Nuclear IkappaBbeta maintains persistent NF-kappaB activation in HIV-1-infected myeloid cells.
Monocytic cells exhibit constitutive NF-kappaB activation upon infection with human immunodeficiency virus-1 (HIV-1). Because IkappaBbeta has been implicated in maintaining NF-kappaB.DNA binding, we sought to investigate whether IkappaBbeta was involved in maintaining persistent NF-kappaB activation in HIV-1-infected monocytic cell lines. IkappaBbeta was present in the nucleus of HIV-1-infected cells and participated in the ternary complex formation with NF-kappaB and DNA. In contrast to uninfected cells, the addition of recombinant glutathione S-transferase-IkappaBalpha protein to preformed NF-kappaB.DNA complexes from HIV-1-infected cell extracts did not completely dissociate the complexes, suggesting that IkappaBbeta may protect NF-kappaB complexes from IkappaBalpha-mediated dissociation. Immunodepletion of IkappaBbeta resulted in an NF-kappaB.DNA binding complex that was sensitive to IkappaBalpha-mediated dissociation, thus demonstrating the protective role of IkappaBbeta. In addition, co-transfection studies with an NF-kappaB-dependent reporter construct demonstrated that IkappaBbeta co-expression partially alleviated inhibition of NF-kappaB-mediated gene expression by IkappaBalpha, implying that IkappaBbeta can maintain transcriptionally active NF-kappaB.DNA complexes. Furthermore, constitutive phosphorylation of IkappaBalpha was observed. Immunoprecipitation of the IkappaB 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-kappaB.DNA complex by IkappaBbeta, maintains persistent NF-kappaB activity in HIV-1-infected myeloid cells.
DeLuca
C
C
Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, McGill University, Montreal, Quebec H3T 1E2, Canada.
Petropoulos
L
L
Zmeureanu
D
D
Hiscott
J
J
D001483:Base Sequence; D001854:Bone Marrow Cells / Q000821:virology; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D004247:DNA; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004789:Enzyme Activation; D015497:HIV-1 / Q000302:isolation & purification; D006801:Humans; D051550:I-kappa B Kinase; D020672:I-kappa B Proteins; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; C083487:I kappa B beta protein; D020672:I-kappa B Proteins; D016328:NF-kappa B; D020033:Protein Isoforms; D004247:DNA; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human