Methods of identifying agents for inhibiting lentivirus replication
The present invention provides screening methods for identifying a compound that induces loss of the lentiviral protein Vpr; screening methods for identifying compounds that inhibit the peptidyl-prolyl cis/trans isomerase (PPIase) activity of a protein that catalyzes cis-trans isomerization of cis-peptidylprolyl bonds in Vpr; and compounds identified by the screening methods. The compounds are useful for treating a lentiviral infection. The present invention further provides methods of inducing loss of the lentiviral protein Vpr; methods of inhibiting lentivirus viral replication; and methods of treating a lentivirus infection in an individual. The methods generally involve administering to an individual infected with the lentivirus an effective amount of a compound that induces Vpr loss and/or that inhibits PPIase activity of a protein that catalyzes cis-trans isomerization of cis-peptidylprolyl bonds in Vpr.
1. A method of identifying a candidate agent for treating a lentivirus infection in a cell the method comprising:
a) contacting a cell that produces a Vpr protein with a test agent; and
b) determining the effect, if any, of the test agent on a peptidyl-prolyl cis/trans isomerase (PPIase) activity of a PPIase protein that catalyzes cis-trans isomerization of cis-peptidylprolyl bonds in Vpr, wherein an agent that inhibits PPIase activity of the PPIase protein reduces the level of Vpr in the cell, and wherein a test agent that reduces the level of Vpr in the cell is a candidate agent for treating a lentivirus infection in the cell.
2. The method of claim 1 , wherein said determining comprises detecting PPIase activity using as a substrate a compound of the formula Xaa-Ala-Xaa-Pro-Phe-X, where Xaa is any amino acid, and wherein X is a moiety that provides a detectable signal.
3. The method of claim 1 , wherein the compound is Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide.
4. The method of claim 1 , further comprising determining the effect, if any, of the agent on Vpr-induced cell cycle arrest.
5. The method of claim 4 , wherein said determining step comprises determining the DNA complement of the cell.
6. The method of claim 5 , wherein said determining step comprises staining the cell with a DNA binding dye.
7. The method of claim 6 , wherein the DNA binding dye is propidium iodide.
8. The method of claim 1 , wherein the PPIase protein is a cyclophilin.
9. The method of claim 2 , wherein X is selected from a chromogenic label, a fluorogenic label, a chemiluminescent label, and a radiolabels.