Screening method for the identification of inhibitors of HIV Tat methylation by the lysine methyltransferase Set7/9
The present invention provides isolated methylated Tat peptides; and compositions comprising the peptides. The present invention further provides isolated antibodies specific for a Lys-51-methylated Tat polypeptide. Also provided are methods of identifying agents that inhibit Lys-51 methylation of a Tat polypeptide. The present invention further provides methods of treating an immunodeficiency virus infection in a mammalian subject.
1. An in vitro method of identifying an agent that inhibits the methyltransferase activity of a methyltransferase that catalyzes methylation of Lys-51 of a human immunodeficiency virus (HIV) Tat polypeptide, the method comprising
a) contacting a test agent with a sample comprising the methyltransferase and an HIV Tat polypeptide, forming a test sample,
wherein the methyltransferase comprises an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:31 and
wherein the HIV Tat polypeptide has a length of from 15 amino acids to 100 amino acids and comprises an amino acid sequence having at least 90% amino acid sequence identity to amino acids 41 through 55 of SEQ ID NO:1; and
b) determining the effect, if any, of the test agent on the methylation of the Tat polypeptide, wherein said determining comprises detecting methylation of lysine at a position corresponding to Lys-51 of the amino acid sequence set forth in SEQ ID NO:1.
2. The method of claim 1 , wherein said Tat polypeptide comprises the amino acid sequence SYGRKKRRQR (SEQ ID NO:27).
3. The method of claim 1 , wherein said determining comprises contacting the test sample with an antibody that specifically binds a methylated Tat polypeptide comprising a methylated lysine at a position corresponding to Lys-51 of SEQ ID NO:1.
4. The method of claim 1 , wherein said method is a cell-free method.
5. The method of claim 1 , wherein said Tat polypeptide comprises the amino acid sequence ISYGRKKRRQRR (SEQ ID NO:28).
6. The method of claim 1 , wherein said Tat polypeptide comprises the amino acid sequence ISYGRKKRRQRRRP (SEQ ID NO:26).
7. The method of claim 1 , wherein said Tat polypeptide is a fusion protein comprising a fusion partner selected from an enzyme and an epitope tag.
8. The method of claim 1 , wherein said Tat polypeptide is biotinylated.