HK1015408A - Method for reproducing in vitro the rna-dependent rna polymerase and terminal nucleotidyl transferase activities encoded by hepatitis c virus (hcv) - Google Patents

Description

Method for reproducing in vitro the RNA-dependent RNA polymerase and terminal nucleotidyl transferase activities encoded by Hepatitis C Virus (HCV)

The present invention relates to the molecular biology and virology of Hepatitis C Virus (HCV). More precisely, the subject of the present invention is the RNA-dependent RNA polymerase (RdRp) and terminal nucleotidyl transferase (TNTase) activities produced by HCV, methods for expressing HCV RdRp and TNTase, and methods for the in vitro detection of the RdRp and TNTase activities encoded by HCV for therapeutic purposes by identifying compounds that inhibit these enzymatic activities and thus can interfere with HCV viral replication.

Hepatitis C Virus (HCV) is known to be the major causative agent of non-a, non-b hepatitis (NANB). HCV is estimated to cause at least 90% of post-transfusion NANB viral hepatitis and 50% of sporadic NANB hepatitis. Although great progress has been made in the selection of blood donors and immunological identification of blood for transfusion, HCV infection still occurs in many of those receiving transfusions (one million or more infected individuals worldwide per year). Approximately 50% of HCV-infected people develop cirrhosis of the liver within a period that can be 5 to 40 years. In addition, recent clinical studies have shown that there is some correlation between chronic HCV infection and the development of hepatocellular sarcoma.

HCV is an enveloped virus containing an approximately 9.4kb positive-stranded RNA genome. The virus is a member of the flavivirus family, and the other members of this family are flavivirus and pestivirus. The RNA genome of HCV has recently been mapped. Comparison of HCV genomic sequences isolated throughout the world indicates that these sequences may be very homologous. The majority of the HCV genome is occupied by an Open Reading Frame (ORF) that varies between about 9030 and 9099 nucleotides. This ORF encodes a single viral polyprotein, which can vary in length from 3010 to 3033 amino acids. During the period of viral infection, polyproteins are processed by proteolytic cleavage into individual gene products necessary for viral replication. The gene encoding the HCV structural protein is located at the 5' -end of the ORF, while the region encoding the non-structural protein occupies the remainder of the ORF.

The structural proteins consist of C (core, 21kDa), E1 (envelope, gp37) and E2(NS1, gp 61). C is a 21kDa non-glycosylated protein that is likely to form the viral nucleocapsid. Protein E1 is an approximately 37kDa glycoprotein that is considered to be a structural protein of the outer viral envelope. Another 61kDa membrane glycoprotein E2 may be another structural protein in the outer envelope of the virus.

The nonstructural region begins with NS2(p24), a 24kDa hydrophobin of unknown function. It is postulated that NS3, a 68kDa protein located after NS2 in polyproteins, has two functional domains: a serine protease domain in the first 200 amino-terminal amino acids, and an RNA-dependent atpase domain at the carboxy terminus. The gene region corresponding to NS4 encodes two hydrophobic proteins NS4A (p6) and NS4B (p26) of 6kDa and 26kDa, respectively, the functions of which are unknown. The gene corresponding to NS5 also encodes two proteins NS5A (p56) and NS5B (p65) of 56 and 65kDa, respectively.

Various molecular biological studies have shown that signal peptidase, a protease associated with the endoplasmic reticulum of host cells, is responsible for proteolytic processing in the nonstructural region, that is, at the C/E1, E1/E2 and E2/NS2 sites. One HCV virally encoded protease appears to be responsible for cleavage between NS2 and NS 3. This protease activity is contained in a region comprising part NS2 and part NS3 containing the serine protease domain, but does not utilize the same catalytic mechanism. The serine protease contained in NS3 is responsible for the cleavage points of the linkage between NS3 and NS4A, between NS4A and NS4B, between NS4B and NS5A and between NS5A and NS 5B.

Like other (+) strand RNA viruses, HCV replication is thought to begin by the initial synthesis of a complementary (-) RNA strand, which in turn serves as a template for the production of progeny (+) strand RNA molecules. It is presumed that an RNA-dependent RNA polymerase (RdRp) is involved in these steps. An amino acid sequence present in all RNA-dependent RNA polymerases can be recognized in the NS5 region. This indicates that the NS5 region contains components of the viral replication machinery. Traditionally, the virally encoded polymerase has been considered an important target for the inhibition of antiviral compounds, however, in the specific case of HCV, the study of this substance has been severely hampered by the lack of a suitable model system for viral infection (e.g. infection of cells in culture or in a readily available animal model) and a method for the detection of functional RdRp enzymatic activity.

It has surprisingly been found that this main limitation can be overcome by employing the method according to the invention, which also provides other advantages that will be clear from the following description.

The object of the present invention is a method for reproducing in vitro the RNA-dependent RNA polymerase activity of HCV by means of the sequence contained in the HCV NS5B protein. The terminal nucleotidyl transferase activity, another property of the NS5B protein, was also reproduced by this method. This method exploits the fact that proteins containing the sequence of NS5B can be expressed in eukaryotic or prokaryotic heterologous systems: recombinant proteins containing the sequence of NS5B purified to apparent homogeneity or present in extracts of overexpressed organisms can catalyze the addition of nucleotides to the 3' end of foreign RNA molecules in a template-dependent (RdRp) or template-independent (TNTase) manner.

The invention also extends to a novel composition characterized in that it comprises a protein of the sequence described in sequence No. 1 or contained therein or derived therefrom. It is known that this sequence may differ among different HCV isolates, since all RNA viruses exhibit a high degree of variability. This novel composition has the RdRp activity necessary for HCV to replicate its genome.

It is another object of the present invention to utilize the compositions for therapeutic purposes in the preparation of a compound capable of identifying inhibitors of the enzymatic activity associated with NS5B, including inhibitors of RdRp and TNTase.

A general description of the invention has been given so far. In order that the points, features, advantages and methods of operation thereof may be more clearly understood, a more particular description of specific embodiments thereof will now be given, with the aid of the following examples.

FIG. 1 shows the plasmid structure used to transfer HCV cDNA into a baculovirus expression vector.

FIG. 2 shows a plasmid (pT7-7(DCoH)) for in vitro synthesis of a D-RNA substrate for HCV RNA-dependent RNA polymerase and a plasmid (pT7-7(NS5B)) for expression of HCV RNA-dependent RNA polymerase in E.coli cells, respectively.

FIG. 3 shows a schematic diagram of (+) and (-) strands of D-RNA. The transcript includes the coding region of DCoH mRNA. The DNA-oligonucleotides a, b and c are designed to anneal with newly synthesized antisense RNA and the DNA/RNA hybrids are cleaved with RNase H. The lower part of the figure depicts the expected RNA fragment sizes generated by rnase digestion of RNA (-) hybridized to oligonucleotides a, b and c, respectively.

Coli DH1 Bacteria transformed with plasmids pBac5B, pBac25, pT7.7 DCoH and pT7.7NS5B containing The cDNA transcribed by SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 12 and The cDNA transcribed by SEQ ID NO. 1, respectively, were deposited at 9.5.1995 under The accession numbers NCIMB 40727, 40728, 40729 and 40730 at The National Collection of Industrial and Marine Bacteria (NCIMB), Aberden, Scotland, UK).

Example 1 method for expressing HCV RdRp/TNTase in Spodoptera frugiperda clone 9(Sf9) culture cells

Systems for the expression of foreign genes in insect culture cells, such as Spodoptera frugiperda clone 9(Sf9) cells infected with a baculovirus vector, are known in the art (V.A. Luckow, a baculovirus system for the expression of human gene products, (1993) Current Opinion in Biotechnology, p. 564-572). The foreign gene is usually placed under the control of strong promoter of polyhedrin of Bombyx mori nuclear polyhedrosis virus alfalfa silver leaf moth nuclear polyhedrosis virus. Methods for introducing foreign DNA at the desired site in a baculovirus vector by homologous recombination are also known in the art (d.r.o' Reilly, l.k.miller, v.a.lucchow, (1992), "baculovirus expression Vectors-a Laboratory Manual", w.h.freeman and Company, new york).

Plasmid vectors pBac5B and pBac25 were derived from a derivative of pBlueBacIII (Invitrogen) and were constructed to transfer the genes encoding NS4B and other non-structural HCV proteins into baculovirus expression vectors. These plasmids are illustrated in a schematic diagram in FIG. 1, and their construction is described in detail in example 8. The selected cDNA fragments corresponding to the genome of the HCV-BK isolate (HCV-BK; Takamizawa, A., Mori, C., Fuke, I., Manabe, S., Murakami, S., Fujita, J., Onishi, E., Andoh, T., Yoshida, I., and Okayama, H., (1991) the structure and organization of the hepatitis C virus genome isolated from human carriers, [ J.Virol. ] 65, 1105-1113) were cloned under the strong promoter of the polyhedrin of nuclear polyhedrosis virus and flanked by sequences allowing homologous recombination in one baculovirus vector.

