37
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

      Targeted HIV-1 Latency Reversal Using CRISPR/Cas9-Derived Transcriptional Activator Systems

      research-article

      Read this article at

      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          CRISPR/Cas9 technology is currently considered the most advanced tool for targeted genome engineering. Its sequence-dependent specificity has been explored for locus-directed transcriptional modulation. Such modulation, in particular transcriptional activation, has been proposed as key approach to overcome silencing of dormant HIV provirus in latently infected cellular reservoirs. Currently available agents for provirus activation, so-called latency reversing agents (LRAs), act indirectly through cellular pathways to induce viral transcription. However, their clinical performance remains suboptimal, possibly because reservoirs have diverse cellular identities and/or proviral DNA is intractable to the induced pathways. We have explored two CRISPR/Cas9-derived activator systems as targeted approaches to induce dormant HIV-1 proviral DNA. These systems recruit multiple transcriptional activation domains to the HIV 5’ long terminal repeat (LTR), for which we have identified an optimal target region within the LTR U3 sequence. Using this target region, we demonstrate transcriptional activation of proviral genomes via the synergistic activation mediator complex in various in culture model systems for HIV latency. Observed levels of induction are comparable or indeed higher than treatment with established LRAs. Importantly, activation is complete, leading to production of infective viral particles. Our data demonstrate that CRISPR/Cas9-derived technologies can be applied to counteract HIV latency and may therefore represent promising novel approaches in the quest for HIV elimination.

          Related collections

          Most cited references25

          • Record: found
          • Abstract: found
          • Article: not found

          Epigenome editing by a CRISPR/Cas9-based acetyltransferase activates genes from promoters and enhancers

          Technologies that facilitate the targeted manipulation of epigenetic marks could be used to precisely control cell phenotype or interrogate the relationship between the epigenome and transcriptional control. Here we have generated a programmable acetyltransferase based on the CRISPR/Cas9 gene regulation system, consisting of the nuclease-null dCas9 protein fused to the catalytic core of the human acetyltransferase p300. This fusion protein catalyzes acetylation of histone H3 lysine 27 at its target sites, corresponding with robust transcriptional activation of target genes from promoters, proximal enhancers, and distal enhancers. Gene activation by the targeted acetyltransferase is highly specific across the genome. In contrast to conventional dCas9-based activators, the acetyltransferase effectively activates genes from enhancer regions and with individual guide RNAs. The core p300 domain is also portable to other programmable DNA-binding proteins. These results support targeted acetylation as a causal mechanism of transactivation and provide a new robust tool for manipulating gene regulation.
            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

            CRISPR RNA-guided activation of endogenous human genes

            Catalytically inactive CRISPR-associated 9 nuclease (dCas9) can be directed by short guide RNAs (gRNAs) to repress endogenous genes in bacteria and human cells. Here we show that a dCas9-VP64 transcriptional activation domain fusion protein can be directed by single or multiple gRNAs to increase expression of specific endogenous human genes. These results provide an important proof-of-principle that CRISPR-Cas systems can be used to target heterologous effector domains in human cells.
              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              HIV reproducibly establishes a latent infection after acute infection of T cells in vitro.

              The presence of latent reservoirs has prevented the eradication of human immunodeficiency virus (HIV) from infected patients successfully treated with anti-retroviral therapy. The mechanism of postintegration latency is poorly understood, partly because of the lack of an in vitro model. We have used an HIV retroviral vector or a full-length HIV genome expressing green fluorescent protein to infect a T lymphocyte cell line in vitro and highly enrich for latently infected cells. HIV latency occurred reproducibly, albeit with low frequency, during an acute infection. Clonal cell lines derived from latent populations showed no detectable basal expression, but could be transcriptionally activated after treatment with phorbol esters or tumor necrosis factor alpha. Direct sequencing of integration sites demonstrated that latent clones frequently contain HIV integrated in or close to alphoid repeat elements in heterochromatin. This is in contrast to a productive infection where integration in or near heterochromatin is disfavored. These observations demonstrate that HIV can reproducibly establish a latent infection as a consequence of integration in or near heterochromatin.
                Bookmark

