Inhibition of HSV-1 Replication by Gene Editing Strategy

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.

Figure 1. CRISPR/Cas9 introduces mutations in ICP0 gene and reduces HSV-1 replication.

(A) Schematic representation of HSV-1 ICP0 genomic region showing exon II and the ring domain. The positions and nucleotide composition of 2A, 2B and 2C targets including PAM (marked in green) are shown. The positions of various primers (P) used in PCR amplification are illustrated. (B) Gel analysis of DNA fragments amplified by primers P3 and P2 (shown in A) in L7 cells expressing Cas9 (control) or Cas9 plus gRNAs 2A and 2C. The positions of an expected 552 bp amplicon and a smaller DNA fragment of 217 bp caused by cleavage of exon II DNA at targets (A,C), and amplification of the remaining fragments after ligation are shown. (C) Sequence traces of the 217 bp cloned fragment (shown in B) illustrating a blunt end-joining of the two ends of the cleaved exon II DNA after excision of 355 bp. D. DNA gel analysis illustrating amplicons using P3 and P4 primers from several clonal cells (named InDels 1-3) expressing Cas9/gRNA 2A or the control cells with no gRNA 2A. (E) DNA sequencing identified nucleotide insertions (as depicted by black arrowheads) in clones InDel 1 and 2, and insertion of 191 bp DNA in clones InDel 3. The red arrowhead points to the cleavage sites. (F) Representative confocal images of L7 cells (control) and its subclones InDel 3 (shown in E) expressing Cas9/gRNA 2A after immunostaining with an antibody against ICP0. Green is indicative of expression of GFP by HSV-1 and DAPI (blue) depicts the nuclei of the cells. (G) Viral production assay demonstrating the titer of ΔICP0-HSV-1 in the supernatant of parental L7 cells (control) endogenously expressing ICP0 or in InDel 3, InDel 2 (shown in E).

Figure 2. CRISPR/Cas9 by targeting ICP0 expression restores association of PML bodies in L7 cells.

Immunostaining analysis of PML bodies in control, uninfected L7 cells shows the appearance of punctate staining of PML bodies within the nuclei of the cells (lower panel). DAPI staining of nuclei (blue) is shown in the right panels. Infection of Vero L7 cells expressing Cas9 at low MOI with HSV-1/GFP shows complete destruction of the PML and robust replication of HSV-1/GFP as shown in green (middle panels). In L7 cells expressing Cas9/gRNA 2A infection with HSV-1/GFP failed to disrupt formation of punctate PML in the nuclei and significantly reduced the level of viral replication detected in the cells (green, top panels).

Figure 3. Human TC620 cells expressing Cas9/gRNAs does not support HSV-1 replication.

(A) Western blot analysis of protein extracts for detection of ICP0 in TC620 human olidodendroglioma cells and its subclones expressing Cas9 or Cas9/gRNA 2A (clones 6 and 10). Expression of 110 kDa ICP0 is detected in the control cells and cells expressing Cas9, but severely reduced in clones 6 and 10. Expression of housekeeping tubulin is shown. (B) Representative plaque assay using the supernatant from HSV-1/GFP infected control TC620 or clones 6 and 10 at two different dilutions showing a drastic decrease in the number of plaques as a result of suppression of ICP0 by Cas9/gRNA editing of the infected cells. (D) Quantification of HSV-1 production by plaque assay shows drastic suppression of the viral production in TC620 cells with continuous production of Cas9 plus gRNA 2A.

Figure 4. Lentivirus (LV) mediated Cas9/gRNA delivery suppresses HSV-1 infection during the lytic cycle and prevents uninfected cells from new infection.

(A) Western blot analysis of protein extracts from TC260 cells that endogenously express Cas9 and are infected with HSV-1/GFP for 24 hours and thereafter treated with lentivirus expressing gRNAs 2A, 2B or both and harvested 48 hours later for protein extraction. The positions of ICP0, ICP8, glycoprotein C Cas9 and α-tubulin are shown. (B,C) illustrate results from plaque assay in which cells treated with LVgRNA 2A or LVgRNA 2B showed a drastic decrease in virus production. (D) Confocal immunofluorescent evaluation of HSV-1/GFP replication in TC620 cells that were treated with vector (LV) LVCas9, LVCas9 plus LVgRNA 2A, LVCas9 plus gRNA 2B and LVCas9 plus LVgRNA 2A and 2B at 48 hours prior to HSV-1 infection. A decrease in the replication of HSV-1/GFP is shown in green. (D) Western blot analysis of protein extracts from the infected cells as described above. (E) Plaque assay showing a decrease in the titer of the virus released upon HSV-1 infection of cells that were pre-treated with LV producing Cas9 and gRNAs as depicted.