To construct pBac5B, a PCR product containing the cDNA region encoding amino acids 2420 to 3010 of the HCV polyprotein and corresponding to the NS5B protein (SEQ ID NO: 1) was cloned between the BamHI and HindIII sites of pBlueBacIII. The PCR sense oligonucleotide contains a translation initiation signal and the original HCV stop codon serves as a termination of translation.

pBac25 is a derivative of pBlue BacIII (Invitrogen) in which the cDNA region encoding amino acids 810 to 3010 (SEQ ID NO: 2) of the HCV-BK polyprotein was cloned between NcoI and HindIII restriction sites.

Spodoptera frugiperda clone 9(Sf9) cells and baculovirus recombination kit were purchased from Invitrogen. Cells were cultured on plates or in suspension at 27 ℃ in complete Grace insect Medium (Gibco) containing 10% fetal bovine serum (Gibco). Transfection, recombination and selection of baculovirus constructs were performed according to the manufacturer's recommendations. Two recombinant baculovirus clones containing the desired HCV cDNA were isolated, Bac25 and Bac 5B.

For protein expression, recombinant baculovirus Bac25 or Bac5B was used at 2X 10 per ml⁶Sf9 cells were transfected at a density of cells with a proportion of approximately 5 viral particles per cell. Sf9 cells were pelleted 48-72 hours after transfection and washed with phosphate buffer (C)PBS) were washed once and carefully resuspended (7.5 × 10 per ml)⁷Individual cells) buffer A containing 1mM Dithiothreitol (DTT), 1mM phenylmethylsulfonyl fluoride (PMSF, Sigma) and 4mg/ml leupeptin (10mM tris/Cl pH8, 1.5mM MgCl₂10mM NaCl). All the following steps were performed on ice: after swelling for 30 minutes, the cells were broken up by 20 strokes in a Dounce homogenizer using a close-fitting pestle. Glycerol and detergents Nonidet P-40(NP40) and 3- [ (3-Cholamidopropyl) -diethylammonium were added]-1-propanesulfonic acid (CHAPS) to final concentrations of 10% (v/v), 1% (v/v) and 0.5% (v/v), respectively, and incubating the cell extract on ice for a further 1 hour with occasional stirring. Nuclei were pelleted by centrifugation at 1000 Xg for 10 min and the supernatant was collected. The pellet was resuspended in buffer A containing glycerol and detergent at the above concentrations (every 7.5X 10) by 20 strokes in a Dounce homogenizer⁷Core 0.5 ml). Then incubated on ice for 1 hour. After re-precipitation of the nuclei, the two supernatants were mixed and centrifuged at 8000 Xg for 10 min, and the precipitate was discarded. The crude cytoplasmic extract obtained was either used directly to measure the activity of RdRp or further purified with a sucrose gradient (see example 5).

Infection of Sf9 cells with recombinant baculoviruses Bac25 or Bac5B resulted in the expression of the expected HCV proteins. In fact, after infection of Sf9 cells with Bac25, NS2(24kDa), NS3(68kDa), NS4B (26kDa), NS4A (6kDa), NS5A (56kDa) and NS5B (65kDa) proteins of HCV were detectable in cell lysates by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunostaining. After infection of Sf9 cells with Bac5B, only one HCV-encoded protein was detectable by immunostaining or coomassie blue staining after SDS-PAGE, the size of which corresponded to authentic NS5B (65 kDa).

Example 2 method for detecting recombinant HCV RdRp on a synthetic RNA template/substrate

The RdRp detection is based on the detection of labeled nucleotides incorporated into the nascent RNA product. The activity of RdRp was determined in vitro in a total volume of 40ul containing 1-5ul Sf9 crude cytoplasmic extract or purified protein fraction. Is BacUnpurified or purified cytoplasmic extracts of 25 or Bac5B infected Sf9 cells can be used as a source of HCV RdRp. Extracts obtained from Sf9 cells infected with recombinant baculovirus constructs expressing one protein unrelated to HCV served as negative controls. The following additions were added to the reaction mixture (final concentrations): 20mM Tris/Cl pH7.5, 5mM MgCl₂1mM DTT, 25mM KCl, 1mM EDTA, 5-10. mu. Ci³²P]NTP (GTP, 3000Ci/mmol, Amersham used unless otherwise stated), 0.5mM each NTP (i.e., CTP, UTP, ATP, unless otherwise stated), 20U RNase (Promega), 0.5ug RNA-substrate (about 4pmol, 100nM final concentration), 2 ug actinomycin D (Sigma). The reaction was incubated at room temperature for two hours and stopped by adding an equal volume of 2 Xproteinase K (PK, Boehringer Mannheim) buffer (300mM NaCl, 100mM Tris/Cl pH7.5, 1% w/v SDS) followed by treatment with 50ug PK at 37 ℃ for half an hour. RNA products were extracted with PCA, ethanol precipitated and analyzed by electrophoresis on a 5% polyacrylamide gel containing 7M urea.

The RNA substrate (D-RNA) we used for the detection has the sequence reported in SEQ ID No. 12 and is typically obtained by in vitro transcription of the linearized plasmid pT7-7(DCoH) with T7 polymerase as described below.

Plasmid pT7-7(DCoH) (FIG. 2) was linearized with a single Bg1II restriction site contained at the end of the DCoH coding sequence and transcribed in vitro with T7 polymerase (Stratagene) using the procedure described by the manufacturer. Transcription was terminated by adding 5U/10. mu.l of DNase I (Promega). The mixture was incubated for a further 15 minutes and extracted with phenol/chloroform/isoamyl alcohol (PCA). Unincorporated nucleotides were removed by gel filtration through a 1-ml Sephadex G50 spin column. After extraction of PCA and ethanol precipitation, RNA was blown dry, dissolved in water and its concentration determined by optical density at 260 nm.

Any other RNA molecule besides D-RNA can be used for the RdRp detection of the invention, as will be clear from the experiments described below.

The above-described HCV RdRp assay produces a characteristic profile of the radiolabelled reaction product: a labeled product, which co-migrates with the substrate RNA observed in all reactions including the negative control. This RNA was also made visible by silver staining and was therefore considered to be associated with the added substrate RNA and most likely to be labelled by terminal nucleotidyl transferase activity present in cytoplasmic extracts of baculovirus-infected Sf9 cells. In reactions carried out with cytoplasmic extracts of Sf9 cells infected with Bac25 or Bac5B, but not with cytoplasmic extracts of cells infected with recombinant baculovirus expressing a protein unrelated to HCV, an additional band was observed that migrated faster than the substrate RNA. This latter reaction product was found to be labeled as a highly specific activity, since it did not appear in the control reaction without the addition of RNA. This product was found to be derived from the added RNA template, as it was not present in the control reaction without RNA addition. Interestingly, the formation of labeled samples migrating faster than the substrate RNA was observed with a variety of template RNA molecules, whether or not they contained the HCV 3' -untranslated region. The 399 nucleotide mRNA (D-RNA) of the liver-specific transcription helper DCoH has been shown to be a well-accepted substrate in our RdRp assay.

To determine the nature of the nascent samples produced by extracts of Bac25 or Bac5B infected cells in the reaction, we performed the following series of experiments: (i) the product mixture was treated with rnase a or nuclease P1. Since this resulted in the complete disappearance of the radioactive band, we concluded that both of these labeled products were RNA molecules. (ii) Omission of any of the four nucleotide triphosphates in the reaction mixture resulted in labeling of only the input RNA, indicating that the fast migrating sample was the product of the polymerization reaction. (iii) Omission of Mg in detection²⁺The ions lead to complete blocking of the reaction: neither synthesis of nascent RNA nor labeling of input RNA was observed. (iv) When detected with a radiolabeled input RNA and unlabeled nucleotides, the labeled products are indistinguishable from those obtained under standard conditions. From this result we conclude that the nascent RNA product was produced from the initially added RNA molecule.

In summary, our data indicate that extracts from Bac25 or Bac5B infected Sf9 cells contain a novel magnesium ion dependent enzyme activity that catalyzes RNA synthesis. This activity was shown to be dependent on the presence of the input RNA but not on the source of the added primer or input RNA molecule. In addition, since the products produced by extracts of Sf9 cells infected with Bac25 or Bac5B appear to be identical, the experiments just described indicate that the observed RdRp activity is encoded by the HCV NS5B protein.

Example 3 method for identifying HCV RdRp RNA products.

To elucidate the structural features of the newly synthesized RNA product, the following procedure was used. Under our standard electrophoresis conditions (5% polyacrylamide, 7M urea), the size of the nascent RNA product appears to be about 200 nucleotides. This may be due to internal initiation or premature termination of RNA transcription. However, these possibilities seem to be highly unlikely, since it was found that the RdRp assays performed with different RNA substrates all gave products that migrated significantly faster than their respective substrates. Increasing the temperature during electrophoresis and the concentration of acrylamide in the analysis gel resulted in significantly different migration behavior of the RdRp product. For example, using a gel system such as one containing 10% acrylamide, 7M urea, and performing the separation at a higher temperature, the RdRp product migrates slower than the added substrate RNA, at a position corresponding to at least twice the length of the input RNA. When an RNA denaturant such as hydroxymethyl mercury (CH) is added to the RdRp product on a low percentage/low temperature gel prior to electrophoresis₃HgOH, 10mM), a similar effect can be observed. These observations indicate that the RdRp product has extensive secondary structure.

