WO2000001718A2

WO2000001718A2 - Ns4a-ns3 catalytic domain of hepatitis c

Info

Publication number: WO2000001718A2
Application number: PCT/US1999/015035
Authority: WO
Inventors: Ben M. Dunn; Marina Bukhtiyarova
Original assignee: University of Florida
Current assignee: University of Florida
Priority date: 1998-07-02
Filing date: 1999-07-02
Publication date: 2000-01-13
Anticipated expiration: 2001-01-02
Also published as: AU4855299A; WO2000001718A3

Abstract

The subject invention concerns modified domains of the polyprotein of the hepatitis C virus. The polypeptides of the invention are directed to modified forms of the NS3 and NS4A domains of the polyprotein. In one embodiment, the NS4A fragment is covalently attached to the NS3 catalytic domain. The NS4A fragment is attached to the N-terminal end of NS3. The subject invention also concerns polynucleotides that encode the NS3/NS4A polypeptides. The invention also pertains to methods of preparing the subject polypeptides and methods for screening for compounds that inhibit the serine protease activity of the NS3 protein.

Description

DESCRIPTION

NS4A-NS3 CATALYTIC DOMAIN OF HEPATITIS C

This invention was made with government support under United States National

Institutes of Health Grant No. UO1 AI41423. The government has certain rights in this invention.

Background of the Invention Hepatitis C is an infectious disease that infects nearly 2% of Americans, and even higher levels of the populations of other countries. Previously, this virus was spread mainly by transfusion with infected blood, due to the lack of a diagnostic test prior to 1990. The major mechanism of transmission currently is by IV drug use, with sexual transmission a secondary factor. This virus causes chronic disease in the liver, leading to cirrhosis and potential liver failure. In about 10% of infected individuals, carcinoma of the liver is also an outcome. By the year 2015, it is estimated that 400,000 Americans will have advanced stages of liver disease caused by hepatitis C and that 40,000 will die annually. Thus, hepatitis C has emerged as the next significant anti-viral drug target, following the successful development of anti-HIV compounds. The virus responsible for hepatitis C is a positive-stranded RNA virus that produces progeny virus in a mechanism similar to that of the HIV system; the viral message is translated to produce a viral "polyprotein" containing a number of functional proteins connected head-to-tail. Embedded within this large polyprotein are two putative proteolytic enzymes, NS2 and NS3. The NS3 protein includes a "catalytic domain" of 182 amino acids that bears a sequence relationship to members of the trypsin family of serine proteases. NS3 is responsible for cleavage of four of the sites within the viral polyprotein that must be cut to separate the functional units. Viral replication is not possible without these cuts; thus, the activity of NS3 protease is a target for anti-viral drug discovery. U.S. Patent Nos. 5,371,017 and 5,712,145 describe the cloning and expression of hepatitis C protease involved in polyprotein processing. U.S. Patent No. 5,739,002 describes a method for reproducing in vitro the proteolytic activity of the NS3 protease.

The 182 amino acid NS3 catalytic domain, which is only about one-third of the entire NS3 protein, has been cloned and expressed by several groups, and shown to possess the catalytic activity necessary to achieve the four cleavages. However, the efficiency of the processing is very low, and the 182-residue protein is very unstable and insoluble. Thus, working with this system is difficult at best. It has been discovered by a variety of workers (Failla et al., 1984; Lin et al, 1995; Shimizu et al, 1996; and Bianchi et al, 1997) that another portion of the viral polyprotein, the NS4A fragment, is necessary to increase the activity of the NS3 catalytic domain. The crystal structure described in Kim et al. (1996) indicated a non-covalent complex between the NS4A fragment and the NS3 catalytic domain. The NS4A segment of the hepatitis polyprotein is downstream of the NS3 protein, and is separated from the NS3 catalytic serine protease domain by a separate domain that possesses helicase activity. U.S. Patent No. 5,843,752 describes fusion complexes between NS3 and NS4A.

As can be understood from the above, there remains a need in the art for means of expressing the NS3 domain in a stable, soluble form and to provide a means for screening for compositions that can be used in the diagnosis or treatment of hepatitis C infection.

