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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.


Related in: MedlinePlus

Latency reversal using SAM in the HIVis model system.(A) Scheme of replication incompetent HIVis latency reporter virus (left panel). Transcriptional activation results in LTR-dependent BFP expression, while the SFFV promoter drives constitutive expression of Venus fluorescent protein. Upon transduction of Jurkat cells with HIVis reporter, cells with active LTR transcription (II: BFP+, Venus+) can be distinguished from cell populations with a silenced LTR (III: BFP-, Venus+) or non-transduced cells (I: BFP-, Venus-) by flow cytometry (right panel). (B) Components of the SAM system together with gRNA5, gRNA8 or no gRNA (control) were transiently expressed in clonal HIVis cells (HIVisB2). Proviral activation was monitored by flow cytometry at 48, 72, and 96 h post transfection. Two-way ANOVA was used for statistical evaluation (in relation to control); ** signifies p<0.01. (C) Proviral activation levels in HIVisB2 cells after 24 h exposure to different latency reversing agents. (D) Levels of cell-associated tat and gag RNA in HIVisB2 cells as measured by qRT-PCR at 72 h post transfection with SAM system components plus gRNA5, gRNA8 or no gRNA (control). One-way ANOVA was used for statistical evaluation; * signifies p<0.05. Shown are results of three (B, C) and two (D) independent experiments. (E) Number of HIVisB2 cells showing BFP expression (upper panel) and cell-associated p24 expression (lower panel) at 72 h post transfection with SAM system components and gRNA5, gRNA8 or no gRNA (control). Insert shows a flow cytometry contour plot of p24 and BFP co-expression in control (grey) and SAM/gRNA5 transfected (red) cells.
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pone.0158294.g003: Latency reversal using SAM in the HIVis model system.(A) Scheme of replication incompetent HIVis latency reporter virus (left panel). Transcriptional activation results in LTR-dependent BFP expression, while the SFFV promoter drives constitutive expression of Venus fluorescent protein. Upon transduction of Jurkat cells with HIVis reporter, cells with active LTR transcription (II: BFP+, Venus+) can be distinguished from cell populations with a silenced LTR (III: BFP-, Venus+) or non-transduced cells (I: BFP-, Venus-) by flow cytometry (right panel). (B) Components of the SAM system together with gRNA5, gRNA8 or no gRNA (control) were transiently expressed in clonal HIVis cells (HIVisB2). Proviral activation was monitored by flow cytometry at 48, 72, and 96 h post transfection. Two-way ANOVA was used for statistical evaluation (in relation to control); ** signifies p<0.01. (C) Proviral activation levels in HIVisB2 cells after 24 h exposure to different latency reversing agents. (D) Levels of cell-associated tat and gag RNA in HIVisB2 cells as measured by qRT-PCR at 72 h post transfection with SAM system components plus gRNA5, gRNA8 or no gRNA (control). One-way ANOVA was used for statistical evaluation; * signifies p<0.05. Shown are results of three (B, C) and two (D) independent experiments. (E) Number of HIVisB2 cells showing BFP expression (upper panel) and cell-associated p24 expression (lower panel) at 72 h post transfection with SAM system components and gRNA5, gRNA8 or no gRNA (control). Insert shows a flow cytometry contour plot of p24 and BFP co-expression in control (grey) and SAM/gRNA5 transfected (red) cells.

Mentions: Different in vitro models of HIV latency have shown variable responses to LRAs [31]. To test whether SAM-mediated latency reversal also demonstrates variable effects, we used a second, newly established latency system. This system, called HIVis (HIV visible), comprises an HIV-1 reporter variant [29,32] with a dual fluorescent marker cassette inserted into the nef locus within an HIV-1-derived replication-incompetent vector backbone. The marker cassette encodes blue fluorescent protein (BFP) under HIV LTR control and Venus-fluorescent protein under the control of constitutive spleen focus-forming virus (SFFV) promoter (Fig 3A, left). Upon transduction with HIVis reporter, flow cytometry analysis can readily distinguish between non-transduced cells and transduced cell populations with different states of HIVis reporter transcriptional activity, thereby enabling visualization of productive versus latent infection (Fig 3A, right panel). Notably, latent HIVis reporter is responsive to LRA exposure, which induces viral gene expression at RNA and protein level (S3 Fig).


