MXPA97008679A - Dissociable, soluble substrates of the hepatiti virus protease - Google Patents

Abstract

The present invention relates to soluble non-structural substrates of hepatitis C virus of hepatitis C polyprotein.

Description

DISSOCIATED, SOLUBLE SUBSTRATES OF HEPATITIS C VIRUS PROTEASE BACKGROUND OF THE INVENTION Hepatitis C virus (HCV) is considered as the main etialgic agent of non-A hepatitis B NANB), chronic liver disease, and hemo carcinoma. Ocelular (HCC) in the world. Viral infection accounts for more than 90% of hepatitis associated with ransfusions in the United States of America and is the predominant form of hepatitis in 40-year-old adults. Almost all infections result in chronic hepatitis and almost 1 develop liver cyst. The viral part has not been identified due to the lack of a system of rep> i ca > efficient in vitro and the extremely low amount of HCV particles in infected blood or liver tissues. However, molecular cloning of the viral genome has been achieved by isolating the messenger RNA GnAPN) from the serum of infected chimpanzees after carrying out the cloning using the r < _ > ombinante íGrakoui A. et al. J. iro !. 67: i 385- 139? (1993)). It is now known that HCV contains a positive-strand NPC genome which comprehends 9400 nucleotides approximately, whose organization is similar to the organism of flavivirus and pestivirus. The HCV genome, such as the genome of flavi irus and pesti virus, encodes a large protein of approximately 3000 amino acids that is protected! J sis to form mature viral proteins in cells i fec: tadas. Translational studies in viral iprotein cells and expression in cell culture establishes that the polyprotein of HCV is processed by cellular and viral proteases to produce putative structural and non-structural (NS) proteins. . They produce nine mature viral proteins from the polyprotein by means of Psymophilic proteolysis. The order and the nomenclature of the cleavage products are the following: NH2-C-E1-E2-NS2-NS3-NS4A-NB4R-NS A ~ NS5F-C00H. (Figure 1). The three putative amino-terminal structural proteins, C (capsid), El, and E2 (two enveloping coprotein gl * s), are considered dissociated by μeptidases from the host signal of the endo- lysis reticulum (EP). The in? A guest is also responsible for the generation of the end-to-end of S2. The prateolithic processing of non-structural proteins is carried out by means of lav > viral proteases: NS2-3 and NS3, contained within the viral iprotein pol. The NS2-3 protease catalyzes the d isoc ion between NS2 and NS3, it is a metalloprotease and requires either S2 co or the psease domain of NS3, the N3 protease catalyses the rest of the substrates. in the non-structural part of the pol i protein The NS3 protein contains 631 amino acid residues and consists of two enzymatic domains: the protease domain contained within amino acid residues 1-181 and an ATPase helicase domain contained within the rest of the protein It is not known whether the 70 VO NS3 pratein is further dissociated in infected cells to separate the protease domain from the helicase domain, however, no dissociation has been observed in cell culture expression studies. protease is a member of the class of enzymes called serine, contains His, Asp, and Ser as the catalytic triad, Ser is the active site residue, and the mutation of the residue Ser cancels the dissociations in substrates NS3. A, NS4A / 4B, NS4B / 5A, and NS5A / 5B. The dissociation between NS3 and NS4A is intramolecular, whereas dissociations at the NS4A 4B, 4B 5A, 5A 5B sites occur in trans. Experiments employ the transient expression of various forms of HCV polyproteins NS in cells of mammals have established that the NS-3 serine protease is necessary but not sufficient to efficiently process all these dissociations. Like flaviviruses, the NS3 protease of HCV also requires a cofactor to catalyze some of these dissociation reactions. In addition to the serene NS3 protease, the NS4A protein is absolutely required for the dissociation of the substrate at the 4A 5A site and increases the efficiency of the dissociation of the substrate between 5A 5B, and possibly 4A 4B. Because the NS3 protease * »HCV dissociates the non-structural HCV proteins that are necessary for HCV replication, the NS3 protease may be a target for the development of therapeutic agents against the HCV virus. The gene encoding the HCV NS3 protease has been cloned in accordance with that presented in US Pat. No. 5,371,017, however, it has not been expressed in an active, soluble form which is useful for discovering inhibitors of the NS3 protease. The substrates 4A 4B, 4B / 5A and 5A / 5B have themselves been cloned but not expressed in active soluble form useful for discovering inhibitors of the NS3 protease. Because the HCV protease has utility as a target in screening to discover therapeutic agents, either the protease co or the substrates must be in a soluble active form. Accordingly, there is a need for a soluble active form of HCV protease substrates that can be produced in large quantities for use in high performance screening to detect protease inhibitors and for structural studies: . SUMMARY OF THE INVENTION The present invention fulfills this need because it provides soluble di »Hü 'substrates comprising the dissociation sites of non-structural polyprotein of HCV.

The substrate peptides are rendered soluble by fixing a sun motif ub 11 i ion on the peptide. The sequences of the substrates defined by SEQ ID NO: 16, 17, 18, 19, 20 and 21 are particularly claimed. BRIEF DESCRIPTION OF THE Fissures FIG. 1 schematically represents the polyloprotein of HCV. Figure 2 represents the synthesis of the plasmid pB-JlOlS. Figure 3 represents the recom- binant synthesis of plasmid pT56-9. Figure 4 rep resents the rec ommen ting s sten ce of p lsmido p IB1006. Figure 5 represents the recombinant synthesis of the plasmid gone pBJ1022. Figure 6 represents the recombinant synthesis of plasmid pNB (-V) 182A4AHT. Figure 7 represents the recombinant synthesis of plasmid pT5His / HIV / 183. Figure 8 schematically depicts an assay of at or yield to discover inhibitors of the HCV protease by the use of plasmon resonance technology superf l l l. DETAILED DESCRIPTION OF THE INVENTION The teachings of all the aforementioned references are hereby incorporated by reference in their entirety. The present invention is a soluble form of the non-tru cial proteins of HCV which are substrates for the NS3 proteas-i of HCV. The NS3 protease of HCV di or i pratein and separates 4A 4B, 4B / 5A, and 5A 5B regions from Ti to poly HCV iprotein. The undissociated substrates can be used to assay for protease inhibitors. By using the test of • nr, _- t \ í? The scintillation assay or the superficial plasma assay described below, one can determine whether or not the HCV protease has dissociated the substrate used. If the substrate is not dissociated, then the test is an inhibitor of HCV protease. On the other hand, * if the substrate is dissociated, then the test substance is not an inhibitor of HCV protease. On the other hand, if the substrate is dissociated, then the test substance is not a protease inhibitor. The substrates of the present invention are made soluble by the fixation of a solubilisation motif on the substrate, examples of sun motive ubiquating are ionizable amino acids with por1 > . jemp lo a rg i p i na and l i i na. Substrates 5A 5B and 4B 5A can be synthesized by a suitable method such as for example exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. The polipeptide they are preferably prepared by solid phase peptide synthesis according to that described by Memfield, J. Am. Chem. ^ a. 85: 2149 (1963). The synthesis is carried out with protected amino acids at the end of the a-a ino. Non-acidic acids with labile side chains are also protected with suitable groups to avoid undesired chemical reactions during the assembly of the polypeptides. The alpha-ano protection group is selectively removed to allow a subsequent reaction to take place at the animo end.

