Nuclear IκBβ maintains persistent NF-κB activation in HIV-1-infected myeloid cells

Abstract

Monocytic cells exhibit constitutive NF-κB activation upon infection with human immunodeficiency virus-1 (HIV-1). Because IκBβ has been implicated in maintaining NF-κB·DNA binding, we sought to investigate whether IκBβ was involved in maintaining persistent NF-κB activation in HIV-1-infected monocytic cell lines. IκBβ was present in the nucleus of HIV-1-infected cells and participated in the ternary complex formation with NF-κB and DNA. In contrast to uninfected cells, the addition of recombinant glutathione S-transferase-IκBα protein to preformed NF-κB·DNA complexes from HIV-1-infected cell extracts did not completely dissociate the complexes, suggesting that IκBβ may protect NF-κB complexes from IκBα- mediated dissociation. Immunodepletion of IκBβ resulted in an NF-κB·DNA binding complex that was sensitive to IκBα-mediated dissociation, thus demonstrating the protective role of IκBβ. In addition, co-transfection studies with an NF-κB-dependent reporter construct demonstrated that IκBβ co-expression partially alleviated inhibition of NF-κB-mediated gene expression by IκBα, implying that IκBβ can maintain transcriptionally active NF-κB·DNA complexes. Furthermore, constitutive phosphorylation of IκBα was observed. Immunoprecipitation of the IκB kinase (IKK) complex followed by in vitro analysis of kinase activity demonstrated that IKK was constitutively activated in HIV-1-infected myeloid cells. Thus, virus- induced constitutive IKK activation, coupled with the maintenance of a ternary NF-κB·DNA complex by IκBβ, maintains persistent NF-κB activity in HIV-1-infected myeloid cells.