Methods for treating lentivirus infections
The present invention provides methods of identifying an agent that inhibits an activity of a lentiviral Vif protein. The present invention provides methods of identifying an agent that increases the level of active APOBEC3G in a cell. The present invention provides agents identified by a subject screening method; and further provides methods for treating lentivirus infections.
1. A genetically modified eukaryotic cell comprising:
(i) an amount of a nucleic acid consisting of a nucleotide sequence encoding an APOBEC3G polypeptide and operably linked to a nucleic acid comprising regulatory nucleotide sequences; and
(ii) an amount of a nucleic acid consisting of a nucleotide sequence encoding a Vif polypeptide and operably linked to a nucleic acid comprising regulatory nucleotide sequences;
wherein the amount (i) and (ii) is selected to produce an amount of the APOBEC3G polypeptide and an amount of the Vif polypeptide in the cell so that the amount of the APOBEC3G polypeptide and the amount of the Vif polypeptide produced in the cell permit
(a) detecting a Vif polypeptide-mediated reduction in the level of the APOBEC3G polypeptide in the cell;
(b) detecting a reduced incorporation of the APOBEC3G polypeptide into a human immunodeficiency virus virion; or
(c) detecting a shortening of the half-life of the APOBEC3G polypeptide.
2. The genetically modified cell of claim 1 , wherein the cell is permissive to infection with a human immunodeficiency virus.
3. The genetically modified cell of claim 1 , wherein the APOBEC3G polypeptide is a fusion protein.
4. The genetically modified cell of claim 3 , wherein the APOBEC3G fusion protein comprises an immunological tag.
5. The genetically modified cell of claim 3 , wherein the APOBEC3G fusion protein comprises a protein that provides a detectable signal.
6. The genetically modified cell of claim 1 , wherein the cell is an immortalized cell line.
7. The genetically modified cell of claim 6 , wherein the cell is selected from the group consisting of a Supt1 cell, a Jurkat cell, and a 293T cell.
8. The genetically modified cell of claim 1 , wherein the cell is transiently transfected (i).
9. The genetically modified cell of claim 1 , wherein the cell is stably transfected with (i).
10. A genetically modified eukaryotic cell comprising:
(i) a nucleic acid consisting of a nucleotide sequence encoding an APOBEC3G polypeptide and operably linked to a nucleic acid comprising regulatory nucleotide sequences; and
(ii) a nucleic acid consisting of a nucleotide sequence encoding a Vif polypeptide and operably linked to a nucleic acid comprising regulatory nucleotide sequences;
wherein the cell comprises (i) and (ii) at a ratio selected to produce an amount of the APOBEC3G polypeptide and an amount of the Vif polypeptide in the cell; and
wherein the amount of the APOBEC3G polypeptide and the amount of the Vif polypeptide produced in the cell permit
(a) detecting a Vif polypeptide-mediated reduction in the level of the APOBEC3G polypeptide in the cell;
(b) detecting a reduced incorporation of the APOBEC3G polypeptide into a human immunodeficiency virus virion; or
(c) detecting a shortening of the half-life of the APOBEC3G polypeptide.
11. The genetically modified cell of claim 10 , wherein the cell is permissive to infection with a human immunodeficiency virus.
12. The genetically modified cell of claim 10 , wherein the cell is transiently transfected with (i) or (ii) or (i) and (ii).
13. The genetically modified cell of claim 10 , wherein the cell is stably transfected with (i) or (ii) or (i) and (ii).
14. The genetically modified cell of claim 10 , wherein the Vif polypeptide is a fusion protein.
15. The genetically modified cell of claim 14 , wherein the Vif fusion protein comprises Vif and glutathione-S-transferase.
16. The genetically modified cell of claim 10 , wherein the cell is an immortalized cell line.
17. The genetically modified cell of claim 16 , wherein the cell is selected from the group consisting of a Supt1 cell, a Jurkat cell, and a 293T cell.
18. The genetically modified cell of claim 10 , wherein the APOBEC3G polypeptide is a fusion protein.
19. The genetically modified cell of claim 18 , wherein the APOBEC3G fusion protein comprises an immunological tag.
20. The genetically modified cell of claim 18 , wherein the APOBEC3G fusion protein comprises a protein that provides a detectable signal.
21. The genetically modified cell of claim 20 , wherein the protein that provides a detectable signal comprises a luciferase.
22. The genetically modified cell of claim 20 , wherein the protein that provides a detectable signal comprises a fluorescent protein.
23. The genetically modified cell of claim 10 , wherein the nucleic acid encoding the Vif polypeptide is operably linked to an inducible promoter.
24. The genetically modified cell of claim 1 , further comprising a nucleic acid encoding ubiquitin.
25. The genetically modified cell of claim 10 , further comprising a nucleic acid encoding ubiquitin.
26. The genetically modified cell according to claim 1 , wherein the cell is a mammalian cell.
27. The genetically modified cell according to claim 26 , wherein the mammalian cell is a human cell.
28. The genetically modified cell according to claim 6 , wherein the immortalized cell line is a human cell line.
29. The genetically modified cell according to claim 1 , wherein the cell comprises the human immunodeficiency virus.
30. The genetically modified cell according to claim 1 , wherein the cell comprises the human immunodeficiency provirus.
31. The genetically modified cell according to claim 1 , wherein the amount of (i)/(ii) is 0.5 or less.
32. The genetically modified cell according to claim 31 , wherein the amount of (i)/(ii) is 0.2 or less.
33. The genetically modified cell according to claim 31 , wherein the amount of (i)/(ii) is 0.1.
34. The genetically modified cell according to claim 1 , further comprising
(iii) an amino-terminal or carboxy-terminal degradation product of the APOBEC3G polypeptide.
35. The genetically modified cell according to claim 10 , wherein the cell is a mammalian cell.
36. The genetically modified cell according to claim 35 , wherein the mammalian cell is a human cell.
37. The genetically modified cell according to claim 16 , wherein the immortalized cell line is a human cell line.
38. The genetically modified cell according to claim 10 , wherein the ratio of (i)/(ii) is 0.5 or less.
39. The genetically modified cell according to claim 38 , wherein the ratio of (i)/(ii) is 0.2 or less.
40. The genetically modified cell according to claim 38 , wherein the ratio of (i)/(ii) is 0.1.
41. The genetically modified cell according to claim 10 , further comprising
(iii) an amino-terminal or carboxy-terminal degradation product of the APOBEC3G polypeptide.
42. A mammalian cell culture comprising the genetically modified eukaryotic cell of claim 1 .
43. The mammalian cell culture according to claim 42 , wherein the APOBEC3G polypeptide is a fusion protein.
44. The mammalian cell culture according to claim 42 , wherein the APOBEC3G fusion protein comprises an immunological tag.
45. The mammalian cell culture according to claim 42 , wherein the APOBEC3G fusion protein comprises a protein that provides a detectable signal.
46. The mammalian cell culture according to claim 45 , wherein the protein that provides a detectable signal comprises a luciferase.
47. The mammalian cell culture according to claim 45 , wherein the protein that provides a detectable signal comprises a fluorescent protein.
48. The mammalian cell culture according to claim 42 , further comprising
(iii) an amino-terminal or carboxy-terminal degradation product of the APOBEC3G polypeptide.