (7a) and BE116 (8b) had no remarkable inhibitory effects on the protease enzyme. Notably, PMSF had the highest inhibitory effect on the protease enzyme [Figure 5].
|Figure 5: Evaluation of the inhibitory potency of the test compounds on the activity of HCV NS3/4A protease. The activity of the β-galactosidase enzyme was assessed in the absence or presence of 1 M phenylmethane sulfonyl fluoride (PMSF) as well as in the presence of the test compound (BE113 [7a], BE114 [7b], BE115 [8a] and BE116 [8b] at a concentration of 10 mM, at different incubation time points (zero, 15, 30, 45, 60 and 90 minutes). DMSO, dimethyl sulfoxide|
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An alternative assay for measuring β-galactosidase enzyme activity to evaluate the inhibitory potency of the test compounds against HCV NS3/4A protease
The results showed that compound 7b (BE114) had moderate inhibitory efficacy against HCV NS3/4A protease, whereas compounds 7a (BE113) and 8b (BE116) had no remarkable inhibitory effects on the protease enzyme. PMSF and compound 8a (BE115) had the highest inhibitory effects on the protease enzyme [Figure 6].
|Figure 6: An alternative assay for measuring the β-galactosidase enzyme activity to evaluate the inhibitory potency of the test compounds on the activity of HCV NS3/4A protease. The activity of the β-galactosidase enzyme was assessed in the absence or presence of 1 M phenylmethane sulfonyl fluoride (PMSF) as well as in the presence of the test compound (BE113 [7a], BE114 [7b], BE115 [8a] and BE116 [8b] at a concentration of 10 mM, at different incubation time points (zero, 15, 30, 45, 60 and 90 minutes). DMSO, dimethyl sulfoxide |
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| Discussion|| |
The development of therapeutics against HCV has been hampered by the lack of an efficient cell culture system and a small animal model for this virus. Lohmann et al. partially solved this problem by developing a reliable cell culture HCV replication system based on a subgenomic HCV or replicon. Nonetheless, this system was restricted to replicons derived from genotype 1 isolates, thus limiting the ability to evaluate potential inhibitors across a spectrum of clinically relevant genotypes, and the system only mimicked the authentic replication cycle of HCV, without production of infectious particles. In 2005, the first cell culture replication system based on an HCV genome of genotype 2a was developed.,, However, there is not yet a cell culture system for the most prevalent genotypes in Egypt and the Middle East. Although the replicon system has some limitations, it is currently the preferred standard to evaluate HCV antivirals. In contrast with the replicon system, this approach allows the evaluation of the protease activity alone, without interference from other viral components. This is important in order to demonstrate that the effect of inhibitors is exerted on the target enzyme, the NS3/4A protease.
Several alternative systems for monitoring the activity of NS3/4A serine protease and for screening specific enzyme inhibitors have been reported, including cell-based systems based on reporter substrates fused to a cleavage sequence,,,in vitro assay based on recombinant substrate NS5ab and single-chain serine protease, genetic system based on the bacteriophage lambda regulatory circuit, and fluorescence resonance energy transfer-based assay (FRET).
One of the systems that has been developed for monitoring the activity of the NS3-4A serine protease of HCV in mammalian cells relies on coexpression of the protease and of an artificial substrate containing a reporter domain and a NS3-4A-specific cleavage site. One of the reporter domains that have been used is the secreted embryonic alkaline phosphatase (SEAP).
One assay has constructed pCI-neo-NS3/4A-SEAP chimeric plasmid, in which the SEAP was fused in-frame to the downstream of NS4A/4B cleavage site. The protease activity of NS3 was reflected by the activity of SEAP in the culture media of transient or stable expression cells. Stably expressing cell lines were obtained by G418 selection. The rationale for this system was based on the assumption that the secretion of SEAP protein into the culture media depends on the cleavage between NS4A protein and SEAP protein by HCV NS3 protease. Comparably, this cell-based NS3/4A-SEAP expression system is safe, easy to handle, and the report gene of SEAP can be sensitively and quantitatively measured continuously without killing cells.
A disadvantage of AP is, however, the fact that many cells express AP at their cell surface, thus raising the possibility of a significant background signal. Moreover, the production of a soluble form of SEAP results in a dilution of the signal into the volume of supernatant media.
Interestingly, a genetically coded FRET probe that detects NS3/4A protease activity in living cultured human cells has been constructed. This FRET probe consisted of an enhanced cyan fluorescent protein-citrine fusion, with a cleavage site for HCV NS3/4A protease embedded within the linker between them. Expression of the biosensor in mammalian cells resulted in a FRET signal, and cotransfection with the NS3/4A expression vector produced a significant reduction in FRET, indicating that the cleavage site was processed. Western blot and spectrofluorimetry analysis confirmed the physical cleavage of the fusion probe by the NS3/4A protease. The level of FRET decay was a function of the protease activity. This FRET probe could be adapted for high-throughput screening of new HCV NS3/4 protease inhibitors. This system is simple and allows the characterization of large cohorts of samples of different genotypes. Because fluorescence techniques are very sensitive, the assay can potentially be used to evaluate inhibitors against proteases of several genotypes and can be adapted to assay formats suitable for high-throughput pharmacological screening. In contrast with the replicon system, this approach allows the evaluation of the protease activity alone, without interference from other viral components. This is important in order to demonstrate that the effect of inhibitors is exerted on the target enzyme, the NS3/4A protease.