We investigated the sensitivity of the product molecules to a number of ribonucleases of different specificities. The product was completely degraded by treatment with rnase a. On the other hand, it was surprisingly found that it is resistant to the single strand specific nuclease rnase T1. Complete degradation of input RNA after incubation with 60U RNase T1 at 22 ℃ for 10 minutes, silver staining of the same gel confirmed that: not only the template but also other RNAs routinely detectable in cytoplasmic extracts of Sf9 cells were completely hydrolyzed during incubation with rnase T1. While the RdRp product remains unchanged, it is affected only after a long incubation with RNase T1. For example, after 2 hours of treatment with rnase T1, the labeled product molecule can no longer be detected in its original position in the gel. And a new band that electrophoretically migrates similarly to the input template RNA appears. Similar effects were observed with rnase T1 but at different temperatures for 1 hour of digestion: at 22 ℃, the RdRp product remains largely unaffected, while at 37 ℃ it converts to a new product that co-migrates with the original substrate.

The explanation for these observations is that the input RNA serves as a template for HCV RdRp, where the 3' -OH serves to cause synthesis of the complementary strand by a turn-around or "reverse replication" mechanism to produce a double-stranded RNA "hairpin" molecule consisting of the sense (template) strand to which the antisense strand is covalently attached. Such a structure may explain the exceptional electrophoretic mobility of the RdRp product in polyacrylamide gels and its high resistance to single strand specific nucleases. The turn-around loop may not be base paired and should be accessible to nucleases. Treatment with rnase T1 then results in hydrolysis of the covalent bond between the sense and antisense strands to produce a double-stranded RNA molecule. During denaturing gel electrophoresis, the two strands separate and only the newly synthesized antisense strand, similar in length to the original RNA template, remains detectable. This mechanism seems to be more likely, especially considering the fact that this product is produced in vitro by several other RNA polymerases.

To demonstrate that the RNA product labeled during the polymerase reaction and significantly released by treatment with RNase T1 behaves in an antisense orientation to the added template, the following experiment was designed. For this purpose, we synthesized oligodeoxynucleotides corresponding to three independent sequences of the input template RNA molecule (fig. 2): oligonucleotide a (SEQ ID NO: 3) corresponding to nucleotide 170-195 of D-RNA; oligonucleotide b (SEQ ID NO: 4) complementary to nucleotide 286-; and oligonucleotide c (SEQ ID NO: 5) complementary to nucleotide 331 and 354. These are used to form DNA/RNA hybrids with the products of the polymerase reaction in order to digest them with RNase H. Initially, all RdRp products were used for hybridization. However, since this structure is very thermostable, no specific hybridization is formed, and thus hairpin RNA is pretreated with RNase T1 and denatured by boiling for 5 minutes. Then cooled to room temperature in the presence of the respective oligonucleotides. As expected, exposure of the hybrids to RNase H produced specific cleavage products. Oligonucleotide a directed cleavage results in products of about 170 and 200 nucleotides in length, oligonucleotide b produces products of about 290 and 110 nucleotides and oligonucleotide c produces fragments of about 330 and 65 nucleotides. Since these fragments were of the expected size (see FIG. 3), the results suggest that HCV NS 5B-mediated RNA synthesis proceeds via a replication-reverting mechanism that produces hairpin-like RNA duplexes.

EXAMPLE 4 detection of recombinant HCV TNTase on a synthetic RNA substrate

The TNTase assay is based on the detection of a labeled nucleotide that is not template-dependent for incorporation into the 3' hydroxyl group of an RNA substrate. The RNA substrate (D-RNA) used for the detection was typically obtained by in vitro transcription of the linearized plasmid pT7-7DCoH with T7 polymerase as described in example 2. However, any other RNA molecule than D-RNA is used for the TNTase assay of the present invention.

TNTase activity was determined by in vitro assays in a total volume of 40. mu.l containing 1-5. mu.l of crude cytoplasmic extract of Sf9 or purified protein fractions. An uninsulated or purified cytoplasmic extract of Bac25 or Bac5B infected Sf9 cells can be used as a source of HCV TNTase. Sf9 cell extracts obtained from Sf9 cells infected with a recombinant baculovirus expressing a protein unrelated to HCV were used as a negative control. The following additions were added to the reaction mixture (final concentrations): 20mM Tris/ClpH7.5, 5mM MgCl₂1mM DTT, 25mM KCl, 1mM EDTA, 5-10. mu. Ci³²P]NTP (used as UTP, 3000Ci/mmol, Amersham, unless otherwise stated), 20U RNase (Promega), 0.5. mu.g of gRNA-substrate (about 4pmol, 100nM final concentration), 2. mu.g of actinomycin D (Sigma). The reaction was incubated at room temperature for two hours by adding an equal volume of 2 Xproteinase K (PK, Boehringer Mannheim) buffer (300mM NaCl, 100mM Tris/Cl pH7.5, 1% w/v SDS) followed by 50. mu.L SDS at 37 ℃gPK was treated for half an hour to terminate the reaction. RNA products were extracted with PCA, ethanol precipitated and analyzed by electrophoresis on a 5% polyacrylamide gel containing 7M urea.

Example 5 purification of HCV RdRp/TNTase by sucrose gradient sedimentation

A linear 0.3-1.5M sucrose gradient was prepared in buffer A (see example 1) containing detergent. Up to 2ml of an extract of Bac5B or Bac25 infected Sf9 cells (equivalent to about 8X 10)⁷Individual cells) were loaded onto a 12ml gradient. Centrifugation was carried out using a Beckman SW40 rotor at 39000 Xg for 20 hours. Fractions of 0.5ml were collected and assayed for activity. The NS5B protein identified by western blotting was found to migrate in the density gradient with an unexpectedly high sedimentation coefficient. Viral proteins and nucleoprotein bodies were found to co-settle in the same gradient fractions. This unique behavior allows us to separate viral proteins from the major part of the cytoplasmic proteins that remain in the upper part of the gradient. Detection of RdRp activity revealed that RdRp activity co-precipitated with NS5B protein. A terminal nucleotidyl transferase activity (TNTase) is also present in these fractions.

Example 6 method for purification of HCV TNTase/RdRp from Sf9 cells

Whole cell extracts were prepared from 1g of Sf9 cells infected with Bac5B recombinant baculovirus. Frozen cells were thawed on ice in 10ml of buffer containing 20mM Tris/HCl pH7.5, 1mM EDTA, 10mM DTT, 50% glycerol (N buffer) supplemented with 1mM PMSF. To facilitate cell disruption, Triton X-100 and NaCl were then added to final concentrations of 2% and 500mM, respectively. After adding MgCl₂(10mM) and DNase I (15ug/ml), the mixture was stirred at room temperature for 30 minutes. The extract was then clarified by ultracentrifugation at 40,000rpm for 30 minutes at 4 ℃ in a Beckman centrifuge using a 90Ti rotor. To adjust the NaCl concentration to 300mM, the clarified extract was diluted with a buffer containing 20mM Tris/HCl pH7.5, 1mM EDTA, 10mM DTT, 20% glycerol, 0.5% Triton X-100(LG buffer) and incubated in bulk with 5ml DEAE-Sepharose Fast Flow equilibrated in LG buffer containing 300mM NaCl. The matrix was then poured onto a column and washed with twice the volume of the same buffer. The effluent and the first wash of a DEAE-Sepharose Fast Flow column were diluted 1: 3 with LG buffer and added to a heparin-Sepharose CL6B column (10ml) equilibrated with LG buffer containing 100mM NaCl. heparin-Sepharose C16B was washed thoroughly and bound proteins were eluted with a linear 100ml gradient of 100mM to 1M NaCl in LG buffer. According to the identification of silver staining and immunostaining by SDS-PAGE, fractions containing NS5B were mixed and diluted with LG buffer to adjust the NaCl concentration to 50 mM. The diluted fractions were then applied to a Mono Q-FPLC column (1ml) equilibrated with LG buffer containing 50mM NaCl. The protein was eluted with a linear gradient (20ml) from 50mM to 1M NaCl in LG buffer. According to the identification of silver staining and immunostaining by SDS-PAGE, fractions containing NS5B were mixed and dialyzed against LG buffer containing 100mM NaCl. After extensive dialysis, the pooled fractions were applied to PoyU-Sepharose CL6B (10ml) equilibrated with LG buffer containing 100mM NaCl. PoyU-Sepharose CL6B was washed thoroughly and bound proteins were eluted with a linear 100ml gradient from 100mM to 1M NaCl in LG buffer. According to the identification of silver staining and immunostaining by SDS-PAGE, the fractions containing NS5B were pooled, dialyzed against LG buffer containing 100mM NaCl and stored in liquid nitrogen before activity detection.

