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. 2014 Jun;34(11):1911-28.
doi: 10.1128/MCB.01013-13. Epub 2014 Mar 17.

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

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Free PMC article

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

Julie K Jadlowsky et al. Mol Cell Biol. .
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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.

Figures

FIG 1
FIG 1
Silencing of the NELF subunits activates latent proviral transcription. (A) Flow cytometry. Clones of E4 cells superinfected with lentiviral vectors expressing shRNA targeting individual NELF subunits (A, B, C/D, and E) were analyzed by two-color flow cytometry. mCherry, shRNA marker; d2EGFP, HIV expression marker. (B) HIV reactivation in clones infected with lentiviral vectors expressing shRNAs targeting NELF subunits. (C) Western blot analysis of NELF subunit expression in the N1C6 clone expressing shRNA to NELF-E. Scr, scrambled shRNA.
FIG 2
FIG 2
Knockdown of NELF-E by shRNA inhibits resilencing of HIV proviruses. (A) Flow cytometry using 2D10 cell-derived clones expressing NELF-E shRNA (N2G10) and a scrambled control (N2F2). mCherry expression was used as an shRNA marker, and d2EGFP expression was used as a marker of HIV transcription. Max, maximum. (B) Western blot analysis of NELF subunit expression in cells transfected with shRNA to NELF-E (N2G10) or a scrambled RNA control (N2F2). (C) Silencing of activated cells. 2D10 cells and clones carrying NELF-E shRNA (N2G10) and its scrambled control (N2F2) were stimulated overnight with 10 ng/ml TNF-α, 5 μg/ml anti-CD3, or 5 μg/ml anti-CD3 and 1 μg/ml anti-CD28. d2EGFP expression was monitored by flow cytometry between 0 and 10 days after removal of the activators. The percentage of d2EGFP-expressing cells immediately after activation was normalized. α, anti.
FIG 3
FIG 3
RNAP II accumulates on the 5′ LTR but not on the 3′ LTR in latently infected cells. (A) Location of primers used for ChIP assays (Table 1 gives primer sequences). (B) ChIP analysis of RNAP II levels on the 5′ and 3′ proviral LTR using LTR-specific primers. Analysis was performed on E4, 2D10, and 3C9 cells induced for 0, 0.5, or 16 h with 2 ng/ml TNF-α. (C) Analysis of the same samples as used for the experiment shown in panel B using genomic primers on the 5′ and 3′ LTR. (D) RNAP II levels on the 5′ and 3′ LTR of E4 cells induced for 0, 0.5, or 8 h using LTR-specific primers. (E) Analysis of the same samples as described for panel D using nonspecific primers for the Nuc-0 and Nuc-1 regions of the LTR. Readout for this assay was from Ion Torrent sequencing of PCR products. Error bars represent the standard deviations of triplicate ChIP assays (21, 46). Fwd: forward genomic primer; Rvs, reverse genomic primer.
FIG 4
FIG 4
Distribution of RNAP II on the HIV genome following knockdown of NELF-E. ChIP analyses were performed using NELF-E shRNA (clone N2G10) and its scrambled control (clone N2F2) in unstimulated cells (minus TNF-α) (A), cells stimulated with 2 ng/ml TNF-α for 1 h (B), and cells stimulated with 2 ng/ml TNF-α for 10 h (C). Error bars represent the standard deviations of triplicate real-time PCR determinations for each primer set.
FIG 5
FIG 5
Distribution of RNAP II and associated transcription factors on the HIV genome following TNF-α activation. ChIP assays were used to measure the density of RNA polymerase along the proviral genome from the core promoter region in the LTR (shaded in blue) to 600 nt downstream of the transcription start site. (A) Schematic map of the HIV LTR showing nucleosomes and transcription factor binding sites. (B) Micrococcal nuclease protection assay. (C to G) ChIP analyses were performed using antibodies against the indicated proteins: RNAP II, CycT1, CDK9, NELF-E, and NELF-A. Error bars represent standard deviations of triplicate real-time PCR determinations for each primer set.
FIG 6
FIG 6
