title: Transdominant mutants of I kappa B alpha block Tat-tumor necrosis factor synergistic activation of human immunodeficiency virus type 1 gene expression and virus multiplication.
abstract: The human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) contains two binding sites for the NF-kappa B/Rel family of transcription factors which are required for the transcriptional activation of viral genes by inflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha) and interleukin-1. In the present study, we examined the effect of transdominant mutants of I kappa B alpha on the synergistic activation of the HIV-1 LTR by TNF-alpha and the HIV-1 transactivator, Tat, in Jurkat T cells. The synergistic induction of HIV-1 LTR-driven gene expression represented a 50- to 70-fold stimulation and required both an intact HIV-1 enhancer and Tat-TAR element interaction, since mutations in Tat protein (R52Q, R53Q) or in the bulge region of the TAR element that eliminated Tat binding to TAR were unable to stimulate LTR expression. Coexpression of I kappa B alpha inhibited Tat-TNF-alpha activation of HIV LTR in a dose-dependent manner. Transdominant forms of I kappa B alpha, mutated in critical serine or threonine residues required for inducer-mediated (S32A, S36A) and/or constitutive (S283A, T291A, T299A) phosphorylation of I kappa B alpha were tested for their capacity to block HIV-1 LTR transactivation. I kappa B alpha molecules mutated in the N-terminal sites were not degraded following inducer-mediated stimulation (t1/2, > 4 h) and were able to efficiently block HIV-1 LTR transactivation. Strikingly, the I kappa B alpha (S32A, S36A) transdominant mutant was at least five times as effective as wild-type I kappa B alpha in inhibiting synergistic induction of the HIV-1 LTR. This mutant also effectively inhibited HIV-1 multiplication in a single-cycle infection model in Cos-1 cells, as measured by Northern (RNA) blot analysis of viral mRNA species and viral protein production. These experiments suggest a strategy that may contribute to inhibition of HIV-1 gene expression by interfering with the NF-kappa B/Rel signaling pathway.
authors.affiliation: Lady Davis Institute for Medical Research, Montreal, Quebec, Canada.
authors.forename: P
authors.initials: P
authors.lastname: Beauparlant
authors.forename: H
authors.initials: H
authors.lastname: Kwon
authors.forename: M
authors.initials: M
authors.lastname: Clarke
authors.forename: R
authors.initials: R
authors.lastname: Lin
authors.forename: N
authors.initials: N
authors.lastname: Sonenberg
authors.forename: M
authors.initials: M
authors.lastname: Wainberg
authors.forename: J
authors.initials: J
authors.lastname: Hiscott
mesh_terms: D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D015967:Gene Expression Regulation, Viral; D015696:Gene Products, tat / Q000096:biosynthesis / Q000378:metabolism*; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D006801:Humans; D020672:I-kappa B Proteins; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000378:metabolism*; D014779:Virus Replication; D054322:tat Gene Products, Human Immunodeficiency Virus
chemical_list: D004268:DNA-Binding Proteins; D015696:Gene Products, tat; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D054322:tat Gene Products, Human Immunodeficiency Virus; D000072000:NF-KappaB Inhibitor alpha