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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

Conservation of optimal gRNAs and the effect of sequence variation on SAM-mediated LTR activation.(A) Sequence conservation of HXB2-derived gRNA3, -4, -5 and -6 based on the Los Alamos HIV Sequence Database considering all HIV subtypes (upper panel), subtype B only (middle panel) or subtype C only (lower panel). Percentages of sequence hits are shown, allowing for 0 to 9 point mutations in the indicated gRNA (see Materials and Methods for details). (B) Frequency distribution of gRNA5 mutations observed in database hits according to nucleotide (nt) position. Different colors indicate the number of mutations (1–9). (C) The effect of sequence variations in gRNA5 on SAM-mediated LTR activation was tested by transient expression of gRNA mutants (gRNA *X, where X is the mutated nucleotide position) with SAM system components in TZM-bl cells. Activation levels were measured at 48 h post transfection by luciferase assays performed on whole cell lysates. Activation is shown as fold increase (light units/transfected cell) over negative control (no gRNA expression). Shown are results of two independent experiments.
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pone.0158294.g005: Conservation of optimal gRNAs and the effect of sequence variation on SAM-mediated LTR activation.(A) Sequence conservation of HXB2-derived gRNA3, -4, -5 and -6 based on the Los Alamos HIV Sequence Database considering all HIV subtypes (upper panel), subtype B only (middle panel) or subtype C only (lower panel). Percentages of sequence hits are shown, allowing for 0 to 9 point mutations in the indicated gRNA (see Materials and Methods for details). (B) Frequency distribution of gRNA5 mutations observed in database hits according to nucleotide (nt) position. Different colors indicate the number of mutations (1–9). (C) The effect of sequence variations in gRNA5 on SAM-mediated LTR activation was tested by transient expression of gRNA mutants (gRNA *X, where X is the mutated nucleotide position) with SAM system components in TZM-bl cells. Activation levels were measured at 48 h post transfection by luciferase assays performed on whole cell lysates. Activation is shown as fold increase (light units/transfected cell) over negative control (no gRNA expression). Shown are results of two independent experiments.

Mentions: We first determined to what extent gRNA3, -4, -5 and -6 sequences are conserved within the optimal LTR target region based on the Los Alamos National Laboratory HIV sequence database (Fig 5A; www.hiv.lanl.gov). Among all HIV-1 subtypes, as well as subtype B or C alone, the gRNA5 sequence is the most conserved, present in 5% of all deposited sequences, and in 13% of subtype B or 0.5% of subtype C isolates. Allowing for point mutations within the 20 nt gRNAs, tolerating two mutations increases sequence conservation of gRNA5 to 26% for all subtypes, 34% for subtype B and 31% for subtype C. For all retrieved HIV sequences, our database searches revealed that gRNA3, -4 and -6 are much less conserved than gRNA5, with at least nine mutations required for a sequence match of 50% or more, as opposed to four mutations for gRNA5 (Fig 5A).


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)

Conservation of optimal gRNAs and the effect of sequence variation on SAM-mediated LTR activation.(A) Sequence conservation of HXB2-derived gRNA3, -4, -5 and -6 based on the Los Alamos HIV Sequence Database considering all HIV subtypes (upper panel), subtype B only (middle panel) or subtype C only (lower panel). Percentages of sequence hits are shown, allowing for 0 to 9 point mutations in the indicated gRNA (see Materials and Methods for details). (B) Frequency distribution of gRNA5 mutations observed in database hits according to nucleotide (nt) position. Different colors indicate the number of mutations (1–9). (C) The effect of sequence variations in gRNA5 on SAM-mediated LTR activation was tested by transient expression of gRNA mutants (gRNA *X, where X is the mutated nucleotide position) with SAM system components in TZM-bl cells. Activation levels were measured at 48 h post transfection by luciferase assays performed on whole cell lysates. Activation is shown as fold increase (light units/transfected cell) over negative control (no gRNA expression). Shown are results of two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4920395&req=5

pone.0158294.g005: Conservation of optimal gRNAs and the effect of sequence variation on SAM-mediated LTR activation.(A) Sequence conservation of HXB2-derived gRNA3, -4, -5 and -6 based on the Los Alamos HIV Sequence Database considering all HIV subtypes (upper panel), subtype B only (middle panel) or subtype C only (lower panel). Percentages of sequence hits are shown, allowing for 0 to 9 point mutations in the indicated gRNA (see Materials and Methods for details). (B) Frequency distribution of gRNA5 mutations observed in database hits according to nucleotide (nt) position. Different colors indicate the number of mutations (1–9). (C) The effect of sequence variations in gRNA5 on SAM-mediated LTR activation was tested by transient expression of gRNA mutants (gRNA *X, where X is the mutated nucleotide position) with SAM system components in TZM-bl cells. Activation levels were measured at 48 h post transfection by luciferase assays performed on whole cell lysates. Activation is shown as fold increase (light units/transfected cell) over negative control (no gRNA expression). Shown are results of two independent experiments.
Mentions: We first determined to what extent gRNA3, -4, -5 and -6 sequences are conserved within the optimal LTR target region based on the Los Alamos National Laboratory HIV sequence database (Fig 5A; www.hiv.lanl.gov). Among all HIV-1 subtypes, as well as subtype B or C alone, the gRNA5 sequence is the most conserved, present in 5% of all deposited sequences, and in 13% of subtype B or 0.5% of subtype C isolates. Allowing for point mutations within the 20 nt gRNAs, tolerating two mutations increases sequence conservation of gRNA5 to 26% for all subtypes, 34% for subtype B and 31% for subtype C. For all retrieved HIV sequences, our database searches revealed that gRNA3, -4 and -6 are much less conserved than gRNA5, with at least nine mutations required for a sequence match of 50% or more, as opposed to four mutations for gRNA5 (Fig 5A).

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