The method of reproduction in vitro proteolytic activity of ns3 protease of hepatitis c virus (nso)


(57) Abstract:

The invention relates to biotechnology and can be used for the development of anti-NPC therapeutic agents. A reproduction of the proteolytic activity of the NS3 protease of hepatitis C virus (NSO) is carried out by introducing into the reaction mixture together with the NS3 protease amino acid sequences of NS4A in the ratio of 1:1. NS3 and NS4A can be entered as the predecessor of the NS3 - NS4A. The invention allows to increase the activity of the NS3 serine protease in vitro. 3 C.p. f-crystals, 1 Il.

The aim of the present invention is a method of conversion activity of serine protease associated with the NS3 protein NPC that allows you to use the ability of NS4A protein of the NSA, or the contained sequence in the form of the cofactor activity of serine proteases, or, in a broader sense, enzyme activities associated with NS3.

It is well known that hepatitis C virus (NSA) is the main etiological agent of hepatitis nia, NIV (NANB). It is established that the NSA calls, at least 90% of viral hepatitis NANB, after blood transfusion and 50% of sporadic NANB hepatitis. Although the selection of blood donors and immunological analysis of blood used for transfusions, d is carrying a blood transfusion (in the world there are a million or more such infections annually). Approximately 50% of infected NPC patients demonstrate the development of cirrhosis of the liver over a period of time from 5 to 40 years. In addition, recent clinical studies have confirmed the fact that there is a correlation between chronic infection with the NSA and the development of hepatocellular carcinoma.

The NSA is an enveloped virus that contains RNA-positive genome size of approximately 9.4 kb. This virus is a member of the family Flaviridal, other members of which are flavivirus and pestivirus. The RNA genome of the NSA has recently been mapped. Comparison of sequences of the GCS-genomes allocated in different parts of the world have shown that such sequences can be extremely heterogeneous. A large part of the GCS-genome has an open reading frame (ORF), which may contain from 9030 to 9099 nucleotides. This ORF encodes a single viral polyprotein, the length of which can vary from 3010 to 3033 amino acids. During the cycle of viral infection such polyprotein proteoliticeski processed in individual gene products necessary for viral replication. Genes encoding structural proteins of the NPC, located at the 5' end of the ORF, while the section that encodes non-structural proteins, occupies the entire OST is is neglikolizirovanny protein 21 kDa, which probably forms the viral nucleocapsid. Protein E1 provides a glycoprotein of approximately 37 kDa, and it is assumed that it is a structural protein of the outer shell of the virus. E2, another membrane glycoprotein, 61 kDa, apparently, is the second structural protein in the outer membrane of the virus.

Non-structural region begins with NS3 (p24), a hydrophobic protein of 24 kDa, the function of which is unknown. The NS3 protein of 68 kDa, following the NS2 in polyprotein, in accordance with predictions, has two functional domains: the domain of the serine protease in the first 200 amino-terminal amino acids and the domain of the RNA-dependent ATP-ASE at the end. Gene site corresponding to NS4, encodes NS4 (p6) and NS4B (p26), two hydrophobic protein, 6, and 24 kDa, respectively, which to date has not been elucidated. The gene corresponding to the NS5, also encodes two proteins; 5A (p56)NS and NS5B (p65), 56 and 65 kDa, respectively. Amino acid sequence present in all RNA-dependent RNA-polymerase, can be recognized in the field of NS5. This fact confirms that the site NS5 contains part of the apparatus virus replication.

Various molecular biological studies have shown that the signal peptidase, protease, with uctures region, i.e., on the websites of the C/E1, E1/E2 and E2/NS2 serine protease contained in NS3, responsible for the breakdown in the joints between NS3 and NS4A, between NS4A and NS4B, between NS4B and NS5A, and between NS5A and NS5B. It was found that as a result of decomposition under the action of serine proteases cysteine residue or threonine remains from amino end (position P1) and the residue is alanine or serine remains from-end (position P1') split connection. Second proteasa activity of the NSA, apparently, responsible for the cleavage between NS2 and NS3. This proteasa activity is contained in the field, including as part of NS2 and part NS3 containing domain serine proteases, however, are different catalytic mechanisms.

In light of the foregoing, the NS3 protease is considered as a potential target for development of anti-NPC therapeutic agents. However, the search for such agents is complicated by the fact that the activity of serine proteases, showing NS3 in vitro, is too small for the implementation of screening inhibitors.