Knockdown of NELF-E expression selectively enhances elongation from the HIV LTR. The distribution of RNA polymerase on HIV was measured by ChIP-Seq assays performed using the parental line E4 and clones carrying the NELF-E shRNA (N1C6) and its scrambled control (N1D9) in unstimulated control (N1D9) cells (A), control cells stimulated by 10 ng/ml TNF-α for 60 min (B), unstimulated cells expressing NELF-E shRNA (N1C6) (C), and cells expressing NELF-E shRNA and activated by 10 ng/ml TNF-α for 60 min (D). Note that the downstream distribution of RNAP II uniformly increases when NELF-E is knocked down, but there is only minimal impact of NELF-E depletion on promoter-proximal pausing.
FIG 7
FIG 7
Promoter-proximal pause sites on the HIV LTR. (A) The average RNAP II distribution relative to the transcription start site for the 500 genes that are most highly activated by TNF-α in unstimulated control (N1D9) cells (A) and control cells stimulated by TNF-α (B). (C to F) RNAP II accumulation in HIV promoter region in unstimulated control (N1D9) cells (C), control (N1D9) cells stimulated by TNF-α (D), unstimulated cells expressing NELF-E shRNA (N1C6) (E), and cells expressing NELF-E shRNA and stimulated by TNF-α (F). Black vertical lines indicate the sites of RNAP II accumulation on averaged cellular genes. Blue vertical lines indicate additional sites of RNAP II accumulation on the HIV LTR. The major peaks were fitted to a series of Gaussian curves (white lines) and summed (yellow lines).
FIG 8
FIG 8
Relative RNAP II accumulation on cellular genes and the HIV provirus. RNAP II reads from the ChIP-Seq data sets were mapped to individual gene coordinates and then totaled within each gene boundary. Two experimental conditions were compared in each analysis and plotted using log-log plots. (A to D) RNAP II distribution per gene region in the presence of NELF-E shRNA (N1C6) and its scrambled control (N1D9) under unstimulated conditions (A), in the presence and absence of TNF-α in scrambled control cells (B), in the presence of NELF-E shRNA and its scrambled control after stimulation by TNF-α (C),and in the presence of NELF-E shRNA in the presence and absence of TNF-α (D). For each condition the HIV provirus (red square) and the top 10 highest-scoring genes are indicated. The HIV “gene” has highly upregulated RNAP II binding patterns compared to those of cellular genes when the provirus is stimulated with TNF-α in the presence and absence of NELF.
FIG 9
FIG 9
Kinetic model for the restriction of HIV transcription from latent proviruses by NELF. (A) Latent HIV provirus. In latent proviruses transcription elongation is very inefficient due to absence of the transcription elongation factor NF-κB as well as chromatin restrictions (not shown for simplicity). However, a significant number of proviruses carry RNAP II paused in the promoter-proximal region. The small number of transcription complexes that are able initiate and elongate through TAR are subject to additional elongation restrictions by NELF which forces premature termination. (B) Partially activated HIV provirus in the absence of the NELF. In the absence of NELF, promoter-proximal pausing remains relatively constant, but there is enhanced escape of paused complexes. (C) NF-κB and Tat-activated transcription. Initiation is strongly induced by NF-κB, which removes chromatin restrictions near the promoter through recruitment of histone acetyltransferases. Under these circumstances promoter clearance is also much more efficient, and there is an enhanced accumulation of elongation complexes in the promoter-proximal region. After the transcription through the TAR element, both NELF and the Tat/P-TEFb complex (the superelongation complex factors are not shown for simplicity) are recruited to the elongation complex via binding interactions with TAR RNA. This activates the CDK9 kinase and leads to hyperphosphorylation of the CTD of RNA polymerase II, Spt5, and NELF-E. The phosphorylation of NELF-E leads to its release. Although the promoter is transcribing more rapidly than in the latent condition, there is relatively little change in the amount of RNAP II that accumulates in the promoter-proximal region due to its rapid replacement by newly initiated transcription complexes.

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