Fig 2: Immortalized human skin fibroblasts harboring mutations in NER genes are defective in repair of the BFP_AFB1-Fapy reporter plasmid. (A) Illustration of FM-HCR plasmids used in Fig. 2 created in BioRender. Undamaged cocktail: pMax_GFP transfection control, pMax_BFP_2, and pMax_mOrange. Damaged cocktail: pMax_GFP transfection control, BFP_AFB1-Fapy, and mOrange_UV (randomly distributed UV damage induced by irradiation with 800 J/m2 UV light). (B) Representative flow cytometry scatter plots of FM-HCR experiments in XP2OS (top four plots) and XP2OS + XPA (bottom four plots) cells transfected with damaged plasmid cocktail. The leftmost plot for each sample (forward scatter area versus side scatter area) is gated to separate cells from debris. Cells are then displayed on the other three plots and gated for expression of BFP, mOrange, or GFP. The number of cells within each gate is displayed on the plot. A detailed gating strategy is provided in Supplementary Fig. S12. (C) FM-HCR in XP2OS/XP2OS + XPA cell line pair. (D) FM-HCR in XP2YO (ERCC4 mutant) cells and XP2YO complemented with wild-type XPF or catalytically inactive mutant, XPF D676A. (E) FM-HCR in XP3BRSV (ERCC5 mutant) and cells complemented with XPGWT or catalytically inactive mutant, XPG E791A. In panels (B)–(D), each data point is from an independent experiment (n = 3), error bars show the standard deviation, and P-values are from unpaired two-tailed t-test.

Fig 3: Knockout of NER factors XPA and XPF suppresses AFB1-Fapy dG repair in human cell lines. (A) BFP_AFB1-Fapy expression in TK6 wild-type and XPA-knockout (XPA−/−) cells. (B) Western blot for XPA in U2OS wild type or XPA−/− cells stably expressing either empty vector or XPA. (C) FM-HCR in U2OS cells from panel (B) transfected with undamaged cocktail (pMax_BFP, pMax_mOrange, and pMax_mPlum) or damaged cocktail (BFP_AFB1, mOrange_UV, and pMax_mPlum). (D) Representative flow cytometry plots from cells transfected with the damaged cocktail from panel (C). These plots show three different representations of data from the same sample to illustrate the gating strategy. The leftmost plot gated for BFP-expressing cells and the rightmost plot gated for mPlum-expressing cells. The title above the plot indicates the population being gated. (E) Western blot for XPF in wild-type U2OS cells transfected with non-targeting sgRNA (sgNT) or U2OS XPF-knockout cells (two independent clones). (F) FM-HCR in cells from panel (E). In panels (A), (C), and (F), each data point is from an independent experiment (n = 3) and error bars show the standard deviation. P-values are from an unpaired two-tailed t-test with comparison to wild type unless otherwise indicated.

Fig 4: Reduced resistance to GCV of Exo-mutant HCMV in XPA deficient cells.(A-B) Control cells (A) and XPA− cells (B) were infected at MOI 2 with WT HCMV, the Exo-mutant virus F412V or its rescued virus F412V_R, respectively. After 2 h incubations, the viruses were removed and media containing various concentrations of GCV were added. At 5 dpi, the supernatants were harvested. The viruses were titrated on HFF cells and the yield of virus at each concentration of GCV was calculated using standard plaque assay. The ED50 values for GCV inhibition and their 95% confidence intervals (CI) presented in these two panels were determined from triplicate experiments by least squares regression with variable slope. (C) Curves were similarly fitted for WT and F412V in each independent experiment to obtain three ED50 values for each cell type. Those ED50 values from three biological replicate experiments were used for a ratio paired t test comparing viruses within each cell type and the Holm-Šídák method was used to correct for multiple comparisons. ED50 mean values are plotted with standard deviation error bars. (D) From each experiment, for each cell type, the ratio of the ED50 for WT virus to the ED50 for F412V was calculated. Because ED50 ratios typically do not follow a normal distribution, which is necessary for t tests, while logarithms of such values typically do follow a normal distribution(74), we plotted log2 values and used them for an unpaired t test with Welch’s correction. Mean log2(ED50 ratio) values are shown with 95% CI error bars.

Fig 5: XPA rescue in XPA− deficient fibroblasts restores F412V resistance to GCV.(A) Whole cell lysates from XPA− fibroblasts transduced with GFP-expressing control vector lentivirus (+GFP, left lane) or one expressing XPA-GFP (+XPA-GFP, right lane) were collected to determine XPA expression by immunoblotting. Tubulin serves as a loading control. (B) XPA− fibroblasts were infected at an MOI of 1 with WT or F412V. At 2 hpi, virus was washed out and media was replaced containing various concentrations of GCV. At four days post infection, cells and supernatant were collected for analysis by TCID50. Virus yield at each GCV concentration was compared to the DMSO control for each condition and plotted as percent yield reduction for three biological replicate experiments. Curves were similarly fitted for WT and F412V in each individual experiment to obtain ED50 values and CIs for each condition. (C) Those ED50 values from three biological replicate experiments were used for a ratio paired t test comparing viruses within each cell type and WT in the two cell types and the Holm-Šídák method was used to correct for multiple comparisons. ED50 mean values are plotted with standard deviation error bars.