Recombinant Hepatitis C virus RNA replicase

Abstract

A recombinant RNA-dependent RNA polymerase of hepatitis C virus (r-HCV-RDRP) coding DNA was cloned and expressed yielding active enzyme in vitro. The r-HCV-RDRP can include up to 20 added amino acids and up to nine deleted or substituted amino acids at the NH 2 -terminus of the encoded amino acid sequence. The invention provides method to solubilize r-HCV-RDRP from a host cell lysate and purified r-HCV-RDRP. Methods for screening for inhibitors of r-HCV-RDRP in vitro, for making stably transfected mammalian cells expressing r-HCV-RDRP and for in vivo testing of r-HCV-RDRP inhibitors in vivo are disclosed. The invention provides antibodies to r-HCV-RDRP and methods for detecting antibodies to HCV-RDRP in serum of human patients.

What is claimed is:  
     
         1 . An isolated and purified recombinant hepatitis C virus RNA-dependent RNA polymerase (r-HCV-RDRP) having an amino acid sequence encoded by an isolated NS5B coding sequence modified to encode one or more of the following amino acids substituted for the corresponding amino acid encoded by the isolated NS5B sequence: Ser or Glu at amino acid position 21; Arg or Lys at amino acid position 67, Lys at amino acid position 100, Lys at amino acid position 116, Glu or Val at amino acid position 133, Ser at amino acid position 220, Ser at amino acid position 302, or Ala at amino acid position 340, said r-HCV-RDRP having in vitro RDRP activity.  
     
     
         2 . The r-HCV-RDRP of  claim 1  further comprising a carboxy-terminal deletion of from 18 to 57 amino acids.  
     
     
         3 . The r-HCV-RDRP of  claim 2  further comprising a sequence selected from the group LeuGlu(His) 6 , (Ala) n Ser(His) 6  or (Gly) n Ser(His) 6  when n is 1-5, substituted for said deletion.  
     
     
         4 . The r-HCV-RDRP of  claim 1  further comprising one or more of the following amino acid substitutions: Arg at amino acid position 533, Thr at amino acid position 209, Leu at amino acid position 49, Asn at amino acid position 233, Arg at amino acid position 512 and Ser at amino acid position 551, said r-HCV-RDRP having in vitro RDRP activity.  
     
     
         5 . The r-HCV-RDRP of  claim 4  further comprising a carboxy-terminal deletion of from 18 to 57 amino acids.  
     
     
         6 . The r-HCV-RDRP of  claim 5  further comprising a sequence selected from the group LeuGlu(His) 6 , (Ala) n Ser(His) 6  or (Gly) n Ser(His) 6  when n is 1-5, substituted for said deletion.  
     
     
         7 . The r-HCV-RDRP of  claim 4  further comprising one or more of the following amino acid substitutions: Trp at amino acid position 575, Phe at amino acid position 576, Ala at amino acid position 582 and Lys at amino acid position 583, said r-HCV-RDRP having in vivo RDRP activity.  
     
     
         8 . An isolated and purified recombinant hepatitis C virus RNA-dependent RNA polymerase (r-HCV-RDRP) having an amino acid sequence encoded by an isolated NS5B coding sequence modified to encode one or more of the following amino acids substituted for the corresponding amino acid encoded by the isolated NS5B sequence: Ser or Glu at amino acid position 21, Arg at amino acid position 67, Lys at amino acid position 116, Val at amino acid position 133, Thr or Asn at amino acid position 215, Arg at amino acid position 256, Thr or Arg at amino acid position 302, Cys at amino acid position 318, Ala at amino acid position 340, Gln at amino acid position 466, Arg or Ala at amino acid position 512, or R at amino acid position 533, said r-HCV-RDRP having in vitro RDRP activity.  
     
     
         9 . The r-HCV-RDRP of  claim 8  further comprising a carboxy-terminal deletion of from 18 to 57 amino acids.  
     
     
         10 . The r-HCV-RDRP of  claim 8  further comprising a sequence selected from the group LeuGlu(His) 6 , (Ala) n Ser(His) 6  or (Gly) n Ser(His) 6  when n is 1-5, substituted for said deletion.  
     
     
         11 . The r-HCV-RDRP of  claim 8  further comprising a carboxy-terminal deletion of from 18 to 57 amino acids.  
     
     
         12 . The r-HCV-RDRP of  claim 8  further comprising a sequence selected from the group LeuGlu(His) 6 , (Ala) n Ser(His) 6  or (Gly) n Ser(His) 6  when n is 1-5, substituted for said deletion.  
     
