Sustained induction of NF-kappa B is required for efficient expression of latent human immunodeficiency virus type 1.
Cells harboring infectious, but transcriptionally latent, human immunodeficiency virus type 1 (HIV-1) proviruses currently pose an insurmountable barrier to viral eradication in infected patients. To better understand the molecular basis for HIV-1 latency, we used the J-Lat model of postintegration HIV-1 latency to assess the kinetic relationship between the induction of NF-kappaB and the activation of latent HIV-1 gene expression. Chromatin immunoprecipitation analyses revealed an oscillating pattern of RelA recruitment to the HIV-1 long terminal repeat (LTR) during continuous tumor necrosis factor alpha (TNF-alpha) stimulation. RNA polymerase II (Pol II) recruitment to the HIV-1 LTR closely mirrored RelA binding. Transient stimulation of cells with TNF-alpha for 15 min induced only a single round of RelA and RNA Pol II binding and failed to induce robust expression of latent HIV-1. Efficient formation of elongated HIV-1 transcripts required sustained induction by NF-kappaB, which promoted de novo synthesis of Tat. Cyclin-dependent kinase 9 (CDK9) and serine-2-phosphorylated RNA Pol II were rapidly recruited to the HIV-1 LTR after NF-kappaB induction; however, these elongating polymerase complexes were progressively dephosphorylated in the absence of Tat. Okadaic acid promoted sustained serine-2 phosphorylation of the C-terminal domain of RNA Pol II and stimulated efficient transcriptional elongation and HIV-1 expression in the absence of Tat. These findings underscore important differences between NF-kappaB and Tat stimulation of RNA Pol II elongation. While NF-kappaB binding to the HIV-1 LTR induces serial waves of efficient RNA Pol II initiation, elongation is impaired by the action of an okadaic acid-sensitive phosphatase that dephosphorylates the C-terminal domain of RNA Pol II. Conversely, the action of this phosphatase is overcome in the presence of Tat, promoting very efficient RNA Pol II elongation.
Williams
Samuel A
SA
Gladstone Institute of Virology and Immunology, University of California San Francisco, 1650 Owens Street, San Francisco, CA 94158, USA.
Kwon
Hakju
H
Chen
Lin-Feng
LF
Greene
Warner C
WC
D002460:Cell Line; D015967:Gene Expression Regulation, Viral / Q000502:physiology*; D015696:Gene Products, tat / Q000096:biosynthesis / Q000235:genetics; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D016328:NF-kappa B / Q000096:biosynthesis* / Q000502:physiology; D011533:Proviruses / Q000502:physiology; D012319:RNA Polymerase II / Q000502:physiology; D051996:Transcription Factor RelA / Q000502:physiology; D017735:Virus Latency / Q000502:physiology*; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D016328:NF-kappa B; C498143:RELA protein, human; D051996:Transcription Factor RelA; D054322:tat Gene Products, Human Immunodeficiency Virus; D012319:RNA Polymerase II