CRISPR/Cas9-mediated genome editing of the thymidine kinase gene in a clinical HSV-1 isolate identifies F289S as novel acyclovir-resistant mutation

• Published in: Antiviral research Vol. 228; p. 105950
• Main Authors: Zheng, Shuxuan, Verjans, Georges M.G.M., Evers, Anouk, van den Wittenboer, Ellen, Tjhie, Jeroen H.T., Snoeck, Robert, Wiertz, Emmanuel J.H.J., Andrei, Graciela, van Kampen, Jeroen J.A., Lebbink, Robert Jan
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.08.2024
• Subjects: Acyclovir resistance; CRISPR/Cas9; Herpes simplex virus 1; Recurrent herpetic eye disease; Thymidine kinase; Herpes simplex virus 1; Recurrent herpetic eye disease; Thymidine kinase; CRISPR/Cas9; Acyclovir resistance; acyclovir resistance; recurrent herpetic eye disease; thymidine kinase
• ISSN: 0166-3542; 1872-9096; 1872-9096
• DOI: 10.1016/j.antiviral.2024.105950

Herpes simplex virus type 1 (HSV-1) is a neurotropic alphaherpesvirus that establishes a lifelong infection in sensory neurons of infected individuals, accompanied with intermittent reactivation of latent virus causing (a)symptomatic virus shedding. Whereas acyclovir (ACV) is a safe and highly effective antiviral to treat HSV-1 infections, long-term usage can lead to emergence of ACV resistant (ACVR) HSV-1 and subsequently ACV refractory disease. Here, we isolated an HSV-1 strain from a patient with reactivated herpetic eye disease that did not respond to ACV treatment. The isolate carried a novel non-synonymous F289S mutation in the viral UL23 gene encoding the thymidine kinase (TK) protein. Because ACV needs conversion by viral TK and subsequently cellular kinases to inhibit HSV-1 replication, the UL23 gene is commonly mutated in ACVR HSV-1 strains. The potential role of the F289S mutation causing ACVR was investigated using CRISPR/Cas9-mediated HSV-1 genome editing. Reverting the F289S mutation in the original clinical isolate to the wild-type sequence S289F resulted in an ACV-sensitive (ACVS) phenotype, and introduction of the F289S substitution in an ACVS HSV-1 reference strain led to an ACVR phenotype. In summary, we identified a new HSV-1 TK mutation in the eye of a patient with ACV refractory herpetic eye disease, which was identified as the causative ACVR mutation with the aid of CRISPR/Cas9-mediated genome engineering technology. Direct editing of clinical HSV-1 isolates by CRISPR/Cas9 is a powerful strategy to assess whether single residue substitutions are causative to a clinical ACVR phenotype. •We isolated an acyclovir resistant (ACVR) HSV-1 with a novel F289S mutation in UL23.•CRISPR/Cas9-technology was used to revert F289S in the genome of the isolate.•The edited clinical isolate (S289F) became sensitive to ACV (ACVs).•Conversely, F289S introduction in an ACVs reference rendered it ACVR.•CRISPR/Cas9 technology simplifies directed mutagenesis of clinical isolates.