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Development and validation of a cell-based assay system to assess human immunodeficiency virus type 1 integrase multimerization.

Development and validation of a cell-based assay system to assess human immunodeficiency virus type 1 integrase multimerization.
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Nakamura T, Campbell JR, Moore AR, Otsu S, Aikawa H, Tamamura H, Mitsuya H,


Nakamura T, Campbell JR, Moore AR, Otsu S, Aikawa H, Tamamura H, Mitsuya H, (click to view)

Nakamura T, Campbell JR, Moore AR, Otsu S, Aikawa H, Tamamura H, Mitsuya H,

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Journal of virological methods 2016 7 26() pii 10.1016/j.jviromet.2016.07.023

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.

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