Brief Summary of Invention The subject invention concerns compositions and methods of use of modified domains of the polyprotein of the hepatitis C virus. The polypeptides of the invention are directed to modified forms of the NS3 and NS4A domains of the polyprotein covalently linked together. In one embodiment, an NS4A fragment is covalently attached to the N-terminal end of an NS3 domain. The subject invention also concerns polynucleotides that encode the NS3/NS4A polypeptides of the invention. The invention also pertains to methods of preparing the subject polypeptides and methods for screening for compounds that inhibit the serine protease activity of the NS3 protein. Brief Description of the Drawings Figure 1 shows the amino acid sequence of an NS3 clone of the IB genotype. Amino acid residues deleted for the NS4A/NS3 construct of the subject invention are shown underlined. Figure 2 shows the amino acid sequence of an NS4A/NS3 fusion construct according to the subject invention.

Figure 3A and 3B show an NS3 nucleic acid sequence and an amino acid sequence encoded by the nucleic acid sequence, respectively. The polypeptide shown in

Figure 3B includes a histidine tag sequence consisting of about the first 23 amino acids which is attached to allow for easier purification of the protein using Ni Agarose matrix.

Figure 4A and 4B show an NS4 S3 fusion construct nucleic acid sequence and an amino acid sequence encoded by the nucleic acid sequence, respectively. The polypeptide shown in Figure 4B includes a histidine tag sequence consisting of about the first 23 amino acids which is attached to allow for easier purification of the protein using Ni Agarose matrix.

Detailed Description of the Invention The subject invention concerns polynucleotides which encode polypeptides comprising an NS3 catalytic domain, or a catalytic fragment or variant thereof, of hepatitis C serine protease covalently attached to a peptide derived from an NS4A domain. The subject invention also concerns the NS4A/NS3 polypeptides encoded by the polynucleotides of the invention. In one embodiment, an NS4A/NS3 construct was prepared using an oligonucleotide primer to produce a polynucleotide encoding the NS4A/NS3 polypeptide that had several amino acid residues of an NS4A fragment added to the amino terminus of an NS3 catalytic domain (Figure 2). In an exemplified embodiment, one amino acid of the NS3 sequence was changed, based on the structure, to introduce a more polar amino acid to improve solubility. Five residues of the NS3 catalytic domain were also removed in the exemplified construct. Thus, one aspect of the present invention concerns constructs where an NS4A fragment, which is normally associated with the C-terminal end of the NS3 fragment, is placed on the N-terminal end of NS3. Although the IB genotype was utilized in one of the constructs exemplified herein, all other hepatitis C genotypes are contemplated within the scope of the present invention.

In another exemplified embodiment, a polynucleotide was prepared that encodes a fusion construct comprising a histidine tag sequence for Ni Agarose purification covalently linked to the amino terminus of an NS3 catalytic domain. From that fusion construct, an NS4A peptide sequence (SEQ ID NO. 7) was inserted between the histidine tag sequence and the start of the NS3 domain sequence (Figure 4B). The histidine tag sequence can be optionally removed or deleted from the NS4A/NS3 construct.

A plasmid encoding the modified polypeptide of the invention was expressed in E.coli in high levels in the form of insoluble inclusion bodies. The preparation of inclusion bodies included isolation via centrifugation through sucrose gradients. The resulting material was solubilized and the protein was re-folded by dialysis in several steps to yield a solution at pH 8.0. An aliquot of this preparation was able to cleave a peptide substrate based on the hepatitis 5A/5B cleavage site. A similar procedure of expression and re-folding using a construct containing only the NS3 catalytic domain

(i.e., lacking the NS4A piece) yielded inactive protein. Thus, refolding of the NS3 catalytic domain without the N-terminal modification is significantly less successful, and yields protein of reduced stability.

The methods and compositions of the present invention yield several milligrams of protein per liter of expression medium. The NS4A NS3 constructs of the present invention exhibit properties of the non-covalent NS3 and NS4A complex with respect to catalytic activity and ligand binding, but with superior stability and solubility. Thus, the subject invention provides for the expression of the hepatitis C NS3 serine protease domain in greater amounts and in higher purity with fewer steps. In addition, the NS4/NS3 protein produced according to the present invention is more stable and considerably more soluble than the NS3 catalytic domain alone. Thus, the NS4A/NS3 polypeptides of the present invention can be used for direct screening of potential antiviral drug candidates.