The conditions for the removal of the alpha-amino protection group do not remove the side chain protection groups. The lfa-amino protection groups are those known to be useful in the technique of the progressive synthesis of the polypeptides. Acyl-type protection groups (for example, formyl, tp f luoroacetyl, acetyl), aplo-type protection groups (for example, biotinyl), aromatic urethane type protection groups (for example, benzylaccarbomla) are included. Ctaz), benz i lox i carbom substituted and 9-f luorenylmethyl-carbon (F? IO)), aliphatic urethane protection groups (for example, t-but i lo icarboni lo (tBoc) , isapropiic, icarboni 1 or, cyclohexylloxibboni) and alkyl type protection groups (for example, benzyl, trifepillieset). The preferred protection groups are tBoc and Fmoc, therefore it is said that the peptides are synthesized by the chemistry of tBoc and Fa.o, respectively. The selected side chain protection groups must remain intact >; During the coupling, they must not be removed during the protection of the end-of-protection protection group or during the coupling operations. The protein chain protecting groups must also be removed at the terminal1 synthesis, employing reaction conditions that do not alter the finished polypeptide. In tBoc chemistry, side chain protection groups for functional amino acids are mainly based on benzyl. Er. The chemistry of Fmoc, are mainly based on tert-butyl or trityl. In tBoc chemistry, the preferred side chain protection groups are tosyl for Arg, cyclohexyl for Asp, 4-met i lben i lo (and acetamidometho lo) for Cys, benzyl for Glu, Ser and Thr, fc > En J lox imet i lo (y di ni trof n i 1 o) for His, 2-Cl-benz lo ica rfooni lo for Lys, formyl for Trp and 2-bramobenci 1 or for Tyr. In the chemistry of Fmoc, the preferred side chain protection groups are 2,2,5,7,8-pentamethyl rom n-6-sulonyl (Pmc) or 2,2,4,6,7- pentameti ldi h iclrot? enzof? r3n-5- ul foni 1 o (Pbf) for Arg, trityl for Asn, Cys, Gln and His, tert-butyl for Asp, Glu, Ser, Thr and Tyr, tBoc p ra l .ys and Trp. For the synthesis of the peptides, either the direct or post-membrane addition of the phosphate group is used. In the strategy of direct incorporation, the phosphate group on Ser, Thr or Tyr must be protected by methyl, benzyl or tert-butyl in the chemistry of Fmoc or by methyl, benzyl or f nil in the chemistry of tBoc. Direct incorporation of phosphosine without phosphate protection can also be used in Fmoc chemistry. In the strategy d * = > After incorporation of the ion, the protected hydroxyl group of Ser, Thr or Tyr was derived in solid phase with di-t rt-bu ti 1 -, dibenzyl- or dimethyl-N, N'-di isopropyl 1 and oxidized by tert-buti 1hidropero id. The solid phase synthesis is usually carried out from the cprb nil end by coupling the amino acid produced by alpha-amino (protected in side chain) on a suitable solid support. An ester bond is formed when the binding is carried out on a retinue of chlorine ethyl, clortptyl or hydroxy, and the resulting polypeptide will have a free carboxyl group at the C-terminus. Finally, when an amide resin is used, for example, a henide and a lamellar resin, or a polybenzene, a lamine (for tBoc chemistry) and a pin amine resin} < or PAL (for the Fmoc chemistry), an amide bond is formed and the resulting polypeptide will have a carboxyl group at the C-terminus. These resins, either based on palstyrene or polya ida or inserted into polyethylene glycol, with or without a linker, with or without the first fixed amino acid, they are usually available, and their prepared ions have been described by Stewart et al. (1984), "Solid Phase Peptide Synthesis" (2nd edition), Pierce Chemical Co., Rocl-ford, IL; and Bayer? / Rap (1986) Chem. Pept. Prot. 3.3; and Atherton, et al. (1989) Sun id Phase Peptide Synthesis: A Practical Approach (Synthesis of solid phase peptide a practical approach), IRl, Press, Oxford. The C-terminal amino acid, protected in the side chain if necessary and in the alpha-a ino group, is fixed on a hydroxymethyl resin using various activation agents including dicy lohex i 1 > . arbocli imi da (DCC), N, N '~ di isoprop i 1 ca rbodi i mida (DIPCDI) and carbom Idi imidazol (CDI). It can be fixed on chloromethylamide or chlorotryril resin directly in its cesarean tetrametammon salt form or either in the presence of tpeti lamina (TEA) or diisopropy let i lamí na (DIEA). The first fixation of amino acids on an amide resin is the same as the formation of amide bond during coupling reactions. After 1 * fi xation on the resin support, the alpha-ami protection group is removed using various reagents according to protection chemistry (for example, tBoc, Fmoc). The magnitude of a. Removal of Fmoc: can be monitored at 300-320 nm, or by a conductivity cell. After removal of the alpha-a mo protection group, the remaining protected amino acids are progressively coupled in the order required to obtain the desired sequence. Various activation agents can be employed for coupling reactions, including DCC, DIPCDI, 2-cl-3, 3-d? Hexafluorophosphate. me 11 i idí or (CIP), benzotr? azole hexafluorophosphate? 1-cj ?? -tps- (di-ethylamine) -phosph-om (BOP) and its pyrrolidine analog (PyBOP), hexaf luorof osf a '<; of bromine-1 ri sp ol ol non-phosphonium (PyBroP), hexaf luorophosphate of 0-ibenzot-azo] -1-yl) -l, 1, 3,3-tet r amet i 1 u ron io (HBTU ) and its tetrafluoroboron analogue (TBTU) or its pyrrolidine analogue (HBPyU), hexaf 1 orof sfa to 0- (7-azabenzot r lazol -li 1) -1, 1, 3,3-tet ra et i luroni or (HATU) and its tetrafluorobor analogue (TATU) or well analogous to pyrrolidine (HAPyll). The most common catalytic additives used in coupling reactions include 4-di met i laminopí ridina (DMAP), 3-h 'droxi -3,4-d? H? Dro-4-oxo-l, 2,3-benzotr i ina (HODhbt ?, N-hydroxybenzot pazol (HOBt) and lh? Drox? -7-azabenzofer la ol (HOAt). Cacia protected amino acid is in excess (more than 2.0 equi alents), and the coupling is carried The actual effect of the coupling reaction can be monitored at each stage, for example, in N-me and Iprol ideal (NMP) or DMF, CH2C12 or mixtures of the same. by the ninhydpna reaction in accordance with that described by Kaiser et al., An., Biochem 34: 595 (1970) In cases where incomplete coupling is encountered, the coupling reaction is extended and repeated and can be added chaotropic salts, L =? s coupling reactions can be carried out automatically with commercially available instruments such as, for example, ABI model 430A, 4 1 and 433A interceptors, after the total assembly of the desired peptide , the peptide-re ina was dissociated with a reagent with its own re ecurors. Fmoc's are dissociated and deprotected habitually by means of TFA with removers (for example, H20, ethanedithiol, phenol and thioanisole). The tBoc peptides are dissociated and deprotected usually by liquid HF for 1-2 hours at a temperature of -5 to O'C, which dissociates the peptide from the resin and removes most of the side chain protection groups. . Peptides such as anisole, dimethyl sulfide and p-thioc resol are commonly used with liquid HF to prevent the cations formed during dissociation from alkylating and acylating the amino acid residues present in the polypeptide. The formyl group of Trp and the di nitrophenol group of His require their removal, respectively, by piperid na and thiophenol fii DMF before dissociation > : nn HF, The acetamidomethyl group of Cys can be removed with mercury acetate (II) and alternatively by iodine, thallium trifluoroacetate (III) or silver tetrafluoroborate with simultaneous oxidation of cysteine with cystine. Other strong acids employed for isocycling and deprotection of the tBoc peptide include trifluoromethanesulfonic acids (TFMSA) and tri-ethyl-1-trifluoroacetate (TMSOTf). A recombinase DNA methodology may also be employed to prepare the polypeptide substrates. The known, suitable and desired genetic code with preferred codons known to a more efficient expression in a given host organism can be used to synthesize oligonucleotides encoding the desired amino acid sequences. The phosphorous solid support method of Atteucci et al., J. Am. Chem. Soc. 103: 3185 (1981) or other known methods can be employed for such syntheses. The resulting oligonucleotides can be inserted into an appropriate vector and expressed in a comparable host organism. The peptides of the present invention should be purified using HPLC, gel fl uid, blunt or ion exchange and ion exchange, countercurrent distribution as well as other well known methods. The production of NS3 p.otease of HCV in soluble form is also presented. the NS3 protease of HCV must e = > In a soluble form it can be used in a sieving to detect compounds that prevent the protease from dissociating its white set. We have found that if a peptide comprising a sunscreen motif on any of the NS3 protease, preferably on the carboxyl terminus, the NS3 protease becomes readily soluble. The amino acid sequence of the catalytic domain of NS3 protease appears in SEQ ID NO: 1. Prior to the present invention, the NS3 protease is not present? in a cell in a soluble form in sufficient amounts for extraction and pu ification. In addition, NS3 soluble HCV protease could not be produced in bacteria-soluble form. This is important because bacterial expression is the preferred method of expressing large amounts of HCV protease. NS3 soluble HCV protease of the present invention can be produced in various ways. A reason for solubilization can be fused on the protein ju results in a soluble protein. One reason for solubilization is any chemical portion bound to the HCV NS3 protease that results in the NS3 protease becoming soluble in a regulated solution. Examples of reasons for solubilization and ionization of this type are chains of amino acids which have polar side chains, preferably positively charged amino acids. The amino acid chains must have a length of approximately 4-30 amino acid residues. The preferred amino acids are argmin and lysine. Another example of motif of sslnb i 11 ac i ón is an amphipathic portion. The solubilization motif can be fused either on the in-end or on the carboxy end of the NS3 protease. A sequence that has been successfully merged soL >The carboxyl end of the NS3 soluble protease is -Arg-Lys-Lys-Lys-Arg-Arg- (SEQ ID NO: 2). This sequence has been fused on the ex emo carboxylic of the NS3 protease p _. r. produce the pal ipé tidos of SEO ID NO; 3, SEQ ID N0: 4, SEQ ID N0: 8, SEO ID N0: 27. Other examples of NS3 soluble HCV protease that have the hydrophilic amino acid residue tail that were made are SEQ ID NO: 9 and SFQ ID NOs 10. It can also produce NS3 soluble HCV protease that does not have a ubiquitous sun motif 1 such as, for example, the proteases illustrated in SEQ ID NO: l and SEQ ID N0: 7. Preferably, the NS3 protease will have a histidine tag fused to its end of the membrane for use in the purification of the protein on a resin coated with nickel (Ni ++). See SEO ID NO: 5. In this modality, the protease is produced in the form of insoluble aggregates or co or inclusion bodies in bacteria- as for example in E. coli. The NS3 roteaí-a of HCV 11.soluble s > * extract first of the bacteria by homogen i z c i n or sound of the bacteria. The aggregates that contain the bacteria are then solubilized in a 5 M solution of guanidine idpoclaride (GuHCl). The NS-3 protease - > purify afterwards from the aggregates dt? high molecular weight by size exclusion chromatography, blunt for example by applying the solution to a gel column by extrusion of size SEPHACPYl S-300. Fractions containing the NS3 protease in 5 M GuCl are combined and diluted to SuHCl of approximately 0.1 M in a redouble regulator comprising dithiofcreHol and lactoid high. The solution di luid-3 is apli > Then add to a reverse phase chromatography column and collect com 1 na i onßb containing the NS3 protease. The pH of the protease fractions is then raised progressively to about 7.4 to produce an active, soluble, suitably replenished NS3 pratease. It has also been discovered that the MSV protease of HCV is much more effective in dissociating the non-structural proteins of HCV if the source of the NS4A protein is present (SEQ ID NO: 6). Next, the present invention also comprises a fusion of the cofactor domain protein of NS4A with the NS3 protease, particularly the fusion of the NS3 protease of the NS4A cofactor where the NS4A is mutated in such a way that the NS3 protease and the Cafac or NS4A do not dissociate by NS3 protease. Examples of the fused NS3 and NS4A constructs appear in SEQ ID NOs: 7, 8, 9, 10 and 27. The DNA encoding the NS3 protease of this invention can be prepared by chemical synthesis employing the known nucleic acid sequence, (Ratner et al. al., N? cleic Acids Res. 13: 5007 (1985)) and standard methods such as Matteuc's fasforacinid solid support method. i et al. (A. Chem. Soc. 103: 3385 (1981)) or the method of Yoo et al. (T. Biol. Chem. 764 17078c (1989)). See also Gl i > _l-, Bernard P, and Pasternak, Molecular Biotechnology (Biotechnol or i Molecular): pages 55-63, (ASM Press, Washington, D.C. 1994). The gene encoding the pretease can also be obtained using the plasmid presented in Grakoui, A., Wychat-ist i, C, Lin, C, Femstone, SM, and Rice, CM, Expression and Identifies ion of Hepatitis C Virus polyprotein Cleavage Products (Expression and Identification of the Products of the Dissociation of Polypeptides of the Hepatitis C Virus), J. Virol 67; 1385-1395 (1993). Likewise, the nucleic acid encoding the HCV protein can be isolated, amplified and cloned from patients infected with the HCV virus). In addition, the HCV genome has been presented in PCT WO 89/04669 and is available from the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rocfcville, MD under the number of access 40394. And, rightly, due to the degeneration of the genetic code, there are many func lically equivalent nucleic acid sequences which can encode the mature human HCV protease in accordance with that defined herein. Such functionally uniform sequences, which can be readily prepared using known methods such as for example chemical synthesis, polymerase chain reaction using modified primers and site-directed mutagenesis, are within the scope of this invention. of expression to express the DNA encoding the NS3 protease of HCV. Conventional vectors for the protection of recombinant proteins in prokaryotic or eukaryotic cells can be used. Preferred sites include the pcD vectors described by O ^ ayamaet al, Mol. Cell. Bio. Vol. 3: 280-289 (1983); and Tal-ebe et al., Mol. Cell. Bial. Vel. 8: 466-472 (1988). Other mammalian expression vectors based on SV40 include the vectors presented in Kaufman et al., Mol. Cell., Biol., Val. 2: 1304-3319 (1982) and in U.S. Pat. No. 4,675,285. These SV40-based screens are especially useful in C0S7 monkey cells (ATCC No. CRL 1651), as well as cells from other mammals such as, for example, mouse l cells and CHO cells. Standard methods of transfection can be employed to produce eukaryotic cell lines that express large amounts of the polypeptide. Eukaryotic cell lines include mammalian, yeast and insect cell lines. Exemplary mammalian cell lines include COS-7 cells, mouse L cells and Chinese Hamster ovary cells (CHO). See Sambrool et al. supra and Ausebel et al., supra. As used herein, the term "transformed bacteria" refers to bacteria that have been genetically engineered to produce a mammalian protein. Such genetic engineering usually includes the introduction of an expression vector into a bacterium. The expression vector can be replicated autonomously and can express protein in relation to genes in the baterian genome. The construction of bacterial expression is well known in the art, provided that the nucleotide sequence encoding a desired protein is known or otherwise available. For example, DeBoer in the North American patent no. 4,553,433 presents promoters for its use in bacterial expression vectors; Goeddel et al. in 1 = ¡. non-American patent no. 4,603,980 and Piggs, in the North American patent no. 4,433,739 present the production of amifeio proteins by E.coli expression systems; and Riggs supra, Ferretti e al. Proc. Nati Acad. Sci.83: 599 (3986), Sproat et al., Nucleic Acid Research 13: 2959 (19B5) and Mullenbach et al., J. Bipl. Chem 263: 719 (3986) show how to construct synthetic genes for their expression in bacteria. Many vectors of bacterial expression are found spontaneously and through the American Type Culture Collective (ATCC), Rockville, Maryland. The insertion of DNA encoding the human HCV protease into a vector is easily achieved when the ends of DNA and the vector comprise the same restriction site. If this is not the case, it may be necessary to modify the ends of the DNA and / or vector by the digestion of the single-stranded DNA produced by restriction endonuclease dissociation to produce blunt ends, or to achieve the same result by filling the ends single strand with an appropriate DNA polymerase. Alternatively, any desired site may be produced by ligating nucleic acid sequences (linkers) on the ends. Such linkers can comprise specific sequences of oligonucleotides that define the desired restriction sites. The dissociated vector and the DNA fragments can also be modified if required by homopolymer addition.