Fractions containing purified NS5B protein were tested for the presence of both activities. In the same fractions, RdRp and TNTase activity were found. These results suggest that both RNA-dependent RNA polymerase and terminal nucleotidyl transferase activities are functions of the HCV NS5B protein.

We tested the terminal nucleotidyl transferase activity of purified NS5B at unsaturated substrate concentrations using each of the 4 nucleotide triphosphates. The results clearly show that UTP is the preferred TNTase substrate, followed by ATP, CTP and GTP, regardless of the source of the input RNA.

Example 7 detection of recombinant HCV RdRp on a homopolymeric RNA template

We have described to date that HCV NS5B has an RNA-dependent RNA polymerase activity and that synthesis of the complementary RNA strand is a template-initiated reaction. Interestingly, using the unseparated cytoplasmic extract of Bac5B or Bac25 infected Sf9 cells as a source of RdRp, we could not observe the synthesis of complementary strand RNA using an exogenously added oligonucleotide as a primer. We explain that this may be due to the large amount of ATP-dependent RNA duplex enzyme that is positively present in our non-isolated extract. We therefore wanted to answer this question with purified NS 5B.

First, we wanted to determine if purified NSSB polymerase could synthesize RNA in a primer-dependent manner on a homotypic poly-RNA template that could not form an intramolecular hairpin, and therefore we wanted that complementary strand RNA synthesis be strictly primer-dependent. Thus we determined the poly (A) template dependent UMP infiltration. And evaluated oligo (rU)₁₂And oligo (dT)_12-18As primers for the polymerase reaction. The infiltration of radioactive UMP was determined as follows. In a medium containing 20mM Tris/HClpH7.5, 5mM MgCl₂、1mM DTT、25mM KCl、1mM EDTA、20U RNasin(Promega)、1μCi[³²P]UTP (400Ci/mmol, Amersham) or 1. mu. Ci³H]UTP (55Ci/mmol, Amersham), 10. mu.MUTP and 10ug/ml poly (A) or poly (A)/oligo (dT)_12-18The standard reaction (10-100. mu.l) was performed in the buffer of (1). Adding oligo (U)₁₂(1. mu.g/ml) was used as a primer. PolyA and PolyA/oligo (dT)_12-18Purchased from Pharmacia. oligo (U)₁₂Obtained from Genset. The final NS5B enzyme concentration was 10-100 nM. The reaction proceeds straight through for up to 3 hours under these conditions. After 2 hours incubation at 22 ℃ the reaction was stopped by adding the sample to a DE81 filter (Whatman) and 1M Na₂HPO₄/NaH₂PO₄The filters were rinsed thoroughly at pH7.0, rinsed with water, blown dry and the filter-bound radioactivity was determined on a scintillation beta counter. In addition, in 0.2M sodium pyrophosphate by 10% trichloroacetic acid and 100ug carrier tRNA precipitation in vitro synthesis of radioactive products, collected in 0.45um Whatman GF/C filter, vacuum drying and scintillation liquid counting.

Although some are in the absence of a primer³²P]UMP or [ 2 ]³H]UMP incorporation was detectable and probably due to terminal nucleotidyl transferase activity associated with our purified NS5B, but only when oligo (rU) was added to the reaction mixture₁₂As primers up to 20% product infiltration was observed. Unexpectedly, oligo (dT)_12-18Although less efficient, it also functions as a primer for poly (A) -dependent poly (U) synthesis.

Other templates/primers suitable for determining RdRp activity of NS5B include poly (C)/oligo (G) or poly (C)/oligo (dG) in the presence of radioactive GTP, poly (G)/oligo (C) or poly (G)/oligo (dC) in the presence of radioactive CTP, poly (U)/oligo (A) or poly (U)/oligo (dA) in the presence of radioactive ATP, poly (I)/oligo (C) or poly (I)/oligo (dC) in the presence of radioactive CTP.

Example 8 method for expressing HCV RdRp/TNTase in E.coli

In order to allow expression of the HCV protein fragment having the sequence reported in SEQ ID NO. 1 in E.coli, plasmid pT7-7(NS5B) described in FIG. 2 and example 8 was constructed. This protein fragment contains RdRp and TNTase of NS5B as discussed above. The HCV eDNA fragment encoding the NS5B protein was then cloned downstream of the phage T7 φ 10 promoter and in frame with the 1 st ATG codon of the phage T7 gene 10 protein using methods known in molecular biology practice and described in detail in example 8. The pT7-7(NS5B) plasmid also contained a selectable marker that could be used as a selection marker for E.coli cells transformed with the plasmid pT7-7(NS 5B).

The plasmid pT7-7(NS5B) was then transformed into E.coli strain BL21(DE53), which was generally used for high level expression of genes cloned into an expression vector containing the T7 promoter. In this strain of E.coli, phage 1 DE53 carries a T7 gene polymerase, which is integrated into the chromosome of BL21 cells (Studier and Moffatt, use of phage T7RNA polymerase to direct selective high-level expression of cloned genes, (1986), J.Mol.biol., 189, page 113-130). Expression of the gene of interest was induced by addition of Isopropylthiogalactoside (IPTG) in the growth medium according to the method already described (Studier and Maffatt). Recombinant NS5B protein fragments containing RdRp are then produced in inclusion bodies of the host cells. The recombinant NS5B protein can be purified from the pellet fraction of E.coli BL21(DE53) extracts and refolded according to methods known in the art (D.R. Thatcher and A.Hichcok, protein folding in Biotechnology (1994) protein folding Mechanism (Mechanism of protein folding) eds R.H.Pain, TRL PRESS, p. 229-255). Recombinant NS5B protein can also be produced as a soluble protein by lowering the temperature of the bacterial growth medium to below 20 ℃. Soluble proteins can then be purified from E.coli lysates essentially as described in example 5.

Example 9 detailed construction of plasmids in the accompanying drawings

The selected cDNA fragment corresponding to the genome of the HCV-BK isolate (HCVBK) was cloned under the strong promoter of nucleopolyhedrosis of the nucleopolyhedrosis virus and flanked by sequences allowing homologous recombination in a baculovirus.

pBac5B contains the HCV-BK sequence comprised between nucleotides 7590 and 9366 and encodes the NS5B protein reported in SEQ ID NO. 1. To obtain this plasmid, cDNA fragments were generated by PCR using synthetic oligonucleotides having the sequences 5 '-AAGGATCCATGTCAATGTCCTACACATGGAC-3' (SEQ ID NO: 6) and 5 '-AATATTCGAATTCATCGGTTGGGGAGCAGGTAGATG-3' (SEQ ID NO: 7), respectively. The PCR product was then treated with Klenow DNA polymerase, digested at the 5' end with BamHI, and subsequently cloned between the BamHI and SmaI sites of Bluescript SK (+) vector. Subsequently, the objective cDNA fragment was completely digested with restriction enzymes BamHI and HindIII and religated at the same site in pBlueBacIII vector (Invitrogen).

pBac25 contains the HCV-BK cDNA region contained between nucleotides 2759 to 9416 and encodes amino acids 810 to 3010 of the HCV-BK polyprotein (SEQ ID NO: 2). The plasmid was obtained according to the following method. First, an 820bp cDNA fragment containing the HCV-BK sequence included between nucleotides 2759-3578 was obtained from pCD (38-9.4) (Tomei, L., Faillea, C., Santolini, E., De France sco, R., and La Monica, N. (1993) NS3, a serine protease required for the processing of hepatitis C virus polyprotein, by NcoI digestion, and cloned in the NcoI site of pBlueBacIII vector (Invitrogen) to give a plasmid called pBacNCO. A cDNA fragment containing the HCV-BK sequence included between nucleotide 1959 and 9461 was obtained from pCD (38-9.4) (Tomei et al, 1993) by digestion with NotI and XbaI and cloned in the same site of Bluescript SK (+) vector to generate a plasmid called pBlsNX. A cDNA fragment containing the HCV-BK sequence included between nucleotides 3304 to 9461 was obtained from pBlsNX by SacII and HindIII digestion and cloned at the same site on the pBlsNX plasmid to produce the pBac25 plasmid.

pT7-7(DCoH) contains the entire coding region (316 nucleotides) for the dimerization cofactor of rat hepatocyte nuclear factor 1a _ (DCoH; Mendel, D.B., Khavari, P.A., Conley, P.B., Graves, M.K., Hansen, L.P., Admon, A. and Crabtree, G.R. (1991) for the identification of an cofactor that modulates dimerization of mammalian homeodomain proteins, Science 254, 1767 @, gene bank accession number M83740). A cDNA fragment corresponding to the coding sequence of rat DCoH was amplified by PCR using synthetic nucleotides Dprl and Dpr2 having the sequences TGGCTGGCAAGGCACACAGGCT (SEQ ID NO: 8) and AGGCAGGGTAGATCTATGTC (SEQ ID NO: 9), respectively. The cDNA fragment thus obtained was cloned into the SmaI restriction site of E.coli expression vector pT 7-7. The pT7-7 expression vector is a derivative of pBR322 which contains, in addition to the beta-lactamase gene and the Col E1 origin of replication, the T7 polymerase promoter phi 10 and the translation initiation site of the T7 gene 10 protein (Tabor S. and Richardson C.C (1985) a T7 phage RNA polymerase/promoter system for the controlled unique expression of specific genes, Proc. Natl. Acad. Sci. USA 82, 1074-1078).

pT7-7(NS5B) contains the HCV sequence from nucleotide 7590 to 9366 and encodes the NS5B protein reported in SEQ ID NO. 1.

