title: Development and validation of a cell-based assay system to assess human immunodeficiency virus type 1 integrase multimerization.
abstract: Multimerization of HIV-1 integrase (IN) subunits is required for the concerted integration of HIV-1 proviral DNA into the host genome. Thus, the disruption of IN multimerization represents a new avenue for intervening HIV-1 infection. Here, we generated a cell-based assay system to assess IN multimerization using a newly constructed bimolecular fluorescence complementation (BiFC-IN) system. BiFC-IN proteins were efficient in emitting fluorescence, and amino acid (AA) substitutions associated with IN multimerization attenuated fluorescence, suggesting that the BiFC-IN system may be useful for evaluating the profile of IN multimerization. A recently reported non-catalytic site IN inhibitor (NCINI), which allosterically induces IN over-multimerization/aggregation, significantly increased fluorescence in the BiFC-IN system. An IN's substitution, A128T, associated with viral resistance to NCINIs, decreased the NCINI-induced increase of fluorescence, suggesting that A128T reduces the potential for IN over-multimerization. Moreover, E11K and F181T substitutions known to inhibit IN tetramerization also reduced the NCINI-induced fluorescence increase, suggesting that NCINI-induced IN over-multimerization was more likely to occur from tetramer subunits than from dimer subunits. The present study demonstrates that our cell-based BiFC-IN system may be useful in elucidating the profile of IN multimerization, and also help evaluate and identify novel compounds that disrupt IN multimerization in living cells.
authors.affiliation: Departments of Infectious Diseases and Hematology, Kumamoto University Graduate School of Medical Sciences, Kumamoto 860-8556, Japan.
authors.forename: Tomofumi
authors.initials: T
authors.lastname: Nakamura
authors.affiliation: Departments of Infectious Diseases and Hematology, Kumamoto University Graduate School of Medical Sciences, Kumamoto 860-8556, Japan.
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Campbell
authors.affiliation: Departments of Infectious Diseases and Hematology, Kumamoto University Graduate School of Medical Sciences, Kumamoto 860-8556, Japan.
authors.forename: Amber R
authors.initials: AR
authors.lastname: Moore
authors.affiliation: Departments of Infectious Diseases and Hematology, Kumamoto University Graduate School of Medical Sciences, Kumamoto 860-8556, Japan.
authors.forename: Sachiko
authors.initials: S
authors.lastname: Otsu
authors.affiliation: Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo, 101-0062, Japan.
authors.forename: Haruo
authors.initials: H
authors.lastname: Aikawa
authors.affiliation: Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo, 101-0062, Japan.
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Tamamura
authors.affiliation: Departments of Infectious Diseases and Hematology, Kumamoto University Graduate School of Medical Sciences, Kumamoto 860-8556, Japan; Experimental Retrovirology Section, National Center for Global Health and Medicine Research Institute, Shinjuku, Tokyo 162-8655, Japan; HIV and AIDS Malignancy Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address: mitsuyah@helix.nih.gov.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Mitsuya
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D003584:Cytological Techniques / Q000379:methods*; D019427:HIV Integrase / Q000378:metabolism*; D019428:HIV Integrase Inhibitors / Q000032:analysis*; D015497:HIV-1 / Q000201:enzymology*; D061848:Optical Imaging / Q000379:methods*; D055503:Protein Multimerization
chemical_list: D019428:HIV Integrase Inhibitors; D019427:HIV Integrase
keywords: BiFC; HIV-1; HIV-1 integrase multimerization; NCINIs