Many vectors of e. Pressure compatible with E.coli can be used to produce soluble NS3 protease of HCV, including, but not limited to, vectors comprising bacterial promoters or bacteriophages such as, for example, Tac, La--, Trp, Lac VS, 1 promoters. Pr 1 PL. Preferably, a vector is selected that has expression control sequences that allow the regulation of the rate of expression of the 3a HCV protease. Then, the production of HCV protease can be regulated to avoid the overproduction that could cause the host cells. A vector comprising, from 5 * to 3 * (upstream to downstream), a Tac promoter, a lac Iq repressor gene and DNA encoding mature human HCV protease is preferred. Vectors selected for their use in this invention may also encode secretory leaders such as for example ompA or proin A, insofar as such leaders are dissociated during post-translational processing to produce rote of mature HCV, or if the leaders are not dissociated, the leaders do not interfere with the enzymatic activity of the pr-oteasa. Typically, the fusion peptide will be prepared either by recombinant nucleic acid methods or by intimate polypeptide methods. Techniques for the manipulation and expression of nucleic acids are generally described, for example, in Sambroo, et al. (3989) Molecular Cloning; A Laboratory Manual (Molecular Cloning: A Laboratory Manual) (2nd edition), volumes 1-3, Cold Spring Harbor Labaratory; and Ausubel, et al. (editions) (1993) Current Protocola m Molecular Biology (Current Protocols in Molecular Biology), re n and Wiley, NY. Techniques for the synthesis of polypeptides are described, for example in Merpfield (3963) J. Amer. Che. Soc. 85: 2149-2156; Merrifield (1986) Science 232: 343-347; and Stewart et al (3984)., "Sun id Phase Peplide Synthesis" (2nd Synthesis), Pierce Chemical Co., Borkfard, IL.; and Atherton, et al. (1989) Exit Phase Peptide Synthesiss A Practice! Approach (Solid Phase Peptide Synthesis: A Practical Approach), IPL Press, Oxford; and Gt ant (1992) Synthetic Paptides A User's Guide (Synthetic Peptides: A User's Guide), W.H. Freeman, NY. The NS3 protease, the cofactor of NS4 and the peptide substrates, either 4B / 5A or 5A / 5B, can be used to develop high throughput assays. These can be used to screen compounds that inhibit the prateolytic activity of the protease. This is achieved through the development of techniques to determine whether or not a compound will inhibit dissociation by NS3 from the viral substrates. Examples of such synthetic substrates are SEO ID NOs 16, 17, 38, 19, 2 and 23. If the substrates are not dissociated, the virus does not replicate. An example of a high performance test of this type is the scintillation proximity assay (SPA). SPA technology includes the use of scintillation coated beads. On the beads, accepting molecules are fixed, for example, antibodies, receptors or substrates of in ima that interact with lysates or enzymes in reversible ways. For a protease assay, the substrate peptide is biotinylated at one end and the other end is irradiated with low energy emitters, for example 1251 or 3H. The labeled substrate is then incubated with the enzyme. SPA gloves coated with avidin are then added, which are bonded onto the biotin. When the peptide ele substrate is dissociated by the protease, the radioactive emitter is no longer in the vicinity of the scintillation count and no light emission is observed. Protease inhibitors will leave the substrate intact and may be identified by the resulting light emission that takes place in its presence. Another type of protease assay employs the phenomenon of surface plasflion resonance (SPR). A novel high-throughput epzimatieo assay employing surface plason resonance technology has been developed successfully. Using this trial, and a special instrument? JlAcore (R), can be sieved at least 3000 samples per week either to determine its enzymatic activity or its effects * - »inhibi o ios r» relationship with the activity in: imática, in a plate format of 96 μo: o =. This methodology is easily adaptable to any enzyme-substrate reaction. The sale of this assay so comparison with the SPA assay is that it does not require a peptide substrate. The i utes and employ ... _, influences in order to illustrate the present invention but not and limit it. EXAMPLE 1 PRODUCTION OF HCV NS3 PPOTEASE A. PI.ASMID CONSTRUCTIONS Several plasmids were designed and constructed using standard DNA techniques (Sambok, Fritsch? - Maniatis) to express the HCV protease in E coli (figures 2-7), All the sequences specific for HCV originated from the plasm gone párente! pBRTM / HCV 3-3011 (Gra-oui the al.3993). To express the N-terminal 383 amino acid versions of the protease, a stop codon was inserted into the HCV using no synthetic Igonuclides (Figure 3). The plasmics designed to express them 246 residues from the year of; The N-strains were bound in the natural restriction site N-C at the C-terminus. i) CONSTRUCTION DF PL SMID0 pBJ3035 (Figure 2) The plasmid gone pBRTM / HCV 1-3 (U containing the entire HCV genome < Gral < ou? A., et al., T. Vi., 67: 3385-3 95) was digested with the restriction enzymes S a I and Hpa I and the DNA fragment of 7338 bp (par. ses) was isolated and cloned on the S to I site of pSF72 (Promega ^ to produce the plasmid pRT201.) Plasmid pRJ 201 was digested with Msc I and s isolated and cloned the 10 bp Msc I fragment at the Sms I site of the pl ^ smido pBD7 The plasmid resulted.te pMBM48 was digested with ace I and NCO 1, and the DNA fragment of 734 bp after formation of blunt end with pal im rasa of Kenom was isolated and cloned into the expression plasmid of pTrc HIS B seq treated with p > __) klenatu limerase, digested with or Ii Invi trogep). The ligation regenerated a Neo T site at the 5 'end and a si 3 Nsi I at the 3' end of the HCV sequence. The pTHB HCV NS3 plasmid can be digested with Neo I and Nsc I, and treated with klenoi polymerase. and T4 DNA palmitase to produce a 738 bp blunt-ended DNA fragment that was isolated and cloned into a Henoiii polyerase-treated expression plasmid, cut with Asp T, p0E3 < "> (HIV) The resulting plasmid pBJ K? m to the NS3 protease of HVC (246 amino acids) iii) CONSTRUCTION OF PLASMIDE pTS 56 ~ 9 WITH A RETENTION CODON AFTER THE AMINO ACID 183 Fig. 3 a) Plasmid pTHB HCV NS3 was digested with N or I, treated with full-length polymerase ", then digested with Bst YT, and the ADM fragment containing the HCV sequence was isolated and cloned into pSF72 digested with Sitia I and B l II The resulting plasmid? SP72 was then digested with Bgl II and Hpa I and ligated with double-stranded oligonucleotide to: AG TCA CCG GTC TAG ATCT T GGC CAG ATC TAGA (SEO ID NO 11) to produce pTS 56-9. A retention codon was placed directly at the end of the DNA encoding the protease catalytic domain of the NS3 protein. This allowed the HCV protease to be expressed and depend on the helicase domain of the S3 protein. (lil) CONSTRUCTION OF PLASMIDE pJB 1006 FUSED WITH A PEPTIDE OF AMINO CIDOS POSITIVELY LOADED IN THE EXTREME CARBOXI OF NS3 383 (figure 4). Plasmid pTS 56-9 was digested with Sph I and Bgl II and the DNA fragment containing the HCV sequence was isolated and cloned into a pSF72 cut cor. Sph I, Bgl II. The resulting plasmid J 1002 digested with Age I and Hpa I and ligated on an oligo gonuc leot. double-stranded, CCG GTC CGG AAG AAA AAG AGA CGC TAG C AG GCC TTC TTT TTC TCT GCG ATC G (SEQ ID NO 32), to build pJB 30 6. This fu - »oned the solubili ain motif hydrophilic on NS3 dv) CONSTRUCTION OF Fl ASMTD0 pBJ t? 2 EXPRESSING His-NS3 (183) -HT IN E. coli (figure 5) Plasmid pJB 3006 was digested with NgoM i and Nhe I and the DNA fragment of 216 bp was isolated and cloned into pBT 1035 cut with Ngo MT, Nhe I to construct the plasmid pBJ 3019. The plasmid pBJ 3039 was digested with t-ia v I and Pvu II, and treated with nsnom polymerase to fill the 5 'ends of the Nar I. fragments. The expression plasmid pQE33 (Invitrogen) was digested with Ba HI, and then tenderly turned blunt with the palmi merasa. The DNA fragment of 717 bp Nar I-Pvu II was isolated and ligated onto the 2787 bp fragment of BamH I -Msc I (B-tl I) subjected to Klenow of the pOE33 expression plasmid (T nv i t royen). The recombinant plafsmt, pRT 1022, obtained after transformation in E. coli expresses His NS (2-183) -HT which does not contain any HIV protease dissociation site sequence. The plasmid also contains a large removal in the CAT gene (Cl oranfem col Acet i 1 transierase). (v) PLASMID CONSTRUCTION p B1-) 182- ¿4A HT (Figure 6) Plasmid pMBM 48 was ingested with Fag I and Xho I, treated with Klenow polyeraserase and the 320 bp DNA fragment was isolated and cloned in blunt-ended pSP 72, cut with BamH I to construct the plasmid or pJB1004. The 320 bp fragment encodes 7 amino acids from the former carboxy moiety of NS3C631), tocios and NS4A, and the 46 amino acids of the former amino oar of WS4B. The recombinant plasmid pJBl? 04 was digested with Eag I and Cel 2, an e was formed; tremo romo with polymer * by Klenow. The 22 bp DNA fragment was isolated and cloned into the plasmid pG) E30 which was digested with Ba H I and s > - formed a former roman eino with polymerase from Klenow before the league- ion. The resulting plasmid pJB 1011 was digested > to NgoM I and Hing III and linked on the igonuc e t i do of heb? a dob 1 e, CCG GCA ATT ATA CCT GAC AGG GA ': GTT CTC TAC CAG GAA TTC GT TAA TAT GGA CTG TCC C < CAÁ GAG ATG GTC CTT AAG CAT «C ATG CAA GAG TGC CGG AAG AAA AAG AGA CGC A CIA CIC TAC CTT CTC ACG QCC TTC TTT TTC TCT GCG TTC GA (SEQIDN013) To construct the plasmid pNB 4A HT. The plasmid gone pNB 4AHT was digested with Msl I and Xba I. The 3238 bp DNA fragment was isolated and cloned into vector pBJ3039 DNA cut with Xba I, treated with polymer -. * Of ll now, cut with Age I The ligation results in a substitution of amino acid residue 183, valine, for a glycine residue in NS3, and a removal of three amino acid residues from the NS4A at the junction. The recombinant plasmid P 38? HT q? E comprises NS3 (382aa-G-NS4 A (4-54 amino acids) does not contain the NS3 NS4A cleavage site sequence in the junction and is not dissociated by 1. acti idad a? Toque a 1 í ii Finally, the plasmid pNB182 &4A HT (SEO TD NO 8) was> digested with Stu I and Nhe I, the DNA fragment> 803 bp was isolated and cloned into the plasmid pBJ 1022 cut with Stu I and Nhe I. The resulting plasmid pN (-V) 182- ^ 4A HT contains an amino acid and HIV removal of the amino terminus of the frequency of NS3 and the e CAT (SEQ ID NO 27). (Vi) Construction of plasmid pT5 His HIV-NS3 (figur 7) Plasmid pTS56-9 was digested with Byl II, and treated with .1 enoi polymerase. i to fill the ends 5 '. The plasmid was then digested with Ny > DM I and the Bgl II NgoMI fragment of the ex-blunt oar containing the sequence NS3 was isolated and ligated onto the NgmMI cut-off subjected to polymerase ci Klenow, SglTI, and pBT 1 1 subjected to Klenow polymerase cut with Sal I. The resulting plasmid is named pT-5Hi% HIV 183. E TEMPLE 7 Purification of HCV NS3 protease having a sun motif 11 zac ion Purification of Hisl82HT (SEQ ID NO 4) and His (-V) 382 4AHT (SEQ TD NO 8) Recombinant plasmids p > E: T1022 and pNB (-V) 182 ^ 4A were used to transform separate cultures of strain M15 (pREP4) of E. coli (Qiayen), qu > They express the r ^ pi lor lac according to methods recommended by the manufacturer. The M35 bacteria (pREP4) that have the plasmids reeombina t.es were grown overnight in broth containing 20 g / l bac to ri pton, lOg L bacto-yeast extract, 5g / L NaCl (broth 20 -10-5) and supplemented with lOOμg / ml ampicillin and 25μg / ml kanamycin. The cultures were diluted to 0.D.60 of 0.3, then cultivated at a temperature of 30 ° C until O.D. 00 from 0.6 to 0.8, and from which IPT8 was added until a final concm of ImM. 2 to 3 hours after induction, the cells were harvested by pelletizing, and the cell pellets were treated with 300 mM Tris, pH 7.5. The cells were repaired in the following ways, each nil equivalent of fermentation broth in which was added 50μl of sonification buffer of 50 M sodium phosphate, pH 7.8, 0.3 M NaCl, with 1 mg. / ml of lisa-ri a; the cell suspension was placed in ice > _3 for 30 minutes. The suspension was then brought to a final concentration of 0.2 * 4 Tween-20, 10mM dithiothreitol (DTT) and was sonicated until the total cell rupture *. The insoluble material was formed into pellets at 12.0 0 - • g in n my crocentri fugarant for 35 minutes, the soluble portion was moved to a separate tube and the soluble lysate was brought to a concentration final of 10 * 4 of gl icol. The soluble lixides from the cells expressing the plasmids produce strongly i nmu and iv bands of the predicted molecular weight. soluble lysates prepared for purification on a Ni ++ column were pyrepals with lOmM of beta-71. mercaptoethanol (BMF) in v z of DTT. the lysates were stored at a temperature of -80 ° C. Purification using Nitrogen + Nitrate + Nitric Acid (NTA) agaro ^ a) Proteins were purely > : those by placing the root l a s on a NTA agarose column. It was chromed or affixed on the NTA agarose column because the marker and histi that it was fused. about the] trema N d »r > The membrane is easily bonded on the nickel column. This produces a potent affinity cratatogenic technique for rapid purification of the soluble protease. Column chromatography was carried out in a 3 > : tte. The Ni ++ NTA resin (-3 ml) was washed twice with 50 ml of regulator A (50 mM phosphatide: sodium j, pH 7.8, containing 3% glycerol, 0.2% Tween-20, lOmM of BME). Number 3 ? d > j obtained from a fermentation of 250 ml (32.5 ml) was incubated with the resin for 1 hour at a temperature of 4 ° C. -It was collected by cen ion rifle. The fcte resin packed in a 1.0 x 4 cm column and washed with A Insta regulator reaches the baseline. The bound protein was then eluted with a gradient of 20 ml of? M? Da? Ol (0-0.5 mM) in regulator A. Eluid fractions were evaluated by SDS-PAGE and by wester t blot analysis using a rabbit polyclonal antibody piara His-HIV 183.

Purification using column affinity meta 1 -q e] a t > : t PORES In an alternative method to purify the 5 proteins, the lysate containing the prot * »na *, was added to an affinity column of m * such -qu oate POROS. It's 33 > - .. < or a perfusion chromatography in a meta-1-quel column.

