An essential interaction between distinct domains of HIV-1 integrase mediates assembly of the active multimer.
Integrase mediates integration of the retroviral genome into a host cell chromosome, an essential step in the viral life cycle. In vitro, a stable complex containing only purified human immunodeficiency virus (HIV) integrase and a model viral DNA substrate processively executes the 3'-end processing and DNA joining steps in the integration reaction. We examined the relationship of three essential components of the HIV integrase: the HHCC domain, a putative zinc-finger near the N terminus; the phylogenetically conserved "DD35E" motif, which defines the catalytic domain; and a feature recognized by its sensitivity to the alkylating agent N-ethylmaleimide (NEM). HIV integrase is a multimer, and these three components can be distributed among at least two subunits of the multimeric enzyme. The components function asymmetrically in the active multimer; the DD35E motif and NEM-sensitive site are required in trans to the HHCC region. A divalent cation-dependent interaction involving the NEM-sensitive site of one integrase subunit and the HHCC region of another subunit points to a role for these two features of integrase in multimer assembly. Deletion of the HHCC domain, or modification of integrase with NEM, impaired the assembly of a stable complex between integrase and viral DNA, suggesting that this initial step in the integration pathway requires assembly of the active integrase multimer.
Department of Biochemistry, Stanford University, California 94305.
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Ellison
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Gerton
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Vincent
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Brown
D000595:Amino Acid Sequence; D001483:Base Sequence; D002712:Chlorides / Q000494:pharmacology; D004254:DNA Nucleotidyltransferases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005816:Genetic Complementation Test; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D019426:Integrases; D007700:Kinetics; D046911:Macromolecular Substances; D015636:Magnesium Chloride / Q000494:pharmacology; D017895:Manganese Compounds / Q000494:pharmacology; D008961:Models, Structural; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D017354:Point Mutation; D017384:Sequence Deletion; D013379:Substrate Specificity; D016662:Virus Integration
D002712:Chlorides; D046911:Macromolecular Substances; D017895:Manganese Compounds; D009838:Oligodeoxyribonucleotides; D015636:Magnesium Chloride; D004254:DNA Nucleotidyltransferases; D019426:Integrases; D005033:Ethylmaleimide; C025340:manganese chloride; D004229:Dithiothreitol