To obtain this plasmid, a cDNA fragment was generated by PCR using synthetic oligonucleotides having the sequences 5 '-TCAATGTCCTACACATGGAC-3' (SEQ ID NO: 10) and 5 '-GATCTCTAGATCATCGGTTGGGGGAGGAGGTAGATGCC-3' (SEQ ID NO: 11), respectively. The PCR product was then treated with Klenow DNA polymerase. This was subsequently ligated into the E.coli expression vector pT7-7 after linearization with EcoRI and filling in the ends with Klenow DNA polymerase. In addition, cDNA fragments were generated by PCR using synthetic oligonucleotides containing the sequences 5 '-TGTCAATGTCCTACACATGG-3' (SEQ ID NO: 13) and 5 '-AATATTCGAATTCATCGGTTGGGGAGCAGGTAGATG-3' (SEQ ID NO: 14), respectively. The PCR product was then treated with Klenow DNA polymerase and subsequently ligated into E.coli expression vector pT7-7 after NdeI linearization and filling in the ends with Klenow DNA polymerase.

Sequence listing

General information:

(i) the applicant: ISTITUTO DI RICERCHE DI biologica molecolar p.angele tti s.p.a.

(ii) The invention provides a subject: method for reproducing in vitro the RNA-dependent RNA polymerase and terminal nucleotidyl transferase activities encoded by Hepatitis C Virus (HCV)

(iii) Sequence number: 14

(iv) Contact address:

(A) the addressee: societa Italiana Brevetti

(B) Street Piazza di Pietra

(C) City: rome

(D) The state is as follows: italy (chemical vapor deposition)

(E) And (3) post code: 1-00186

(V) computer readable form:

(A) media type: 3.5' 1.44 Mbyte floppy disk

(B) A computer: IBM PC compatibility

(C) Operating the system: PC-DOS/MS-DOS Rev.6.22

(D) Software: microsoft Word 6.0

(viii) Lawyer/attorney information:

(A) name: DI CERBO, Mario (Dr.)

(C) Reference numbers: RM/X88530/PCT-DC

(ix) Communication information:

(A) telephone: 06/6785941

(B) Faxing: 06/6794692

(C) Electric transmission: 612287 ROPAT (1) data for sequence number 1:

(i) sequence characteristics: length (A): 591 amino acids (B) type: amino acid (C) linear: single chain (D) topology: linear (ii) molecular type: protein (iii) pseudo structure: (iii) no (iv) antisense: no (v) fragment type: c-terminal fragment (vi) originally derived: (A) an organism: hepatitis c virus (B) isolate: bk (vii) direct source: the pCD (38-9.4) (ix) feature of the cDNA clone described by Tomei et al, 1993: (A) name: NS5B non-structural polyprotein (C) identification method: description of the experimental (xi) sequence: sequence number 1Ser Met Ser Tyr Thr Trp Thr Gly Ala Leu Ile Thr Pro Cys Ala Ala 151015 Glu Glu Ser Lys Leu Pro Ile Asn Ala Leu Ser Asn Sar Leu Arg

20 25 30His His Asn Met Val Tyr Ala Thr Thr Ser Arg Ser Ala Gly Leu Arg

35 40 45Gln Lys Lys Val Thr Phe Asp Arg Leu Gln Val Leu Asp Asp His Tyr

50 55 60Arg Asp Val Leu Lys Glu Met Lys Ala Lys Ala Ser Thr Val Lys Ala65 70 75 80Lys Leu Leu Ser Val Glu Glu Ala Cys Lys Leu Thr Pro Pro His Ser

85 90 95Ala Lys Ser Lys Phe Gly Tyr Gly Ala Lys Asp Val Arg Asn Leu Ser

100 105 110Ser Lys Ala Val Asn His Ile His Ser Val Trp Lys Asp Leu Leu Glu

115 120 125Asp Thr Val Thr Pro Ile Asp Thr Thr Ile Met Ala Lys Asn Glu Val

130 135 140Phe Cys Val Gln Pro Glu Lys Gly Gly Arg Lys Pro Ala Arg Leu Ile145 150 155 160Val Phe Pro Asp Leu Gly Val Arg Val Cys Glu Lys Met Ala Leu Tyr

165 170 175Asp Val Val Ser Thr Leu Pro Gln Val Val Met Gly Ser Ser Tyr Gly

180 185 190Phe Gln Tyr Ser Pro Gly Gln Arg Val Glu Phe Leu Val Asn Thr Trp

195 200 205Lys Ser Lys Lys Asn Pro Met Gly Phe Ser Tyr Asp Thr Arg Cys Phe

210 215 220Asp Ser Thr Val Thr Glu Asn Asp Ile Arg Val Glu Glu Ser Ile Tyr225 230 235 240Gln Cys Cys Asp Leu Ala Pro Glu Ala Arg Gln Ala Ile Lys Ser Leu

245 250 255Thr Glu Arg Leu Tyr Ile Gly Gly Pro Leu Thr Asn Ser Lys Gly Gln

260 265 270Asn Cys Gly Tyr Arg Arg Cys Arg Ala Ser Gly Val Leu Thr Thr Ser

275 280 285Cys Gly Asn Thr Leu Thr Cys Tyr Leu Lys Ala Ser Ala Ala Cys Arg

290 295 300Ala Ala Lys Leu Gln Asp Cys Thr Met Leu Val Asn Gly Asp Asp Leu305 310 315 320Val Val Ile Cys Glu Ser Ala Gly Thr Gln Glu Asp Ala Ala Ser Leu

325 330 335Arg Val Phe Thr Glu Ala Met Thr Arg Tyr Ser Ala Pro Pro Gly Asp

340 345 350Pro Pro Gln Pro Glu Tyr Asp Leu Glu Leu Ile Thr Ser Cys Ser Ser

355 360 365Asn Val Ser Val Ala His Asp Ala Ser Gly Lys Arg Val Tyr Tyr Leu

370 375 380Thr Arg Asp Pro Thr Thr Pro Leu Ala Arg Ala Ala Trp Glu Thr Ala385 390 395 400Arg His Thr Pro Val Asn Ser Trp Leu Gly Asn Ile Ile Met Tyr Ala

405 410 415Pro Thr Leu Trp Ala Arg Met Ile Leu Met Thr His Phe Phe Ser Ile

420 425 430Leu Leu Ala Gln Glu Gln Leu Glu Lys Ala Leu Asp Cys Gln Ile Tyr

435 440 445Gly Ala Cys Tyr Ser Ile Glu Pro Leu Asp Leu Pro Gln Ile Ile Glu

450 455 460Arg Leu His Gly Leu Ser Ala Phe Ser Leu His Ser Tyr Ser Pro Gly465 470 475 480Glu Ile Asn Arg Val Ala Ser Cys Leu Arg Lys Leu Gly Val Pro Pro

485 490 495Leu Arg Val Trp Arg His Arg Ala Arg Ser Val Arg Ala Arg Leu Leu

500 505 510Ser Gln Gly Gly Arg Ala Ala Thr Cys Gly Lys Tyr Leu Phe Asn Trp

515 520 525Ala Val Lys Thr Lys Leu Lys Leu Thr Pro Ile Pro Ala Ala Ser Arg

530 535 540Leu Asp Leu Ser Gly Trp Phe Val Ala Gly Tyr Ser Gly Gly Asp Ile545 550 555 560Tyr His Ser Leu Ser Arg Ala Arg Pro Arg Trp Phe Met Leu Cys Leu

565 570 575Leu Leu Leu Ser Val Gly Val Gly Ile Tyr Leu Leu Pro Asn Arg

580585590 (2) data of sequence number 2:

(i) sequence characteristics:

(A) length: 2201 amino acids

(B) Type (2): amino acids

(C) Line type: single strand

(D) Topological structure: linearity

(ii) Molecular type: polypeptides

(iii) Pseudo structure: whether or not

(iv) Antisense: whether or not

(v) Fragment type: c-terminal fragment

(vii) The direct source is as follows: the cDNA clone pCD (38-9.4) described by Tomei et al, 1993

(ix) The method is characterized in that:

(A) name: NS2-NS5B precursor of nonstructural protein

(C) The identification method comprises the following steps: experimental results

(xi) Description of the sequence: sequence number 2Met Asp Arg Glu Met Ala Ala Ser Cys Gly Gly Ala Val Phe Val Gly 151015 Leu Val Leu Leu Thr Leu Ser Pro Tyr Tyr Lys Val Phe Leu Ala Arg

20 25 30Leu Ile Trp Trp Leu Gln Tyr Phe Thr Thr Arg Ala Glu Ala Asp Leu 35 40 45His Val Trp Ile Pro Pro Leu Asn Ala Arg Gly Gly Arg Asp Ala Ile