     
         13 . An isolated and purified recombinant hepatitis C virus RNA-dependent RNA polymerase r-HCV-RDRP having an amino acid sequence encoded by the isolated NS5B coding sequence of SEQ ID NO:23 modified to encode one or more of the following amino acids substituted for the corresponding amino acid encoded by the isolated NS5B sequence: Ser or Glu at amino acid position 21, Arg at amino acid position 67, Lys at amino acid position 116, Val at amino acid position 133, Thr or Asn at amino acid position 215, Arg at amino acid position 256, Thr or Arg at amino acid position 302, Cys at amino acid position 318, Ala at amino acid position 340, Gln at amino acid position 466, Arg or Ala at amino acid position 512, or R at amino acid position 533, said r-HCV-RDRP having in vitro RDRP activity.  
     
     
         14 . The r-HCV-RDRP of  claim 13  further comprising a carboxy-terminal deletion of from 18 to 57 amino acids.  
     
     
         15 . The r-HCV-RDRP of  claim 14  further comprising a sequence selected from the group LeuGlu(His) 6 , (Ala) n Ser(His) 6  or (Gly) n Ser(His) 6  when n is 1-5, substituted for said deletion.  
     
     
         16 . The r-HCV-RDRP of  claim 13  further comprising a carboxy-terminal deletion of from 18 to 57 amino acids.  
     
     
         17 . The r-HCV-RDRP of  claim 16  further comprising a sequence selected from the group LeuGlu(His) 6 , (Ala) n Ser(His) 6  or (Gly) n Ser(His) 6  when n is 1-5, substituted for said deletion.  
     
     
         18 . A method of enhancing enzyme properties of an isolated recombinant hepatitis C virus RNA-dependent RNA polymerase (r-HCV-RDRP) comprising 
 modifying the amino acid sequence of said r-HCV-RDRP by substituting at the designated amino acid position one or more of the following amino acids: Ser or Glu at amino acid position 21; Arg or Lys at amino acid position 67, Lys at amino acid position 100, Lys at amino acid position 116, Glu or Val at amino acid position 133, Ser at amino acid position 220, Ser at amino acid position 302, or Ala at amino acid position 340, whereby enzyme properties of the isolated r-HCV-RDRP are enhanced.    
     
     
         19 . A method of enhancing enzyme properties of an isolated recombinant hepatitis C virus RNA-dependent RNA polymerase (r-HCV-RDRP) comprising 
 modifying the amino acid sequence of said r-HCV-RDRP by substituting at the designated amino acid position one or more of the following amino acids: Ser or Glu at amino acid position 21, Arg at amino acid position 67, Lys at amino acid position 116, Val at amino acid position 133, Thr or Asn at amino acid position 215, Arg at amino acid position 256, Thr or Arg at amino acid position 302, Cys at amino acid position 318, Ala at amino acid position 340, Gln at amino acid position 466, Arg or Ala at amino acid position 512, or R at amino acid position 533, whereby enzyme properties of the isolated r-HCV-RDRP are enhanced.    
     
     
         20 . A method for testing a compound for inhibitory activity against recombinant hepatitis C virus RNA-dependent RNA polymerase (r-HCV-RDRP) in vivo comprising preparing a mammalian cell line capable of expressing active r-HCV-RDRP comprising DNA encoding r-HCV-RDRP, 
 transfecting said cell line with a reporter gene whose translation depends on the presence of active r-HCV-RDRP in the same cell, whereby a reporter cell line is prepared,    incubating the reporter cell line in the presence or absence of a test compound, whereby inhibitor of r-HCV-RDRP by the test compound results in inhibition of translation of the reporter gene.    
     
     
         21 . The method of  claim 20  wherein the reporter gene is selected from the group: luciferase, secreted alkaline phosphatase, chloramphenicol acetyl transferase, or fluorescent green protein.  
     
     
         22 . The method of  claim 21  wherein the reporter gene is transcribed to yield a messenger RNA encoding the antisense of the reporter, whereby synthesis of a complementary RNA thereto by r-HCV-RDRP produces a sense messenger RNA translatable to yield active reporter.  
     
     
         23 . The method of  claim 22  wherein the transcribed messenger RNA of the reporter further comprises an internal ribosome entry site from HCV located 3′ from the reporter antisense coding region, whereby synthesis of a complementary RNA thereto by r-HCV-RDRP produces a messenger RNA having the internal ribosome entry site located 5′ from the sense messenger RNA coding region thereby providing cap-independent translation of the reporter.  
     
