Inhibition of HSV-1 Replication by Gene Editing Strategy.

Pamela C Roehm,Anna Bellizzi,Kamel Khalili,Lifan He,Masoud Shekarabi,Julian Salkind,Hassen S Wollebo

doi:10.1038/srep23146

Abstract

HSV-1 induced illness affects greater than 85% of adults worldwide with no permanent curative therapy. We used RNA-guided CRISPR/Cas9 gene editing to specifically target for deletion of DNA sequences of the HSV-1 genome that span the region directing expression of ICP0, a key viral protein that stimulates HSV-1 gene expression and replication. We found that CRISPR/Cas9 introduced InDel mutations into exon 2 of the ICP0 gene profoundly reduced HSV-1 infectivity in permissive human cell culture models and protected permissive cells against HSV-1 infection. CRISPR/Cas9 mediated targeting ICP0 prevented HSV-1-induced disintegration of promonocytic leukemia (PML) nuclear bodies, an intracellular event critical to productive HSV-1 infection that is initiated by interaction of the ICP0 N-terminus with PML. Combined treatment of cells with CRISPR targeting ICP0 plus the immediate early viral proteins, ICP4 or ICP27, completely abrogated HSV-1 infection. We conclude that RNA-guided CRISPR/Cas9 can be used to develop a novel, specific and efficacious therapeutic and prophylactic platform for targeted viral genomic ablation to treat HSV-1 diseases.

Highlights

Herpes simplex virus type 1 (HSV-1) is a human neurotropic virus that infects the majority of the human population worldwide[1,2]
The endogenous type II CRISPR system in such microbes apparently defends against foreign DNA, as association of CRISPR RNA and transactivated RNA sequences can trigger targeting of foreign DNA sequences for double-strand cleavage by the DNA endonuclease Cas[9]
Seeking a convenient human cell line in which to further explore the anti-HSV-1 activity of our CRISPR/Cas[9] system, we found that the human oligodendroglioma cell line TC62034 robustly supported HSV-1 replication at low and high multiplicities of infection, as evidenced by expression of infected cell protein 0 (ICP0), the early viral protein ICP8, which is involved in viral DNA replication and late gene transactivation[35,36,37], and the late envelope protein, glycoprotein C, which enhances infectivity of the virus[38] (Fig. S3A–D)

Summary

Introduction

Herpes simplex virus type 1 (HSV-1) is a human neurotropic virus that infects the majority of the human population worldwide[1,2]. To assess its translational potential for application to treat and eliminate HSV-1 infection, we tested the ability of CRISPR/Cas[9] to suppress HSV-1 replication by targeting specific DNA sequences essential to viral protein expression during early and late phases viral infection/reactivation. We targeted DNA sequences of the ICP0 gene and developed guide RNAs to combine with Cas[9], to test their ability to suppress viral gene expression We found this strategy introduced specific InDel mutations in the ICP0 sequence, produced no off-target effects or toxicity to host cells, and rescued antiviral PML bodies from disintegration by ICP0, supporting the promise of this model approach for developing anti-HSV-1 therapies capable of eliminating latent infection

Methods

Results

Conclusion