Sequencing of HSV-1 genomic DNA to identify genomic variation resulting from selective pressure imposed by mutation of the PP1 binding site in the pUL21 gene

The pUL21 gene is essential for efficient replication and dissemination in herpes simplex virus 1 (HSV-1) but the molecular basis for its function is not well understood. Experimental work revealed that pUL21 is a virus-encoded adaptor of protein phosphatase 1 (PP1). Mutation of the specific motif in pUL21 responsible for PPI recruitment, followed by propagation in cell culture, was performed in order to identify the impact of preventing PP1 recruitment on the viral genome. This allowed identification of compensatory mutations disrupting the viral kinase gene pUS3, which restored viral growth and spread, revealing that pUL21 directly antagonises pUS3. Three mutant strains of HSV-1 were generated, mutating the conserved residues F242 and V243 in the PP1 binding sequence, individually and in combination (pUL21F242E, pUL21V243D, pUL21FV242AA), along with a knockout strain lacking pUL21 (ΔpUL21). These four virus stocks, plus wild-type HSV-1, were propagated in Vero cells for three passages. Genomic DNA was extracted from the P3 viruses and sequenced with Illumina. Reads were trimmed and mapped to an edited version of the HSV-1 genome (based on JQ673480.1) and read depth and variants were quantified.