A CRISPR-based rapid DNA repositioning strategy and the early intranuclear life of HSV-1
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Curated by eLife
eLife assessment:
In this important work, the authors describe a recombinant CRISPR/Cas9 construct, CRISPR-nuPin, that has the reported capacity to rapidly tether DNA to the inner nuclear membrane of cells. They then evaluate the effect of this construct on Herpes Simplex virus type 1 infection, identifying different phases of viral replication susceptible to inner nuclear membrane tethering. This work provides convincing evidence for the effects of intranuclear DNA localization on viral gene expression and replication, using a method applicable to nonviral genes as well.
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Abstract
The relative positions of viral DNA genomes to the host intranuclear environment play critical roles in determining virus fate. Recent advances in the application of chromosome conformation capture-based sequencing analysis (3 C technologies) have revealed valuable aspects of the spatiotemporal interplay of viral genomes with host chromosomes. However, to elucidate the causal relationship between the subnuclear localization of viral genomes and the pathogenic outcome of an infection, manipulative tools are needed. Rapid repositioning of viral DNAs to specific subnuclear compartments amid infection is a powerful approach to synchronize and interrogate this dynamically changing process in space and time. Herein, we report an inducible CRISPR-based two-component platform that relocates extrachromosomal DNA pieces (5 kb to 170 kb) to the nu clear p eriphery in minutes (CRISPR-nuPin). Based on this strategy, investigations of herpes simplex virus 1 (HSV-1), a prototypical member of the human herpesvirus family, revealed unprecedently reported insights into the early intranuclear life of the pathogen: (I) Viral genomes tethered to the nuclear periphery upon entry, compared with those freely infecting the nucleus, were wrapped around histones with increased suppressive modifications and subjected to stronger transcriptional silencing and prominent growth inhibition. (II) Relocating HSV-1 genomes at 1 hr post infection significantly promoted the transcription of viral genes, termed an ‘Escaping’ effect. (III) Early accumulation of ICP0 was a sufficient but not necessary condition for ‘Escaping’. (IV) Subnuclear localization was only critical during early infection. Importantly, the CRISPR-nuPin tactic, in principle, is applicable to many other DNA viruses.
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eLife assessment:
In this important work, the authors describe a recombinant CRISPR/Cas9 construct, CRISPR-nuPin, that has the reported capacity to rapidly tether DNA to the inner nuclear membrane of cells. They then evaluate the effect of this construct on Herpes Simplex virus type 1 infection, identifying different phases of viral replication susceptible to inner nuclear membrane tethering. This work provides convincing evidence for the effects of intranuclear DNA localization on viral gene expression and replication, using a method applicable to nonviral genes as well.
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Joint Public Review:
The authors employ a range of microscopy, biochemical, and virologic techniques to evaluate the efficacy of CRISPR-nuPin to relocalize DNA and the subsequent impact of HSV-1 replication. There are many compelling experiments that utilize solid approaches to HSV-1 transcription, replication, and histone association. The microscopy images are particularly stunning, strongly supported by biochemical evaluation, and consistent with most of the authors' interpretations. Overall, the manuscript presents data that suggests the dCas9-emerin fusion protein can be used to manipulate the nuclear localization of smaller DNA elements like the HSV-1 viral genome. Chromosomal DNA, as tested by telomere targeting, reveal reduced capacity and elongated kinetics for retargeting. Using this system, authors find differing effects on …
Joint Public Review:
The authors employ a range of microscopy, biochemical, and virologic techniques to evaluate the efficacy of CRISPR-nuPin to relocalize DNA and the subsequent impact of HSV-1 replication. There are many compelling experiments that utilize solid approaches to HSV-1 transcription, replication, and histone association. The microscopy images are particularly stunning, strongly supported by biochemical evaluation, and consistent with most of the authors' interpretations. Overall, the manuscript presents data that suggests the dCas9-emerin fusion protein can be used to manipulate the nuclear localization of smaller DNA elements like the HSV-1 viral genome. Chromosomal DNA, as tested by telomere targeting, reveal reduced capacity and elongated kinetics for retargeting. Using this system, authors find differing effects on HSV-1 replication based on the timing of sgRNA electroporation post-infection. Further experiments support that the transcriptional effects of either inhibitory or enhancing treatments may be related to chromatin modifications and expression of the viral protein ICP0.
There are many strengths to both the methodology and analysis in this work. That said, there are several areas where a more expansive explanation of methods and data analysis combined with tempered interpretations and language will greatly improve the manuscript.
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