Method to confer cell culture replication activity to different Hepatitis C virus isolates
View Patent ↗The present invention features methods for producing HCV replicons using HCV encoding sequences from different isolates. The featured methods are based on the discovered importance of NS3 amino acid position 470 in conferring cell culture replication activity to different HCV isolates.
1. A method of making a Hepatitis C virus (HCV) replicon having an increased replication activity in a Huh7 cell or derivative thereof comprising modifying a HCV replicon construct to encode an amino acid substitution at a position corresponding to amino acid 470 of NS3 and to encode an isoleucine at the position corresponding to amino acid 232 of NS5A, using SEQ ID NO: 1 as a reference sequence, wherein a replicon that has been modified has an increased replication activity in said Huh7 cell or derivative thereof compared to said HCV replicon construct prior to said modifying.
2. The method of claim 1 , further comprising the step of modifying said HCV replicon construct to encode an amino acid substitution at a position corresponding to amino acid 196 of NS3 using SEQ ID NO: 1 as a reference sequence.
3. The method of claim 1 , wherein said amino acid modification is preformed on a replicon construct derived from HCV genotype 1 a strain.
4. The method of claim 1 , wherein said amino acid modification is preformed on a replicon construct derived from HCV-BK, and said modification results in a methionine at said position corresponding to amino acid 470 of NS3.
5. The method of claim 1 , wherein said amino acid modification is preformed on a replicon construct derived from HCV-H77, and said modification results in a leucine at said position corresponding to amino acid 470 of NS3.
6. A method for identifying a Hepatitis C virus (HCV) replicon that grows in cell culture comprising the steps of:
(a) producing a modified replicon construct comprising modifying a HCV replicon construct to encode an amino acid substitution at a position corresponding to amino acid 470 of NS3 and to encode an isoleucine at the position corresponding to amino acid 232 of NS5A, using SEQ ID NO:1 as a reference sequence;
(b) introducing said modified replicon construct into a Huh7 cell or derivative thereof; and
(c) measuring replication activity of said modified replicon construct, wherein increased replication activity as compared to an unmodified HCV replicon indicates the ability of said modified HCV replicon to grow in cell culture.
7. The method of claim 6 , wherein said cell is an Huh7 cell.
8. A replicon comprising a nucleotide sequence encoding for SEQ ID NO: 1.
9. The replicon of claim 8 , wherein said replicon consists of the nucleic acid sequence of SEQ ID NO: 3.
10. A method of measuring the ability of a compound to affect HCV replicon activity comprising the steps of;
a) providing said compound to a Huh7 cell or derivative thereof comprising a replicon made by the method of claim 1 ; and
b) measuring HCV replicon activity in said cell in the presence and absence of the compound.
11. A method of measuring the ability of a compound to affect HCV replicon activity comprising the steps of;
a) providing said compound to a Huh7 cell or derivative thereof comprising a replicon made by the method of claim 2 ; and
b) measuring HCV replicon activity in said cell in the presence and absence of the compound.
12. A method of measuring the ability of a compound to affect HCV replicon activity comprising the steps of;
a) providing said compound to a Huh7 cell or derivative thereof comprising a replicon made by the method of claim 3 ; and
b) measuring HCV replicon activity in said cell in the presence and absence of the compound.
13. A method of measuring the ability of a compound to affect HCV replicon activity comprising the steps of;
a) providing said compound to a Huh7 cell or derivative thereof comprising a replicon made by the method of claim 4 ; and
b) measuring HCV replicon activity in said cell in the presence and absence of the compound.
14. A method of measuring the ability of a compound to affect HCV replicon activity comprising the steps of;
a) providing said compound to a Huh7 cell or derivative thereof comprising a replicon made by the method of claim 5 ; and
b) measuring HCV replicon activity in said cell in the presence and absence of the compound.
15. A method of measuring the ability of a compound to affect HCV replicon activity comprising the steps of;
a) providing said compound to a Huh7 cell or derivative thereof comprising the replicon of claim 8 ; and
b) measuring HCV replicon activity in said cell in the presence and absence of the compound.
16. A method of measuring the ability of a compound to affect HCV replicon activity comprising the steps of;
a) providing said compound to a Huh7 cell or derivative thereof comprising the replicon of claim 9 ; and
b) measuring HCV replicon activity in said cell in the presence and absence of the compound.
17. A replicon comprising a nucleotide sequence encoding for SEQ ID NO: 2.
18. The replicon of claim 17 , wherein said replicon consists of the nucleic acid sequence of SEQ ID NO: 4.