Negative Elongation Factor Is Required for the Maintenance of Proviral Latency but Does Not Induce Promoter-Proximal Pausing of RNA Polymerase II on the HIV Long Terminal Repeat

Abstract

The role of the negative elongation factor (NELF) in maintaining HIV latency was investigated following small hairpin RNA (shRNA) knockdown of the NELF-E subunit, a condition that induced high levels of proviral transcription in latently infected Jurkat T cells. Chromatin immunoprecipitation (ChIP) assays showed that latent proviruses accumulate RNA polymerase II (RNAP II) on the 5′ long terminal repeat (LTR) but not on the 3′ LTR. NELF colocalizes with RNAP II, and its level increases following proviral induction. RNAP II pause sites on the HIV provirus were mapped to high resolution by ChIP with high-throughput sequencing (ChIP-Seq). Like cellular promoters, RNAP II accumulates at around position +30, but HIV also shows additional pausing at +90, which is immediately downstream of a transactivation response (TAR) element and other distal sites on the HIV LTR. Following NELF-E knockdown or tumor necrosis factor alpha (TNF-α) stimulation, promoter-proximal RNAP II levels increase up to 3-fold, and there is a dramatic increase in RNAP II levels within the HIV genome. These data support a kinetic model for proviral transcription based on continuous replacement of paused RNAP II during both latency and productive transcription. In contrast to most cellular genes, HIV is highly activated by the combined effects of NELF-E depletion and activation of initiation by TNF-α, suggesting that opportunities exist to selectively activate latent HIV proviruses.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.01013-13.

ACKNOWLEDGMENTS

We thank Sheldon Bai for help in the ChIP-Seq analysis, Mudit Tyagi for help in preparing ChIP-Seq samples, David Alvarez and Michael Greenberg for help in the development of the sequence-based ChIP assay, and our colleagues Nick Funderburg and Doug Bazdar for assistance in the multicolor flow cytometry. We also thank present and former Karn laboratory members Hongxia Mao, Richard Pearson, Joseph Hokello, Julia Friedman, and Kara Lassen for their help and useful discussions.

This work was supported by Public Health Service grants R01-AI067093 and DP1-DA028869 to J.K. and the Martin Delaney CARE Collaboratory (U19-AI096113). We also thank the Case Western Reserve University/University Hospitals Center for AIDS Research (NIH grant P30-AI036219) for provision of flow cytometry services.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.