50 55 60Ile Leu Leu Met Cys Ala Val His Pro Glu Leu Ile Phe Asp Ile Thr65 70 75 80Lys Leu Leu Ile Ala Ile Leu Gly Pro Leu Met Val Leu Gln Ala Gly

85 90 95Ile Thr Arg Val Pro Tyr Phe Val Arg Ala Gln Gly Leu Ile His Ala

100 105 110Cys Met Leu Val Arg Lys Val Ala Gly Gly His Tyr Val Gln Met Ala

115 120 125Phe Met Lys Leu Gly Ala Leu Thr Gly Thr Tyr Ile Tyr Asn His Leu

130 135 140Thr Pro Leu Arg Asp Trp Pro Arg Ala Gly Leu Arg Asp Leu Ala Val145 150 155 160Ala Val Glu Pro Val Val Phe Ser Asp Met Glu Thr Lys Ile Ile Thr

165 170 175Trp Gly Ala Asp Thr Ala Ala Cys Gly Asp Ile Ile Leu Gly Leu Pro

180 185 190Val Ser Ala Arg Arg Gly Lys Glu Ile Leu Leu Gly Pro Ala Asp Ser

195 200 205Leu Glu Gly Arg Gly Leu Arg Leu Leu Ala Pro Ile Thr Ala Tyr Ser

210 215 220Gln Gln Thr Arg Gly Leu Leu Gly Cys Ile Ile Thr Ser Leu Thr Gly225 230 235 240Arg Asp Lys Asn Gln Val Glu Gly Glu Val Gln Val Val Sar Thr Ala

245 250 255Thr Gln Ser Phe Leu Ala Thr Cys Val Asn Gly Val Cys Trp Thr Val

260 265 270Tyr His Gly Ala Gly Ser Lys Thr Leu Ala Ala Pro Lys Gly Pro Ile

275 280 285Thr Gln Met Tyr Thr Asn Val Asp Gln Asp Leu Val Gly Trp Pro Lys

290 295 300Pro Pro Gly Ala Arg Ser Leu Thr Pro Cys Thr Cys Gly Ser Ser Asp305 310 315 320Leu Tyr Leu Val Thr Arg His Ala Asp Val Ile Pro Val Arg Arg Arg

325 330 335Gly Asp Ser Arg Gly Ser Leu Leu Ser Pro Arg Pro Val Ser Tyr Leu

340 345 350Lys Gly Ser Ser Gly Gly Pro Leu Leu Cys Pro Phe Gly His Ala Val

355 360 365Gly Ile Phe Arg Ala Ala Val Cys Thr Arg Gly Val Ala Lys Ala Val

370 375 380Asp Phe Val Pro Val Glu Ser Met Glu Thr Thr Met Arg Ser Pro Val385 390 395 400Phe Thr Asp Asn Ser Ser Pro Pro Ala Val Pro Gln Ser Phe Gln Val

405 410 415Ala His Leu His Ala Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro

420 425 430Ala Ala Tyr Ala Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser

435 440 445Val Ala Ala Thr Leu Gly Phe Gly Ala Tyr Met Ser Lys Ala His Gly

450 455 460Ile Asp Pro Asn Ile Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ala465 470 475 480Pro Val Thr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys

485 490 495Ser Gly Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Set Thr

50θ 505 510Asp Ser Thr Thr Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu

515 520 525Thr Ala Gly Ala Arg Leu Val Val Leu Ala Thr Ala Thr Pro Pro Gly

530 535 540Ser Val Thr Val Pro His Pro Asn Ile Glu Glu Val Ala Leu Ser Asn545 550 555 560Thr Gly Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro Ile Glu Ala Ile

565 570 575Arg Gly Gly Arg His Leu Ile Phe Cys His Ser Lys Lys Lys Cys Asp

580 585 590Glu Leu Ala Ala Lys Leu Ser Gly Leu Gly Ile Asn Ala Val Ala Tyr

595 600 605Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Thr Ile Gly Asp Val Val

610 615 620Val Val Ala Thr Asp Ala Leu Met Thr Gly Tyr Thr Gly Asp Phe Asp625 630 635 640Ser Val Ile Asp Cys Asn Thr Cys Val Thr Gln Thr Val Asp Phe Ser

645 650 655Leu Asp Pro Thr Phe Thr Ile Glu Thr Thr Thr Val Pro Gln Asp Ala

660 665 670Val Ser Arg Ser Gln Arg Arg Gly Arg Thr Gly Arg Gly Arg Arg Gly

675 680 685Ile Tyr Arg Phe Val Thr Pro Gly Glu Arg Pro Ser Gly Met Phe Asp

690 695 700Ser Ser Val Leu Cys Glu Cys Tyr Asp Ala Gly Cys Ala Trp Tyr Glu705 710 715 720Leu Thr Pro Ala Glu Thr Ser Val Arg Leu Arg Ala Tyr Leu Asn Thr

725 730 735Pro Gly Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Ser Val

740 745 750Phe Thr Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr Lys

755 760 765Gln Ala Gly Asp Asn Phe Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val

770 775 780Cys Ala Arg Ala Gln Ala Pro Pro Pro Ser Trp Asp Gln Met Trp Lys785 790 795 800Cys Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro Thr Pro Leu Leu

805 810 815Tyr Arg Leu Gly Ala Val Gln Asn Glu Val Thr Leu Thr His Pro Ile

820 825 830Thr Lys Tyr Ile Met Ala Cys Met Ser Ala Asp Leu Glu Val Val Thr

835 840 845Ser Thr Trp Val Leu Val Gly Gly Val Leu Ala Ala Leu Ala Ala Tyr

850 855 860Cys Leu Thr Thr Gly Ser Val Val Ile Val Gly Arg Ile Ile Leu Ser865 870 875 880Gly Arg Pro Ala Ile Val Pro Asp Arg Glu Leu Leu Tyr Gln Glu Phe

885 890 895Asp Glu Met Glu Glu Cys Ala Ser His Leu Pro Tyr Ile Glu Gln Gly

900 905 910Met Gln Leu Ala Glu Gln Phe Lys Gln Lys Ala Leu Gly Leu Leu Gln

915 920 925Thr Ala Thr Lys Gln Ala Glu Ala Ala Ala Pro Val Val Glu Ser Lys

930 935 940Trp Arg Ala Leu Glu Thr Phe Trp Ala Lys His Met Trp Asn Phe Ile945 950 955 960Ser Gly Ile Gln Tyr Leu Ala Gly Leu Ser Thr Leu Pro Gly Asn Pro

965 970 975Ala Ile Ala Ser Leu Met Ala Phe Thr Ala Ser Ile Thr Ser Pro Leu

980 985 990Thr Thr Gln Ser Thr Leu Leu Phe Asn Ile Leu Gly Gly Trp Val Ala

995 1000 1005Ala Gln Leu Ala Pro Pro Ser Ala Ala Ser Ala Phe Val Gly Ala Gly1010 1015 1020Ile Ala Gly Ala Ala Val Gly Ser Ile Gly Leu Gly Lys Val Leu Val1025 1030 1035 1040Asp Ile Leu Ala Gly Tyr Gly Ala Gly Val Ala Gly Ala Leu Val Ala

1045 1050 1055Phe Lys Val Met Ser Gly Glu Met Pro Ser Thr Glu Asp Leu Val Asn

1060 1065 1070Leu Leu Pro Ala Ile Leu Ser Pro Gly Ala Leu Val Val Gly Val Val

1075 1080 1085Cys Ala Ala Ile Leu Arg Arg His Val Gly Pro Gly Glu Gly Ala Val1090 1095 1100Gln Trp Met Asn Arg Leu Ile Ala Phe Ala Ser Arg Gly Asn His Val1105 1110 1115 1120Ser Pro Thr His Tyr Val Pro Glu Sar Asp Ala Ala Ala Arg Val Thr

1125 1130 1135Gln Ile Leu Ser Ser Leu Thr Ile Thr Gln Leu Leu Lys Arg Leu His

1140 1145 1150Gln Trp Ile Asn Glu Asp Cys Ser Thr Pro Cys Ser Gly Ser Trp Leu

1155 1160 1165Arg Asp Val Trp Asp Trp Ile Cys Thr Val Leu Thr Asp Phe Lys Thr1170 1175 1180Trp Leu Gln Ser Lys Leu Leu Pro Gln Leu Pro Gly Val Pro Phe Phe1185 1190 1195 1200Ser Cys Gln Arg Gly Tyr Lys Gly Val Trp Arg Gly Asp Gly Ile Met

1205 1210 1215Gln Thr Thr Cys Pro Cys Gly Ala Gln Ile Thr Gly His Val Lys Asn

1220 1225 1230Gly Ser Met Arg Ile Val Gly Pro Lys Thr Cys Ser Asn Thr Trp His

1235 1240 1245Gly Thr Phe Pro Ile Asn Ala Tyr Thr Thr Gly Pro Cys Thr Pro Ser1250 1255 1260Pro Ala Pro Asn Tyr Ser Arg Ala Leu Trp Arg Val Ala Ala Glu Glu1265 1270 1275 1280Tyr Val Glu Val Thr Arg Val Gly Asp Phe His Tyr Val Thr Gly Met