POPOS MC (4.6 x 0? I ??. ?, 1.7 ml) μr r. gada with N ++. The sample was applied. 30 l go »and the column was washed with regulator A. l column was of pué-ñ luida in steps with die; volumes of c to a > Regulator A containing 25 mM of imidazole. The column was further eluted with a gradient of v »: > 3ume .. column of 25-250 M > : ie imidol the regulator A. All the f eldings are strong! evaluated by SDS-PA8E and by analisys weslern blot using poly antibody the »m l of» one jo. EXAMPLE 3 Synthesis cieμμlido n I »..}. -? substrates 5A / 5B 4B / 5A Peptides from substrates 5A / 5B and 4B / 5A (SEO ID NOs 36, 18, 1, ^ 3 and 21) were synthesized using the Fmoc chemistry in a peptide detector AE; T model 433 A. The strategy FastMoc activation (r) (HßTl / HOB) recommended by the manufacturer for the ini of activator peptide 4A was used. lin ac ador m --_ potent-1, HATU with or without HOAt added to the couple to assemble substrate fibers 5A / 5B n a Wang μ re ar a a resin. The peptides were dissociated from the resin and protected by means of a standard TFA dissociation protocol. The peptides were purified in reverse phase HPLC and co-signature by means of mass spectrometric analyzes. EXAMPLE 4 HPLC Assay Using 5T / 5B Synthetic Peptide Substrate uT To test the protololytic activity of NS3 protease HCV, the DTEDVVCC SMSYTWTG < SEQ ID NO 36) and soluble HCV NS3 ^ »(SEO ID NO 27) together in a test regulator. The regulator of the assay was 50mM phosphate. sodium, pH 7.8, containing 15% glycol, 10mM DTT, 0.2% Tuteen 20 and 200 mM NaCl). The act,? Vi »_Jad protease > SEQ ID NO 27 dissociated the substrate in d > ^ Peptides of your product, say 5A and 5E- :. The substrate and the by-product peptides were separated on an HPLC column from phase i ^ ev & a. (Dynamax, 4.6 t <250 mm) with a pore size of 300 Angstrams and a particle size of 5 μm. The column was equilibrated with 0.1% TFA (Solvent A) at a flow rate of 1 roll per minute. The peptide standards of product and substrate were applied to the equilibrated column n A. An elution was carried out with a gradient of a > - etum tr i lo (Solvent B = 100% ucetorutrilo in A). Two gradients were used for elution (5% to 70% B in 50 minutes followed by 70% to 10% B in 10 minutes). In another experiment, SEO ID NO. 27 easily purified or rec control was incubated with 1 μM substrate for 3.7 and 24 hours at 30 ° C. The mixture of 1 = t reaction was turned off by a > TFA at 0. 3% and applied to the reverse phase HPLC column, ls racci ne- * provenienter »> Each xp i in or were evaluated »by mass spectrometry and sequence > _ lamiente). EXAMPLE 5 Analysis of the activity of NS3 protease by assay from 1 to s 1 -i c 1 on i rt v 1 'ro To detect l a. As an activity of NS3 protease of HCV in trans, we expressed a pratef na of 40 1-D containing the cleavage site NS5A / 5B in a cell-free translation system and used it as a substrate for the enzyme. The substrate protein produces two protein products of apparent molecular weight of 12.5 tD (NS 5A ') and 27 1 < DINS5B ') upon dissociation by means of the NS3 protease of HCV. Plasmid pTSl 2 qα encoding substrate 5A / 5B was linearized by digestion with EcoP I and was transcribed using T7 RNA poly erasa ín vi tro. The RNA was transfected in the presence of 35S in 1 isolates of rabbit retinitis at confi dence with the manufacturer's protocol (Promega) to produce a specific protein for HCV. In a mixture of total action > d 2 μl containing lOmM Tris, pH 7.5, I M DTT, 0.5mM EDTA, and 30% glycerol were placed from? to 8 μl of transferred 5A / 5B substrate labeled with 35S ionin. The reaction was started with the addition of 10 μl of MS3 protease of HCV n solubilization regulator (50mM Na phosphate, pH 7.8, NaCl 0.3M, T et 20 0.2%, 10mM DTT or BME, 10% glycerol ), and incubated at a temperature of 3"C d >; i n nio the specified type. The reactions were stopped by the addition of an equal volume of 2X Laemmli sample buffer (Enprotei 1> Inc.) and slow down at 100 ° C for 3 minutes, the products *. The reaction was separated by the ect rófore i SDS FAGE, the gels were fixed, dried and subjected to aut ography. The translated substrate in vitro was used to test the proton-jd NS3 of HCV expressed by E. coli containing plasmids pB71022 and pNB í-V) 382¿4A (SEO ID NOs 4 and 27). Er, a two-hour assay incubated at 30 ° C, the soluble 1 iao cuda of pBJ1022 ert 3, 6 and 3 Oμl was able to dissociate the 5 5B substrate in a manner corresponding to the dose, producing the expected dissociation products: 5A ( 12.5 kD) and 5B (27 VO) as shown by SDS PAGE analysis. The control lysate of the corresponding vector did not show any dissociation activity - > > in the background. The crude soluble lysate derived from-pNB182 ^ 4 was much active in this assay. After only 3 minutes of incubation, the dissociation products of 5A and 5B were detected using 0.125μl of 1 cellulose, and increasing amounts of 11 sado showed an increasing dissociation, which at > -an_ró urt máxim a lμl. We performed a temporal course study of the NS3 protease activity of pNB182 ^ 4A in an in vitro annealing assay for a further characterization of the activity, at a temperature of 30 ° C, in a reaction containing the substrate 5A / 5B tr When soluble in B182 ^ 4A in lμl per 2 μJ of the reaction volume, dissociation products 5 and 5B will appear at 1 minute and increase with the passage of time to 2.5, 5? It and minutes. Since we had demonstrated the activity of NS3 protease and HCV using the crude cell phones of pBJ3022 and pNB182A4A, we wanted to purify at least partially expressed proteins in an effort to remove the bacterial proteases from these preparations. For this purpose, it was found that the chromography was similar in column using ligands linked to N? ++ effective t, they bind the marker of h? S > In the former we will rely on the expressed protein, and subsequently releasing the linked proteins by elution. The fractions eluted with imidazsl resulted from the purification of pNB382 4A on a Ni-NTA column were tested for their activity in the ip vi tro translation assay. The resulting fractions were able to dissociate the 5A / 5F substrate; moved, producing l i. expected products 5A and 5B. No bacterial protrusion background activity was detected in these fractions the uide. As described above, pBJ3 [Delta] 22 was purified by means of another Ni-++ chelate chromatography method, using a POROS Ni ++ chelate resin and perfusion chromatography. The fractions eluted with a clear role that were positive for the immu oreac 11 vity • ">" > u antibody 3 NS3 383 were tested for their HCV protease activity, by in vi tro translation assay. In order to optimize the detection of activity in this assay for HCV protease, the reactions were complemented with truncated peptide urt derived from the cofactor NS4A which was shown to increase dissociation at the 5A 5B site by NS3 protease. The coffer was supplied in the form of a synthetic peptide containing ami ods 22 to 54 of NS4A (strain HCV-B) in a final concentration of IμM. All the fractions tested were active in this translation test. EXAMPLE 6 POP PEPTIDE INCREMENT 4A NS4A can increase the activity of NS3 serine proLea at the NS5A / 5B site in mammalian cells that express transiently NS, NS4, and the various sites of current dissociation. ab or containing the non-structural polyprotein of HCV. We have studied this incrementing activity in a well-defined cell-free biochemical assay, using pBJ3022 in purified E. coli as a source of NS3 protease, and synthetic peptides containing several truncated NS4A ions. In our first experiment, we used a crude cell lysate of pBJ1022 or the truncated NS4A peptide and the amino acid 22 to the amino acid 54, the carboxy terminus in the transient dissociation reaction. io i rt itro. The peptide of 33 C-terminal amino acids S4A was able to increase the activity of the catalytic domain of NS3 in a deperted manner at IB doses of 0.03 μM to 1.0 μM of peptide, yielding the expected products of 5A (12.5 1 < D), and 5B (27kD) from the transferred substrate 5A / 5B of 40kD. Without the 4A peptide, a dissociation activity was observed relative to the protease srtlar in the short perloid. of incubation for 30 minutes. Peptide 4A itself or with the combination of crude lysate produced from cells having the vector plasmid did not dissociate the substrate. To characterize additional Try the a > "t i vi"; l d of increase of NS4A was reo.1 i. ' < • &rd runcan, i-j additional to the sequence of NS4A. * - ". peptides were evaluated to determine their ai id in the transla tion test in vi tro empleartd > _? pB! 1022 purified on N? + - »- chelate column (catalytic domain of NS3). We observed that in addition to the peptide 33 amino acids of trem C, a peptide of 18 amino acids containing the sequence of S4A from amino acid 19 to 36 was able to increase the dissociation activity mediated by NS3. Other peptides, including the 21 amino acid peptide. Extremely long. »C and two shorter trun acons at the end of the former paddle box, iM 22 > tter and a 15mer, they did not have an efe? likewise e found that a heterologous peptide d? 38 amino acids did not have any incidental activity either. Given the experiments described in this report, it is clear that the HCV piotease expresses bacteri anamertt catalyses the dissociation of i) pul i proteí rías > .ie HCV and ii) synthetic peplid substrates in the trache biochemical assay, the catalytic domain processing activity of NS3 et »increased by NS4A and its derivatives. The activity of the fusion protein containing the mini catalytic NS3 and NS4A is very its activity in the catalytic domain activity of NS3 alone. The analysis of the hydrophobicity of the catalytic domain of NS3 protease reveals that the protein is very hydrophobic and also contains seven cysteine residues. To neutralize the fob idity and therefore to improve the solubility we have added six amino acid residues f argado-a pos i 11 varnente co í.) Mo iu de solubi 11 zac i n. The addition of a solubility motive seems to improve 1. - »olub i 1 i -u] if it affects the activity in? i át ic3. We have also shown that the NS4A HCV from the Japanese strain BK--reverses the dissociation measured by NS3 of HCV-H in the 5A / 5B site. This suggests that essential elements must be preserved between several strains of HCV. It is clear from the previous experimental findings that the fixation of a hi-fi tail (ici of water-dissolving structures) at the end of the carboxy-based oar. of the catalytic domain of NS3 fused rust tustidine improved the expression of soluble protein in F. cal i. In these experiments they were set, amino acid residues »loaded posi ively at the end of the cai oxy end of the protein. It should be noted that other fusions containing no Sii st idi na, GST (Blutathione S transferase), MBP (maltose binding protein), thior doxin alone did not show an increased salinity of -NS3. Other examples of salubilization motif are a helical amphipathic tail (peptides that have residues): charged amino acids and carbohydrate, to form r and to the f or b or c a). l ation »: an amphipathic helix at the carboxy end of such fusion proteins will be an alternative to achieve solubility mea- sure without delaying enzyme activity. The hydrophobic tail is the same as this one, with six innocents. The length and length d >;? the amino acids hi drof i 1 i. They can vary to achieve optimal expression of soluble origin. Accordingly, the size of the motif and solubility and the nature of the charged residues can achieve the expression of NS3 soluble in E. coli. The position of this structure that attracts the water / motif n both enter ", in xro (end to ino or extreme carboxy) or insertion into the catalytic domain of NS3 and fusion protein (domain ca tal ít? Co) -4A of NS3 can improve the solubility of the pro sein to affect l ac ti ./idad. Based on homology of sequestrations with the members of the superfamily of trypsins and the protease of other members of the flav irus, amino acid 381 of the extreme end of NS3 is predicted to be the catalytic domain of NS3 protease. of HCV. Recently, it has also been shown that a protein of 189 amino acids that contains a removal of the amino acids from the amino-2-amylazole at the end of the domain was so intact that it retains all its e ertivity. i a t ica. The model that we have developed predicts that YOU? A 150-amino acid protein that retains a 26-amino acid removal from the ex-animo moiety and a 2-amino acid emotion from the caibooxyl oar retains its even enrichment activity. the 5A / 5B user. Analysis of the amino acid semen from the catalytic domain of NS3 protease reveals that the domain contains the residues of is read. , an odd number that can cause aggregation. the mutation of a cysteine residue (located in the supe fi les of the molecule and not involved in the activity) can improve the solubility of the protein without affecting the protease activity. By using the cell-free biochemical assay, we have shown that the synthetic peptide containing 18 amino acids of HCV ions and NS4A is sufficient to increase the isocyon in the NS5A / 5B mediated by the catalytic domain of NS3. EXAMPLE 7 Enhancement of NS3 Insoluble HCV Protease The present example is a novel process for the redoubling of an NS3 protease of HCV that does not have a solubility motif from a pellet in the body of inclusion of E. coli. This procedure can be used to generate purified purification for activity tests and studies is ruc tur 1 e- ». Extra-tion and fiupfication of His-HTV 183 3 from body pellet > and inclusion of E. coli and E. cali cells that contained the plAsmiclo for H? sHIV383 were used to transform a culture from the M15 strain of E. coli (pREP44) (Oiagen), which overlays the er "lac, in accordance with recnene methods re1ected by the commercial source, the bacteria (" 115 (? PEP4) u> ont the plasmids rec obiu rtti-s were ul ti vadi dui before n iclie in a broth "" - 10-5 complete with lOOucf'ml of ampie i lina and 25μg / l canamic ina d. The cultures were diluted until OD600 of Ol, then cultured at 37 'C h ss 0.D.6 0 from 0.6 to 0.8, and from which -> TPTG up to a final concentration of 3 M. From 2 to 3 hours after the induction, the cells were harvested by pelletizing, and the cell pellets were washed with the Tris OmM, pH 7.5, and they formed pellets by centrifugation. 10 ml of 0.1 M Tris-HCl, 5 mM EDTA, pH 8.0 (regulator A) was added to each gm of wet weight, and the pellet was homogenized and resuspended using a huiriogeiiei: Dounce, The suspension was enlighten broken by cent i leak i n to 20, 0 \ > j for 30 minutes at a temper ur of 4 ° C. The pellet was subsequently washed with the following five regulators: 1. regulator A 2. sodium chloride 3.0f1 (N-iCl) in regulator A 3. 1.0 * 4 triton X-100 in regulator A 4. regulator A 5. guan i na HCl (GuHCl) 3. M n regulator A. The washed pellet was solubilized by 5 M r OuHCl, beta mercaptoethanol at VA in gul3fl »-)? A (3 ml per gm wet weight of pellet) em cleaves a hom gene i _. Dounce and centrifuged to 10, OO > "Jura rite 30 minutes at a temperature of 4 ° C. The purification of denatured H? S HlV183 from high molecular weight agathodes was achieved by size exclusion on a SEPHACRYI gel filtration column. S-300. Particularly, an 8 ml uer tra of an extract of E. t ol f GuHCl, 0M was applied to a column of 360 ml of Pharmacia S-3 0 í 3.? 3 cm) at a flow rate of 1.0 l / in. The regulator e lumna statia conformed by GuHCl 5.0M, Tria-HC 0.311, pH 8. O, and 5.0 M EDTA. The size of the fractions * was 5.0 ml, appropriate fractions were combined ", in b-as the results of SDS- ASE, thus blunt by ^ n l i = - »!. = • > The N-terminal sequence of the protein transferred to a Protil or > »Redouble assisted? HCV Protease Protein The protein was concentrated by means of a pneumatic reaction. an Autii ort YM30 membrane of 43 mm at 1.0 ttvj by > ? tl in GuHCl 5, 0.3 M Tris-HCl, pH 8.0 1.0 mM EDTA, 1.0 * 4 beta-ercaptoetanal. It was then diluted 5 times with GuHCl O.tM in regrind regulator (300 M phosphate of s > -> or pH 8.0, lOmM of DTT, 0.1 * 4 of poor cough gone laur il), and 1. "M arla was incubated ert ice dur-.nl at least one hour. A 25 ml mu sto containing 50 μg of the redissolved regulator protein was applied on a ProPPC HP 3/5 reverse phase chromatography column. The applied sample contained 0 μg of protein and 25 ml of redouble regulator. A B >solution was then applied to the column. What consisted of 99. 14 of H20 + 0.1 * 4 of trifluoroacetic trifluoric acid (TFA), A volume of 3 ml of solution C (30 * 4 of H20, ^ 0 * 4 of acetonitrile (AcN) + .3 * 4 e TFA) was applied. to the column, in a gradient of 0-60 * 4 n a solution B at a flow rate of 0.5 ml / min and a fraction size of 0.5 ml. The fractions were monitored at A234f 2. Total scale absorbance units (AUUFF,). Fractions that contained the protein (corresponding to type 1> were timed for frog 1 by progressive dialysis) The fi ftings were 1 J "first in 0.1 * 4 TFA, in li epoi at 25 * 4 during the non-temperature 4 ° C, then were dialysed at 0.01 * 4 TFA in glyceral at 25 * 4 for 3.0 hour 5> J were then digested for 3 h.jr at a temperature of 4 ° C. in 50 M aP04, pH 6.0, 10 M dithiothreitol (DTT) in glycerol at 25 * 4.

The protein was then dial- ized for 3.0 hours at a temperature of 4'C in 5? M NaP04, pH 7.0, 0.15 M NaCl, 10 M DTT in glycerol at 25 * 4;; and finally dialed in 50 M NaP04, pH 7.8, 0.3 M NaCl, 10 mM DTT, 0. * 4 Tween 2 in glycerol at 25 * 4. This resulted in an active, soluble, redoubled, purified NS3 protease. An analysis of dirroi was used. or circular (CD) of distant UV to monitor - redoubling from a denatured state μ > > r A i > D to a doubled state at a neutral pH. The recovery of the protein was monitored by an explorer of UV and a 3i? He? is SDS-PAGE. Resulted: Redoubled detergent-assisted His-HIV183 H? SHIV383 was extracted from an inclusion body pellet of E. culi. The SDS-PAGE analysis in the various stages of extraction shows that sequential sequencing is essential to remove significant quantities of proletarian data. It ex tr * .un-. H? SHíV183 from the washed inclusion u ^ rp pellet in the presence of 5M OuHCl. The 5M GuHCl extract was applied to a SEPHACPY1 column. S-300 and the appropriate fractions were -combinated based on the SDS-PAGE analysis. The ami noér sequence of the first 30 res and duos will be seen. The bend was carried out at low proton concentrations, in the presence of DTT, poor coughing of lauryl, and glycerol at 4 * C. The diluted protein was incubated in a Pro-RPC reverse phase column. Two types were obtained based on the protein- * and UV profile. Only type 1 provided a soluble protein after > dialysis p. aggressive The spectral analysis of distant UV CDs was used to monitor the redoubling from a denatured state at an acid pH until LUÍ is a or b at a neutral pH. At a pH of 7.4, the protein was found to have secondary enzymes, which is consistent with that of the beta-sheet protein. At a pH of aunt jo, the CD spectrum showed that e, > a totally random spiral which has a minimum molar epilepticity at 2 0m.t. Do you provide this minimum at 200f,? with the shoulder at 220 rtm is about 4: 1. This proportion decreased when the formation of the secondary structure occurred in a nentium. An exploration > ie UV n »; Dialysis step »showed that the recovery of the protein was supet mr si 9« 4 to a pH of 7.4, and that there was no effect of light scattering due to protein aggregates. . Fl náli is SDS - FAGE also indicated that there was no river »? protein up to a pH of 7.0 ciur nor the doubled r. The last 3-protein ion occurred in the last case of dialysis, and the soluble protein was cleared by centrifugation ion. The total recovery of olein was approximately 0.30 * 4. It was found that the active re ¬ dblazer in a trial of ¯ i ¯. =; oc? ac i n trans erttp! A substrate 5A-'5F: transladado vi tro pts »l of péμt? d. 4A in accordance with the writing in the following e.ie plu, F TEMPLE 8 Analysis of ac ti i ad of NS3 ote a redoubled by ns and n of t. ns 1 c i o u i u v 1 1 o To detect the activity of NS3 protein »HC HCV in trans, we have expressed a protein cie 4 | < D containing the cleavage ion NS5A / 5B in a cell-free translational system, and used as the substrate for the enzyme. The protein of substrate produces protein products of apparent molecular weight. Je 12.5 \, D UJS 5A ') and 27 1. D < NS5B ') when dissociated by NS3 ptotease of HCV, the plasmid pTS3 2 that binds the substrate 5A / 5B was 1 inedized by digestion with E oP T and was transcribed using T7 APN polymerase-a in vitro. The RNA was transferred in the presence of 35S met ion in rabbit isolates from the rabbit in accordance with the manufacturer's protocol.

(Fromega) to produce a specific protein of HCV. In a mixture of the 20 μ c / e etal reaction it contained 3 Tris, pH 7.5, lmM of DTT; O.SmM of EDTA, and glycerol at 10 * /, were placed from 2 to 8 μl > Je substrate 5A / 5F: transferred marked with 35S me ion ina. The reaction was initiated with the addition of 10 μl of NS3 protease of HCV SEQ TD NO: 5) with an amount approximately tJq? one l t < 2 uh) of the end-carbohydrate NS4A 33-mer cofactor (SEO TD N0: 29) in solubilization solution (5 mM Na phosphate, pH 7.8, 0.3 M NaCl, 0, * 4 Tween 20, 10 mM DTT or RME, at 30 * 4), go up to a temperature of 30ßC dprantr? * «Next to 3 hours. The reactions were suspended by the addition of an equal volume of 2X teguldor of sample to emitted li (Enprotech Tin.) And I warmed up to 100 ° C for 3 minutes. The reaction products were separated by electrophoresis SDS-PAGE; The gels were fixed, dried and subjected to autoradiography. The assay was able to dissociate the 5A / 5B substrate in a co-responsive manner to the dose, producing the pipeline. is »3C expected: (12.5 ID) and 5B (27kD romo is shown by SDS-PAGE analysis) The production of polypeptides 5A and 5B cli soc. It proves that the active, soluble, redoubled HCV supply was produced by the method of Example 7. EXAMPLE 9 Surface plasmon test: This example illustrates a method to determine if a compound can be used in the field. It is useful as an HCV protease inhibitor using the super plasmon or plasmid resonance assay. FIGS. 8A and 8B illustrate the Leem -t.

