CHARACTERIZATION OF A DNA-BINDING DOMAIN IN THE C-TERMINUS OF HIV-1 INTEGRASE BY DELETION MUTAGENESIS

The integrase (IN) protein of human immunodeficiency virus type 1 (HIV-1) catalyzes site-specific cleavage of 2 bases from the viral long terminal repeat (LTR) sequence yet it binds DNA with little DNA sequence specificity. We have previously demonstrated that the C-terminal half of IN (amino acids 154 - 288) possesses a DNA binding domain. In order to further characterize this region, a series of clones expressing truncated forms of IN as N-terminal fusion proteins in E. coli were constructed and analyzed by Southwestern blotting. Proteins containing amino acids 1 - 263, 1 - 248 and 170 - 288 retained the ability to bind DNA, whereas a protein containing amino acids 1 - 180 showed no detectable DNA binding. This defines a DNA binding domain contained within amino acids 180 - 248. This region contains an arrangement of 9 lysine and arginine residues each separated by 2 - 4 amino acids (KxxxKxxxKxxxxRxxxRxxRxxxxKxxxKxxxK), spanning amino acids 211 - 244, which is conserved in all HIV-1 isolates. A clone expressing full-length IN with a C-terminal fusion of 16 amino acids was able to bind DNA comparably to a cloned protein with a free C-terminus, and an IN-specific monoclonal antibody which recognizes an epitope contained within amino acids 264 - 279 was unable to block DNA binding, supporting the evidence that a region necessary for binding lies upstream of amino acid 264.