Anna Kula

Fast transcription rates of RNA polymerase II in human cells

Averaged estimates of RNA polymerase II (RNAPII) elongation rates in mammalian cells have been shown to range between 1.3 and 4.3 kb min−1. In this work, nascent RNAs from an integrated human immunodeficiency virus type 1‐derived vector were detectable at the single living cell level by fluorescent RNA tagging. At steady state, a constant number of RNAs was measured corresponding to a minimal density of polymerases with negligible fluctuations over time. Recovery of fluorescence after photobleaching was complete within seconds, indicating a high rate of RNA biogenesis. The calculated transcription rate above 50 kb min−1 points towards a wide dynamic range of RNAPII velocities in living cells.

Characterization of the HIV-1 RNA associated proteome identifies Matrin 3 as a nuclear cofactor of Rev function

BackgroundCentral to the fully competent replication cycle of the human immunodeficiency virus type 1 (HIV-1) is the nuclear export of unspliced and partially spliced RNAs mediated by the Rev posttranscriptional activator and the Rev response element (RRE).ResultsHere, we introduce a novel method to explore the proteome associated with the nuclear HIV-1 RNAs. At the core of the method is the generation of cell lines harboring an integrated provirus carrying RNA binding sites for the MS2 bacteriophage protein. Flag-tagged MS2 is then used for affinity purification of the viral RNA. By this approach we found that the viral RNA is associated with the host nuclear matrix component MATR3 (Matrin 3) and that its modulation affected Rev activity. Knockdown of MATR3 suppressed Rev/RRE function in the export of unspliced HIV-1 RNAs. However, MATR3 was able to associate with Rev only through the presence of RRE-containing viral RNA.ConclusionsIn this work, we exploited a novel proteomic method to identify MATR3 as a cellular cofactor of Rev activity. MATR3 binds viral RNA and is required for the Rev/RRE mediated nuclear export of unspliced HIV-1 RNAs.

Transcriptional competence of the integrated HIV‐1 provirus at the nuclear periphery

Spatial distribution of genes within the nucleus contributes to transcriptional control, allowing optimal gene expression as well as constitutive or regulated gene repression. Human immunodeficiency virus type 1 (HIV‐1) integrates into host chromatin to transcribe and replicate its genome. Lymphocytes harbouring a quiescent but inducible provirus are a challenge to viral eradication in infected patients undergoing antiviral therapy. Therefore, our understanding of the contribution of sub‐nuclear positioning to viral transcription may also have far‐reaching implications in the pathology of the infection. To gain an insight into the conformation of chromatin at the site of HIV‐1 integration, we investigated lymphocytes carrying a single latent provirus. In the silenced state, the provirus was consistently found at the nuclear periphery, associated in trans with a pericentromeric region of chromosome 12 in a significant number of quiescent cells. After induction of the transcription, this association was lost, although the location of the transcribing provirus remained peripheral. These results, extended to several other cell clones, unveil a novel mechanism of transcriptional silencing involved in HIV‐1 post‐transcriptional latency and reinforce the notion that gene transcription may also occur at the nuclear periphery.

Hyperthermia Stimulates HIV-1 Replication

HIV-infected individuals may experience fever episodes. Fever is an elevation of the body temperature accompanied by inflammation. It is usually beneficial for the host through enhancement of immunological defenses. In cultures, transient non-physiological heat shock (42–45°C) and Heat Shock Proteins (HSPs) modulate HIV-1 replication, through poorly defined mechanisms. The effect of physiological hyperthermia (38–40°C) on HIV-1 infection has not been extensively investigated. Here, we show that culturing primary CD4+ T lymphocytes and cell lines at a fever-like temperature (39.5°C) increased the efficiency of HIV-1 replication by 2 to 7 fold. Hyperthermia did not facilitate viral entry nor reverse transcription, but increased Tat transactivation of the LTR viral promoter. Hyperthermia also boosted HIV-1 reactivation in a model of latently-infected cells. By imaging HIV-1 transcription, we further show that Hsp90 co-localized with actively transcribing provirus, and this phenomenon was enhanced at 39.5°C. The Hsp90 inhibitor 17-AAG abrogated the increase of HIV-1 replication in hyperthermic cells. Altogether, our results indicate that fever may directly stimulate HIV-1 replication, in a process involving Hsp90 and facilitation of Tat-mediated LTR activity.

HIV-1 pre-mRNA commitment to Rev mediated export through PSF and Matrin 3.

Human immunodeficiency virus gene expression and replication are regulated at several levels. Incompletely spliced viral RNAs and full-length genomic RNA contain the RRE element and are bound by the viral trans-acting protein Rev to be transported out of the nucleus. Previously we found that the nuclear matrix protein MATR3 was a cofactor of Rev-mediated RNA export. Here we show that the pleiotropic protein PSF binds viral RNA and is associated with MATR3. PSF is involved in the maintenance of a pool of RNA available for Rev activity. However, while Rev and PSF bind the viral pre-mRNA at the site of viral transcription, MATR3 interacts at a subsequent step. We propose that PSF and MATR3 define a novel pathway for RRE-containing HIV-1 RNAs that is hijacked by the viral Rev protein.

Reactivation capacity by latency-reversing agents ex vivo correlates with the size of the HIV-1 reservoir.

