Comparison of metal-dependent catalysis by HIV-1 and ASV integrase proteins using a new and rapid, moderate throughput assay for joining activity in solution
June 30th, 2009 Posted by: admin
Background:
HIV-1 integrase (IN) is an attractive target for the development of drugs to treat AIDS, and inhibitors of this viral enzyme are already in the clinic. Nevertheless, there is a continuing need to devise new approaches to block the activity of this viral protein because of the emergence of resistant strains. To facilitate the biochemical analysis of wild-type IN and its derivatives, and to measure the potency of prospective inhibitory compounds, a rapid, moderate throughput solution assay was developed for IN-catalyzed joining of viral and target DNAs, based on the detection of a fluorescent tag.
Results:
A detailed, step-by-step description of the new joining assay is provided. The reactions are run in solution, the products captured on streptavidin beads, and activity is measured by release of a fluorescent tag. The procedure can be scaled up for the analysis of numerous samples, and is substantially more rapid and sensitive than the standard radioactive gel methods. The new assay is validated and its utility demonstrated via a detailed comparison of the Mg++- and Mn++-dependent activities of the IN proteins from human immunodeficiency virus type 1 (HIV-1) and the avian sarcoma virus (ASV). The results confirm that ASV IN is considerably more active than HIV IN, but with both enzymes the initial rates of joining, and the product yields, are higher in the presence of Mn++ than Mg++. Although the pH optima for these two enzymes are similar with Mn++, they differ significantly in the presence of Mg++, which is likely due to differences in the molecular environment of the binding region of this physiologically relevant divalent cation. This interpretation is strengthened by the observation that a compound that can inhibit HIV-1 IN in the presence of either metal cofactors is only effective against ASV in the presence of Mn++.
Conclusion:
A simplified, assay for measuring the joining activity of retroviral IN in solution is described, which offers several advantages over previous methods and the standard radioactive gel analyses. Based on comparisons of signal to background ratios, the assay is 10-30 times more sensitive than gel analysis, allows more rapid and accurate biochemical analyses of IN catalytic activity, and moderate throughput screening of inhibitory compounds. The assay is validated, and its utility demonstrated in a comparison of the metal-dependent activities of HIV and ASV IN proteins.
Similar Posts:
- Expression, crystallization and preliminary crystallographic study of mouse hepatitis virus (MHV) nucleocapsid protein C-terminal domain
- Comparative study of the persistence of anti-HIV activity of deoxynucleoside HIV reverse transcriptase inhibitors after removal from culture
- Persons With Moderate Alzheimer’s Disease Improve Activities and Mood via Instruction Technology
- Design, Synthesis, and Biological Application of Fluorescent Sensor Molecules for Cellular Imaging
- Beer-Induced Pancreatic Enzyme Secretion: Characterization of Some Signaling Pathways and of the Responsible Nonalcoholic Compounds
- Dynamic Visualization of Cellular Signaling
- An Assay for Evoked Locomotor Behavior in Drosophila Reveals a Role for Integrins in Ethanol Sensitivity and Rapid Ethanol Tolerance
- Effect of a Web Site Intervention on Physical Activity of College Females
- Crystallization of human complement component C3b in the presence of a staphylococcal complement-inhibitor protein (SCIN)
- Effect of BSA Antigen Sensitization during the Acute Phase of Influenza A Viral Infection on CD11c+ Pulmonary Antigen Presenting Cells.