Spatiotemporal dynamics of HSV genome nuclear entry and compaction state transitions using bioorthogonal chemistry and super-resolution microscopy
Cells were mock-treated or treated with MG132 (10 μM), Leptomycin B (20 nM) or nocodazole (2 μM) as indicated. Inhibitors were added to cells for 1 hr prior to infection with HSVEdC (moi 10). Cells were analysed at 0.5 hpi for the localisation of EdC-labeled genomes as described for other figures. For MG132 we also analysed genome localisation at 1 hr. (a) Each panels shows a representative image at high magnification (x63 objective) together with histograms of quantitative evaluation of the frequency of numbers of genomes/nucleus observed for each condition (at least 200 nuclei for each). (b) Box and whisker plots for data in (a). Box shows 2nd and 3rd quartiles with a horizontal bar in the middle showing the median, while whiskers show up to 5–95% of the total population. ‘+’ denotes the mean value. Unpaired two-tailed t-tests were used for statistical results (ns = not statistically significant, *** = p<0.0001). In this experiment infection even in the untreated sample was somewhat less efficient than standard, but there was no significant difference with either MG132 or Leptomycin B at 30 min and no diference for MG132 at 1 hr. In contrast, Nocodazole treatment resulted in a substantial and significant reduction in accumulation of uncoated nuclear genomes as discussed in the text.