The authors of the invention it was unexpectedly found that such an important restriction may be removed as a result of application of the method according to the present invention, which also provides dopolnitelnye, the method of reproduction in vitro proteolytic activity of the protease NS3 NPC is characterized by using in the reaction mixture as sequences contained in NS3 and sequences contained in NS4A.

The optimum activity of the serine protease is achieved in the case when NS4A is present in a ratio of 1:1 against NS3.

NS3 and NS4A can also be introduced into the reaction mixture in the form of NS3 - NS4A predecessor, if such predecessor in autoproteolytic conditions forms an equimolar amount of NS3 and NS4A.

There is also the possibility of mutation of the cleavage site between NS3 and NS4A in the precursor so that NS4A remains covalently associated with NS3. Sequence, without affecting the proteolytic activity of NS3, can be further removed from such not capable of proteolysis predecessor.

The present invention also relates to a new composition of matter, characterized in that it includes proteins, the sequence of which is described by the sequence SEQID No. 1 and SEQID No. 2, or contained sequences or their derivatives. It should be borne in mind that such sequences can vary in different isolates of political activity, required for proteolytic maturation of several of the non-structural NPC proteins.

Another objective of the present invention is the use of such compositions for carrying out enzymatic assays capable for therapeutic purposes to identify compounds that inhibit the enzymatic activity associated with NS3, including inhibitors of the interaction between NS3 and NS4A.

Above was the description of the present invention. Now with the help of the following examples will give a detailed description of the specific technical solution of the invention, aimed at a better understanding of its objectives, differences, advantages and method of its implementation.

The figure illustrates the plasmid vectors used in the method of the invention for the activation of GCS NS3 protease in cultured cells and in vitro (example 1 and example 2).

Example 1

The activation method NS3 serine protease NPC in cultured cells.

Designed plasmid vectors for the expression of NS3 4A and other non-structural proteins of the NPC in HEla cells. Constructed plasmids is shown schematically in the drawing. Selected cDNA fragments corresponding to the genome NPC BK (GCS-BK), cloned below promo the entry of ribosomes virus encephalomyocarditis with the to ensure efficient translation of mRNAs, transcarbamylase with T7 promoter even in the absence of a CAP structure.

Various fragments of GCS-VK cDNA cloned in the plasmid pCite-1Rusing methods known in the practice of molecular biology. pCite /NS3/ includes part of the GCS-VK genome contained between nucleotides 3351 and 5175 (amino acids 1007-1615 polyprotein). pCite/NS4B/5A includes part of the HCE-VK genome contained between nucleotides 5652, 7467 (amino acids 1774-2380). pCite/NS3/4A includes part of the GCS-VK genome contained between nucleotides 3711 and 5465 (amino acids 991-1711). pCite/NS4A/ includes part of the GCS-VK genome contained between nucleotides 5281 and 5465 (amino acids 1649-1771). pCite/NS5AB/ includes part of the GCS-VK genome contained between nucleotides 6224 and 9400 (amino acids 1965-3010). The above numbering is consistent with the sequences of the genome and polyprotein provided for GCS-VK article Jakamizava with TCS. "Structure and organization of the genome of hepatitis C virus isolated from human carriers, (1991), J. Virol. 65, 1105-1113.

In order to achieve efficient expression of different parts of the NSA polyprotein Hela cells were infected VTF7-3, recombinant vaccinia virus, which allows to have sinceraly plasmid vectors, selected from among those shown on the drawing. Infected and transfetsirovannyh thus Hela cells then were metabolically labeled (35S) methionine, and recombinant proteins encoded by different plasmids, could have been identified by the method of immunoassay using rabbit polyclonal antibodies that recognize NS3, NS4 or NS5A. The method described in the present example, is designed for the analysis of recombinant NPC proteins, has already been published Z. Jomei, al. in the article "NS3 is a serine protease required for processing of polyprotein of hepatitis C virus", J. Virol (1993) 67,1017-1026 and in the bibliography.