     
         24 . A method for testing a compound for inhibiting activity against recombinant hepatitis C virus RNA-dependent RNA polymerase (r-HCV-RDRP) in vivo comprising 
 preparing a mammalian cell line containing DNA encoding r-HCV-RDRP and capable of expressing active r-HCV-RDRP,    transfecting said cell line with an RNA template comprising a 5′-non-coding region and a 3′-non-coding region of HCV-RNA, either as a (+)RNA or (−) RNA, whereby a transfected cell is produced, and    measuring the amount of said RNA template or its complement produced by the transfected cell in the presence and in the absence of a compound being tested for activity against r-HCV-RDRP in vivo, whereby inhibition of r-HCV-RDRP by the compound results in inhibition of template RNA replication.    
     
     
         25 . A method for measuring in vivo activity of a r-HCV-RDRP in a mammalian cell 
 comprising preparing mammalian cells containing DNA encoding the r-HCV-RDRP and capable of expressing active r-HCV-RDRP, and    incubating the cells under conditions that express active r-HCV-RDRP.    
     
     
         26 . The method of  claim 25  comprising additionally the steps of 
 further incubating the cells in the presence or absence of a putative inhibitor compound,  
 washing the cells to remove any extracellular putative inhibitor,  
 preparing an extract of the cells whereby any RDRP activity is measurable in the extract,  
 measuring RDRP activity in the cell extract, and  
 comparing the activity of RDRP in the extracts of cells incubated in the presence or in the absence of the putative inhibitor, whereby any inhibition by the putative inhibitor can be detected.  
 
     
     
         27 . A method of purifying an RDRP enzyme, the enzyme being r-HCV-RDRP ΔC21-60-oligoHis, from  E. coli  cells containing and capable of expressing DNA encoding the enzyme comprising 
 incubating the cells under conditions that permit expression of the enzyme,  
 preparing a lysate of the cells,  
 fractionating the lysate by chromatography through a column of chelated Co ++  and collecting samples of RDRP activity,  
 fractionating the samples having RDRP activity by chromatography on a heparin Sepharose FPLC column and collecting samples eluted from the column having RDRP activity, said samples being at least 95% pure.  
 
     
     
         28 . The method of  claim 27  wherein the step of incubating the cells under conditions that express the enzyme is carried out at a temperature of from 24° C.-30° C.  
     
     
         29 . The method of  claim 27  wherein the temperature is from 26° C.-28° C.  
     
     
         30 . The method of  claim 27  wherein the temperature is 27° C.  
     
     
         31 . A polyclonal or monoclonal antibody to r-HCV-RDRP in partially purified form.  
     
     
         32 . Antibody of  claim 31  having the capability to inhibit the enzyme activity of HCV-RDRP  
     
     
         33 . A method for testing a compound for inhibitory activity against HCV-RDRP comprising measuring RNA product in an in vitro RNA synthesis reaction catalyzed by r-HCV-RDRP, and comparing amounts of RNA synthesized in the presence and absence of the compound, whereby comparative reduction of the amount of RNA synthesized in the presence of the compound indicates that the compound has inhibitory activity against HCV-RDRP.  
     
     
         34 . The method of  claim 33  further comprising comparing the activity of a control enzyme in the presence and absence of the test compound, whereby inhibition of the control enzyme by the test compound indicates absence of a specific effect on r-HCV-RDRP.  
     
     
         35 . A method for measuring or detecting antibody to HCV-RDRP in serum of an infected patient comprising 
 contacting the antibody with r-HCV-RDRP whereby an antigen-antibody complex is formed,    separating the antigen-antibody complex from any unbound antibody or r-HCV-RDRP, and    measuring or detecting the antigen-antibody complex.    
     
     
         36 . The method of  claim 20  wherein r-HCV-RDRP is localized by gel electrophoresis, the gel is contacted with serum containing antibody to HCV-RDRP, and the antigen-antibody complex is detected or measured by labeled anti-human antibody.  
     
     
         37 . A method for modifying NS5B protein of HCV, having an N-terminus that is not methionine and having N-terminal amino acids, to provide an N-terminal methionine therefor, comprising the steps of: 
 adding to the N-terminus of said protein a peptide of from 1-20 amino acids, said peptide having an N-terminal methionine,    deleting up to 9 amino acids at the N-terminus of said protein,    substituting an amino acid for any of 0-5 amino acids within the first nine N-terminal amino acids of NS5B,    said steps being carried out in the alternative or in any combination whereby the NS5B protein is provided with an N-terminal methionine.    
     
     
         38 . The method of  claim 37  wherein the substituting amino acid of step (c) is alanine.  
     
     
         39 . The method of  claim 37  wherein the substituting amino acid is histidine.  
     
     
         40 . The method of  claim 37  wherein the peptide of step (a) is a polyalanine having an N-terminal methionine.  
     
     
         41 . The method of  claim 37  wherein the peptide of step (a) comprises a specific proteolytic cleavage site.