The subject invention also concerns methods for screening for compounds that can be used in diagnostic or therapeutic applications for persons infected with the hepatitis C virus. For example, the recombinant polypeptides of the invention can be used to screen for compounds that inhibit the hepatitis C serine protease. In one embodiment, a method of the present invention comprises contacting an NS4A/NS3 polypeptide of the subject invention with a selected composition and then determining whether the composition binds to or inhibits the activity of the NS4/NS3 polypeptide. Also contemplated within the scope of the present invention are compounds that inhibit hepatitis C serine protease. These compounds can be readily identified using the compositions and screening methods according to the present invention.

As those of ordinary skill in the art will appreciate, any of a number of different nucleotide sequences can be used, based on the degeneracy of the genetic code, to produce the modified proteins described herein. Accordingly, any nucleotide sequence which encodes the proteins described herein comes within the scope of this invention. Also, as described herein, fragments and variants of the subject NS3 domain covalently linked to the NS4A peptide are an aspect of the invention so long as such fragments and variants retain serine protease activity substantially the same as wild type NS3 protein. Variants include those sequences in which amino acid substitutions, insertions and/or deletions have been introduced that change the variant sequence from the wild type sequence. Such fragments and variants can easily and routinely be produced by techniques well known in the art. For example, time-controlled BaBl exonuclease digestion of the full-length DNA followed by expression of the resulting fragments and routine screening can be used to readily identify expression products having the desired activity (Wei et al., 1993). Variants can also be readily produced using, for example, site-directed mutagenesis methods. NS4A peptides within the scope of the invention include those peptides specifically exemplified herein, as well as peptides having additional amino acids added to or deleted from either or both ends of the peptide sequence. For example, 10 to 20 amino acids can be added to either or both ends of the peptide. Preferably, the amino acids added are the same as the corresponding amino acids in the NS4A domain.

As used herein, the terms "nucleic acid" and "polynucleotide sequence" refer to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, would encompass known analogs of natural nucleotides that can function in a similar manner as naturally-occurring nucleotides. The polynucleotide sequences include both the DNA strand sequence that is transcribed into RNA and the RNA sequence that is translated into protein. The polynucleotide sequences include both full-length sequences as well as shorter sequences derived from the full-length sequences. It is understood that a particular polynucleotide sequence includes the degenerate codons of the native sequence or sequences which may be introduced to provide codon preference in a specific host cell. Allelic variations of the exemplified sequences also come within the scope of the subject invention. The polynucleotide sequences falling within the scope of the subject invention further include sequences which specifically hybridize with the exemplified sequences under stringent conditions. The nucleic acid includes both the sense and antisense strands as either individual strands or in the duplex.

The terms "hybridize" or "hybridizing" refer to the binding of two single-stranded nucleic acids via complementary base pairing. The phrase "hybridizing specifically to" refers to binding, duplexing, or hybridizing of a molecule to a nucleotide sequence under stringent conditions when that sequence is present in a preparation of total cellular DNA or RNA.

The term "stringent conditions" refers to conditions under which a polynucleotide will hybridize to another sequence, but not to sequences having little or no homology to the sequence. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a complementary polynucleotide. Typically, stringent conditions will be those in which the salt concentration is at least about 0.1 to 1.0 N Na ion concentration at a pH of about 7.0 to 7.5 and the temperature is at least about 60°C for long sequences (e.g., greater than about 50 nucleotides) and at least about 42 °C for shorter sequences (e.g., about 10 to 50 nucleotides).

A further aspect of the present invention are antibodies that are raised by immunization of an animal with a purified protein of the subject invention. Both polyclonal and monoclonal antibodies can be produced using standard procedures well known to those skilled in the art using the proteins of the subject invention as an immunogen (see, for example, Monoclonal Antibodies: Principles and Practice, 1983; Monoclonal Hybridoma Antibodies: Techniques and Applications, 1982; Selected Methods in Cellular Immunology, 1980; Immunological Methods, Vol. II, 1981 ; Practical Immunology, and Kohler et al., 1975.

