Development and validation of a cell-based assay system to assess human immunodeficiency virus type 1 integrase multimerization.
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.
['Nakamura|Tomofumi|T|', 'Campbell|Joseph R|JR|', 'Moore|Amber R|AR|', 'Otsu|Sachiko|S|', 'Aikawa|Haruo|H|', 'Tamamura|Hirokazu|H|', 'Mitsuya|Hiroaki|H|']
D000818:Animals D019556:COS Cells D002522:Chlorocebus aethiops D003584:Cytological Techniques D019427:HIV Integrase D019428:HIV Integrase Inhibitors D015497:HIV-1 D061848:Optical Imaging D055503:Protein Multimerization
HIV-1 NCINIs BiFC HIV-1 integrase multimerization