BIAcore (r) is a processing unit for Biospec ific Interaction Ana - = »is (Analysis and Interaction Bi oespec 1 f i o) _ l a unida »! »The process integrates a system of» lei, e > Optimal with a self-propeller and an idle system. BTAf ov ímr) uses optical phenomena, surface plas ion resonance for iitonitoivai 'the interaction n r'e b inmol "; ul as. SPR is a phenomenon of resonance between incoming photons and the signal on the surface of the film, the> -jag> 1a of mel .. Observed resonance at a precisely defined angle of lur. At an angle, called the resonance angle, the energy is transferred to the electrons in the metallic film, read in a diminished intensity of the reflected light.The SPP response depends on a change in the refractive index. in close proximity to the sensor surface, and proportional to the mass of the analyte attached to the surface, BIAcore continuously follows the resonance angle by means of a relative scale of resonance units (Rt!) and! : > presents as a SPR signal in urt sensorgram, where l = > units of resonance are plotted as a function of time.In addition, BIAcore (r) reads a technology of continuous flow. irreversibly on clef flake enso, which comprises a non-crosslinked dextran carbide which provides a hydrolic environment for the b i omoleeul i interaction. A solution containing the operating medium flows through the surface of the sensor flake evenly. As the molecules of the solution bind on the ligand, the resonance angle changes resulting in one registered by the instrument. In this methodology, the enzymatic reactions were carried out outside the BT ore, that is to say, in reaction tubes of 96-well culture and tissue dishes, but it is carried out co-operatively. any of the trials »: 3e high performance currently available. The SPR is used only as a means of detection to determine the amount of intact substrate that remains in a range with and without the: 1 i a »Je = > The reaction is off. With the object of the amount of substrate inta.es ard of the addition of enci, a device had to be established to cater the substrate in the sensor flake. In addition, to fulfill the requirements for a high-performance test, it is the BTAcore, the _ »nst? Att? it had to be removed from the surface subsequently at the end of the analysis. This is required post >; u the same surface e? will be used for subsequent reactions. To achieve these two requirements, an internal phosphot is fixed intimately on one end of the substrate. The pho- pherosine was chosen because of commercial availability. "N Antiphosphotyrosine monoclonal antibody A-P-MAb. The antibody is covalently fixed on the sensor flake (L-S) by means of a standard amine coupling chemistry. The antiphosphotyrosine antibody, permanently bound on the flake, is used to capture the substrate containing phosphotyrosine (P) reversibly. The antibody-phosphotyrosine interaction is ultimately used to capture and release the peptide substrate when desired by regenerating the surface with various reagents, for example 2 M MgCl2. The introduction of the intact peptide on the antibody surface results in a larger mass that is detected by the instrument. To follow the magnitude of the peptide dissociation, a mixture of peptide and enzyme substrate is incubated for the desired time and then quenched. The introduction of this mixture containing the dissociated peptide and the intact peptide to a regenerated antibody surface results in a lower mass value than that detected for a sample containing only intact peptide. The difference between the two values is then used to calculate the exact amount of intact peptide remaining after dissociation by enzyme. Even when the mass reduction can be followed directly with many large substrates, due to the small mass of a typical peptide substrate (10-20 amino acids, 1-3 daltons), the difference in mass, and consequently the signal difference between the intact peptide and the dissociated peptide is very small within the signal to noise ratio of the instrument. To avoid this low sensitivity, we fix a biotin (B) at the N-terminus of the peptide. By adding and consequently marking peptide with streptavidin (E) before injection of the labeled peptide on the antibody surface of the flake, the signal caused by the presence of streptavidin will be higher. Using this approach, a dissociated peptide that does not have half the N-terminus, labeled with streptavidin will result in a much smaller signal. The peptide substrate 5A-5B (S-5A / 5B) of HCV protease (HCV-P), DTEDVVACSMSYT TGK (SEQ ID NO 18) was synthesized with an additional phosphotyrosine at the C-terminus and biotin at the N-terminus. Biotin (B) was then labeled with streptavidin (E). An anti-phosphotyrosine monoclonal antibody, A-P-MAb, 4G10 (Upstate Biotechnology Inc., Lake Placid, New ) was coupled with the sensor flake (L-S). In the absence of HCV protease, intact streptavidin-labeled biotinylated phosphotyrosine peptide results in a large signal (large unit mass (GMU) / large signal (SG) through its interaction with the monoclonal antibody antiphosphotyrosine (Mab). The protease-catalyzed hydrolysis of the biotinylated peptide by phosphotyrosine was performed in a 96-well dish.The reaction was then suspended with an equal volume of mercury benzoate.The dissociated peptide that does not have the labeled streptavidin (smaller mass) results in the loss of response units (minor signal) Reduced mass unit (RMU) with a reduced signal (SR) Using this method, numerous compounds can be tested for their inhibitory activity since the antibody surface can be regenerated repeatedly with MgC12 2 M. Procedure for coupling Mab of anti-f osf otirosin on the sensor flake The Mab of anti-f osf otirosin is coupled to the surface of a carboxymethylated dextran matrix (MDC) of a sensor chip (L-S) in the following manner. The flow rate used in the coupling procedure is 5 μl / min. The surface is first activated with an injection of 35 μl of NHS / EDC (N-hydroxysuccinimide / N-dimethylaminopropyl-N '-ethylcarbodiimide-HCL). This is followed by a 40 ml injection of Mab 4G10 at 50 μl / ml in 10 mM sodium acetate buffer, pH = 4.0. The remaining activated esters are then blocked by injection of 35 μl of 1 M ethanolamine. These conditions result in the immobilization of approximately 7,500 response units (420 μM) of antibody.