Also included within the scope of the invention are binding fragments of the antibodies of the subject invention. Fab', F(ab')₂, and Fv fragments may be obtained by conventional techniques, such as proteolytic digestion of the antibodies by papain or pepsin, or through standard genetic engineering techniques using polynucleotide sequences that encode binding fragments of the antibodies of the subject invention.

The one-letter symbol for the amino acids used in the sequences shown herein is well known in the art. For convenience, amino acids and their corresponding one-letter symbols are indicated as follows:

Alanine A

Arginine R

Asparagine N

Aspartic Acid D

Cysteine C

Glutamine Q

Glutamic Acid E

Glycine G

Histidine H

Isoleucine I

Leucine L

Lysine K

Methionine M

Phenyalanine F

Proline P

Serine s

Threonine T

Tryptophan w

Tyrosine Y

Valine V All publications and patents cited herein are hereby incorporated by reference.

Following are examples which illustrates the procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

Materials and Methods

Viral RNA was isolated from lOOμL of human serum obtained from patients at Shands Teaching Hospital Liver Unit. Reverse Transcription PCR was carried out using an upper primer beginning (5' end) at nucleotide number 2920 of the full sequence, and a lower primer beginning (3' end) at nucleotide number 3897. Amplification was the achieved by PCR with Taq polymerase using primers to produce a product of 990bp. This fragment was cloned into the pGEM-T vector. Individual clones were selected for DNA sequence analysis using standard dideoxy sequencing techniques.

PCR amplification using sequence-specific primers was used to obtain a 0.58kb DNA fragment coding for 181 amino acids of the NS3 region. This region contains the catalytic residues of the serine protease domain. This was initially cloned into the pETl 1 vector using the BamHl and Ndel restriction sites. This vector was used for further amplification as described below.

In an effort to achieve a more stable expression of the NS3 domain, the regions of the NS3 and NS4A proteins were genetically engineered to produce a recombinant protein having portions of the NS3 and NS4A domains covalently connected into one continuous protein chain. In one embodiment (see Figure 2), five residues from the amino terminus of the NS3 catalytic domain were deleted. The sequence of the original NS3 clone of the IB genotype is shown in Figure 1. The amino acid residues deleted are MAPIT. In addition, the sixth amino acid of the NS3 sequence, proline (P), was changed to a lysine (K). A sequence of the NS4A fragment (amino acid sequence: MSVVIVGRIVLS; SEQ ID NO. 12) was also added.

Example 1- Expression of protein A recombinant NS4A/NS3 construct encoding the amino acid sequence shown in Figure 2 was constructed by PCR amplification from the pETl 1 vector by using a primer designed for the 5' end that included the additional amino acids and restriction sites. The resulting DNA was cloned into the pET28b vector, which also contains the His(6) tag and a thrombin cleavage site at the amino terminal end. This vector was transformed into BL21DE3 cells, and ampicillin-resistant clones were picked and grown in liquid culture. Glycerol was added to 20% and the samples frozen.

For expression, some of the frozen culture was scraped out using a spatula and used to inoculate a culture of M9CA media for overnight growth in a 37 °C shaker. Twenty mis of the overnight culture were used to inoculate a 1L Luria Broth media containing 50μl/ml ampicillin and grown in a 4L flask in a 37°C shaker to an O.D. of 0.5-1.0.

Induction of expression was achieved by adding l .OmM IPTG, and growth was continued for three hours. The cultures were transferred to 250ml polypropylene bottles and spun in a J14 rotor for 10 min at 8000 rpm to pellet the cells. The supernatant was poured off and the pellets were re-suspended in 10 ml of 50mM Tris pH 7.4, 160ml NaCl, ImM MgCl₂ (TN buffer). Cells were spun again and re-suspended in the same buffer, using 4.2 mis per gram of cells. DNase stock (60U/ml) was added to re-suspend cells at a concentration of 80U/ml. Cells were lysed in a French Pressure cell two times, and the lysed material was applied over 10 mis of 27% sucrose in Corex tubes (30ml). The tubes were spun in a JS-13.1 rotor (swinging bucket) for 30 min at 8500 rpm. The pellets were re-suspended in 5 mis of 50mM Tris pH 7.4, 150 mM NaCl, 1 %

Triton X-100 (TN + Triton Buffer) and again applied over a 27% sucrose cushion in two Corex tubes. The tubes were spun in a JS-13.1 rotor for 30 min at 8500 rpm. The supernatant was discarded and the tubes inverted over Kimwipes to drain. The inclusion bodies were stored at -20°.

