A biochemical analysis of topoisomerase II α and β kinase activity found in HIV-1 infected cells and virus

Abstract

Human topoisomerase II plays a crucial role in DNA replication and repair. It exists in two isoforms: topoisomerase II alpha (α) and topoisomerase II beta (β). The α isoform is localized predominantly in the nucleus, while the β isoform exhibits a reticular pattern of distribution both in the cytosol and in the nucleus. We show that both isoforms of topoisomerase II are phosphorylated in HIV infected cells and also by purified viral lysate. An analysis of the phosphorylation of topoisomerase II isoforms showed that extracts of HIV infected cells at 8 and 32 h. post-infection (p.i.) contain maximal phosphorylated topoisomerase II α, whereas infected cell extracts at 4 and 64 h p.i. contain maximum levels of phosphorylated topoisomerase II β. In concurrent to phosphorylated topoisomerase II isoforms, we have also observed increased topoisomerase II α kinase activity after 8 h p.i and topoisomerase β kinase activity at 4 and 64 h p.i. These findings suggest that both topoisomerase II α and β kinase activities play an important role in early as well as late stages of HIV-1 replication. Further analysis of purified virus showed that HIV-1 virion contained topoisomerase II isoform-specific kinase activities, which were partially isolated. One of the kinase activities of higher hydrophobicity can phosphorylate both topoisomerase II α and β, while lower hydrophobic kinase could predominantly phosphorylate topoisomerase II α. The phosphorylation status was correlated with catalytic activity of the enzyme. Western blot analysis using phosphoamino-specific antibodies shows that both the kinase activities catalyze the phosphorylation at serine residues of topoisomerase II α and β. The catalytic inhibitions by serine kinase inhibitors further suggest that the α and β kinase activities associated with virus are distinctly different. © 2005 Elsevier Inc. All rights reserved.