Recombinant Hepatitis C virus RNA replicase
OwnerCurt H. Hagedorninventor
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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 NH2-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.
Claims41
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.
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.
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.
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.
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.
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.
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.
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.
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.
A polyclonal or monoclonal antibody to r-HCV-RDRP in partially purified form.
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.
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.
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.
Dependencies are inferred from the claim text.