HCV replicons containing NS5B from genotype 2B
View Patent ↗The present invention features methods for enhancing the ability of a genotype 2b NS5B sequence to function in a replicon, for producing replicons containing a functional genotype 2b NS5B, and for using replicons to measure the ability of a compound to affect HCV replication that is sustained with the genotype 2b polymerase. Also featured is a genotype 1b NS4B adaptive mutation. The ability to produce replicons containing a functional genotype 2b NS5B is illustrated by the production of chimeric replicons based on HCV genotype 1b where substantially all the NS5B sequence is replaced with a genotype 2b NS5B.
1. A method of enhancing the ability of a genotype 2b NS5B sequence to function in a replicon comprising the step of altering either, or both
(a) said genotype 2b NS5B sequence to encode one or more adaptive mutations selected from the group consisting of:
serine corresponding to position 24 of SEQ ID NO: 1;
isoleucine corresponding to position 31 of SEQ ID NO: 1;
leucine corresponding to position 392 of SEQ ID NO: 1; or
(b) a genotype 1b NS4B sequence to encode an adaptive mutation of alanine corresponding to position 218 of SEQ ID NO:28.
2. The method of claim 1 , wherein altering said genotype 2b NS5B sequence comprises at least isoleucine corresponding to position 31 of SEQ ID NO: 1, or at least serine corresponding to position 24 of SEQ ID NO: 1 in combination with leucine corresponding to position 392of SEQ ID NO: 1.
3. The method of claim 2 , wherein said genotype 2b NS5B sequence is obtained from a clinical isolate.
4. A method of producing a chimeric replicon comprising the step of replacing substantially all of a NS5B sequence of a HCV replicon comprising a NS3-5B genotype 1b sequence encoding SEQ ID NO: 3 with a genotype 2b NS5B encoding nucleic acid sequence.
5. The method of claim 4 , further comprising the step of enhancing the ability of said genotype 2b NS5B according to the method of claim 1 , wherein said enhancing can be done prior to, or after, said replacing step.
6. The method of claim 5 , wherein said genotype 2b NS5B sequence is obtained from a clinical isolate.
7. A chimeric HCV replicon comprising:
a) a NS3-5A sequence of a genotype 1b replicon or a modified NS3-5A sequence of genotype 1b replicon wherein NS4B contains a V218A modification; and
b) substantially all of a genotype 2b NS5B encoding nucleic acid sequence.
8. The chimeric replicon of claim 7 , further comprising a reporter or selection sequence.
9. The chimeric replicon of claim 7 , wherein said genotype 2b NS5B sequence encodes at least one of the following amino acids in the indicated position:
serine corresponding to position 24 of SEQ ID NO: 1;
isoleucine corresponding to position 31 of SEQ ID NO: 1;
leucine corresponding to position 392 of SEQ ID NO: 1.
10. The chimeric replicon of claim 9 , wherein said genotype 2b NS5B sequence encodes at least isoleucine corresponding to position 31 of SEQ ID NO: 1, or at least serine corresponding to position 24 of SEQ ID NO: 1 in combination with leucine corresponding to position 392 of SEQ ID NO: 1.
11. The chimeric replicon of claim 8 , wherein said genotype 2b NS5B sequence encodes NS5B provided by SEQ ID NO: 1.
12. The chimeric replicon of claim 9 , wherein said NS3-5A sequence consists of SEQ ID NO: 4 and NS5B sequence consists of SEQ ID NO: 2.
13. The chimeric replicon of claim 12 , wherein nucleotide 2711 of SEQ ID NO: 4 is C.
14. A recombinant cell comprising a replicon made the method of claim 1 , wherein said replicon is expressed in said cell.
15. A method of measuring the ability of a compound to inhibit replicon activity comprising the steps of:
a) providing said compound to the recombinant cell of claim 12 , and
b) measuring the ability of said compound to affect replicon activity.
16. A recombinant cell comprising a replicon made by the method of claim 2 , wherein said replicon is expressed in said cell.
17. A recombinant cell comprising a replicon made by the method of claim 4 , wherein said replicon is expressed in said cell.
18. A recombinant cell comprising the replicon of claim 7 , wherein said replicon is expressed in said cell.
19. A recombinant cell comprising the replicon of claim 8 , wherein said replicon is expressed in said cell.
20. A recombinant cell comprising the replicon of claim 9 , wherein said replicon is expressed in said cell.