Example 2 - Refolding of protein

Inclusion bodies (wet weight = lOOmg) were solubilized in 100 ml of fresh 8M urea, 50 mM CAPS buffer pH 10.5, 5 mM EDTA, 100 mM β-mercaptoethanol, and stirred 90 min at room temperature. The samples were centrifuged at 12,000 rpm for 30 min. The resulting supernatant was dialyzed against 4 L 50 mM Tris-HCl pH 10.5 buffer at RT for 2.0 hours, using dialysis membrane with a cut-off of 8-10 kDa. The buffer was changed to a new buffer at pH 9.5, containing 2mM DTT and 5% isopropanol, and dialysis continued at 4° for 5.0 hours. The buffer was changed to one of pH 8.0, containing 2 mM DTT and 5% isopropanol and dialysis continued overnight at 4 ° . Final dialysis was done with 50 mM Tris-HCl pH 8.0, containing 2 mM DTT and 5% isopropanol.

Using these conditions, a clear solution is obtained with little or no precipitation of the protein. Other conditions tried let to significant losses due to precipitation. Samples were analyzed by SDS-PAGE at all stages.

Example 3 - Activity Assay

The material obtained has the ability to cleave substrates of several types, including proteins derived from the hepatitis polyprotein, and peptide substrates either with or without fluorescent groups for protection. The activity obtained is at or above

20,000M 's ¹.

Example 4 - Production of NS3 Protease

Expression of NS3: A cell culture containing vector encoding a recombinant NS4A/NS3 construct of the present invention was grown overnight at 37 °C in LB media containing 25 mg/mL Kanamycin. A 4 L preparation can be prepared from a 150 mL culture (Cell line BL21 DE3, plasmid pET 28b). For each 1 L of LB media (containing kanamycin), 30 mL of an overnight culture (3% inoculation) was added. The cells were grown at 37 °C for about 1.5 hours until the A₆₀₀=0.8-l .0 and then protein expression was induced with 0.2-0.4 mM IPTG (200-400 μL of 1 M). The cells were allowed to grow at 22 °C for 4 hours and then harvested by centrifugation at 8500 rpm for 20 minutes. Once pelleted, cells can be stored at -20 °C, if necessary. The cells were resuspended in Buffer A (0.1 M K. Phosphate, 13% glycerol, 1 M NaCl, 0.05% Triton X-100, 20 mM Imidazole pH 7.5) at 20 mL per 1 L of culture. DNase was added to a final concentration of 80 U/mL. The cells were then French pressed at 1000 psi 3 times. Cell lysate was clarified by centrifugation at 14,000 rpm for 25 minutes using a JA-20 rotor. The cell lysate can be stored at -20 °C until required for Ni-agarose.

Ni Agarose purification: One mL of a Ni-agarose slurry was equilibrated with Buffer A (described above). 20 mL of cell lysate was incubated with the resin for 15-20 minutes with intermittent shaking. The lysate was allowed to elute off, but stored for a consecutive incubation with the resin. The resin was subsequently washed with 20 mL Buffer A at least four times to ensure the removal of contaminating proteins. The protein was initially eluted with 5 mL of Buffer B (0.1 M K. Phosphate, 13% glycerol, 0.2M imidazole), which can be mixed with the resin, and then another 5 mL is used to ensure all protein is eluted. The protein can be stored at -80 °C until required for gel filtration purification.

