title: Characterization of feline immunodeficiency virus integrase and analysis of functional domains.
abstract: The wild-type and mutant derivatives of the integrase protein of feline immunodeficiency virus (FIV) were cloned and expressed in Escherichia coli. The purified proteins were examined using various model DNA substrates for their catalytic activities: 3'-end processing, 3'-end joining, and disintegration. The reactions required the presence of either Mn2+ or Mg2+ as a divalent cation. The N-terminal and C-subterminal domains (residues 1-52 and 189-235, respectively) were necessary for 3'-end processing and joining reactions but not for disintegration. Substitution of asparagine for the highly conserved aspartic acid at position 118 resulted in a complete loss of all three activities, confirming that the catalytic domain resides in the central core region (residues 53-188) of the protein. Deletion of the C-terminus (residues 236-281) resulted in a FIV integrase mutant that had efficient 3'-end processing and disintegration activities but weak 3'-end joining activity, a finding that has not been reported previously with other retroviral integrases. The result suggests that the C-terminus is the primary binding site for target DNA. Attachment of a histidine-tag at the N-terminus of the wild-type and deletion derivatives increased the binding affinity to the DNA substrate, resulting in altered levels of catalytic activities and selection of integration sites. Similar to other retroviral integrases, certain pairs of mutant derivatives of FIV integrase could complement each other to restitute 3'-end processing and joining activities, suggesting that formation of functional multimers is a general feature of proteins in the integrase family.
authors.affiliation: Department of Molecular and Medical Pharmacology, UCLA School of Medicine 90095, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Shibagaki
authors.forename: M L
authors.initials: ML
authors.lastname: Holmes
authors.forename: R S
authors.initials: RS
authors.lastname: Appa
authors.forename: S A
authors.initials: SA
authors.lastname: Chow
mesh_terms: D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002415:Cats; D016089:Immunodeficiency Virus, Feline / Q000201:enzymology*; D019426:Integrases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009154:Mutation; D016324:Sequence Tagged Sites
chemical_list: D019426:Integrases