Zn2+ Promotes the Self-Association of Human Immunodeficiency Virus Type-1 Integrase in Vitro

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Abstract
It has been recently demonstrated that the Mg2+-dependent 3‘-processing activity of purified human immunodeficiency virus type-1 (HIV-1) integrase is stimulated by the addition of exogenous Zn2+ [Lee, S. P., & Han, M. K. (1996) Biochemistry 35, 3837−3844]. This activation was hypothesized to result from integrase self-association. In this report, we examine the Zn2+ content of purified HIV-1 integrase by atomic absorption spectroscopy and by application of a thiol modification reagent, p-(hydroxymercuri)benzenesulfonate, with a metallochromic indicator, 4-(2-pyridylazo)resorcinol. We find that the Zn2+ content of HIV-1 integrase varies from 0.1 to 0.92 equiv of Zn2+ per monomer depending on the conditions of protein purification. In vitro activity assays, time-resolved fluorescence emission anisotropy, and gel filtration chromatographic analyses all indicate that EDTA yields an apoprotein which is predominantly monomeric and less active with Mg2+. Further, sedimentation equilibrium studies reveal that reconstitution of the apoprotein with Zn2+ results in a monomer−tetramer−octamer transition. These results suggest that Zn2+ promotes a conformation with enhanced oligomerization and thereby stimulates Mg2+-dependent 3‘-processing. This may also imply that multimers larger than dimers (tetramers and possibly octamers) are required for in vitro activity of integrase in the presence of Zn2+ and Mg2+. It should be noted, however, that the content of Zn2+ did not significantly affect the 3‘-processing and strand transfer reactions with Mn2+in vitro.