Peptide bond and surface regeneration of Mab 4G10 The flow rate used in the BIAcore analysis is 5μl / min. An injection of 4 μl containing peptide matted with the latter is performed in 2μM, can > : on the basis of if i. stitching link v i ina in *? μM). The amount of peptide marketed with est ept id linked to the surface of antibody in response units) e fli i de seyundc > = »After finishing l a. injection n. SENSOR IASCA SURFACE REGENERATION The regeneration of the surface of f 4G10 is achieved by using an impulse of 4 μl of f1gCl2 M2 after the injection of the pepidoid. Regenerated surfaces tiasta 5 times are still missing, one notch 1 0Ü of the measured residue. DETERMINATION OF THE OPTIMUM CONCENTRATION OF PEPTIDE AND ESTREPTAVIDI A To determine the optimal peptide concentration, a standard curve was generated using several amounts of peptide (0-10 μM) in the presence of the strept and id in e; ceso. 9e chose a value in the 1 i nea 1, 2 μM range, for standard assay conditions > _ > . The amount of this protein was completely determined by the use of a peptide ion concentration of 2.5 μM and titrating the amount of ept idine. (μJI link site). S showed that o or the peptides were totally different when concentrations were used and were higher than Z μM 5óv íapro: - imadamente ecjui olar to the concentration of peptide). A concentration of streμtav id ina of 9 μM (a 4.5-fold excess) was chosen at standard assay conditions. APPLICATION OF METHODOLOGY DESCRIBED TO HCV PROTEASE The peptide time 5A / 5B is synthesized from the HCV protease DTEDVVACSMSYT4TG (SEQ ID NO. 18), with photophosphate in the former pad C and biotyrta in the N-terminus. It coupled a monoclonal anti-phosphthati rosi u, 4G10 antibody on the bursor flake. In the absence of HCV protease, the peptide of biotinylated peptide labeled with int stptavidin resulted in a large signal (large mass unit / uni ad of large response) through its interaction with the antibody onoc 1 onal ant i -fosfot i ros i ría. The protease-catalyzed hydrolysis of the biotinylated peptide with phosphot i rosin was carried out in a 96-well plate. The reaction was suspended with an equal volume of quench buffer containing mercury benzoate. Streptavi was added to mark the peptide that binds on the biotin. The dissociated peptide that does not have the marked streptavidin (minor mass) results in the loss of response units. Using this assay, numerous compounds can be tested for their inhibitory activity since 1A antibody surface can be regenerated repeatedly with 2M MgC12. The averaging of peptide dissociation by HCV protease can be monitored dependently Rail time using a m everything! ogia based on BIAeote. Ufan or the concentrated biotin and the cold substrate, ioli na ~ DTEDVVAC SMSYTWTG - Y (SEQ ID NO 17), a 50% dissolution of the dent 1 substrate or one hour is achieved using the HCV test. based on BIAcore. Based on the amount of enzymes, His-NS3Í183) ^ 4AHT required the para to an, a dissociation of 50 *? Within two hours, a desired time scale for the development of a high-throughput test, we estimate that a liter of processing will be used. 1 or Hi -NSNS3 i 183) A 4AHT results in a sufficient protease to perform at least 100 reactions in BIAcare. STANDARD OPERATING PROCEDURE FOR HCV TEST BASED ON BIArore Reactions were prepared on a tissue culture plot: > of 96 wells using the reaction regulator (50 M HEPES, pH 7.4, glycerol in 20V, 150 mM NaCl, 1 M EDTT, 1 VA Tween 20, 1 M DTT) or diluent. The ol fi of the reaction is 1 OO μl. A sample is prepared with the peptide alone (bio 1 na-DTEDVVACRMSYTWTGkpY) by the addition of 10 μl of the mother peptide? to 10O μl (prepared in the reaction regulator) to 90 μl reaction buffer, so that the final concentration of pe is 10 μn. Samples consisting of peptide and enzyme are prepared by the addition of 10 μl of mother peptide at 100 μM and 10 μl. His ~ NS3 (183) - ^ 4A-HT mother partially purified at 1.7 mg / ml (both prepared in the regulator: the reaction) to 80 μl »: j regulator of the reaction, such that 3a final concentration of After the enzyme is 10 and 0.1 j-tM, respectively, the reaction is maintained at a temperature of 30 ° C for the specified 1 st and then turned off. The reaction can be quenched by transferring a 20 μl aliquoule from the area mixture.A new tissue culture dish containing an equal volume of PMB buffer (50 mM HEPES, pH 7.8, 150 mM NaCl, 5 mM Pyridium hydroxymethyl benzyl acid, and 13 M of EDTA). To prepare the reaction mixture for injection on the sensor surface, s a »grega 3? μl of regulator PMB BIAcore (50 mM of HEPES, pH 7.4, 1 M NaCl) and 30 μl of is i, rep i, v id ina to 0. mg / ml in water at 40 μl of the reaction mixture switched off * until reaching a final volume of 100 μl. In this step, all the peptides are labeled with streptav before the sample injection. Finally, 4 μl of the sample is injected onto the surface of the photoresistors. ± determine the intact peptide versus the peptide, the final concentration of the peptide and the reptile in the BIAcore sample is 2 and 9 JJM, respectively. CONDITIONS OF EXPERIMENT! SUBSTRATE: Biotin-DTEDVVAC SMSYTWTG-gX (SE ID NO. 19) in reaction regulator without DTT CONCENTRATION 170 μM (crude peptide on a weight basis) ENZYMALIUM of His-NS3 (183) - ^ 4A-HT concentrated at 1.7 mg / ml REACTION VOLUME: lOOμL REACTION REGULATOR: 50 M HEPES, pH 7.8 l iceron l 20 * / »1 0 M of 1 M NaCl of 1 M EDTA of DDT 0. VA of TEMPERATURE interface: 30 ° C OFF CONI p-hydroKimercup benzoa to LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: Scher ing Corporation (ii) TITLE OF THE INVENTION: DI-SOCIABLES, SOLUBLE SUBSTRATES OF THE PROTEASE OF THE HEPATITIS C VIRUS (iii) .) SEQUENCE NUMBER: 31 (iv) ADDRESS FOR CORRESPONDENCE: (A) RECIPIENT: Scher ing Corp. < B) STREET: 2000 Ga 11op i ng Hil Ro d (C) CITY: and il? Orth (D) STATE: NJ < E > COUNTRY: USA (F) POSTAL CODE: 07033-0530 (v) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIA: Blast disk (B) COMPUTER: Apple Macintosh (C) SYSTEM OPERATION: Macintosh 7.1 (D> PROGRAM: Microsoft Word 5.1.a (vi) DATA OF THE CURRENT APPLICATION: (A) APPLICATION NUMBER: - (B> DATE OF SUBMISSION: - (C) CLASSIFICATION: - (vii) DATA FROM THE PREVIOUS APPLICATION: (A) NUMBER OF THE APPLICATION: 08 / 439,747 ÍB) DATE OF SUBMISSION: May 12, 1995 íviii) INFORMATION SOBPE LAWYER / AGENT: (A) NAME: Lunn, Paul G. < B) REGISTRATION NUMBER: 32,743 5 íC) REFERENCE NUMBER / CÉDU A JB05O9PCT d; < TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 908-298-5061 (B) TELEFAX: 908-298-5388 (2) PAPA INFORMATION SEQ ID N0: 1: 10 (i) SEQUENCE CHARACTERISTICS; (A) LENGTH: 549? base i (B) TYPE: acid nu > -!and? < -o (OR FABRIC CONFORMATION: simple (D) TOPOLOGY: linear 5 (i.) TTPO DF MOLECULE: cDNA (m) CHARACTERISTICS: (A) NAME / KEY: NS3 P rote a de HCV GCG CCC ATC ACG GCG TAC GCC CAG CAG ACG AGA GGC CTC CTA GGG «5 Ala Pro llß Thr Ala Tyr Ala Gln Gln Thr Arg Gly Leu Leu Gly 1 5 10 15 TGT ATA ATC ACC AGC CTG ACT GGC CGG GAC AAA AAC CAG GTG GAG 90 Cys Xle He Thr Ser Leu Thr Gly Arg Asp Lys Asn Glp Val Glu 20 25 30 GGT CAG CTC CAG ATC GTG TCA ACT GCT ACC ACC TTC CTC GCA 135 Gly Glu Val Cln He Val Ser Thr Wing Thr Gln Thr Phe Leu Wing "- '35 40 45 ACG TGC ATC AAT CGG CTA TGC TGG ACT CTC TAC CAC GGG GCC CGA 180 Thr Cyß He Asn Cly Val Cyß Trp Thr Val Tyr His Cly Ala Cly 50 55 60 ACC AGG ACC ATC CCA TCA CCC ?? G CGT CCT CTC? TC C? G? TG T? T 225 Thr? Rg Thr llm? The Sar Pro Ly? Cly Pro Val Ha Gln Mat Tyr 65 70 75 ? CC ?? T CTG G? C C ?? C? C CTT GTG GGC TGG CCC GCT CCT C ?? CCT 270 Thr? Sn Val? Sp Gln? ßp Leu Val Gly Trp Pro? Pro Gln Gly 1 80 85 90 TCC CCC TC? TTG? C? CCC TGC? CC TCC GGC TCC TCG G? C CTT T? C 315 Ser? Rg Sar Lau Thr Pro Cys Thr Cys Gly Ser Ser? Sp Lau Tyr 95 100 105 CTG GTT? CG? GG C? C GCC G? C GTC? TT CCC GTG CGC CGG CGA GGT 360 Lau Val Thr Arg Hi? Ala? Sp Val He Pro Val Arg Arg Arg Gly 110 115 120 GAT AGC AGG GGT? GC CTG CTT TCG CCC CGG CCC? TT TCC T? C CTA 405? Sp Ser? Rg Gly Ser Leu Leu Ser Pro Arg Pro He Ser Tyr Leu 125 130 135 AAA GGC TCC TCG GGG GGT CCG CTG TTG TGC CCC GCG GGA CAC GCC 450 Lyß Gly S9T Ser Gly Gly Pro Leu Leu Cys Pro Gly His Wing 140 145 150 CTG GGC CT? TTC? GG GCC GCG GTG TGC? CC CGT GGA GTG ACC? AG 495 Val Gly Leu Phe? Rg Wing Wing Val Cye Thr Arg Gly Val Thr Lye 155 160 165 ¿-j CCG CTG CAC TTT ATC CCT GTG G? G AAC CTA GAG AC? ? CC? TG? GA 540? The Val? Sp Phe He Pro Val Glu? Sn Leu Glu Thr Thr Met? Rg 170 175 180 TCC CCG CTG Ser Pro Val (2) INFORMATION PAPA SEO ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 ami noá idus (B) TYPE: amino acid (OR CONFORMATION OF HEBRA: sim le < D > TOPOLOGY: linear íii) TYPE OF MOLECULE: p? gone Arg Lys Lys I. s Aro Arg (2) INFORMATION FOR SEO ID N0: 3: (i) CHARACTERISTICS DF SEQUENCE: (A) LENGTH; 567 base pairs ÍB) TYPE: a.-io nucleic (OR SHAPE CONFORMATION: simpl (D) TOPOLOGY: linear (i: <) MOLECULE TIFF: cIDN i i: <) CHARACTER I ST IS A: ÍA) NAME / KEY: CCC CCC? TC? CG CCG TAC GCC CAG CAG ACG AGA GGC CTC CTA GGG 45 Wing Pro He Thr Wing Tyr Wing Gln Gln Thr Arg Gly Leu Leu Gly 1 5 10 15 TGT AT? CT? CC? GC CTG ACT GGC CGG GAC AAA AAC CAA GTG GAG 9C Cys He He Thr Ser Leu Thr Gly? Rg? Sp Lys Asn Gln Val Glu 20 25 30 CGT CAG CTC CAO ATC CTG TCA? CT GCT? CC CAA ACC TTC CTG GCA 135 Cly Glu Val Gln Ha Val Ser Thr? Thr Cln Thr Phe Leu? La 35 40 45 ? TG TGC? TC ?? T OGG CT? TGC TGG? CT CTC T? C C? C GGG GCC GG? 180 Thr Cyß Zla? Sn Gly Val Cys Trp Thr Val Tyr His Gly? La Cly 50 55 60 ? CG? GG? CC? TC GC? TC? CCC ?? G GGT CCT CTC? TC C? G? TG T? T 225 Thr? Rg Thr He? The Sar Pro Lys Gly Pro Val He Gln Met Tyr 65 70 75 ? CC ?? T CTG C? C C ?? G? C CTT GTG GGC TGG CCC GCT CCT C? A GGT 270 Thr? Sn Val? Sp Gln? Sp Lau Val Gly Trp Pro? The Pro G n Gly 80 85 90 TCC CGC TCA TTC AC? CCC TGC? CC TGC GGC TCC TCG G? C CTT TAC 315 Sar? Rg Sar Leu Thr Pro Cys Thr Cys Gly Ser Ser? Sp Lau T r 95 100 105 CTG GTT? CG? GG C? C GCC G? C GTC? TT CCC CTG CGC CGG CGA GGT 360 Leu Val Thr? Rg His? La? Sp Val He Pro Val? Rg? Rg? Rg Gly 110 115 120 G? T? GC? GG GGT? GC CTG CTT TCG CCC CGG CCC ATT TCC TAC CTA 405? Sp Ser? Rg Gly Ser Leu Leu Ser Pro Arg Pro He Ser Tyr Leu 125 130 135 AAA CGC TCC TCG CGG GGT CCG CTG TTG TGC CCC GCG GGA CAC GCC 450 Lys Cly Ser Ser Cly Gly Pro Leu Leu Cys Pro? Gly His Wing 140 145 150 CTG GCC CT? TTC? GC CCC GCG GTG TGC? CC CGT GGA GTG ACC A? G 495 Val Gly Leu Phe? Rg? The? Val Cys Thr? Rg Gly Val Thr Lys 155 160 165 GCG CTG G? C TTT? TC CCT GTG GAG AAC CTA GAG ACA ACC ATG AGA 540? The Val? Sp Pha He Pro Val Glu? Sn Leu Glu Thr Thr Met? Rg 170 175 180 TCC CCG CTG? G? ?? G ?? G ?? GA? GA AGA Ser Pro Val Arg Lys Lyß Lys? Rg? Rg Í2) INFORMATION FOR SEO TD NO: 4: (i) SEQUENCE CHARACTERISTICS: ÍA) LENGTH: 07. pairs > i ~ Í '> _ &&- ÍR) TYPE: acid r »u» lico ÍC) FABRIC CONFORMATION: im le ÍD) TOPOLOGY: linear < n; TYPE OF MOLECULE; DN iix) CHARACTERISTICS: ÍA) NAME / KEY: pBJ 1022 (Hi s / US3 (182) -'W. T.? TG? G? GC? TCG C? T C? C CAT CAC CAT CAC ACG GAT CCG CCC ATC 45 Met? Rg Gly Ser His His His His His His His Thr Asp Pro Pro He 1 5 10 15 ACG GCG TAC CCC C? GC? G? CG? GA GGC CTC CTA GGG TGT ATA ATC 90 Thr Ala Tyr Ala Gln Gln Thr Arg Gly Leu Leu Gly Cys He He 20 25 30? CC? GC CTC? CT GGC CGG GAC AAA AAC CAG GTG GAG GGT GTC GTC 13E Thr Ser Leu Thr Gly Arg Asp Lys Aen Gln Val Glu Gly Glu Val 35 40 45 CAG ATC GTC TCA ACT GCT ACC ACC TTC CTC GCA ACG TGC ATC 180 Gln He Val Ser Thr? Thr Gln Thr Phe Leu Wing Thr Cye He A A T CCG GTA TGC ACT GTC TAC CAC GGG GCC GGA ACG? GG? CC 225? Sn Cly Val Cyß Trp Thr Val Tyr His Gly Wing Gly Thr Arg Thr 65 70 75 ATC ßC? TC? CCC ?? G CGT CCT CTC? TC C? G? TC T? T? CC ?? T CTC 270 Zla? La Sar Pro Lys Cly Pro Val Zla Gln Met Tyr Thr? Sn Val 80 85 90 G? C C ?? C? C CTT CTG GGC TGG CCC GCT CCT C? A GGT TCC CGC TCA 315? Sp ßln? Sp Lau Val Gly Trp Pro? Pro Gln Gly Ser? Rg Ser 95 100 105 TTG AC? CCC TGC? CC TGC GGC TCC TCG G? C CTT TAC CTG GTT ACG 360 eu thr Pro Cyß Thr Cys Gly Ser Ser? Sp Leu Tyr Leu Val Thr 110 115 120 ? GG C? C GCC G? C GTC? TT CCC GTG CGC CGG CGA GGT GAT? GC? GG 405? Rg Bis? La? Sp Val