FRET-based reagents and methods for identifying anti-HIV compounds
A modified HIV gp120 protein labeled with a fluorophore has been used herein in single-molecule imaging techniques to demonstrate the conformation states of HIV-1 Env. The introduction of small organic fluorophores at selected positions within HIV-1 envelope protein gp120 that do not affect infectivity permits the detection of changes in inter-dye distances as a measure of conformational changes. Implementation of the smFRET-based technologies disclosed herein enable conformational screening for molecules that block, induce or trap HIV-1 Env in specific conformational states. Methods for identifying anti-HIV drugs in a smFRET-based screening, and various reagents useful for implementation of such methods, are provided.
1. A modified HIV gp120 protein comprising a first mutation in a first variable loop, wherein said first mutation comprises an insertion of at least one amino acid in said first variable loop and said at least one amino acid inserted in said first variable loop is labeled with a first fluorophore.
2. The modified HIV gp120 protein of claim 1 , wherein said first variable loop is selected from the group consisting of V1, V2, V3, V4 and V5.
3. A modified HIV gp120 protein comprising a first mutation in a first variable loop, wherein said first mutation comprises an insertion of at least one amino acid in said first variable loop, and wherein said at least one amino acid inserted in said first variable loop is a first unnatural amino acid.
4. A modified HIV gp120 protein comprising a first variable loop, wherein a first peptide is inserted into said first variable loop, wherein said first peptide is selected from the group consisting of the S6 tag, the A1 tag, and the Q3 tag.
5. A modified HIV gp120 protein comprising a first mutation in a first variable loop, wherein said first mutation comprises an insertion of at least one amino acid in said first variable loop, and comprising a second mutation in a second variable loop, wherein said second mutation comprises an insertion of at least one amino acid in said second variable loop for labeling with a first fluorophore.
6. The modified HIV gp120 protein of claim 5 , wherein the second variable loop is different from the first variable loop and is selected from the group consisting of V1, V2, V3, V4 and V5.
7. The modified HIV gp120 protein of claim 6 , wherein the first and second variable loops are V1 and V4.
8. The modified HIV gp120 protein of claim 5 , wherein said at least one amino acid inserted in said second variable loop is an unnatural amino acid.
9. The modified HIV gp120 protein of claim 5 , wherein a peptide is inserted into said second variable loop.
10. The modified gp120 protein of claim 9 , wherein the peptide is selected from the group consisting of the S6 tag, the A1 tag, and the Q3 tag.
11. An isolated HIV Env trimer, comprising the modified HIV gp120 according to claim 1 .
12. An isolated HIV Env trimer comprising a modified HIV gp120, and further comprising an unmodified HIV gp120, wherein the modified HIV gp120 comprises a first mutation in a first variable loop, and wherein said first mutation comprises an insertion of at least one amino acid in said first variable loop, and wherein said at least one amino acid inserted in said first variable loop is labeled with a first fluorophore.
13. The isolated HIV Env trimer of claim 12 , composed of two copies of the unmodified HIV gp120 and one copy of the modified HIV gp120.
14. The isolated HIV Env trimer of claim 13 , wherein the at least one amino acid inserted is labeled with at least one fluorophore.
15. The isolated HIV Env trimer of claim 13 , wherein said first mutation comprises a first peptide inserted in the first variable loop, and wherein the modified HIV gp120 further comprises a second mutation comprising a second peptide inserted in a second variable loop and the peptides are labeled with a first and second fluorophores, respectively.
16. An isolated HIV Env gp140 soluble trimer, comprising a copy of a modified gp140 and two copies of unmodified gp140, wherein said modified gp140 comprises an insertion of at least one amino acid in a first variable loop, and an insertion of at least one amino acid in a second variable loop.
17. The isolated HIV Env gp140 soluble trimer of claim 16 , labeled with a first fluorophore at said at least one amino acid inserted in the first variable loop and a second fluorophore at said at least one amino acid inserted in the second variable loop.
18. An isolated HIV virion, comprising the HIV trimer of claim 13 .
19. An isolated HIV virion, comprising the HIV trimer of claim 15 .
20. An isolated HIV virion, comprising the soluble trimer of claim 16 .
21. An isolated HIV virion, comprising the HIV trimer of claim 17 .
22. The modified HIV gp120 protein of claim 5 , wherein said at least one amino acid inserted in the first variable loop is labeled with a second flourophore, and said at least one amino acid inserted in the second variable loop is labeled with the first flourophore.