Hcv ns5b protease mutants

An isolated polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:

• 516 or at least 90% identical to a portion of SEQ ID NO:
• 516 having Hepatitis C Virus (HCV) NS5B polymerase activity, with the proviso that at least one of the amino acids of the polypeptide that correspond to positions 419, 482, 486, and/or 494 of SEQ ID NO:
• 516 is identical to the corresponding amino acid(s) of SEQ ID NO:
• 516.

A method for detecting the presence of drug-resistant HCV in a sample, wherein the method comprises determining the presence or absence of a polynucleotide in the sample, the polynucleotide comprising a nucleic acid sequence encoding HCV NS5B polymerase containing at least one codon selected from the group consisting of

• a codon encoding cysteine, isoleucine, valine, or proline at a position corresponding to codon position 419 of SEQ ID NO:
• 2;
• a codon encoding alanine, valine, or asparagine at a position corresponding to codon position 482 of SEQ ID NO:
• 2;
• a codon encoding valine, isoleucine, or serine at a position corresponding to codon position 486 of SEQ ID NO:
• 2;
• and
• a codon encoding isoleucine at a position corresponding to codon position 494 of SEQ ID NO:
• 2,
• wherein the presence of the at least one codon indicates the presence of drug-resistant HCV in the sample.

A method for determining whether an HCV-infected patient is infected with an HCV strain that has a decreased sensitivity to VX-222, the method comprising determining the presence or absence of a polypeptide in a biological sample from the patient, the polypeptide comprising an amino acid sequence comprising at least one variation from SEQ ID NO:

• 1, the at least one variation selected from the group consisting of
• cysteine, isoleucine, methionine, serine, valine, or proline at amino acid position 419;
• lysine at amino acid position 422;
• alanine, isoleucine, threonine, or valine at amino acid position 423;
• alanine, leucine, threonine, valine, or asparagine at amino acid position 482;
• valine, isoleucine, threonine, or serine at amino acid position 486;
• and
• isoleucine or alanine at amino acid position 494,
• as the amino acid positions are defined in SEQ ID NO:
• 1, wherein the presence of the polypeptide indicates infection with an HCV strain that has a decreased sensitivity to VX-222.

A method for identifying an agent able to rescue the polymerase-inhibitor activity of VX-222 against an HCV NS5B polymerase having resistance to VX-222, the method comprising:

• a) performing an HCV NS5B polymerase reaction with a polypeptide comprising an amino acid sequence comprising at least one variation from SEQ ID NO:
• 1, the at least one variation selected from the group consisting of
• cysteine, isoleucine, methionine, serine, valine, or proline at amino acid position 419;
• lysine at amino acid position 422;
• alanine, isoleucine, threonine, or valine at amino acid position 423;
• alanine, leucine, threonine, valine, or asparagine at amino acid position 482;
• valine, isoleucine, threonine, or serine at amino acid position 486;
• and
• isoleucine or alanine at amino acid position 494,
• as the amino acid positions are defined in SEQ ID NO:
• 1, in the presence of an agent and VX-222;
• and
• b) comparing polymerase activity of the polypeptide in the presence of the agent with polymerase activity of the polypeptide in the absence of the agent, wherein a decrease in HCV polymerase activity in the presence of the agent is indicative of the ability to rescue the polymerase-inhibitory activity of VX-222 against an HCV NSSB polymerase having resistance to VX-222.

A method for determining whether an HCV-infected patient is infected with an HCV strain that has a decreased sensitivity to VX-222, the method comprising determining the presence or absence of a polynucleotide in a biological sample from the patient, the polynucleotide comprising a nucleic acid sequence encoding HCV NS5B polymerase containing at least one codon selected from the group consisting of a codon encoding cysteine, isoleucine, methionine, serine, valine, or proline at a position corresponding to codon position 419 of SEQ ID NO:

• 2;
• a codon encoding lysine at a position corresponding to codon position 422 of SEQ ID NO:
• 2;
• a codon encoding alanine, isoleucine, threonine, or valine at a position corresponding to codon position 423 of SEQ ID NO:
• 2;
• a codon encoding alanine, leucine, threonine, valine, or asparagine at a position corresponding to codon position 482 of SEQ ID NO:
• 2;
• a codon encoding valine, isoleucine, threonine, or serine at a position corresponding to codon position 486 of SEQ ID NO:
• 2;
• and
• a codon encoding isoleucine or alanine at a position corresponding to codon position 494 of SEQ ID NO:
• 2,
• wherein the presence of the polynucleotide indicates infection with an HCV strain having a decreased sensitivity to VX-222.