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

Bialek JK, Dunay GA, Voges M, Schäfer C, Spohn M, Stucka R, Hauber J, Lange UC - PLoS ONE (2016)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Heinrich Pette Institute - Leibniz Institute for Experimental Virology, Hamburg, Germany.

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.

No MeSH data available.


Identifying an optimal target region for SunTag and synergistic activation mediator (SAM) systems in the HIV 5‘LTR.(A) Scheme of SunTag and SAM activation systems. In both systems binding of a guide RNA (gRNA) to a specific target DNA sequence results in recruitment of multiple activator domains (VP64, HSF1, p65; see text for details). (B) HIV 5’LTR (HXB2) sequence with complimentary gRNAs1-9. (C, D) The effect of SunTag (C) and SAM (D) systems on LTR activation was tested by transient expression of gRNAs and system components in TZM-bl cells carrying a luciferase reporter under HIV LTR control. Activation levels were measured 48 h post transfection using a luciferase assay on whole cell lysates. Activation is shown as fold increase (light units/transfected cell) over the negative control (no gRNA expression). Shown are results of three (C; D, left panel) and two (D, right panel) independent experiments.
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pone.0158294.g001: Identifying an optimal target region for SunTag and synergistic activation mediator (SAM) systems in the HIV 5‘LTR.(A) Scheme of SunTag and SAM activation systems. In both systems binding of a guide RNA (gRNA) to a specific target DNA sequence results in recruitment of multiple activator domains (VP64, HSF1, p65; see text for details). (B) HIV 5’LTR (HXB2) sequence with complimentary gRNAs1-9. (C, D) The effect of SunTag (C) and SAM (D) systems on LTR activation was tested by transient expression of gRNAs and system components in TZM-bl cells carrying a luciferase reporter under HIV LTR control. Activation levels were measured 48 h post transfection using a luciferase assay on whole cell lysates. Activation is shown as fold increase (light units/transfected cell) over the negative control (no gRNA expression). Shown are results of three (C; D, left panel) and two (D, right panel) independent experiments.

Mentions: We explored two CRISPR/Cas9-derived activator systems for HIV transcriptional induction (Fig 1A). In the SunTag system synthetic transcriptional activator VP64 (derived from four copies of the herpes virus transcriptional activation domain VP16) is fused to single chain variable fragment (scFv) antibodies with binding specificity for peptides derived from the general control protein 4 (GCN4) [26]. Co-expression of these antibodies together with dCas9 (Cas9 nuclease-derived enzymatically inactive protein) fused to GCN4-containing peptide tags (SunTag) and gRNAs, leads to spatial recruitment of multiple VP64 domains to the gRNA-complementary target sites. The SAM (synergistic activation mediator) system [25] comprises three components: dCas9-VP64 fusion protein, modified gRNA (mod. gRNA) containing aptamers bound by MS2 bacteriophage coat protein, and MS2 proteins fused to NF-κB trans-activating subunit p65 and heat-shock factor 1 (HSF1) activation domain. Co-expression of these components establishes a multi activation domain-containing SAM complex mediating transcriptional activation at the gRNA-determined target site.


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

Bialek JK, Dunay GA, Voges M, Schäfer C, Spohn M, Stucka R, Hauber J, Lange UC - PLoS ONE (2016)

Identifying an optimal target region for SunTag and synergistic activation mediator (SAM) systems in the HIV 5‘LTR.(A) Scheme of SunTag and SAM activation systems. In both systems binding of a guide RNA (gRNA) to a specific target DNA sequence results in recruitment of multiple activator domains (VP64, HSF1, p65; see text for details). (B) HIV 5’LTR (HXB2) sequence with complimentary gRNAs1-9. (C, D) The effect of SunTag (C) and SAM (D) systems on LTR activation was tested by transient expression of gRNAs and system components in TZM-bl cells carrying a luciferase reporter under HIV LTR control. Activation levels were measured 48 h post transfection using a luciferase assay on whole cell lysates. Activation is shown as fold increase (light units/transfected cell) over the negative control (no gRNA expression). Shown are results of three (C; D, left panel) and two (D, right panel) independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4920395&req=5

pone.0158294.g001: Identifying an optimal target region for SunTag and synergistic activation mediator (SAM) systems in the HIV 5‘LTR.(A) Scheme of SunTag and SAM activation systems. In both systems binding of a guide RNA (gRNA) to a specific target DNA sequence results in recruitment of multiple activator domains (VP64, HSF1, p65; see text for details). (B) HIV 5’LTR (HXB2) sequence with complimentary gRNAs1-9. (C, D) The effect of SunTag (C) and SAM (D) systems on LTR activation was tested by transient expression of gRNAs and system components in TZM-bl cells carrying a luciferase reporter under HIV LTR control. Activation levels were measured 48 h post transfection using a luciferase assay on whole cell lysates. Activation is shown as fold increase (light units/transfected cell) over the negative control (no gRNA expression). Shown are results of three (C; D, left panel) and two (D, right panel) independent experiments.
Mentions: We explored two CRISPR/Cas9-derived activator systems for HIV transcriptional induction (Fig 1A). In the SunTag system synthetic transcriptional activator VP64 (derived from four copies of the herpes virus transcriptional activation domain VP16) is fused to single chain variable fragment (scFv) antibodies with binding specificity for peptides derived from the general control protein 4 (GCN4) [26]. Co-expression of these antibodies together with dCas9 (Cas9 nuclease-derived enzymatically inactive protein) fused to GCN4-containing peptide tags (SunTag) and gRNAs, leads to spatial recruitment of multiple VP64 domains to the gRNA-complementary target sites. The SAM (synergistic activation mediator) system [25] comprises three components: dCas9-VP64 fusion protein, modified gRNA (mod. gRNA) containing aptamers bound by MS2 bacteriophage coat protein, and MS2 proteins fused to NF-κB trans-activating subunit p65 and heat-shock factor 1 (HSF1) activation domain. Co-expression of these components establishes a multi activation domain-containing SAM complex mediating transcriptional activation at the gRNA-determined target site.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Heinrich Pette Institute - Leibniz Institute for Experimental Virology, Hamburg, Germany.

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.

No MeSH data available.