Replication competent hepatitis C virus and methods of use

A method for making an isolated polynucleotide encoding a replication competent hepatitis C virus genotype la replicon comprising:

• providing a polynucleotide comprising a 5′ NTR, 3′ NTR, and a first coding sequence present between the 5′ NTR and 3′ NTR and encoding a hepatitis C virus polyprotein, wherein the polyprotein comprises an amino acid sequence having at least about 95% identity to amino acids 1-3011 of SEQ ID NO:2, and wherein the amino acid sequence of the polyprotein comprises a serine at about amino acid 2204 of the polyprotein, a glutamine at about amino acid 1067 of the polyprotein, a lysine at about amino acid 1691 of the polyprotein, a phenylalanine at about amino acid 2080 of the polyprotein, a valine at about amino acid 1655 of the polyprotein, a lysine at about amino acid 2040 of the polyprotein, or a glycine at about amino acid 1188 of the polyprotein;
• and
• altering the coding sequence such that the polyprotein encoded thereby comprises at least three adaptive mutations relative to the amino acid positions using SEQ ID NO:
• 2 as a reference sequence, wherein the adaptive mutations comprise an isoleucine at about amino acid 2204 of the polyprotein and at least two adaptive mutations selected from the group consisting of an arginine at about amino acid 1067 of the polyprotein, an arginine at about amino acid 1691 of the polyprotein, a valine at about amino acid 2080 of the polyprotein, an isoleucine at about amino acid 1655 of the polyprotein, an arginine at about amino acid 2040 of the polyprotein, an arginine at about amino acid 1188 of the polyprotein, and a combination thereof.

A method for identifying a compound that inhibits replication of a replication competent hepatitis C virus genotype 1a replicon, the method comprising:

• contacting a cell comprising a replication competent hepatitis C virus genotype 1a replicon with a compound, the replication competent hepatitis C virus genotype 1a replicon comprising a 5′ NTR, 3′ NTR, and a first coding sequence present between the 5′ NTR and 3′ NTR and encoding a hepatitis C virus polyprotein, wherein the hepatitis C virus polyprotein comprises an amino acid sequence having at least about 95% identity to amino acids 1-3011 of SEQ ID NO:2, and wherein the amino acid sequence of the polyprotein comprises at least three adaptive mutations relative to the amino acid positions using SEQ ID NO:
• 2 as a reference sequence, wherein the adaptive mutations comprise an isoleucine at about amino acid 2204 of the polyprotein and at least two adaptive mutations selected from the group of an arginine at about amino acid 1067 of the polyprotein, an arginine at about amino acid 1691 of the polyprotein, a valine at about amino acid 2080 of the polyprotein, an isoleucine at about amino acid 1655 of the polyprotein, an arginine at about amino acid 2040 of the polyprotein, an arginine at about amino acid 1188 of the polyprotein, and a combination thereof;
• incubating the cell under conditions wherein the replication competent hepatitis C virus genotype 1a replicon replicates in the absence of the compound;
• and
• detecting the replication competent hepatitis C virus genotype 1a replicon, wherein a decrease of the replication competent hepatitis C virus genotype 1a replicon in the cell contacted with the compound compared to the replication competent hepatitis C virus genotype 1a replicon in a cell not contacted with the compound indicates the compound inhibits replication of the replication competent hepatitis C virus genotype 1a replicon.

A method for selecting a replication competent hepatitis C virus genotype 1a replicon, the method comprising:

• incubating a cell in the presence of a selecting agent, wherein:
• the cell comprises a polynucleotide comprising a 5′ non-translated region (NTR), a 3′ NTR, and a first coding sequence present between the 5′ NTR and 3′ NTR and encoding a hepatitis C virus polyprotein, and a second coding sequence, wherein the polyprotein comprises an amino acid sequence having at least about 95% identity to amino acids 1-3011 of SEQ ID NO:2, and wherein the amino acid sequence of the polyprotein comprises at least three adaptive mutations relative to the amino acid positions using SEQ ID NO:
• 2 as a reference sequence, wherein the adaptive mutations comprise an isoleucine at about amino acid 2204 of the polyprotein and at least two adaptive mutations selected from the group of an arginine at about amino acid 1067 of the polyprotein, an arginine at about amino acid 1691 of the polyprotein, a valine at about amino acid 2080 of the polyprotein, an isoleucine at about amino acid 1655 of the polyprotein, an arginine at about amino acid 2040 of the polyprotein, an arginine at about amino acid 1188 of the polyprotein, and a combination thereof;
• the second coding sequence encodes a selectable marker conferring resistance to the selecting agent;
• and
• the selecting agent inhibits replication of a cell that does not express the selectable marker;
• and
• detecting a cell that replicates in the presence of the selecting agent, wherein the presence of such a cell indicates the polynucleotide is replication competent.

A method for detecting a replication competent hepatitis C virus genotype 1a replicon, the method comprising:

• incubating a cell comprising a replication competent hepatitis C virus genotype 1a replicon, wherein:
• the replication competent hepatitis C virus genotype 1a replicon comprises a 5′ non-translated region (NTR), a 3′ NTR, and a first coding sequence present between the 5′ NTR and 3′ NTR and encoding a hepatitis C virus polyprotein, and a second coding sequence encoding a transactivator, wherein the polyprotein comprises an amino acid sequence having at least about 95% identity to amino acids 1-3011 of SEQ ID NO:2, and wherein the amino acid sequence of the polyprotein comprises at least three adaptive mutations relative to the amino acid positions using SEQ ID NO:
• 2 as a reference sequence, wherein the adaptive mutations comprise an isoleucine at about amino acid 2204 of the polyprotein and at least two adaptive mutations selected from the group of an arginine at about amino acid 1067 of the polyprotein, an arginine at about amino acid 1691 of the polyprotein, a valine at about amino acid 2080 of the polyprotein, an isoleucine at about amino acid 1655 of the polyprotein, an arginine at about amino acid 2040 of the polyprotein, an arginine at about amino acid 1188 of the polyprotein, and a combination thereof;
• the cell comprises a transactivated coding region and an operator sequence operably linked to the transactivated coding region;
• and
• the transactivated coding region encodes a detectable marker, wherein the transactivator alters transcription of the transactivated coding region;
• and
• detecting the detectable marker, wherein the presence of the detectable marker indicates the cell comprises a replication competent hepatitis C virus genotype 1a replicon.