Spatiotemporal dynamics of HSV genome nuclear entry and compaction state transitions using bioorthogonal chemistry and super-resolution microscopy
(a) RPE-1 cell monolayers were infected with 50 pfu of HSV-1 and incubated in the presence of EdC at various concentrations (added at 2 hpi). Plaques were fixed and stained at 48 hr (scale bar 1 mm). Plaque area (approximately 40 plaques) and plaque numbers at each EdC concentration were quantitated relative to untreated cells (set to 100%). (b) Single-step growth yield assay of HSV-1 in the presence of EdC. Cells were infected (moi 5) and incubated with EdC (added at 2 hpi). Supernatant and cell-associated virus was harvested at 20 hpi and titrated on RPE-1 cells. (c) Multi-step growth yield assay. Cells were infected (moi 0.005) and incubated with EdC added at 2 hpi. Virus was harvested at 72 hpi and titrated on RPE-1 cells. (d) Particles/pfu ratios of HSV and HSVEdC. Virus released into the medium and purified by ultracentrifugation was titrated and equal pfu applied to a defined area on coverslips and stained for VP5+ve particles. Multiple fields were imaged and tiled so that all particles in the samples were quantified. The graph indicates total particle counts from the accumulated individual fields and the SD of particle counts within individual fields.