As a result of transfection with the plasmid pCite (NS3) in Hela cells infected yTF7-3, one can observe the fusion protein containing the catalytic domain NPC NS3 protease. pCite (NS4B5A) encodes part of the NSA of polyprotein containing the peptide bond at the junction between NS4B and NS5B, which, as you might expect, is hydrolyzed under the influence of the activity of the serine protease associated with NS3. However, the implementation of cotransfection pCite NS3 from pCite NS4B5A there were no signs of proteolytic cleavage. In contrast, when the expression of NS3 serine proteases domain in combination with NS4A proteolytic cleavage prego domain and 4A can be achieved, for example, by transfection with equimolar amounts of plasmid pCite (NS3) and pCite (NS4A), transfection of a plasmid that encodes a precursor containing both NS3 and NS4A (pCite(NS34A), or by transfection derived the latter plasmid, in which the entire sequence, not related to proteolysis, was subjected to deletions [pCite(NS34A)] , or by transfection of a derivative of the latter plasmid, in which the entire sequence, not related to proteolysis, was subjected to deletions [pCite(int NS3 12371635)]. Thus, NS4A, briefly expressed in Hela cells, can activate the proteolytic activity associated with NS3, which is not observed in other cases,

Example 2.

The activation method NS3 serine protease GCS in vitro translation assay.

The plasmids shown in the drawing, can also be used for in vitro synthesis of mRNA that encodes the appropriate NPC proteins using purified RNA polymerase enzyme phage T7 (Promega).

Typically, a plasmid derived pCite-1R, linearized using suitable restricts and transcribers using instructions of the manufacturer (Promega). Such synthetic mRNA can be alamannic membranes of the pancreas of the dog. Reticulocyte extracts, microsomal membranes dog pancreas, and other required materials were purchased from Promega, and the manufacturer also provides instructions for the above-described method of protein synthesis in vitro.

In the programming in vitro translation mixture mRNA transcribed from pCite (NS3), you can observe the fusion protein with the expected molecular weight (68 kDa), containing the complete NS3 serine proteiny domain. mRNA, transcribed from pCite (NS5AB), regulates the synthesis of the precursor with a molecular weight of 115 kDa, which contains NS5A and NS5B and, thus, serves as a substrate for proteolytic activity associated with NS3.

However, when two such proteins containing NS3 serine proteiny domain and the substrate with the site corresponding to the junction between NS5A and NS5B, are synthesized in the same reaction mixture, a clear indication of the proteolytic activity of NS3 is missing.

On the other hand, mRNA, transcribed from pCite (NS34A), broadcast in the precursor protein weight of about 76 kDa, which semiprocessed proteoliticeski in vitro with the formation of equimolar amounts of the two proteins weight of 70 kDa and 6 kDa, stevanna from pCite (NS5AB), introduced in in vitro translation mixture, in addition to chemoproteomics on the site between NS3 and NS4A, one can observe the formation of two new proteins weight of 55 kDa and 65 kDa, which contain NS5A and NS5B, respectively. Such proteins are the product of the proteolysis of the precursor containing NS5A and NS5B, under the action of NS3. Similarly protein products with a weight of 56 kDa and 65 kDa, proteoliticeski formed from NS5AB predecessor, obtained in the case of mRNA transcribed from pCite (int NS3 1237-1635), kontroliruetsya with mRNA, translated from pCite (NS5AB).

Summarizing this result, we can conclude that in vitro proteiny NS3 domain by itself is not able to show protease activity on the substrate containing the NS5A and NS5B. However, the serine-proteina activity of NS3 is obvious, if another protein sequence containing NS4A, is in addition NS3 proteases domain.

Example 3

The activation method NS3 protease GCS using a synthetic peptide containing the NS4A sequence.

A synthetic peptide containing the sequence SEQ ID No. 3, was synthesized on solid phase. This sequence is derived C-terminal part of the placenta is out there in this area. This peptide carboxyterminal cysteine substituted at the alpha-aminobutyric acid (Abu).

This peptide was added to in vitro translation environment, simultaneously programmable mRNA transcribed from the plasmid pCite (NS3) and pCite (NS5AB).

Thus, had the opportunity to observe proteoliticescuu activity associated with serine proteases the NS3 domain, resulting in proteolytic cleavage of the substrate two product containing proteins NS5A and NS5B. This activity depends on the simultaneous presence of NS3 serine proteases domain and a synthetic peptide with the sequence SEQ ID No. 3.

Example 4

The method of analysis of recombinant NS3 serine protease NPC on the peptide substrate.

Plasmid RT-7 NS3 (1027-1206), shown in the drawing and described in example 4, was designed with the purpose of expression in E. coli protein fragment, located between amino acid 1 and amino acid 180 of the sequence SEQ ID No. 1. This fragment contains a serine proteiny NS3 domain that is established experimentally. The cDNA fragment NPC encoding described NS3 fragment cloned in plasmid RT-7, the expression vector containing the T7 RNA polymerase, promote is to ensure selective expression of cloned genes (1986), J. ol. Biol, 189, pages 113-130). The cDNA fragment encoding the NS3 serine proteiny domain, as described above, cloned after promoter of bacteriophage T7 and in frame with the first ATG-codon of the gene 10 protein T7 using methods known in the molecular-biological practice. Plasmid RT-7 also contains a gene-lactamase enzyme that can be used as a marker for selection of E. coli cells transformed with plasmids derived RT-7.