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)

Latency reversal using SAM in the HIVis model system.(A) Scheme of replication incompetent HIVis latency reporter virus (left panel). Transcriptional activation results in LTR-dependent BFP expression, while the SFFV promoter drives constitutive expression of Venus fluorescent protein. Upon transduction of Jurkat cells with HIVis reporter, cells with active LTR transcription (II: BFP+, Venus+) can be distinguished from cell populations with a silenced LTR (III: BFP-, Venus+) or non-transduced cells (I: BFP-, Venus-) by flow cytometry (right panel). (B) Components of the SAM system together with gRNA5, gRNA8 or no gRNA (control) were transiently expressed in clonal HIVis cells (HIVisB2). Proviral activation was monitored by flow cytometry at 48, 72, and 96 h post transfection. Two-way ANOVA was used for statistical evaluation (in relation to control); ** signifies p<0.01. (C) Proviral activation levels in HIVisB2 cells after 24 h exposure to different latency reversing agents. (D) Levels of cell-associated tat and gag RNA in HIVisB2 cells as measured by qRT-PCR at 72 h post transfection with SAM system components plus gRNA5, gRNA8 or no gRNA (control). One-way ANOVA was used for statistical evaluation; * signifies p<0.05. Shown are results of three (B, C) and two (D) independent experiments. (E) Number of HIVisB2 cells showing BFP expression (upper panel) and cell-associated p24 expression (lower panel) at 72 h post transfection with SAM system components and gRNA5, gRNA8 or no gRNA (control). Insert shows a flow cytometry contour plot of p24 and BFP co-expression in control (grey) and SAM/gRNA5 transfected (red) cells.
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Related In: Results  -  Collection

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pone.0158294.g003: Latency reversal using SAM in the HIVis model system.(A) Scheme of replication incompetent HIVis latency reporter virus (left panel). Transcriptional activation results in LTR-dependent BFP expression, while the SFFV promoter drives constitutive expression of Venus fluorescent protein. Upon transduction of Jurkat cells with HIVis reporter, cells with active LTR transcription (II: BFP+, Venus+) can be distinguished from cell populations with a silenced LTR (III: BFP-, Venus+) or non-transduced cells (I: BFP-, Venus-) by flow cytometry (right panel). (B) Components of the SAM system together with gRNA5, gRNA8 or no gRNA (control) were transiently expressed in clonal HIVis cells (HIVisB2). Proviral activation was monitored by flow cytometry at 48, 72, and 96 h post transfection. Two-way ANOVA was used for statistical evaluation (in relation to control); ** signifies p<0.01. (C) Proviral activation levels in HIVisB2 cells after 24 h exposure to different latency reversing agents. (D) Levels of cell-associated tat and gag RNA in HIVisB2 cells as measured by qRT-PCR at 72 h post transfection with SAM system components plus gRNA5, gRNA8 or no gRNA (control). One-way ANOVA was used for statistical evaluation; * signifies p<0.05. Shown are results of three (B, C) and two (D) independent experiments. (E) Number of HIVisB2 cells showing BFP expression (upper panel) and cell-associated p24 expression (lower panel) at 72 h post transfection with SAM system components and gRNA5, gRNA8 or no gRNA (control). Insert shows a flow cytometry contour plot of p24 and BFP co-expression in control (grey) and SAM/gRNA5 transfected (red) cells.
Mentions: Different in vitro models of HIV latency have shown variable responses to LRAs [31]. To test whether SAM-mediated latency reversal also demonstrates variable effects, we used a second, newly established latency system. This system, called HIVis (HIV visible), comprises an HIV-1 reporter variant [29,32] with a dual fluorescent marker cassette inserted into the nef locus within an HIV-1-derived replication-incompetent vector backbone. The marker cassette encodes blue fluorescent protein (BFP) under HIV LTR control and Venus-fluorescent protein under the control of constitutive spleen focus-forming virus (SFFV) promoter (Fig 3A, left). Upon transduction with HIVis reporter, flow cytometry analysis can readily distinguish between non-transduced cells and transduced cell populations with different states of HIVis reporter transcriptional activity, thereby enabling visualization of productive versus latent infection (Fig 3A, right panel). Notably, latent HIVis reporter is responsive to LRA exposure, which induces viral gene expression at RNA and protein level (S3 Fig).

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.


Related in: MedlinePlus