ZN2-ASSOCIATION OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 INTEGRASE IN-VITRO( PROMOTES THE SELF)

It has been recently demonstrated that the Mg2+-dependent 3'-processin g activity of purified human immunodeficiency virus type-1 (HIV-I) int egrase is stimulated by thr addition oi exogenous Zn2+ [Lee, S. P., & Han, M. K. (1996) Biochemistry 35, 3837-3844]. This activation was hyp othesized to result from integrase self-association. In this report, w e examine the Zn2+ content of purified HIV-1 integrase by atomic absor ption 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 int egrase 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 chromat ographic analyses all indicate that EDTA yields an apoprotein which is predominantly monomeric and less active with Mg2+. Further, sedimenta tion equilibrium studies reveal that reconstitution of the apoprotein with Zn2+ results in a monomer-tetramer-octamer transition. These resu lts suggest that Zn2+ promotes a conformation with enhanced oligomeriz ation and thereby stimulates This may also imply that multimers larger than dimers (tetramers and possibly octamers) are required for in vit ro activity of integrase in the presence of Zn2+ and Mg2+. It should b e noted, however, that the content of Zn2+ did not significantly affec t the 3'-processing and strand transfer reactions with Mn2+ in vitro.