HCMV-Infected Cells Maintain Efficient Nucleotide Excision Repair of the Viral Genome while Abrogating Repair of the Host Genome
Figure 5
CPDs were selectively removed from viral DNA.
Cells were seeded and infected as described in Figure 1. At 48 hpi, cells were irradiated (or unirradiated) with 75 J/m2 and harvested at the indicated times post irradiation. Cells were pulse-labeled with BrdU just prior to irradiation to enable viral RC visualization. After harvesting, cells were treated as described in Materials and Methods unless otherwise noted. A) Cells were stained for localization of CPDs (green) and DNA (BrdU - red) and imaged using confocal microscopy. Top panels show virus-infected cells with no irradiation to show the specificity of the CPD Ab. Middle panels show CPD staining of M+UV cells at 0 and 24 h post irradiation. Bottom panels show V+UV cells at 0 and 24 h post irradiation. Overlays show selective removal of CPDs from viral DNA over time. Far right panels illustrate how the regions were mapped for analysis in 5C and D. B) Parallel coverslips were harvested at 0 and 24 hp irradiation and fixed using standard “fix first” conditions. These coverslips were stained for visualization of cellular histone H3 (red) and viral processivity factor UL44 (green) to mark viral RCs. Left panels show V+UV samples, right panels show H3 staining in M+UV samples. Images were taken using confocal microscopy as in A. C) Quantitation of total nuclear INTINT at 0 and 24 hp irradiation for three separate experiments was performed as described in Materials and Methods. Average total nuclear INTINT for separate experiments are plotted as different symbols. Grey bars represent the average of these experimental points. The asterisk indicates the statistical significance (p-value<0.0001) between the CPD signal for the entire nucleus at 0 hp irradiation and at 24 h post irradiation (by mixed effects ANOVA). D) Further analysis of the components of the total INTINT (viral DNA and host DNA) by mixed effects ANOVA showed statistical significant differences in the viral DNA INTINT between 0 and 24 h (p<0.0002), but not the host INTINT (p>0.16). NS = not significant.