Sustained Induction of NF-κB 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.