Then the plasmid RT-7 NS3 (1027-1206) transformed E. coli strain (DE53), which is usually used for high-level expression of genes cloned into expression vectors containing the T7 promoter. In this strain of E. coli polymerase T7 gene is located on the bacteriophage DE53, which is integrated into the chromosome B21. The expression of the studied gene is induced by the addition of isopropylthioxanthone (IPTG) in the plant environment in accordance with the above described method (Studier and Moffat). The use of RNA polymerase of bacteriophage T7 for direction selective high-level expression of cloned genes, (1986), J. Mol. Biol. - 189, pages 113-130).

Fragment of recombinant NS3 containing serine proteiny domain, can be purified from E. coli BL21 (DE53), transformed with plasmid RT-7 NS3 (102706) plasmid, grow at 37oC to an optical density at wavelength 600 nm of approximately 0.8 units of absorption. After that, the medium is cooled to 22oC and the production of the desired protein induce the addition of IPTG to a final concentration of 0.4 mm. After 4-6 hours at 22oC in the presence of IPTG, the cells were collected and subjected to lysis using presealing device (French-pressure cell) in buffer containing 20 mm sodium phosphate, pH 6.5, and 0.5% (wt./volume) (3-/(3-cholamidopropyl)-dimethylammonio/1-propanesulfonate (CHAPS), 50% (vol./about.) glycerol, 10 mm dithiothreitol and 1 mm EDTA (litany buffer). Cellular debris was removed by centrifugation (1 hour at a speed of 120,000 xg) and the resulting precipitate resuspendable in Lisina buffer was digested with Ducati 1, rehomogenization and re-centrifuged as described above. Ion-exchange resin S-Sepharose Fast Flow (Pharmacia), previously equilibrated in Lisina buffer, was added to the merged supernatant (30% vol. /about. ) and the resulting suspension was stirred for 1 hour at 4oC. the Resin was sedimentable and thoroughly washed Lisinym buffer and then loaded into a chromatographic column. NS3 protease was suirable with resin, using O-1M NaCl gradient. The fractions containing the protease, orangestone this stage protein had a purity of 90-95%. Clean to an extent more than 98% was achieved, followed by chromatography on Heparin-Sepharose, equilibrated with 50 mm Tris pH 7.5 10% (vol./about.) glycerol and 0.5% (wt./about.) CHAPS and 2 mm dithiothreitol. Elution NS3 protease with this column was carried out using linear O-1M NaCl gradient.

The concentration of purified protein was determined using the Bio-Rad (Bio-Rad) analysis for protein (Bio-Rad category 500-0006).

Recombinant NS3 serine protease, obtained according to the above method in E. coli, should be subjected to analysis on the activity of cleavage of the substrate, providing a detected amount of fragmentation products. The detection signal pre-exercise methods colorimetry or fluorometry. You can apply techniques such as HPLC, etc.

For example, we used as a substrate of synthetic peptides corresponding to the NS4A/4B connection GCS of polyprotein and containing the amino acid sequence of SEQ ID No. 4 or a part of it.

On the other hand, peptide esters having the General structure shown by the sequence SEQ ID No. 5, may also be used in such operations.

Analysis of the activity was carried out by incubation 5-1000 μm substrate and 0,for 1-3 hours at 22oC. the Reaction was stopped by adding triperoxonane acid to achieve a final concentration of about 0.1% (wt./vol.).

Then the reaction products were separated by method HPLC on C18 back-phase column and have them quantify their absorption in the far UV region.

Proteolytic activity displayed recombinant NS3 serial protease purified from E. coli, was found to be very low in the case when the analysis on the activity carried out by the method described above. However, it was found that increasing amounts of synthetic peptide represented by the sequence SEQ ID No. 3, stimulates the proteolytic activity of recombinant NS3 serine protease, increasing it 20 times. Maximum activity is reached in the case, when recombinant NS3 serine protease and the synthetic peptide are present in equimolar amounts.

The above analysis can be used to search for protease inhibitors. Since the activity of the NS3 protease in this analysis depends on the interaction of NS3 serine proteases domain with amino acid sequences derived NS4A, also with the use described in the shape of proteolytic activity, associated with NS3.

