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.
Woerner
A M
AM
Division of Viral Products, FDA, Bethesda, MD 20892.
Marcus-Sekura
C J
CJ
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D015139:Blotting, Southern; D015153:Blotting, Western; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004254:DNA Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015342:DNA Probes; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D015497:HIV-1 / Q000201:enzymology*; D019426:Integrases; D016296:Mutagenesis; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection
D000911:Antibodies, Monoclonal; D015342:DNA Probes; D011993:Recombinant Fusion Proteins; D004247:DNA; D004254:DNA Nucleotidyltransferases; D019426:Integrases