                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                24 June 2016
                2016
                : 11
                : 6
                : e0158294
                Affiliations
                [1 ]Heinrich Pette Institute – Leibniz Institute for Experimental Virology, Hamburg, Germany
                [2 ]Department of Anesthesiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
                [3 ]Friedrich-Baur-Institute, Department of Neurology, Ludwig Maximilian University Munich, Munich, Germany
                [4 ]German Center for Infection Research (DZIF), partner site, Hamburg, Germany
                George Mason University, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: JH UCL. Performed the experiments: JKB UCL GAD MV CS MS RS. Analyzed the data: JKB UCL GAD. Wrote the paper: JH UCL.

                Article
                PONE-D-16-13330
                10.1371/journal.pone.0158294
                4920395
                27341108
                6cbfe477-9108-408e-acc0-5f3181733922
                © 2016 Bialek et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 1 April 2016
                : 13 June 2016
                Page count
                Figures: 5, Tables: 0, Pages: 19
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/100009139, Deutsches Zentrum für Infektionsforschung;
                Award ID: TI07.001
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100009139, Deutsches Zentrum für Infektionsforschung;
                Award ID: TI07.002
                Award Recipient : Gabor A Dunay
                UCL was supported by a Clinical Leave Stipend from the German Center of Infection Research (grant TI07.001; http://www.dzif.de). GAD was supported by a MD/PhD stipend from the German Center of Infection Research (grant TI07.002; http://www.dzif.de). This work received support from the “Viral Latency” program at the Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany ( http://www.hpi-hamburg.de). The publication of this article was funded by the Open Access Fund of the Leibniz Association. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Microbiology
                Medical Microbiology
                Microbial Pathogens
                Viral Pathogens
                Immunodeficiency Viruses
                HIV
                Medicine and Health Sciences
                Pathology and Laboratory Medicine
                Pathogens
                Microbial Pathogens
                Viral Pathogens
                Immunodeficiency Viruses
                HIV
                Biology and Life Sciences
                Organisms
                Viruses
                Viral Pathogens
                Immunodeficiency Viruses
                HIV
                Biology and Life Sciences
                Organisms
                Viruses
                Immunodeficiency Viruses
                HIV
                Biology and life sciences
                Organisms
                Viruses
                RNA viruses
                Retroviruses
                Lentivirus
                HIV
                Biology and Life Sciences
                Microbiology
                Medical Microbiology
                Microbial Pathogens
                Viral Pathogens
                Retroviruses
                Lentivirus
                HIV
                Medicine and Health Sciences
                Pathology and Laboratory Medicine
                Pathogens
                Microbial Pathogens
                Viral Pathogens
                Retroviruses
                Lentivirus
                HIV
                Biology and Life Sciences
                Organisms
                Viruses
                Viral Pathogens
                Retroviruses
                Lentivirus
                HIV
                Biology and life sciences
                Genetics
                Gene expression
                DNA transcription
                Research and Analysis Methods
                Database and Informatics Methods
                Biological Databases
                Sequence Databases
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Sequencing Techniques
                Sequence Analysis
                Sequence Databases
                Research and Analysis Methods
                Molecular Biology Techniques
                Sequencing Techniques
                Sequence Analysis
                Sequence Databases
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Transfection
                Research and Analysis Methods
                Molecular Biology Techniques
                Transfection
                Biology and Life Sciences
                Microbiology
                Virology
                Viral Persistence and Latency
                Biology and Life Sciences
                Microbiology
                Virology
                Viral Replication
                Research and Analysis Methods
                Spectrum Analysis Techniques
                Spectrophotometry
                Cytophotometry
                Flow Cytometry
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Cloning
                Research and Analysis Methods
                Molecular Biology Techniques
                Cloning
                Custom metadata
                All relevant data are within the paper and its Supporting Information files.

                Uncategorized
                Uncategorized

                Comments

                Comment on this article