Host restriction mechanisms in HSV-1 replication have been explored through the analysis of viral mutants, particularly those lacking the ICP0 E3-ubiquitin ligase function, critical for counteracting restriction processes. These mutants are profoundly defective in initiation of early infection. Since the defect is in an immediate-early (IE) protein, current proposals suggest that restriction acts downstream, after IE transcription and is due to the activity of cell factors that would normally be degraded by the E3-ligase activity. Many observations regarding these pathways are derived from mouse models or specialised cell types, potentially limiting their relevance to primary infections, and results are occasionally contradictory.

This study focuses on ICP0 E3-ligase mutant infection in physiologically relevant skin keratinocytes, employing quantitative spatial dynamics of transcription at both the population RNA and individual cell levels. Findings indicate that, in a restrictive environment, there is a selective failure to transcribe the IE ICP4 gene despite efficient transcription of ICP0. This challenges the conventional view that IE genes are expressed as a co-ordinately regulated group and highlights the need for an understanding of the host factors required for this restriction.

The roles of cytoplasmic cGAS/STING and nuclear IFI16, proposed to play major roles in HSV-1 detection and promotion of cellular responses. The absence of cGAS or STING had little impact on HSV-1 initiation and spread in skin keratinocytes, even for an ICP0-defective mutant. Lack of IFI16 did result in increased initiation and progression of infection by the ICP0-defective mutant, but only partial rescue of the ICP0-mutant was observed. Results also indicated that differences in the structural repertoire of wild-type (WT) and ICP0 mutant virions may contribute to the defect observed after ICP0 mutant infection. Furthermore, results highlight differences in transfected DNA vs viral infection and could potentially lead to some revision of interpretations of the DNA sensing pathways.