Gel Filtration: A Sephacryl S-300 column was equilibrated with 25 mM HEPES, 10% glycerol, 5mM DTT, pH 7.0. The column is run at 2 mL/min and fractions are collected for 100 minutes. Activity Assay of NS3 and NS4-NS3 activity: M2235 substrate from Bachem and a Cytoflur fluorescence plate reader is used. For assaying NS3, a total reaction volume of 100 uL was used. A typical assay consisted of 55uL of Assay buffer (0.1 M K. Phosphate, 30% glycerol, 0.5M NaCl, 0.1% Triton X-100, pH 7.5), 5 uL of a 200 uM solution of delta NS4 peptide (single letter amino acid sequence: GSVVIVGRIILS; SEQ ID NO. 7) in the assay buffer, and 20 uL of NS3 enzyme. This mixture is incubated at

37 °C for 3-5 minutes before the reaction is initiated by the addition of 20 uL of M2235 substrate (400 uM) in the assay buffer. For activity with NS4 NS3, the assays were performed the same way except buffer is substituted for the NS4A peptide.

It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. References

U.S. Patent No. 5,371,017 issued December 6, 1994.

U.S. Patent No. 5,712,145 issued January 27, 1998. U.S. Patent No. 5,739,002 issued April 14, 1998.

U.S. Patent No. 5,843,752 issued December 1, 1998.

Bianchi, E., Urbani, A., Biastol, G., Brunetti, M., Pessi, A., DeFrancesco, R., Steinkuhler, C. (1997) Biochemistry. 36: 7890-7897.

Failla, C, Tomei, L., DeFrancesco, R., (1994) J. Virology. 68: 3753-3760.

Kim, J.L., Morgenstem, K.A., Lin, C, Fox, T., Dwyer, M.D., Landro, J.A., Chambers, S.P., Markland, W., Lepre, C.A., O'Malley, E.T., Harbeson, S.L., Rice, CM., Murcko, M.A., Caron, P.R., and Thomson, J.A. (1996) Cell. 87: 343-355.

Kohler, G.C. et al. (1975) Nature 256: 495-497.

Lin, C, Thomson, J.A., Rice, CM. (1995) J. Virology. 69: 4373-4380.

Shimizu, Y., Yamaji, K., Masuho, Y., Yokota, T., Inoue, H., Sude, K., Satoh, S.,

Shimotohno, K., (1996) J. Virology. 70: 127-132.

Wei, C-F. et al. (1993) The Journal of Biological Chemistry. 258(22): 13506-13512.

Claims

1. A polypeptide having protease activity, said polypeptide comprising an NS3 domain, or a fragment or variant thereof, of the hepatitis C virus and an NS4A peptide sequence of said virus, wherein said NS4A peptide sequence is covalently attached to the amino terminus of said NS3 domain.

2. The polypeptide according to claim 1, wherein said NS4A peptide sequence has a peptide sequence selected from the group consisting of SEQ ID NO. 7 and SEQ ID NO. 12.

3. The polypeptide according to claim 1, wherein said NS3 domain comprises an amino acid sequence shown in SEQ ID NO. 1 , SEQ ID NO. 4 or SEQ ID NO. 9, or a fragment or variant thereof.

4. The polypeptide according to claim 1, wherein said polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 6 or SEQ ID NO. 11 , or a fragment or variant thereof.

5. A polynucleotide encoding the polypeptide of claim 1,

6. The polynucleotide according to claim 5, wherein said NS3 domain encoded by said polynucleotide comprises a nucleotide sequence of SEQ ID NO. 3 or SEQ ID NO. 8.

7. The polynucleotide according to claim 5, wherein said polynucleotide encodes an NS4A peptide sequence selected from the group consisting of SEQ ID NO. 7 and SEQ ID NO. 12.

8. The polynucleotide according to claim 5, wherein said polynucleotide comprises a nucleotide sequence shown in SEQ ID NO. 5 or SEQ ID NO. 10.

9. A method for screening for a composition useful for diagnosis or therapy of hepatitis C infection, said method comprising contacting a polypeptide of claim 1 with a selected composition and determining whether said selected composition binds to or inhibits the activity of said polypeptide of claim 1.

10. A composition useful for diagnosis or therapy of hepatitis C infection identified by the method of claim 9.