Ha Pro Val? Rg? Rg? Rg Gly? Sp Ser? Rg 125 130 135 GGT 1GC CTG CTT TCG CCC CGG CCC? TT TCC TAC CTA AAA GGC TCC 450 Gly Ser Leu Leu Ser Pro? Rg Pro He Ser Tyr Leu Lys Gly Ser 140 145 150 TCC CGG CGT CCG CTG TTG TGC CCC GCG GGA CAC GCC GTG GGC CTA 495 Ser Cly Cly Pro Leu Leu Cye Pro? Gly His? Val Gly Leu 155 160 165 TTC AGG GCC GCG GTG TCC? CC CGT GGA GTG ACC? AG GCG GTC GAC 5 0 Phe Arg? La? The Val Cye Thr? Rg Gly Val Thr Lye Wing Val Aep 170 175 180 TTT ATC CCT GTC GAG AAC CTA GAG ACA ACC ATC AGA TCC CCG GTC 585 Phe He Pro Val Glu Asn Leu Glu Thr Thr Met Arg Ser Pro Val 185 190 195 ? G? ?? G ?? G ?? G? G? ? GA (2) INFORMATION FOR SEO ID NO:: ii) CHARACTERISTICS DF SECUFNCI: / A) LENGTH: (R) TIFO: ci o ni je le ico (OR CONFORMATION OF HEP: RA: _ i pl ÍD) TOPOLOGY: linear di TYPE OF MOLECULE: cDNA (i; - * CHARACTERISTICS: (A) NAME / KEY: pT '? Hi s HI 183 without mofc or? TC? GA GGA TCG CAT CAC CAT CAC CAT CAC GGA TCC CAT AAG GCA 5 Met Arg Gly Ser His His Hie His His His Gly Ser His Lys Ala 1 5 10 15 ? C? CTT TTC GCT G? A GCA ATC? GC CAT GGT ACC? TG GCG CCC? TC 90? Rg Val Leu? Glu? The Met Ser Hie Gly Thr Met Ala Pro He 20 25 30 ACG CCG TAC GCC CAG CAG ACG AGA GGC CTC CTA GGG TGT ATA ATC 135 Thr? Tyr? Gln Gln Thr? Rg Gly Leu Leu Gly Cys He He 35 40 45 ACC AGC CTC? CT GGC CGG GAC AAA AAC CAA GTC GAG GGT GAG GTC 180 Thr Ser Leu Thr Gly Arg Asp Lye Asn Gln Val Glu Gly Glu Val 50 55 60 CAG? TC CTC TC? ? CT GCT? CC CAA ACC TTC CTG GCA ACG TGC ATC 225 Cn? Lß Val Ser Thr? Thr Gln Thr Phe Leu? Thr Cys He 65 70 75 ?? T OGG GT? TGC TGG ACT CTC TAC C? C GGG GCC GG? CG? GG? CC 270? Sn Cly Val Cyß Trp Thr Val Tyr His Gly? The Gly Thr? Rg Thr 80 85 90 ? TC CC? TC? CCC ?? G GGT CCT GTC? TC C? G? TG T? T? CC? AT GTG 315 Zla? The Pro Pro Lys Gly Pro Val He Gln Met Tyr Thr? Sn Val 95 100 105 C? C C ?? G? C CTT GTC GGC TGG CCC GCT CCT C ?? GGT TCC CGC TCA 360? Sp Gln? Sp Lau Val Gly Trp Pro? Pro Gln Gly Ser? Rg Ser 110 115 120 TTG? C? CCC TGC? CC TGC GGC TCC TCG GAC CTT TAC CTG GTT? CG 405 Leu Thr Pro Cye Thr Cys Gly Ser Ser Aep Leu Tyr Leu Val Thr 125 130 135 AGG CAC GCC GAC GTC? TT CCC GTG CGC CGG CGA GGT GAT AGC AGG 450 Arg His Ala? Sp Val He Pro Val? Rg Arg Arg Gly Asp Ser Arg 140 145 150 GGT AGC CTC CTT TCG CCC CGG CCC ATT TCC TAC CTA AAA GGC TCC 495 Gly Ser Lau Leu Ser Pro? Rg Pro He Ser Tyr Leu Lys Gly Ser 155 160 165 TCC GGG GGT CCG CTC TTC TCC CCC GCG GGA CAC GCC GTC GGC CTA 540 Ser Gly Gly Pro Leu Leu Cys Pro Wing Gly His Wing Val Gly Leu 170 175 180 TTC AGG GCC GCG GTG TCC ACC CGT GGA GTG ACC AAG GCG GTC GAC 585 Phe Arg Ala? The Val Cye Thr? Rg Gly Val Thr Lys Ala Val Aep 185 190 195 TTT ATC CCT GTC GAG AAC CTA GAG ACA ACC ATG AGA TCC CCG GTG 630 Phe He Pro Val Glu Aen Leu Glu Thr Met Arg Ser Pro Val 200 205 210 < 2 > INFORMATION FOR SEO ID NO:: í? * SEQUENCE CHARACTERISTICS: íAI LONG I TUD: 162 pa ¡de de b. =, e¿ íB) TIFO: 5! .. _. d »_j fiücleLco tr-i FABRIC CONFORMATION: simple íD) TOPOLOGY: linear íii TYPE OF MOUIA: cDNA (i *» CHARACTERISTICS: I NOMBR / CL VE: NS4A AGC ICC TGG GTG CTC GTT GGC GGC GTC CTG GCT GCT CTG GCC GCG 45 Ser Thr Trp Val Leu Val Gly Gly Val Leu Ala Ala Leu Ala? 1 5 10 15 TAT TGC CTG TCA ACA GGC TCC GTG GTC ATA GTG GGC AGG ATT CTC 90 Tyr Cys Leu Ser Thr Gly Cye Val Val He Val Gly Arg He Val 20 25 30 TTC TCC OGG A? G CCG GC? ? TT? T? CCT GAC AGG GAG GTT CTC TAC 135 Leu Sar Cly Lys Pro? The He He Pro? Sp? Rg Glu Val Leu Tyr 35 40 45 CAG GAG TTC GAT GAG ATG GAA GAG TCC 162 Cln Glu Phe? Sp Glu Met Glu Glu Cys 50 Í2) INFORMATION FOR SEO ID N0: 7: ii) CHARACTER I TT T OF SEQUENCE: ÍA5 LENGTH: 702 stop ?, of bases IB) TYPE: 5 i do nuc le i. - C) FABRIC CONFORMATION: simple (D) TOPOLOGY: linear íii) MOLECULE TIFF: cDNA? < ) CHARACTERISTICS: ÍA) NAME / KEY: NS3 + NS A GCG CCC? TC? GC GCG T? C GCC C? GC? G? CG? GA GGC CTC CTA GGG 45? The Pro He Thr? Tyr? Gln Gln Thr? Rg Gly Leu Leu Gly 1 5 10 15 TCT? T? CT? CC? CTG? CT? GGC CGG GAC AAA AAC CA? GTC GAG 90 Cys He He Thr Ser Leu Thr Gly? Rg? Sp Lys Asn Gln Val Glu 20 25 30 CGT C? G CTC C? G? TC GTC TCA ACT GCT ACC ACC ACC TTC CTC GCA 135 Cly Clu Val Cln He Val Ser Thr? Thr Gln Thr Phe Leu? La 35 40 45 ? CG TCC? TC ?? T GGG GTA TCC TGG ACT GTC TAC CAC GGG GCC GGA 180 Thr Cys He? Sn Gly Val Cys Trp Thr Val Tyr Hie Gly Ala Gly 50 55 60 ACG? GG ACC ATC GCA TCA CCC AAG GGT CCT GTC ATC CAG ATG TAT 225 Thr Arg Thr He? The Ser Pro Lys Gly Pro Val He Gln Met Tyr? CC? AT GTC GAC CA? GAC CTT GTC GGC TCG CCC GCT CCT CA? GGT 270 Thr? Sn Val? Sp Gln? Sp Leu Val Gly Trp Pro? The Pro Gln Gly 80 85 90 TCC CGC TCA TTC ACA CCC TGC ACC TCC GGC TCC TCG GAC CTT TAC 315 Ser Arg Ser Leu Thr Pro Cys Thr Cye Gly Ser Ser Aep Leu Tyr 95 100 105 CTG CTT ACG AGG CAC GCC G? C CTC? TT CCC CTC CGC CGG CGA GGT 360 Leu Val Thr Arg His Wing Asp Val He Pro Val Arg Arg Arg Gly 110 115 120 G? T * BC? GG CGT? GC CTG CTT TCG CCC CGG CCC? TT TCC TAC CTA 405? ßp Ser? Rg Cly Sar Lau Lau S »t Pro? Rg Pro Zla Ser Tyr Lau 125 130 135 ??? OBC TCC TCG GGG OGT CCG CTC TTC TCC CCC GCG GG? C? C GCC 450 Lys Cly S »t Ser Gly Gly Pro Leu Leu Cys Pro? Gly His? 140 145 150 GTC OBC CT? TTC? GG CCC GCG CTG TCC? CC CGT GG? GTG? CC ?? G 495 Val Gly Leu Phe? Rg? The? Val Cys Thr? Rg Gly Val Thr Lys 155 160 165 GCG CK C? C TTT? TC CCT GTC G? G AAC CTA GAG? C? ? CC? TC? GA 540? The Val? Sp Phe He Pro Val Glu? Sn Leu Glu Thr Thr Met? Rg 170 175 180 TCC OS GGG CTC CTC CTT GGC GGC GTC CTC GCT GCT CTC GCC GCG 585 Ser P »Gly Val Leu Val Gly Gly Val Leu Ala Ala Leu Ala? 185 190 195 T? T 1GC CTG TC? ? C? GCC GTC GTC GTC GTC GGC AGG ATT GTC 630 Tyr Cys Leu Ser Thr Gly Cye Val Val He Val Gly Arg He Val 200 205 210 TTC TCC GGG AAG CCG GCA ATT? TA CCT GAC AGG GAG GTT CTC TAC 675 Leu Ser Gly Lye Pro Wing He He Pro Pro Arg Glu Val Leu Tyr 215 220 225 CAG CAS TTC GAT GAG ATC GAA GAG TCC 702 ~ 'Gln Ca Ptie? Sp Glu Met Glu Glu Cys 230 I2) INFORMATION FOR SEQ ID NO; 8: (i) CHARACTERISTICS OF SFCUE CIA: íA) LENGTH: B55 pairs of as s (B) TIFO: nucleic acid íC) COBFOPMACIÓN DE BRAB: simpl íD) TOPOLOGY: 1 i nea 3 iii) TYPE OF MOLECULE: cDNA íi. - > «FACTEPI TICAS: ÍA) tlO R? / KEY: pNBlf.2.J l 4AHT ATC AGftßS? TCG C? T C? C C? T CAC CAT CAC GGA TCC CAT AAG GCA 45 Met ArffGly Ser His His Hi? Hi? His His Gly Ser His Lys? La 1 5 10 15 ? G? «I CCT G ?? GC? ATC AGC C? T GsT? CC? TG GCG CCC? TC 90? Rg «if fie»? The Glu? The Met Ser His Gly Thr Met? The Pro He 20 25 30 ? CC GCG1MC CCC C? G C? G? CG? GA GGC CTC CTA GGG TGT ATA ATC 135 Thr Alaiyr? Gln Gln Thr? Rg Cly Leu Leu Gly Cys He He 35 40 45 ? CC? GC C3S? CT GGC CGG GAC AAA AAC CA CA GTC GAG GGT GAG GTC 180 Thr Sar Leu Thr Gly? Rg? Sp Lye? Sn Gln Val Glu Gly Glu Val 50 55 60 C? G? TC CTG TCA ACT GCT? CC CAA ACC TTC CTG GCA ACG TGC ATC 225 Cln He Val Ser Thr Wing Thr Gln Thr Phe Leu Wing Thr Cye He 65 70 75 A? T GGG GTA TGC TCG ACT GTC TAC CAC GGG GCC GGA ACG AGG ACC 270? Sn Gly Val Cys Trp Thr Val Tyr Hie Gly Wing Gly Thr Arg Thr 80 85 90? TC GC? TCA CCC AAG GGT CCT GTC ATC CAG? TC TAT ACC? AT GTC 315 He Wing Being Pro Lyß Gly Pro Val He Gln Met Tyr Thr Asn Val 95 100 105 GAC CA? C? C CTT CTC CGC TGG CCC GCT CCT C ?? GGT TCC CGC TC? 360? Sp Gln? Sp Lau Val Gly Trp Pro? Pro Gln Gly Sar? Rg Sar 110 115 120 TTO? C? CCC TGC? CC TCC OCC TCC TCG C? C CTT TAC CTG GTT ACG 405 Lau Thr Pro Cys Thr Cys Gly Ser Ser? Sp Leu Tyr Leu Val Thr 125 130 135 ? GG C? C GCC G? C GTC? TT CCC GTC CGC CGC CGA GGT GAT AGC AGG 450? Rg His? La? Sp Val He Pro Val? Rg? Rg? Rg Gly? Sp Ser? Rg 140 145 150 CCT? CC CJB CTT TCG CCC CGG CCC? TT TCC T? C CTA A ?? GGC TCC 495 Cly Ser Lau Lau Ser Pro? Rg Pro He Ser Tyr Leu Lys Gly Ser 155 160 165 TCG GGG GGT CCG CTC TTG TCC CCC GCG GGA CAC GCC GTG GGC CTA 5 0 Ser Gly Gly Pro Leu Leu Cys Pro Wing Gly Hie? The Val Gly Leu 170 175 180 TTC AGG GCC GCG GTG TGC? CC CGT GGA GTC ACC AAG GCG GTG GAC 585 Phß Arg Ala? Val Cye Thr? Rg Gly Val Thr Lys? Val? Sp 185 190 195 TTT ATC CCT GTC GAG AAC CTA GAG ACA ACC? TC AGA TCC CCG GGG 630 Phe Zle Pro Val Glu Asn Leu Clu Thr Thr Met Arg Ser Pro Gly 200 205 210 CTC CTC CTT GGC GGC GTC CTC GCT GCT CTC GCC GCG TAT TGC CTG 720 Val Leu Val Gly Gly Val Leu? The? Leu? The? Tyr Cys Leu 215 220 225 TC? ACA CCC TGC CTG GTC AT? GTG GGC AGG? TT GTC TTC TCC GGG 765 S * t _ta Cly Cyß Val Val He Val Cly? Rg He Val Leu Ser Gly 230 235 240 ?? G COB OC? ? TT? T? CCT C? C? GG GAG GTT CTC TAC CAG CAG TTC 810 Ly? Pro? La Zla Zla Pro? Sp? Rg Clu Val Leu Tyr Cln Clu Pha 245 250 255 C? T GIC ATC G ?? G? G TGC CGG ?? G ??? ?? G? G? CGC ?? G CTT ?? T 855? Sp Wave Mßt Glu Clu Cyß? Rg Lyß Lys Ly?? Rg? Rg Ly? Leu? Sn 260? 2) INFORMATION FOR SEO ID NO: 9: - (i) SEQUENCE CHARACTERISTICS: ( A) LENGTH: 711 base pairs., B) TYPE: A nucleic acid (OR HEBREW CONFORMATION: sim le (D) lOFOtOGTA: 1 i ea 3 (ii> TYPE OF MOtECULA: DNA (i?> CHARACTERISTICS: (A) NAME / CLA E: GCG CCC? TC? GC GCG T? C GCC C? G CAG ACG AGA GGC CTC CTA GGG 45? The Pro He Thr? Tyr? Gln Gln Thr? Rg Gly Leu Leu Gly 1 5 10 15 TGT? TA ATC ACC AGC CTG ACT GGC CGG GAC AAA AAC CAA GTC GAG 90 Cys He He Thr Ser Leu Thr Gly Arg Aep Lye Asn Gln Val Glu 20 25 30 OGT C? G GTC CAG ATC GTG TCA ACT GCT ACC ACC TTC CTG GCA 135 Cly Clu Val Gln He Val Ser Thr Ala Thr Gln Thr Phe Leu Ala 35 40 45 ACC TCC? TC ?? T GGG GT? TCC TCC? CT GTC T? C CAC GGG GCC GGA 180 Thr Cyß Zla? ßn Gly Val Cyß Trp Thr Val Tyr His Gly? The Gly 50 55 € 0 ACG AOG ACC? TC GC? TC? CCC ?? G GGT CCT GTC? TC C? G? TC T? T 225 Thr? Rg Thr Zla? La Sar Pro Lys Gly Pro Val Zle Gln Met Tyr 65 70 75 ? CC A? T OTC G? C C ?? G? C CTT GTC OGC TCG CCC GCT CCT CAA GGT 270 Thr Aßn Val? Sp Gln? Sp Lau Val Gly Trp Pro? Pro Gln Gly 80 85 90! TCC CGC TC? TTC? C? CCC TCC? CC TGC GGC TCC TCG GAC CTT TAC 315 Sar? Rg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser? Sp Leu Tyr 95 100 105 CTC CTT? CG? GG C? C GCC G? C GTC? TT CCC GTG CGC CGG CGA GGT 360 Leu Val Thr? Rg His? La? Sp Val He Pro Val? Rg Arg Arg Gly 110 115 120 I GAT AGC AGG GGT AGC CTC CTT TCG CCC CGG CCC ATT TCC TAC CTA 405? Sp Ser? Rg Gly Ser Leu Leu Ser Pro? Rg Pro He Ser T r Leu 125 130 135 AA? GGC TCC TCG GGG GGT CCG CTG TTC TCC CCC GCG GGA CAC GCC 450, Lyß Gly Ser Ser Gly Pro Leu Leu Cys Pro Wing Gly His Wing 140 145 150 GTG GGC CTA TTC AGG GCC GCG GTC TCC ACC CGT GGA GTG ACC AAG 495 Val Gly Leu Phe Arg Ala Wing Val Cys Thr? Rg Gly Val Thr Lye 155 160 165 GCG GTC G? C TTT? TC CCT GTC GAG AAC CTA GAG ACA ACC ATC AGA 540? The Val? Sp Phe He Pro Val Glu? Sn Leu Glu Thr Thr Met? Rg 170 175 180 TCC CCG GGG GTG CTC GTT GGC GGC GTC CTC GCT GCT CTC GCC GCG 585 Ser Pro Gly Val Leu Val Gly Gly Val Leu? La? Leu? La? 185 190 195 T? T TGC CTC TC? ? C? OGC TGC GTG GTC? T? GTC GGC? GG? TT GTC 630 Tyr Cys Lau Ser Thr Gly Cys Val Val Zla Val Gly? Rg He Val 200 205 210 TTG TCC OGG ?? G CCG GC? ? TT? T? CCT G? C? GG G? G GTT CTC T? C 675 Leu Sar Gly Lys Pro? The He Zle Pro? Sp? Rg Glu Val Leu Tyr 215 220 225 C? GG? G TTC GAT GAG ATG GAA GAG AAG GAG ACA GAG Gln ßlu Mie ksp Glu Met Glu Glu Lys Glu Thr Glu 230 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH : 855 base pairs (B) TYPE: nucleic acid (OR SHAPE CONFORMATION: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) CHARACTERISTICS: (A) NAME / KEY:? TC? GA GGA TCG CAT CAC CAT CAC CAT CAC ACG GAT CCG GCG CCC Met? Rg Gly Ser His His Hi? Hie His His? Thr? Sp Pro? La Pro 1 5 10 15 TC? CG GCG TAC GCC C? G C? G? CG? GA GGC CTC CTA GGG TCT ATA Zle Thr Ala Tyr Ala Gln Gln Thr Arg Gly Leu Leu Gly Cye He 20 25 30 ATC? CC? GC CTC ACT GGC CGG GAC? AA AAC CA? GTC GAG GGT GAG 90 Zle Thr Ser Leu Thr Gly? Rg? Sp Lys? Sn Gln Val Glu Gly Glu 35 40 45 CTC CAG ATC CTC TC? ? CT OCT ACC C ?? ? CC TTC CTG CC? ? CC TCC 135 Val Cln Zla Val Sar Thr? Thr Cln Thr Phe Leu? Thr Cy? 50 55 60 ? TC A? T OGG GT? TGC TOG? CT GTC T? C C? C GGG GCC GG? CG? GG 180 llß? ßn Gly Val Cys Trp Thr Val Tyr His Gly? The Gly Thr? Rg 65 70 75? CC JOC GC? TCA CCC AAG GGT CCT GTC ATC CAG ATG T? T? CC? T 225 Thr? Le? The Pro Pro Lys Gly Pro Val He Gln Met Tyr Thr? Sn 80 85 90 GTC C? C C ?? G? C CTT GTC GGC TGG CCC GCT CCT C? A GGT TCC CGC 270 Val? Sp Gln? Sp Leu Val Gly Trp Pro Wing Pro Gln Gly Ser Arg 95 100 105 TCA TTC ACA CCC TCC ACC TCC GGC TCC TCG GAC CTT TAC CTG GTT 315 / Ser Leu Thr Pro Cye Thr Cye Gly Ser Ser Asp Leu Tyr Leu Val 110 115 120 ACG AOG CAC GCC G? C GTC? TT CCC GTC CGC CGG CGA GGT GAT AGC 360 Thr? Rg His? La? Sp Val He Pro Val? Rg Arg Arg Gly? Sp Ser 0 125 130 135 GG CGT? GC CTC CTT TCG CCC CGG CCC ATT TCC TAC CTA AAA GGC 405 Arg Gly Ser Leu Leu Ser Pro Arg Pro Zle Ser Tyr Leu Lye Gly 140 145 150 TCC TCG GGG GGT CCG CTC TTC TCC CCC GCG GGA CAC GCC GTC GGC 450 5 be Ser Gly Gly Pro Leu Leu Cye Pro Wing Gly His Wing Val Gly 155 160 165 CT? TTC? GG GCC GCG GTC TGC? CC CGT GGA GTC ACC? AG GCG GTG 4 5 Lau Phe Arg Wing Wing Val Cys Thr Arg Gly Val Thr Lys? Val 170 175 180 GAC TTT? TC CCT GTC G? G AAC CTA G? G ACA ACC? TG? G? TCC CCG 540 ? 