A limitation for assessing protease activity in transfected mammalian cells is that the relative concentration of substrate sensor and NS3/4A enzyme will likely vary from cell to cell.
Beside the cell-basedin vivo assays described above, there was an assayin vitro for HCV NS3 serine protease based on recombinant substrate and single-chain protease. Based on the crystallographic structure of HCV serine protease, a novel single-chain serine protease was designed, in which the central sequence of cofactor NS4A was linked to the N-terminus of NS3 serine protease domain via a flexible linker GSGS. The fusion gene was cloned into the prokaryotic expression vector. The single-chain recombinant protease was expressed with IPTG and the expression conditions were optimized to produce large amount of soluble protease. The recombinant substrate NS5ab that covers the cleavage point NS5A/B was also expressed in E. coli. Both the protease and substrate were purified by using Ni-NTA agarose metal affinity resin, then they were mixed together in a specific buffer, and the mixture was analyzed by SDS-PAGE. The purified single-chain serine protease could cleave the recombinant substrate NS5ab into two fragments that were visualized by SDS-PAGE. In this assay system, the results were analyzed by SDS-PAGE and visualized by Coomassie brilliant blue R-250 staining (CBB R-250), which make it an attractive system because of its convenience, low cost, and intuitiveness. Furthermore, this assay can be used in high-throughput screening of the potential inhibitors of HCV protease., This cleavage system was used to evaluate some compounds for their inhibitory activity on serine protease.
The genetic screen system that is based on the bacteriophage lambda cI-cro regulatory circuit, the viral repressor cI is specifically cleaved to initiate the switch from lysogeny to lytic infection. An HCV protease-specific target, NS5A-5B, was inserted into the lambda phage cI repressor. The target specificity of the HCV NS5A-5B repressor was evaluated by coexpression of this repressor with a β-galactosidase (β-gal)-HCV NS32-181/421-34 protease construct. Upon infection of E. coli cells containing the two plasmids encoding the cI.HCV5AB-cro and the β-gal-HCV NS32-181/421-34 protease constructs, lambda phage replicated up to 8,000 fold more efficiently than in cells that did not express the HCV NS32-181/421-34 protease. This simple, rapid, and highly specific assay can be used to monitor the activity of the HCV NS3 serine protease, and it has the potential to be used for screening specific inhibitors.
Previous studies have shown that there are two methods to produce fully active HCV serine protease. The first method is to express full length NS31-631 which was fused to the N-terminal of NS4A1-54 in eukaryotic cells, and the second one is to express protease domain in E. coli and mix them with excessive synthetic NS4A core peptide to form heterodimer. Although the two methods work, there still are many shortages. The former can only provide little amount of protein and the enzymatic activity of protein produced by later method is very low., According to Yan's report, the reason that poor activity of the heterodimer formedin vitro by synthetic peptide and protease domain lies in that the affinity of synthetic peptide is much lower than that of the full-length NS4A, which makes the complex unstable. So it is reasonable to express the fusion protein of NS3 protease domain and NS4A in E. coli. Inoue et al. expressed NS3 protease fused at the amino terminal of NS4A in E. coli, which showed ideal stability and activity, but the products existed in inclusion bodies that lead to the poor productivity. In addition, the crystallographic structure of NS3 serine protease domain complexed with synthetic peptide indicated that it might be better for NS4A fused to amino terminal of NS3 protease domain., Since the central part of NS4A is embedded in the cleft formed by NS3 protease domain and its C-terminus lies close to the amino terminus of NS3, it is reasonable to fuse the NS4A central peptide to amino terminal of NS3 via a flexible linker. This fusion format will increase the stability of NS4A-NS3N complex.
The reason that protease can express in a soluble form at high level in E. coli lies in two points: (1) the low concentration of inducer (IPTG) and low temperature culture reduced the synthesis speed of peptide, which enhance the folding of protein;, (2) the central sequences of NS4A may increase the solubility of the single-chain serine protease. Previous studies have confirmed that NS3 protease's N-terminus would be changed from a disordered structure to a regular one when it forms a complex with the NS4A peptide, that is why the NS4A central peptide is capable of promoting the folding of protein.
We established a simple and cheapin vivo assay for HCV NS3 protease. In this assay system, the results were analyzed colorimetrically which make it an attractive system because of its simplicity and low cost. The inherent difficulties in the purification and characterization of the HCV NS3 protease byin vitro classical methodologies prompted us to explore this genetic system as a simple alternative approach for the characterization of HCV NS3 protease activity. To assess the applicability of this assay system in identification of serine protease inhibitors, we examined the effect of a well-known serine protease inhibitor, PMSF, on the activity of HCV protease. The results confirmed that the PMSF could inhibit the proteolytic activity of HCV serine protease completely which have been observed by other researchers.
In conclusion, our system can be seen as a complement to the classical biochemical approach for monitoring NS3 proteolytic activity. This simple, rapid, and highly specific assay can be used to monitor the activity of the HCV NS3 serine protease, and it has the potential to be used for screening specific inhibitors.
This research has been funded by a research grant from Ain Shams University to Amr M. Karim.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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Mohamed A M. Ali
Department of Biochemistry, Faculty of Science, Ain Shams University, Abbassia - 11566, Cairo
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]