Figure 4.

Impact of homing endonuclease (HE)-mediated mutagenesis on herpes simplex virus (HSV) reactivation. (a) Timeline of HSV latency establishment in human fibroblasts (HF), exposure to scAAV2 delivery vectors and reactivation following CMV infection. Triplicate dishes of latently-infected HF transduced at a multiplicity of infection (MOI) of 10⁴ genomes/cell/vector with scAAV2 delivery vectors expressing the indicated transgene were reactivated at day 20 (8 dpt) by infection with HCMV AD169 at a MOI of 1 for 2 days in the absence of ACV. At 2 days postinfection with human cytomegalovirus (HCMV) (day 22; 10 dpt). ACV, acyclovir; IFN, interferon. (b) The presence of virion progeny in culture supernatants was assessed by plaque assay titration and (c) the number of intracellular HSV genomes was measured by ddPCR. Bars indicate the mean from the triplicate samples. (d) The HSV region containing the target site was PCR amplified from total genomic DNA obtained from triplicate dishes of reactivated HSV infected HF treated with HSV1m5 and Trex2. The PCR amplicons were pooled, cloned, and sequenced from individual bacterial colonies. The nature and number of the mutations found are indicated on the right side of each sequence. Δ: deletion; wt, wild-type sequence of the HSV1m5 target site. Bold indicates the four nucleotides constituting the 3′ overhang generated by the DNA DSB; - indicates a deleted nucleotide.