Domains of the integrase protein of human immunodeficiency virus type 1 responsible for polynucleotidyl transfer and zinc binding.

Abstract

The integrase protein of human immunodefi- (1)(2)(3)(4)(5)(6). Integrase thenjoins the recessed 3' ends to protruding 5' ends of breaks made by integrase in the target DNA (the "strand transfer" reaction) (3,4,(7)(8)(9)(10). According to our present understanding, host DNA repair enzymes process and connect the remaining unjoined ends, thereby completing the formation of a provirus (for reviews see refs. 11 and 12). The polynucleotidyl transfer reactions carried out by the integrase proteins of retroviruses are similar to the reactions carried out by the integrases of retrotransposons and also the reactions carried out by the transposase proteins of several prokaryotic transposons (13). The integrase protein of the yeast retrotransposon Tyl is known to join 3' ends of the element DNA to 5' ends in the host DNA (14). For the case of the transposons, the transposase proteins of phage Mu, TnlO, and Tn7 each direct the hydrolysis of phosphodiester bonds that connect element DNA to host DNA and then join the free 3' ends generated by this cleavage to target DNA (15-17).Two conserved amino acid sequence motifs have been found in the integrase proteins of retroviruses and retrotransposons. One motif, found also in the transposase proteins of some bacterial transposons, consists of three invariant acidic residues in the arrangement DX39-58DX35E with various interdigitated conserved residues (the D,D-35-E motif) (18)(19)(20). Another amino acid sequence motif, HX3_7HX23-32CX2C (the HHCC motif), is present near the amino terminus (21-23). This sequence is reminiscent of the "zinc finger" motif that is known to mediate metal binding in many nucleic acid-binding proteins (for review see ref. 24).To explore the function ofthe various parts ofthe integrase protein of human immunodeficiency virus type 1 (HIV-1), we generated and characterized a series of deletion mutants. We find that a polypeptide of only 137 amino acid residues containing the D,D-35-E motif can carry out a subset of the reactions carried out by the complete protein. Our assays of Zn2+ binding by a series ofintegrase derivatives bearing point mutations indicate that the HHCC motif does in fact bind Zn2+. The activities of the integrase mutants put constraints on the possible roles of the various parts ofthe HIV integrase protein. MATERIALS AND METHODSConstruction of Deletion Mutants. Deletion derivatives of the HIV-1 integrase protein were prepared by constructing plasmids encoding the deletion proteins. Procedures for manipulating DNA molecules were essentially as described (25). DNA fragments encoding each deletion derivative were prepared by PCR using pINSD (26) as template. PCR primers were designed to provide an Nde I site in the DNA encoding the amino terminus, and a stop codon followed by a BamHI site in the DNA encoding the carboxyl terminus. Amplification was carried out for 15 cycles using Vent DNA polymerase (New England Biolabs) under the manufacturer's suggested conditions. PCR products were cleaved with BamHI and Nde I and ligated to pA...

Polynucleotidyl transfer reactions in site‐specific DNA recombination

“…In the case of retroviral integrase, the core domain can carry out a reaction called disintegration which mimics the reversal of the normal strand transfer step, although the normal reaction requires the intact protein (Chow et al 1992;Drelich et al 1992;Bushman et al 1993;Vink et al 1993). The cause of the difference in specificity of the reaction direction between the core domain and the intact integrase is currently not well Polynucleotidyl transfer reactions in site-specific DNA recombination…”

FIV: from lentivirus to lentivector

“…-Class I FIV integrase mutants. Certain catalytic triad mutations (termed class I) in HIV-1 integrase mutations prevent the integrase reaction but leave other Gag/Pol functions undisturbed [152][153][154][155]. We have now developed the first class I mutants of FIV integrase by targeting such residues for mutation, and have validated their class I properties [62,156].…”

Human Immunodeficiency Virus Type 1 (HIV-1) Integrase: Resistance to Diketo Acid Integrase Inhibitors Impairs HIV-1 Replication and Integration and Confers Cross-Resistance to l -Chicoric Acid

“…In disintegration, IN is able to correctly resolve into its respective parts an oligonucleotide resembling viral DNA joined to host DNA (12). While the full-length IN protein is required for the 3Ј-end processing and strand transfer reactions (60), the catalytic core domain of IN is sufficient for disintegration (7). Whether the disintegration reaction occurs in vivo is unknown.…”

“…This triad coordinates a divalent metal ion; either magnesium or manganese is required for catalytic activity and is absolutely conserved among INs from retroviruses and retrotransposons (38). The N-terminal domain is believed to be involved in protein multimerization (40) and stabilization of protein folding and contains a His-His-Cys-Cys zinc finger-like motif, which coordinates zinc (6,7). The C-terminal domain of IN has nonspecific DNA binding activity; thus, it is thought to play a role in binding to host DNA (23).…”