Objective: HIV-1 reservoirs are the major hurdle to virus clearance in combination antiretroviral therapy (cART)-treated patients. An approach to eradicating HIV-1 involves reversing latency in cART-treated patients to make latent cells visible to the host immune system. Stimulation of patient cell cultures with latency-reversing agents (LRAs) ex vivo results in heterogeneous responses among HIV-infected patients. Determinants of this heterogeneity are unknown and consequently important to determine. Design and methods: Here, we grouped and retrospectively analyzed the data from our two recent HIV-1 reactivation studies to investigate the role of the HIV-1 reservoir size in the reactivation capacity by LRAs in ex vivo cultures of CD8+-depleted peripheral blood mononuclear cells (PBMCs) isolated from 54 cART-treated patients and of resting CD4+ T cells isolated from 30 cART-treated patients. Results: Our results established a statistically relevant positive correlation between the HIV-1 reservoir size measured by total cell-associated HIV-1 DNA and the frequency of positive HIV-1 recovery measurements in response to various LRAs in ex vivo cultures of cells isolated from cART-treated HIV+ aviremic patients. HIV-1 reservoir size also correlated with the extracellular HIV-1 RNA median level measured in supernatants of cell cultures following LRA treatments. However, we identified HIV+ patients whose positive measurements frequency and median level of extracellular HIV-1 RNA deviated from linearity relative to their corresponding HIV reservoir size. Conclusion: We demonstrated that the reservoir size is one predictive marker of LRA effectiveness but this parameter alone is not sufficient. The identification of other predictive markers is necessary to predict the success of HIV anti-latency approaches.

Dynamic Post-Transcriptional Regulation of HIV-1 Gene Expression

Gene expression of the human immunodeficiency virus type 1 (HIV-1) is a highly regulated process. Basal transcription of the integrated provirus generates early transcripts that encode for the viral products Tat and Rev. Tat promotes the elongation of RNA polymerase while Rev mediates the nuclear export of viral RNAs that contain the Rev-responsive RNA element (RRE). These RNAs are exported from the nucleus to allow expression of Gag-Pol and Env proteins and for the production of full-length genomic RNAs. A balance exists between completely processed mRNAs and RRE-containing RNAs. Rev functions as an adaptor that recruits cellular factors to re-direct singly spliced and unspliced viral RNAs to nuclear export. The aim of this review is to address the dynamic regulation of this post-transcriptional pathway in light of recent findings that implicate several novel cellular cofactors of Rev function.

Novel pathways of transcriptional and post-transcriptional regulation of post-integrative HIV-1 latency

16‐17 July 2010, International AIDS Society’s Workshop “Towards a Cure”: HIV Reservoirs and Strategies to Control Them, Vienna, Austria

Repression of Human T-lymphotropic virus type 1 Long Terminal Repeat sense transcription by Sp1 recruitment to novel Sp1 binding sites.

Human T-lymphotropic Virus type 1 (HTLV-1) infection is characterized by viral latency in the majority of infected cells and by the absence of viremia. These features are thought to be due to the repression of viral sense transcription in vivo. Here, our in silico analysis of the HTLV-1 Long Terminal Repeat (LTR) promoter nucleotide sequence revealed, in addition to the four Sp1 binding sites previously identified, the presence of two additional potential Sp1 sites within the R region. We demonstrated that the Sp1 and Sp3 transcription factors bound in vitro to these two sites and compared the binding affinity for Sp1 of all six different HTLV-1 Sp1 sites. By chromatin immunoprecipitation experiments, we showed Sp1 recruitment in vivo to the newly identified Sp1 sites. We demonstrated in the nucleosomal context of an episomal reporter vector that the Sp1 sites interfered with both the sense and antisense LTR promoter activities. Interestingly, the Sp1 sites exhibited together a repressor effect on the LTR sense transcriptional activity but had no effect on the LTR antisense activity. Thus, our results demonstrate the presence of two new functional Sp1 binding sites in the HTLV-1 LTR, which act as negative cis-regulatory elements of sense viral transcription.

Nuclear organization and the regulation of HIV-1 gene expression

Regulation of gene expression is profoundly involved in HIV-1 pathogenesis. This retrovirus integrates into host chromatin in order to transcribe and replicate its genome. Lymphocytes harboring a quiescent but inducible provirus are a challenge to viral eradication in infected patients undergoing antiviral therapy. Therefore our understanding of the contribution of sub-nuclear positioning to viral gene expression may have far reaching implications also in the pathology of the infection. In order to gain insight in the conformation of chromatin at the site of HIV-1 integration we investigated lymphocytes carrying a single latent provirus. In the silenced state the provirus was consistently found at the nuclear periphery, associated in trans to pericentromeric heterochromatin in a significant number of quiescent cells. After induction of transcription, this association was lost, although the location of the transcribing provirus remained peripheral. These results unveil a novel mechanism of transcriptional silencing involved in HIV-1 post-transcriptional latency and reinforce the notion that gene transcription may occur also at the nuclear periphery. However, transcription is not the only limiting process during viral gene expression. Both unspliced and spliced viral RNAs need to be exported for efficient viral replication. In particular unspliced viral RNAs are retained in the nucleus until the viral Rev proteins mediate their export. Both RNA binding proteins and RNA hyperediting by adenosine deamination have been involved in the process of nuclear retention. In order to gain insights into this still poorly characterized pathway, we explored the proteome associated with the unspliced HIV-1 RNAs that are retained in the nucleus. We show that the viral RNA is associated with the host factors PSF/p54nrb and MATR3. PSF/p54nrb bind the viral RNA co-transcriptionally but MATR3 defines a subnuclear compartment where the viral RNA is delivered. Through this pathway, Rev is able to associate with unspliced HIV RNA directing its nuclear export. Interestingly PSF/p54nrb binding and localization to MATR3 foci occur independently of RNA hyperediting. Our findings reveal a novel cellular mechanism of nuclear retention that is hijacked by a virus.