Differential divalent cation requirements uncouple the assembly and catalytic reactions of human immunodeficiency virus type 1 integrase
| Autor: | J C Hastings, Daria J. Hazuda, B Pramanik, Peter J. Felock, Abigail Wolfe |
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| Jazyk: | angličtina |
| Rok vydání: | 1997 |
| Předmět: |
Cations
Divalent Immunology Integrase inhibitor HIV Integrase Microbiology Cofactor Catalysis Divalent Substrate Specificity Virology Humans Magnesium HIV Integrase Inhibitors chemistry.chemical_classification Exonuclease III Manganese biology Virus Assembly Substrate (chemistry) Cobalt Combinatorial chemistry In vitro Integrase chemistry Biochemistry Insect Science biology.protein HIV-1 Calcium Research Article |
| Popis: | Previous in vitro analyses have shown that the human immunodeficiency virus type 1 (HIV-1) integrase uses either manganese or magnesium to assemble as a stable complex on the donor substrate and to catalyze strand transfer. We now demonstrate that subsequent to assembly, catalysis of both 3' end processing and strand transfer requires a divalent cation cofactor and that the divalent cation requirements for assembly and catalysis can be functionally distinguished based on the ability to utilize calcium and cobalt, respectively. The different divalent cation requirements manifest by these processes are exploited to uncouple assembly and catalysis, thus staging the reaction. Staged 3' end processing and strand transfer assays are then used in conjunction with exonuclease III protection analysis to investigate the effects of integrase inhibitors on each step in the reaction. Analysis of a series of related inhibitors demonstrates that these types of compounds affect assembly and not either catalytic process, therefore reconciling the apparent disparate results obtained for such inhibitors in assays using isolated preintegration complexes. These studies provide evidence for a distinct role of the divalent cation cofactor in assembly and catalysis and have implications for both the identification and characterization of integrase inhibitors. |
| Databáze: | OpenAIRE |
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