Abstract
Herpes simplex virus (HSV) establishes lifelong latent infection and can cause serious human disease, but current antiviral therapies target lytic but not latent infection. We screened for sgRNAs that cleave HSV-1 DNA sequences efficiently in vitro and used these sgRNAs to observe the first editing of quiescent HSV-1 DNA. The sgRNAs targeted lytic replicating viral DNA genomes more efficiently than quiescent genomes, consistent with the open structure of lytic chromatin. Editing of latent genomes caused short indels while editing of replicating genomes produced indels, linear molecules, and large genomic sequence loss around the gRNA target site. The HSV ICP0 protein and viral DNA replication increased the loss of DNA sequences around the gRNA target site. We conclude that HSV, by promoting open chromatin needed for viral gene expression and by inhibiting the DNA damage response, makes the genome vulnerable to a novel form of editing by CRISPR-Cas9 during lytic replication.
Highlights
Herpes simplex virus (HSV) 1 and 2 are prevalent neurotropic DNA viruses that cause significant morbidity and mortality in neonates and adults (Roizman et al, 2013)
As the first step in identifying single gRNA (sgRNA) that could efficiently cleave lytic as well as quiescent HSV genomes in human cells, we measured the endonuclease activity of sgRNA candidates in an in vitro cleavage assay
Targeting HSV-1 with UL26-27 and UL37-38 resulted in significantly less PCR amplification across their respective target sequences compared to non-targeted regions (Figure 8F G compared to Figure 8D). These results suggested that clustered regularly-interspaced short palindromic repeats (CRISPR)/Cas9 targeting of non-coding sites in the HSV-1 genome reduces viral replication by disruption of the viral DNA and/or deletion of neighboring genes
Summary
Herpes simplex virus (HSV) 1 and 2 are prevalent neurotropic DNA viruses that cause significant morbidity and mortality in neonates and adults (Roizman et al, 2013). CRISPR/ Cas editing requires efficient access to target sites within chromatin, and evidence suggests that chromatin modifications and DNA packaging can block genome editing in eukaryotic cells (Bultmann et al, 2012; Chen et al, 2016; Horlbeck et al, 2016; Hsu et al, 2013; Valton et al, 2012; Wu et al, 2014). Differential host cell mechanisms regulating chromatin assembly on viral DNA in permissive (epithelial) versus non-permissive (neuronal) cells contribute to the lytic versus latent infection decision by HSV (Knipe and Cliffe, 2008). Recent studies have demonstrated that CRISPR/Cas can impair active HSV replication in vitro by targeting specific DNA sequences encoding viral proteins (Roehm et al, 2016; van Diemen et al, 2016). Our results define differences in the mechanisms of editing of lytic and quiescent HSV-1 genomes, highlighting the vulnerability of the lytic viral genome due to viral proteins reducing loading of histones on viral DNA, inhibiting host DNA repair mechanisms, and promoting viral lytic DNA replication
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