1285 1290 1295Thr Thr Asp Asn Val Lys Cys Pro Cys Gln Val Pro Ala Pro Glu Phe

1300 1305 1310Phe Ser Glu Val Asp Gly Val Arg Leu His Arg Tyr Ala Pro Ala Cys

1315 1320 1325Arg Pro Leu Leu Arg Glu Glu Val Thr Phe Gln Val Gly Leu Asn Gln1330 1335 1340Tyr Leu Val Gly Ser Gln Leu Pro Cys Glu Pro Glu Pro ASp Val Ala1345 1350 1355 1360Val Leu Thr Ser Met Leu Thr Asp Pro Ser His Ile Thr Ala Glu Thr

1365 1370 1375Ala Lys Arg Arg Leu Ala Arg Gly Ser Pro Pro Ser Leu Ala Ser Ser

1380 1385 1390Ser Ala Ser Gln Leu Ser Ala Pro Ser Leu Lys Ala Thr Cys Thr Thr

1395 1400 1405His His Val 5er Pro Asp Ala Asp Leu Ile Glu Ala Asn Leu Leu Trp1410 1415 1420Arg Gln Glu Met Gly Gly Asn Ile Thr Arg Val Glu Ser Glu Asn Lys1425 1430 1435 1440Val Val Val Leu Asp Ser Phe Asp Pro Leu Arg Ala Glu Glu Asp Glu

1445 1450 1455Arg Glu Val Ser Val Pro Ala Glu Ile Leu Arg Lys Ser Lys Lys Phe

1460 1465 1470Pro Ala Ala Met Pro Ile Trp Ala Arg Pro Asp Tyr Asn Pro Pro Leu

1475 1480 1485Leu Glu Ser Trp Lys Asp Pro Asp Tyr Val Pro Pro Val Val His Gly1490 1495 1500Cys Pro Leu Pro Pro Ile Lys Ala Pro Pro Ile Pro Pro Pro Arg Arg1505 1510 1515 1520Lys Arg Thr Val Val Leu Thr Glu Ser Ser Val Ser Ser Ala Leu Ala

1525 1530 1535Glu Leu Ala Thr Lys Thr Phe Gly Ser Ser Glu Ser Ser Ala Val Asp

1540 1545 1550Ser Gly Thr Ala Thr Ala Leu Pro Asp Gln Ala Ser Asp Asp Gly Asp

1555 1560 1565Lys Gly Ser Asp Val Glu Ser Tyr Ser Ser Met Pro Pro Leu Glu Gly1570 1575 1580Glu Pro Gly Asp Pro Asp Leu Ser Asp Gly Ser Trp Ser Thr Val Ser1585 1590 1595 1600Glu Glu Ala Ser Glu Asp Val Val Cys Cys Ser Met Ser Tyr Thr Trp

1605 1610 1615Thr Gly Ala Leu Ile Thr Pro Cys Ala Ala Glu Glu Ser Lys Leu Pro

1620 1625 1630Ile Asn Ala Leu Ser Asn Ser Leu Leu Arg His His Asn Met Val Tyr

1635 1640 1645Ala Thr Thr Ser Arg Ser Ala Gly Leu Arg Gln Lys Lys Val Thr Phe1650 1655 1660Asp Arg Leu Gln Val Leu Asp Asp His Tyr Arg Asp Val Leu Lys Glu1665 1670 1675 1680Met Lys Ala Lys Ala Ser Thr Val Lys Ala Lys Leu Leu Ser Val Glu

1685 1690 1695Glu Ala Cys Lys Leu Thr Pro Pro His Ser Ala Lys Ser Lys Phe Gly

1700 1705 1710Tyr Gly Ala Lys Asp Val Arg Asn Leu Ser Ser Lys Ala Val Asn His

1715 1720 1725Ile His Ser Val Trp Lys Asp Leu Leu Glu Asp Thr Val Thr Pro Ile1730 1735 1740Asp Thr Thr Ile Met Ala Lys Asn Glu Val Phe Cys Val Gln Pro Glu1745 1750 1755 1760Lys Gly Gly Arg Lys Pro Ala Arg Leu Ile Val Phe Pro Asp Leu Gly

1765 1770 1775Val Arg Val Cys Glu Lys Met Ala Leu Tyr Asp Val Val Ser Thr Leu

1780 1785 1790Pro Gln Val Val Met Gly Ser Ser Tyr Gly Phe Gln Tyr Ser Pro Gly

1795 1800 1805Gln Arg Val Glu Phe Leu Val Asn Thr Trp Lys Ser Lys Lys Asn Pro1810 1815 1820Met Gly Phe Ser Tyr Asp Thr Arg Cys Phe Asp Ser Thr Val Thr Glu1825 1830 1835 1840Asn Asp Ile Arg Val Glu Glu Ser Ile Tyr Gln Cys Cys Asp Leu Ala

1845 1850 1855Pro Glu Ala Arg Gln Ala Ile Lys Ser Leu Thr Glu Arg Leu Tyr Ile

1860 1865 1870Gly Gly Pro Leu Thr Asn Ser Lys Gly Gln Asn Cys Gly Tyr Arg Arg

1875 1880 1885Cys Arg Ala Ser Gly Val Leu Thr Thr Ser Cys Gly Asn Thr Leu Thr1890 1895 1900Cys Tyr Leu Lys Ala Ser Ala Ala Cys Arg Ala Ala Lys Leu Gln Asp1905 1910 1915 1920Cys Thr Met Leu Val Asn Gly Asp Asp Leu Val Val Ile Cys Glu Ser

1925 1930 1935Ala Gly Thr Gln Glu Asp Ala Ala Ser Leu Arg Val Phe Thr Glu Ala

1940 1945 1950Met Thr Arg Tyr Ser Ala Pro Pro Gly Asp Pro Pro Gln Pro Glu Tyr

1955 1960 1965Asp Leu Glu Leu Ile Thr Ser Cys Ser Ser Asn Val Ser Val Ala His1970 1975 1980Asp Ala Ser Gly Lys Arg Val Tyr Tyr Leu Thr Arg Asp Pro Thr Thr1985 1990 1995 2000Pro Leu Ala Arg Ala Ala Trp Glu Thr Ala Arg His Thr Pro Val Asn

2005 2010 2015Ser Trp Leu Gly Asn Ile Ile Met Tyr Ala Pro Thr Leu Trp Ala Arg

2020 2025 2030Met Ile Leu Met Thr His Phe Phe Ser Ile Leu Leu Ala Gln Glu Gln

2035 2040 2045Leu Glu Lys Ala Leu Asp Cys Gln Ile Tyr Gly Ala Cys Tyr Ser Ile2050 2055 2060Glu Pro Leu Asp Leu Pro Gln Ile Ile Glu Arg Leu His Gly Leu Ser2065 2070 2075 2080Ala Phe Ser Leu His Ser Tyr Ser Pro Gly Glu Ile Asn Arg Val Ala

2085 2090 2095Ser Cys Leu Arg Lys Leu Gly Val Pro Pro Leu Arg Val Trp Arg His

2100 2105 2110Arg Ala Arg Ser Val Arg Ala Arg Leu Leu Ser Gln Gly Gly Arg Ala

2115 2120 2125Ala Thr Cys Gly Lys Tyr Leu Phe Asn Trp Ala Val Lys Thr Lys Leu2130 2135 2140Lys Leu Thr Pro Ile Pro Ala Ala Ser Arg Leu Asp Leu Ser Gly Trp2145 2150 2155 2160Phe Val Ala Gly Tyr Ser Gly Gly Asp Ile Tyr His Ser Leu Ser Arg

2165 2170 2175Ala Arg Pro Arg Trp Phe Met Leu Cys Leu Leu Leu Leu Ser Val Gly

2180 2185 2190Val Gly Ile Tyr Leu Leu Pro Asn Arg

21952200 (3) data of sequence number 3:

(i) sequence characteristics:

(A) length: 26 nucleotides

(B) Type (2): nucleotide, its preparation and use

(C) Line type: single strand

(D) Topological structure: linearity

(ii) Molecular type: synthesis of DNA

(iii) Pseudo structure: whether or not

(iv) Antisense: whether or not

(vii) The direct source is as follows: oligonucleotide synthesizer

(ix) The method is characterized in that:

(A) name: oligo a

(C) The identification method comprises the following steps: polyacrylamide gel

(xi) Description of the sequence: serial number 3GCCGAGATGC CATCTTCAAA CAGTTC 26(4) data of serial number 4:

(i) sequence characteristics:

(A) length: 24 nucleotides

(B) Type (2): nucleotide, its preparation and use

(C) Line type: single strand

(D) Topological structure: linearity

(ii) Molecular type: synthesis of DNA

(ii) Pseudo structure: whether or not

(iv) Antisense: whether or not

(vii) The direct source is as follows: oligonucleotide synthesizer

(ix) The method is characterized in that:

