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Development and validation of a cell-based assay system to assess human immunodeficiency virus type 1 integrase
Tomofumi Nakamura1, Joseph R Campbell1, Amber R Moore1
1Departments of Infectious Diseases and Hematology, Kumamoto University Graduate School of Medical Sciences, Kumamoto 860-8556, Japan.
Journal of Virological Methods
|July 31, 2016
Summary
Disrupting HIV-1 integrase multimerization offers a new HIV intervention strategy. A novel bimolecular fluorescence complementation (BiFC-IN) assay effectively monitors integrase multimerization and aids in discovering new drugs.
Area of Science:
- Virology
- Molecular Biology
- Drug Discovery
Background:
- HIV-1 integrase (IN) multimerization is crucial for integrating viral DNA into the host genome.
- Targeting IN multimerization presents a promising strategy for developing novel anti-HIV therapies.
Purpose of the Study:
- To develop and validate a cell-based assay system for assessing HIV-1 integrase multimerization.
- To evaluate the utility of the assay in identifying compounds that modulate IN multimerization.
Main Methods:
- Construction and implementation of a bimolecular fluorescence complementation assay (BiFC-IN) to visualize IN multimerization in living cells.
- Assessment of fluorescence changes in response to known IN multimerization-associated amino acid substitutions and a non-catalytic site IN inhibitor (NCINI).
Main Results:
- The BiFC-IN system successfully detected IN multimerization, with specific amino acid substitutions attenuating fluorescence.
- A NCINI significantly increased BiFC-IN fluorescence, indicating induced IN over-multimerization.
- Viral resistance substitutions (A128T, E11K, F181T) reduced NCINI-induced fluorescence, suggesting interference with tetramer formation.
Conclusions:
- The developed BiFC-IN cell-based assay is a valuable tool for studying HIV-1 integrase multimerization dynamics.
- This system can aid in the evaluation and discovery of novel therapeutic compounds targeting IN multimerization for HIV-1 infection.