Example 5

Detailed design of plasmids in a single picture

pCite (NS3) contains the part of the NSA-BK genome located between nucleotides 3351 and 5175 (amino acids 1007-1615 polyprotein). The design of this plasmid is described in article I. with Tomei al., "NS3 is a serine protease required for processing of polyprotein of hepatitis C virus", J. Virol (1993) 67, 1017-1026.

pCite (NS4B/5A) was obtained by cloning ScaI-BamHI fragment produced from the plasmid pCite (NS4-5) described in the article with Tomei al., in pCite (NS3), which pre-split MSCl and BamH1. pCite (NS4B/5A) contains the part of the NSA genome located between nucleotides 5652, 7467 (amino acids 1774-2380 polyprotein).

pCite (NS5AB) encodes a protein comprising the sequence from amino acid 1965 to amino acids 3010 of polyprotein GCS-BK. To construct a plasmid plasmid pCite (SX), described in the article with Tomei al., (1993), see above, were first digested Asel and processed fragment maple DNA polymerase. After inactivation of the enzyme maple plasmid was digested XbaI. The resulting cDNA fragment containing the region between nucleotides 6224 and 9400, subjected to cleaning and inserted into the BstXI sites and XbaI vector pCite -1Rafter blunting the end of the generating system shall agment cDNA, corresponding to the region between nucleotides 3711 and 5465 GCS-VK genome, synthesized using the polymerase chain reaction (PCR), using as a nucleating oligonucleotides, specific for the sequence. In antisense oligonucleotide appropriately included UAG stop codon. After PCP amplification of the resulting cDNA was digested at the 5'-end with SAL1 and product, consisting of 750 base pairs, directionally cloned in sites SAL2 and NheI plasmid pCite (SX), after blunting Nhel end with fragment maple DNA polymerase. The resulting plasmid encodes part of the GCS-VK polyprotein located between amino acids 991 and 1711.

To construct pCite (NS4A) the cDNA fragment corresponding to a region between nucleotides 5281 and 5465 GCS-BK genome (amino acids 1649-1711), was obtained by amplification polymerase chain reaction (PCR) in the presence of diluted oligonucleotides, specific for the sequence. The resulting PCR amplification of cDNA then cloned into the BstxI sites and Stul plasmid pCite-1Rafter blunting stxI digested end using DNA polymerase of bacteriophage T4.

pCite (int NS3 1237-1635) is a derivative of pCite (NS3/4A), from which all of th what W pCite (NS3/4A) BsteII and partially Scal. The fragment containing interesting for the deletion, then closed using T4 DNA ligase. This plasmid encodes a protein having the same amino - and carboxy-ends that encoded pCite (NS3/4A), however, as it was found experimentally, all amino acid residues between the amino acid 1237 and amino acid 1635, irrelevant to the serine-by NS3 activity and they were subjected to deletions.

pT7-7 /NS3 (1027-1206)/ contains the NSA sequence from nucleotide 3411 to nucleotide 3951 encoding NPC NS3 fragment between amino acid 1027 and amino acid 1206. To obtain this create plasmid DNA garment amplification of cDNA NPC polymerase chain reaction (PCR) using the oligonucleotides, referred to as the sequence SEQ ID No. 6 and SEQ ID No. 7. The cDNA fragment obtained by PCR was fosforilirovanii, was digested with Ndel and then cloned after promoter of bacteriophage T7, immediately after the first ATG codon of the gene 10 T7 in the vector RT-7, pre-digested Ndel and SmaI (Studier and Moffatt). The use of RNA polymerase of bacteriophage T7 for direction selective high-level expression of cloned genes, (1986), J. Mol. Biol. 189, pages 113-130). It should be noted that the amber codon was inserted immediately polease NS3 of hepatitis C virus (NSO), providing for the presence in the reaction mixture, the amino acid sequence of the NS3 protease, characterized in that the reaction mixture is optionally present amino acid sequence of NS4A.

2. The method according to p. 1, characterized in that the ratio of NS4A and NS3 is 1:1.

3. The method according to p. 1 or 2, characterized in that the NS3 and NS4A is introduced into the reaction mixture in the form of NS3 - NS4A predecessor, forming in conditions autoproteolysis equimolar amount of NS3 and NS4A.

4. The method according to p. 1, wherein the NS3 and NS4A is injected in the form of covalently linked mutant NS3 - NS4A predecessor with a mutation that makes it resistant to proteolytic cleavage between NS3 and NS4A.


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