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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

Induction of HIV replication using SAM in the J89 latency model.(A) Scheme of replication-competent HIV reporter virus in J89 clones. Transcriptional activation results in LTR-dependent eGFP expression and viral replication. (B) Components of the SAM system together with gRNA5, gRNA8 or no gRNA (control) were transiently expressed in J89 cells and proviral activation was determined 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<0.001. Shown are results of three independent experiments. (C) Number of J89 cells showing eGFP 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). Inset shows flow cytometry contour plot of p24 and eGFP co-expression in control (grey) and SAM/gRNA5 transfected (red) cells. (D) To assess viral replication after SAM-mediated activation, J89 cells transfected with SAM components and gRNA5, were sorted for eGFP expression and co-cultured at 72 h post transfection with MOLT-4/CCR5 cells using a transwell system. (E) At 7 days post initiation of the co-culture, levels of gag RNA in co-culture supernatants and MOLT-4/CCR5 cells (cell-associated) were measured using ddPCR. Co-culture of non-transfected J89 cells with MOLT-4/CCR5 cells served as control (untreated). Mann-Whitney U test was used for statistical evaluation (in relation to untreated); * signifies p<0.05. (F) At 12 days post initiation of the co-culture, cell-associated LTR DNA content was determined in MOLT-4/CCR5 cells. Levels are shown as fold increase of LTR sequences per cell over untreated control. Shown are results of two independent experiments (E, F).
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pone.0158294.g004: Induction of HIV replication using SAM in the J89 latency model.(A) Scheme of replication-competent HIV reporter virus in J89 clones. Transcriptional activation results in LTR-dependent eGFP expression and viral replication. (B) Components of the SAM system together with gRNA5, gRNA8 or no gRNA (control) were transiently expressed in J89 cells and proviral activation was determined 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<0.001. Shown are results of three independent experiments. (C) Number of J89 cells showing eGFP 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). Inset shows flow cytometry contour plot of p24 and eGFP co-expression in control (grey) and SAM/gRNA5 transfected (red) cells. (D) To assess viral replication after SAM-mediated activation, J89 cells transfected with SAM components and gRNA5, were sorted for eGFP expression and co-cultured at 72 h post transfection with MOLT-4/CCR5 cells using a transwell system. (E) At 7 days post initiation of the co-culture, levels of gag RNA in co-culture supernatants and MOLT-4/CCR5 cells (cell-associated) were measured using ddPCR. Co-culture of non-transfected J89 cells with MOLT-4/CCR5 cells served as control (untreated). Mann-Whitney U test was used for statistical evaluation (in relation to untreated); * signifies p<0.05. (F) At 12 days post initiation of the co-culture, cell-associated LTR DNA content was determined in MOLT-4/CCR5 cells. Levels are shown as fold increase of LTR sequences per cell over untreated control. Shown are results of two independent experiments (E, F).

Mentions: Several currently applied LRAs may only promote incomplete induction of latent provirus, failing to stimulate production of fully replication-competent viral progeny [8]. To test whether robust SAM-mediated activation of HIV LTR-driven transcription can also induce production and release of infectious viral particles in formerly latent cells, we employed the J89 model system [33]. As opposed to JLat6.3 and HIVis systems, Jurkat-derived clonal J89 cells carry a fully replication-competent HIV-derived provirus with an eGFP marker (Fig 4A). This reporter provirus is latent under non-induced culture conditions (<2% GFP positive cells) [33]. Consistent with our results described above, transient expression of SAM and gRNA5 resulted in strong induction of J89 cells, with 66% GFP positive cells after 48 h and up to 89% induced cells at 96 hours (Fig 4B). Induction was also accompanied by up-regulation of cell-associated p24 levels in formerly latent J89 cells (Fig 4C).


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)

Induction of HIV replication using SAM in the J89 latency model.(A) Scheme of replication-competent HIV reporter virus in J89 clones. Transcriptional activation results in LTR-dependent eGFP expression and viral replication. (B) Components of the SAM system together with gRNA5, gRNA8 or no gRNA (control) were transiently expressed in J89 cells and proviral activation was determined 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<0.001. Shown are results of three independent experiments. (C) Number of J89 cells showing eGFP 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). Inset shows flow cytometry contour plot of p24 and eGFP co-expression in control (grey) and SAM/gRNA5 transfected (red) cells. (D) To assess viral replication after SAM-mediated activation, J89 cells transfected with SAM components and gRNA5, were sorted for eGFP expression and co-cultured at 72 h post transfection with MOLT-4/CCR5 cells using a transwell system. (E) At 7 days post initiation of the co-culture, levels of gag RNA in co-culture supernatants and MOLT-4/CCR5 cells (cell-associated) were measured using ddPCR. Co-culture of non-transfected J89 cells with MOLT-4/CCR5 cells served as control (untreated). Mann-Whitney U test was used for statistical evaluation (in relation to untreated); * signifies p<0.05. (F) At 12 days post initiation of the co-culture, cell-associated LTR DNA content was determined in MOLT-4/CCR5 cells. Levels are shown as fold increase of LTR sequences per cell over untreated control. Shown are results of two independent experiments (E, F).
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pone.0158294.g004: Induction of HIV replication using SAM in the J89 latency model.(A) Scheme of replication-competent HIV reporter virus in J89 clones. Transcriptional activation results in LTR-dependent eGFP expression and viral replication. (B) Components of the SAM system together with gRNA5, gRNA8 or no gRNA (control) were transiently expressed in J89 cells and proviral activation was determined 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<0.001. Shown are results of three independent experiments. (C) Number of J89 cells showing eGFP 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). Inset shows flow cytometry contour plot of p24 and eGFP co-expression in control (grey) and SAM/gRNA5 transfected (red) cells. (D) To assess viral replication after SAM-mediated activation, J89 cells transfected with SAM components and gRNA5, were sorted for eGFP expression and co-cultured at 72 h post transfection with MOLT-4/CCR5 cells using a transwell system. (E) At 7 days post initiation of the co-culture, levels of gag RNA in co-culture supernatants and MOLT-4/CCR5 cells (cell-associated) were measured using ddPCR. Co-culture of non-transfected J89 cells with MOLT-4/CCR5 cells served as control (untreated). Mann-Whitney U test was used for statistical evaluation (in relation to untreated); * signifies p<0.05. (F) At 12 days post initiation of the co-culture, cell-associated LTR DNA content was determined in MOLT-4/CCR5 cells. Levels are shown as fold increase of LTR sequences per cell over untreated control. Shown are results of two independent experiments (E, F).
Mentions: Several currently applied LRAs may only promote incomplete induction of latent provirus, failing to stimulate production of fully replication-competent viral progeny [8]. To test whether robust SAM-mediated activation of HIV LTR-driven transcription can also induce production and release of infectious viral particles in formerly latent cells, we employed the J89 model system [33]. As opposed to JLat6.3 and HIVis systems, Jurkat-derived clonal J89 cells carry a fully replication-competent HIV-derived provirus with an eGFP marker (Fig 4A). This reporter provirus is latent under non-induced culture conditions (<2% GFP positive cells) [33]. Consistent with our results described above, transient expression of SAM and gRNA5 resulted in strong induction of J89 cells, with 66% GFP positive cells after 48 h and up to 89% induced cells at 96 hours (Fig 4B). Induction was also accompanied by up-regulation of cell-associated p24 levels in formerly latent J89 cells (Fig 4C).

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