0? Sp Phe He Pro Val Glu? Sn Leu Glu Thr Met? Rg Ser Pro 185 190 195 OGG CTC CTC GTT GGC GGC GTC CTC GCT GCT CTC GCC GCG TAT TGC 585 Gly Val Leu Val Gly Gly Val Leu Ala Ala Leu Ala Ala r Cys 200 205 210 CTC TC? ? C? GGC TGC GTC GTC? TA GTC GGC AGG? TT GTC TTG TCC 630 Lau Ser Thr Gly Cys Val Val Zle Val Gly Arg He Val Leu Ser 215 220 225 OGG A? G CCG GC? ? TT? TA CCT GAC AGG G? G GTT CTC T? C C? G G? G 675 Gly Lys Pro? The He Zle Pro? Sp Arg Glu Val Leu Tyr Gln Glu 230 235 240 TTC GAT GAG ATC GAA GAG AAG GAG ACA GAG 705 Phe? Sp Glu Met Glu Glu Lys Glu Thr Glu 245 250 (2) INFORMATION PAPA SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 pairs of bases (B) TYPE: nucleic acid (OR FABRIC CONFORMATION: double (D) TOPOLOGY: double (ii) TYPE OF MOLECULE: cDNA GA TCA CCG GTC TAG ATCT T GGC CAG ATC TAGA (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) FABRIC CONFORMATION: double (D) TOPOLOGY: linear. (ii) TYPE OF MOLECULE: cDNA (ix CHARACTERISTICS: (A) NAME / KEY: CCG GTC CGG ?? G ?? A ?? G? GA CGC TAG C AG GCC TTC TTT TTC TCT GCG ATC G (2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 79 base pairs (B) TYPE: nucleic acid (C) FABRIC CONFORMATION: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA < ix) FEATURES: (A) NAME / CL VE: CCG GCA ATT AT? CCT GAC AGG GAG GTT CTC TAC CAG GAA TTC GT T ?? T? T GGA CTG TCC CTC CAA GAG? TG GTC CTT ?? G G? T G? G? TG G ?? G? G TCC CGG? AG ?? A AAG AGA CGC A CT? CTC T? C CTT CTC? CG GCC TTC TTT TTC TCT GCG TTC GA (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) HEBREW CONFORMATION: imple (D) TOPOLOGY: linear. (ii) TYPE OF MOLECULE: polypeptide (ix) CHARACTERISTICS: (A) NAME / KEY: NS4A active mutant Gly Cys Val Val HeV Val Gly Arg He Val Leu Ser Gly Lys 5 10 < 2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 amino acids < B) TYPE: amino acid (C) FABRIC CONFORMATION: impl (D) TOPOLOGY: linear iii) TYPE OF MOLECULE: polypeptide (i *) CHARACTERISTICS: (A) NAME / KEY: NS4A active mutant Cys Val Val He Val Gly Ary He V l Leu Ser Gly Lys 5 10 (2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 amino acids (B) TYPE: amino acid (C) HEBREW CONFORMATION: imple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: polypeptide ( ix) CHARACTERISTICS: (A) NAME / KEY: Substrate 5A / 5B soluble Asp Thr Glu Asp Val Val Cys Cys Ser Met Ser Tyr Thr Trp Thr 5 10 15 Gly Lys (2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino acids (B) TYPE: nucleic acid (OR FABRIC CONFORMATION: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: ida palipép (i¡ <;) CHARACTERISTICS: (A) NAME / KEY: Substrate 5A / 5B mutant Asp Thr Glu Asp Val Va Ala Cys Ser Met Ser Tyr Thr Trp Thr 5 10 15 Gly (2) INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 amino acids (B) TYPE: amino acid (C) FABRIC CONFORMATION: simple (D) TOPOLOGY: 1 i ne 1 ( ii) TYPE OF MOLECULE: polypeptide i iK) (CAR CHARACTERISTICS: (A) NAME / KEY: Substrate 5A / 5B soluble mutant Asp Thr Blu Asp Val Val Ala Cys Ser Met Ser Tyr Thr Trp Thr 5 10 15 Gly Lys (2) INFORMATION FOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (OR CONFORMATION OF HEBRA: simple (D) TOPOLOGY: linear. (Ii) TYPE OF MOLECULE: polypeptide (ix) CHARACTERISTICS: (A) NAME / KEY: Substrate 5A / 5B Soluble Asp Thr Glu Asp Val V l Cys Cys Ser Met Ser Tyr Thr Trp Thr 5 10 15 Gly Lys Tyr (2) INFORMATION FOR SEQ ID NO -.20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (C) FABRIC CONFORMATION: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: palpeptide (ix) CHARACTERISTICS: (A) NAME / KEY: Substrate 5A / 5B Soluble ? ßp Thr Glu? sp Val Val? the Cys Ser Met Ser Tyr Thr Trp Thr 5? or 15 (2) INFORMATION FOR SEQ ID NO: 21; (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids (B) TYPE: amino acid (C) HEBREW CONFORMATION: simple (D) TOPOLOGY: linear. (ii) TYPE OF MOLECULE: polypeptide (ix) CHARACTERISTICS: (A) NAME / CL VE: Your time 4B / 5A Soluble Trp He Ser Ser Glu Cys Thr Thr Pro Cye Ser Gly Ser Trp Leu 5 10 15? rg? sp He Trp Asp (2) INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCE CHARACTERISTICS; (A) LENGTH: 13 amino acids (B) TYPE; amino acid (C) FABRIC CONFORMATION: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: polypeptide (i; <CHARACTERISTICS: (A) NAME / KEY: histidine marker Net Arg Gly Ser His Hie Hie His His His Thr Aep Pro 5 10 (2) INFORMATION FOR SEQ ID NO: 23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) FABRIC CONFORMATION: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: polypeptide (ix) CHARACTERISTICS: (A) NAME / KEY: hydrophilic glue Arg Lys Lys Arg Arg Lys Leu Asn 5 (2) INFORMATION FOR SEQ ID NO: 24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 amino acids (B) TYPE: amino acid (OR CONFORMATION OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: polypeptide (ix) CHARACTERISTICS: (A) NAME / KEY: Hydrophilic glue Lys Glu Thr Glu (2) INFORMATION FOR SEQ ID NO: 25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (OR BLOCK CONFORMATION: imple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: polypeptide (i K) CHARACTERISTICS: (A) NAME / KEY: hydrophilic tail Trp Zle Ser Glu Cys Thr Thr Pro Cyß Ser Gly Ser Trp Lau Arg? Sp Zle Trp? Sp 20 (2) INFORMATION FOR SEQ ID NO: 26: 5 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 162 base pairs (B) TYPE: nucleic acid (C) FABRIC CONFORMATION: imple (D) TOPOLOGY: linear 10 (ii) TYPE OF MOLECULE: cDNA (ix) CHARACTERISTICS: (A) NAME / KEY: NS4A mutant CTC CTC GTT GGC GGC GTC CTC GCT GCT CTG GCC GCG TAT TCC CTG 45 Val Leu Val Gly Gly Val Leu Ala? The Leu Ala Ala Tyr Cye Leu 1 5 10. 15 15 TCA ACA GGC TCC GTC GTC ATA GTC GGC AGG ATT GTC TTG TCC GGG 90 Ser Thr Gly Cye Val Val He Val Gly Arg lié Val Leu Ser Gly 20 25 30 A? G CCG GC? ? TT? TA CCT G? C? GG G? G GTT CTC TAC CAG GAG TTC 135 Lyß Pro Wing Zle Zle Pro Asp? Rg Glu Val Leu Tyr Gln Glu Phe 35 40 45 i ? 0 G? T G? G? TG G ?? G? G TGC 50 (2) INFORMATION FOR SEQ ID NO: 27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 810 base pairs (B) TYPE: nucleic acid (C) FABRIC CONFORMATION: simple (D) TOPOLOGY: linear (ii) T MOLECULE IPO: cDNA (ix) CHARACTERISTICS: (A) NAME / KEY: pNB 182de l a4AHT ? TC? G? GGA TCG CAT CAC CAT CAC CAT CAC ACG GAT CCG CCC ATC 45 Met? Rg Gly Ser Hie Hie Hie Hie His Hie Thr Asp Pro Pro He 1 5 10 15 ACG GCG TAC GCC CAG CAG ACG AGA GGC CTC CTA GGG TGT ATA ATC 90 Thr Ala Tyr Ala Gln Gln Thr Arg Gly Leu Leu Gly Cye He He 20 25 30 ACC? GC CTG? CT GGC CGG GAC AAA AAC CAA GTC GAG GGT GAG GTC 135 Thr Ser Leu Thr Gly Arg? ßp Lys? Sn Gln Val Glu Gly Glu Val 35 40 45 C? G? TC GTC TC? ? CT GCT? CC C? A ACC TTC CTC GC? CG TCC TC 180 Gln Zle Val Ser Thr Wing Thr Gln Thr Phe Leu Wing Thr Cys He 50 55 60 ?? T GGG GT? TGC TGG? CT GTC T? C C? C GGG GCC GG? CG? GG? CC 225? Sn Gly Val Cys Trp Thr Val Tyr His Gly? The Gly Thr? Rg Thr 65 70 75 TC TC? TC? CCC ?? G OGT CCT OTC? TC C? G? TC T? T? CC ?? T CTC 270 Zle? The Pro Pro Lys Gly Pro Val Zle Gln Met Tyr Thr? Sn Val 80 85 90 GAC C ?? G? C CTT GTC GGC TGG CCC GCT CCT C? A GGT TCC CGC TCA 315? Sp Gln? Sp Lau Val Gly Trp Pro? Pro Gln Gly Ser? Rg Ser 95 100 105 TTC? C? CCC TCC? CC TCC GGC TCC TCG G? C CTT T? C CTG GTT? CG 360 Leu Thr Pro Cys Thr Cys Gly Ser Ser? Sp Leu Tyr Leu Val Thr 110 115 120 GG C? C GCC G? C GTC? TT CCC GTC CGC CGG CGA GGT GAT? GC? GG 405 i0? Rg His? La? Sp Val He Pro Val? Rg? Rg? Rg Gly? Sp Ser? Rg 125 130 135 GGT? GC CTC CTT TCG CCC CGG CCC? TT TCC T? C CTA AAA GGC TCC 450 Gly Ser Leu Leu Sex Pro Arg Pro He Ser Tyr Leu Lys Gly Ser 140 145 150 TCG OGG OCT CCG CTC TTC TCC CCC GCG GGA CAC GCC GTG GGC CTA 495 Ser Gly Gly Pro Leu Leu Cye Pro.? Gly His? Val Gly Leu 155 160 165 TTC AGG OCC GCG GTC TGC? CC CGT GG? GTG? CC ?? G GCG GTG G? C 540 ^ 0 Phe? Rg? The? The Val Cys Thr? Rg Gly Val Thr Lys? The Val? Sp 170 175 180 TTT? TC CCT GTG G? G ?? C CTA GAG? CA ACC? TG? GA TCC CCG GGG 585 Phe Zle Pro Val Glu Asn Leu Glu Thr Thr Met? Rg Ser Pro Gly 185 190 195 GTG CTC GTT GGC OGC GTC CTG GCT GCT CTC GCC GCG T? T TGC CTG 630 Val Leu Val Gly Gly Val Leu? La? Leu? La? Tyr Cys Leu 200 205 210 TC? ? C? GGC TGC GTG GTC? TA GTC GGC AGG ATT GTC TTG TCC GGG 720 Ser Thr Gly Cye Val Val He Val Gly? Rg He Val Leu Ser Gly 215 220 225 AAG CCG GCA ATT ATA CCT GAC AGG GAG GTT CTC TAC CAG GAG TTC 765 Lys Pro Wing He He Pro Asp Arg Glu Val Leu Tyr Gln Glu Phe 230 235 240 GAT G? G? TC GAA GAG TGC CGG AAG AAA AAG AGA CGC AAG CTT AAT 810? Sp Glu Met Glu Glu Cys? Rg Lys Lys Lye? Rg? Rg Lye Leu? Sn 245 250 255 (2) INFORMATION FOR SEQ ID NO: 28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH; 1.62 base pairs (B) TYPE: nucleic acid (C) FABRIC CONFORMATION: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) CHARACTERISTICS: (A) NAME / KEY: NS4A Na t i vo TC? ? C? TCG OTC CTC GTT GGC GGC GTC CTC GCT GCT CTG GCC GCC 45 Ser Thr Trp Val Leu Val Gly Gly Val Leu? La? La Lau? La? 5 10 15 T? T TGC CTC TCA AC? OGC TCC GTC GTC? T? GTG GGC? GG ATT GTC 90 Tyr Cyß Leu Ser Thr Gly Cye Val Val He Val Gly? Rg Zle Val 20 25 30 TTC TCC GGG ?? G CCG GCA ATT? TA CCT GAC? GG GAG GTT CTC TAC 135 Leu Ser Gly Lys Pro? The He He Pro Asp Arg Glu Val Leu Tyr 35 40 45 CAG G? G TTC GAT GAG ATG GAA GAG TCC Gln Glu Phe Asp Glu Met Glu Glu Cye 50 (2) INFORMATION FOR SEQ ID NO: 29: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 amino acid residues (B) TYPE: nucleic acid (C) HEBREW CONFORMATION: imple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: polypeptide (ix) CHARACTERISTICAB: (A) NAME / KEY: Carboxl 33 ßr of NS4A Cyß Val Val Ha Val Gly? Rg He Val Leu Ser Gly Lys Pro? La 5 10 15 ? lß Zlß Pro? ßp? rg Glu Val Leu Tyr Gln Glu Ph? ßp Glu Mßt 20 25 30 (2) INFORMATION FOR SEQ ID NO: 0: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 amino acid residues (B) TYPE: nucleic acid (C) FABRIC CONFORMATION: ple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: polypeptide (ix) CHARACTERISTICS: (A) NAME / KEY: Carbox 1 33 er of NS4A of BK strain of HCV Ser Val Val He Val Gly? Rg He He Leu Ser Gly Arg Pro Ala 5 10 15 Zlß Val Pro Asp? Rg Glu Leu Leu Tyr Gln Glu Phe? Sp Glu Met 20 25 30 Gl? Glu Cys (2) INFORMATION FOR SEQ ID N0: 31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 residues of ami.nocid id »3s (B) TYPE: nucleic acid (OR CONFORMATION OF HEBRA: sim le ( D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: polypeptide (i; <) CHARACTERISTICS: (A) NAME / KEY: Substrate 5A / 5B Native Asp Thr Gly Asp Val Val Cys Cys Ser Met Tyr Thr Trp Thr 5 10 15 Gly 0 ? 0

Claims (5)

1. CLAIMS 1. A soluble HCV substrate that encodes a non-structural polyprotein of the HCV genome.
2. 2. The Jr'Cv substrate of the rei indication 1 which further comprises a solubilization motif fixed on said substrate.
3. 3. The HCV substrate of rei indication 2, where the solubilization motif is made up of an ionizable amino acid.
4. 4. The HCV substrate of the rei indication 3, where the ionizable substrate is either arginine or lysine. 5. The HCV substrate of the rei indication 4, having a sequence defined by SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21 ,. 6. A nu- meric acid encoding the soluble HCV substrate of claims 1, 2, 3, 4 or 5. 7. A vector containing a nucleic acid encoding a soluble HCV substrate of claims 1, 2, 3, 4 or 5. 8. A cell transfected or alternatively formed with a vector containing a nucleic acid encoding a soluble HCV substrate of the iv ind icac. ions 1, 2, 3, 4, or
5. 5.