title: 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.
authors.affiliation: Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA.
authors.forename: Julie K
authors.initials: JK
authors.lastname: Jadlowsky
authors.forename: Julian Y
authors.initials: JY
authors.lastname: Wong
authors.forename: Amy C
authors.initials: AC
authors.lastname: Graham
authors.forename: Curtis
authors.initials: C
authors.lastname: Dobrowolski
authors.forename: Renee L
authors.initials: RL
authors.lastname: Devor
authors.forename: Mark D
authors.initials: MD
authors.lastname: Adams
authors.forename: Koh
authors.initials: K
authors.lastname: Fujinaga
authors.forename: Jonathan
authors.initials: J
authors.lastname: Karn
mesh_terms: D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D015967:Gene Expression Regulation, Viral; D016325:HIV Long Terminal Repeat / Q000235:genetics; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D019169:Jurkat Cells; D011401:Promoter Regions, Genetic; D011533:Proviruses / Q000235:genetics / Q000502:physiology*; D034622:RNA Interference; D012319:RNA Polymerase II / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D034741:RNA, Small Interfering; D017422:Sequence Analysis, DNA; D014157:Transcription Factors / Q000235:genetics / Q000502:physiology*; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D017735:Virus Latency / Q000235:genetics*; D054321:rev Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism
chemical_list: D034741:RNA, Small Interfering; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; C118437:negative elongation factor; D054321:rev Gene Products, Human Immunodeficiency Virus; D012319:RNA Polymerase II