Herpes simplex virus type 1 (HSV-1) R-loops are targets for APOBEC-mediated mutagenesis

APOBEC3 enzymes are key components of antiviral immunity that introduce mutations into viral genomes. We show that R-loops serve as preferred substrates for APOBEC3 mutagenesis during herpes simplex virus type 1 infection. APOBEC3 enzymes are recruited to viral R-loops, where they generate clustered C-to-T mutations in genes critical for viral assembly. Importantly, R-loops alone are not mutagenic, and APOBECA3 enzymes do not edit HSV-1 without an R-loop; mutagenesis occurs when R-loops and APOBEC3 enzymes interact. These findings identify endogenous R-loops formed during the HSV-1 life cycle as focal vulnerabilities and highlight R-loop–APOBEC3 coupling as a mechanism for antiviral mutagenesis.