(A) name: oligo b

(C) The identification method comprises the following steps: polyacrylamide gel

(xi) Description of the sequence: serial number 4GTGTACAACA AGGTCCATAT CACC 24(5) data of serial number 5:

(i) sequence characteristics:

(A) length: 24 nucleotides

(B) Type (2): nucleotide, its preparation and use

(C) Line type: single strand

(D) Topological structure: linearity

(ii) Molecular type: synthesis of DNA

(iii) Pseudo structure: whether or not

(iv) Antisense: whether or not

(vii) The direct source is as follows: oligonucleotide synthesizer

(ix) The method is characterized in that:

(A) name: oligo c

(C) The identification method comprises the following steps: polyacrylamide gel

(xi) Description of the sequence: serial No. 5GGTCTTTCTG AACGGGATAT AAAC 24(6) data of serial No. 6:

(i) sequence characteristics:

(A) length: 31 nucleotides

(B) Type (2): nucleotide, its preparation and use

(C) Line type: single strand

(D) Topological structure: linearity

(ii) Molecular type: synthesis of DNA

(iii) Pseudo structure: whether or not

(iv) Antisense: whether or not

(vii) The direct source is as follows: oligonucleotide synthesizer

(ix) The method is characterized in that:

(A) name: 5' -5B

(C) The identification method comprises the following steps: polyacrylamide gel

(xi) Description of the sequence: serial number 6AAGGATCCAT GTCAATGTCC TACACATGGA C31 (7) data of serial number 7:

(i) sequence characteristics:

(A) length: 36 nucleotides

(B) Type (2): nucleotide, its preparation and use

(C) Line type: single strand

(D) Topological structure: linearity

(ii) Molecular type: synthesis of DNA

(iii) Pseudo structure: whether or not

(iv) Antisense: whether or not

(vii) The direct source is as follows: oligonucleotide synthesizer

(ix) The method is characterized in that:

(A) name: 3' -5B

(C) The identification method comprises the following steps: polyacrylamide gel

(xi) Description of the sequence: serial number 7AATATTCGAA TTCATCGGTT GGGGAGCAGG TAGATG 36(8) data of serial number 8:

(i) sequence characteristics:

(A) length: 22 nucleotides

(B) Type (2): nucleotide, its preparation and use

(C) Line type: single strand

(D) Topological structure: linearity

(ii) Molecular type: synthesis of DNA

(iii) Pseudo structure: whether or not

(iv) Antisense: whether or not

(vii) The direct source is as follows: oligonucleotide synthesizer

(ix) The method is characterized in that:

(A) name: dpr1

(C) The identification method comprises the following steps: polyacrylamide gel

(xi) Description of the sequence: serial number 8TGGCTGGCAA GGCACACAGG CT 22(9) serial number 9 data:

(i) sequence characteristics:

(A) length: 20 nucleotides

(B) Type (2): nucleotide, its preparation and use

(C) Line type: single strand

(D) Topological structure: linearity

(ii) Molecular type: synthesis of DNA

(iii) Pseudo structure: whether or not

(iv) Antisense: is that

(vii) The direct source is as follows: oligonucleotide synthesizer

(ix) The method is characterized in that:

(A) name: dpr2

(C) The identification method comprises the following steps: polyacrylamide gel

(xi) Description of the sequence: serial number 9AGGCAGGGTA GATCTATGTC 20(10) data of serial number 10:

(i) sequence characteristics:

(A) length: 20 nucleotides

(B) Type (2): nucleotide, its preparation and use

(C) Line type: single strand

(D) Topological structure: linearity

(ii) Molecular type: synthesis of DNA

(iii) Pseudo structure: whether or not

(iv) Antisense: whether or not

(vii) The direct source is as follows: oligonucleotide synthesizer

(ix) The method is characterized in that:

(A) name: NS 5B-5' (1)

(C) The identification method comprises the following steps: polyacrylamide gel

(xi) Description of the sequence: serial number 10TCAATGTCCT ACACATGGAC 20(11) data of serial number 11:

(i) sequence characteristics:

(A) length: 38 nucleotides

(B) Type (2): nucleotide, its preparation and use

(C) Line type: single strand

(D) Topological structure: linearity

(ii) Molecular type: synthesis of DNA

(iii) Pseudo structure: whether or not

(iv) Antisense: is that

(vii) The direct source is as follows: oligonucleotide synthesizer

(ix) The method is characterized in that:

(A) name: HCVA-13

(C) The identification method comprises the following steps: polyacrylamide gel

(xi) Description of the sequence: serial number 11GATCTCTAGA TCATCGGTTG GGGGAGGAGG TAGATGCC 38(12) data for serial number 12:

(i) sequence characteristics:

(A) length: 399 nucleotides

(B) Type (2): nucleotide, its preparation and use

(C) Line type: single strand

(D) Topological structure: linearity

(ii) Molecular type: mRNA

(iii) Pseudo structure: whether or not

(iv) Antisense: whether or not

(vi) The initial sources were:

(A) an organism: rattus Norvegicus

(B) Separating strains: Sprague-Dawley

(vii) The direct source is as follows: pT7-7(DCoH)

(ix) The method is characterized in that:

(A) name: D-RNA

(C) The identification method comprises the following steps: polyacrylamide gel

(xi) Description of the sequence: serial No. 12GGGAGACCAC AACGGUUUCC CUCUAGAAAU AAUUUUGUUU AACUUUAAGA AGGAGAUAUA 60CAUAUGGCUA GAAUUCGCGC CCUGGCUGGC AAGGCACACA GGCUGAGUGC UGAGGAACGG 120GACCAGCUGC UGCCAAACCU GCGGGCCGUG GGGUGGAAUG AACUGGAAGG CCGAGAUGCC 180AUCUUCAAAC AGUUCCAUUU UAAAGACUUC AACAGGGCUU UUGGCUUCAU GACAAGAGUC 240GCCCUGCAGG CUGAAAAGCU GGACCACCAU CCCGAGUGGU UUAACGUGUA CAACAAGGUC 300CAUAUCACCU UGAGCACCCA CGAAUGUGCC GGUCUUUCUG AACGGGAUAU AAACCUGGCC 360AGCUUCAUCG AACAAGUUGC CGUGUCUAUG ACAUAGAUC 399(13) data of serial No. 13:

(i) sequence characteristics:

(A) length: 20 nucleotides

(B) Type (2): nucleotide, its preparation and use

(C) Line type: single strand

(D) Topological structure: linearity

(ii) Molecular type: synthesis of DNA

(iii) Pseudo structure: whether or not

(iv) Antisense: whether or not

(vi) The direct source is as follows: oligonucleotide synthesizer

(ix) The method is characterized in that:

(A) name: NS5B-up

(C) The identification method comprises the following steps: polyacrylamide gel

(xi) Description of the sequence: serial number 13TGTCAATGTC CTACACATGG 20(14) data of serial number 14:

(i) sequence characteristics:

(A) length: 38 nucleotides

(B) Type (2): nucleotide, its preparation and use

(C) Line type: single strand

(D) Topological structure: linearity

(ii) Molecular type: synthesis of DNA

(iii) Pseudo structure: whether or not

(iv) Antisense: is that

(vii) The direct source is as follows: oligonucleotide synthesizer

(ix) The method is characterized in that:

(A) name: 3' -5B

(C) The identification method comprises the following steps: polyacrylamide gel

(xi) Description of the sequence: serial number 14AATATTCGAA TTCATCGGTT GGGGAGCAGG TAGATG 36

Claims (7)

1. A method for reproducing in vitro the RNA-dependent RNA polymerase activity or terminal nucleotidyl transferase activity encoded by hepatitis C virus, characterized in that a sequence containing NS5B (SEQ ID NO: 1) is used in the reaction mixture.

2. The method for reproducing in vitro the RNA-dependent RNA polymerase activity encoded by HCV according to claim 1, wherein NS5B is incorporated into the reaction mixture as NS2-NS5B precursor which generates an enzymatically active form of NS5B by means of a plurality of proteolytic events which occur in the overproducing organism.

3. The method for reproducing in vitro the terminal nucleotidyl transferase activity encoded by HCV according to claim 1, in which NS5B is incorporated in the reaction mixture as NS2-NS5B precursor which, by means of a plurality of proteolytic reactions taking place in the overproducing organism, produces NS5B in an enzymatically active form.

4. A composition characterized in that it comprises NS5B sequence according to claims 1 to 3.

5. A composition according to claim 4, which comprises a protein as described in SEQ ID NO. 1 and sequences contained therein or derived therefrom.

6. Use of a composition according to claim 4 or 5 for the set-up of an enzymatic assay capable of screening for compounds of therapeutic interest which inhibit the enzymatic activity associated with the NS5B protein.

7. The method, composition and use of the composition for reproducing in vitro the RNA-dependent RNA polymerase and terminal nucleotidyl transferase activities of NS5B according to the above description, examples and claims in the creation of an enzymatic assay capable of screening for compounds of therapeutic interest that inhibit the enzymatic activity associated with NS 5B.