Fig 2: RAD23A associates with the catalytic domain of Pol η, Pol κ and REV1. (a) The schematic illustrates the domain structure of the human Y-family polymerases. F = fingers, P = palm, T = thumb, LF = little finger, UBM = ubiquitin-binding motif, UBZ = ubiquitin-binding zinc finger. WT FLAG-RAD23A was immunoprecipitated from cells co-expressing HA-tagged catalytic domains of Pol ι, Pol η, Pol κ and REV1. The eluent and lysate were immunoblotted as indicated. (b, c, d) Immunoprecipitation of WT, V195A and F354A FLAG-RAD23A from cells co-expressing HA-tagged Pol η (b), Pol κ (c) or REV1 (d). Eluent and lysate were immunoblotted as indicated and relative binding calculated based on the ratio of HA polymerase / FLAG-RAD23A in the eluent. Bar graphs illustrate relative binding from three replicates. Error bars indicate the standard deviation. ns = not significant, * = p < 0.5, ** = p < 0.1, *** = p < 0.01, **** = p < 0.001.

Fig 3: UBA1 and UBA2 of RAD23A make distinct interactions with the Pol ι catalytic domain. (a) Immunoprecipitation of the indicated RAD23A truncations from 293 T cells co-expressing WT HA Pol ι. Eluent and WCL (input) were immunoblotted as indicated. Relative binding was calculated based on the ratio of HA to FLAG proteins in the eluent. The bar graph represents the quantification of relative binding from three repeats. Error bars represent standard deviation. Unpaired t-tests were used to assess differences in binding of Pol ι with WT vs mutant RAD23A.**** = p < 0.001. (b) UBA1 and UBA2 domains of RAD23A simultaneously bind to the catalytic domain of Pol ι. 15N-TROSY HSQC spectra of the 15N/2H-labeled UBA2 domain alone (red), after the addition of 1.5 molar excess of unlabeled Pol ι (blue) and after further addition of 5-molar excess of the unlabeled UBA1 domain (green). (c) Comparison of Pol ι regions involved in binding to either UBA1 or UBA2 domains of RAD23A. Representative backbone amide peaks in the 15N-TROSY HSQC spectrum of Pol ι alone (green) and in the presence of either UBA domain at 1:1 (blue), 1:3 (magenta) and 1:6 (red) molar ratios. Identical spectral regions are shown for UBA1 and UBA2 titrations.

Fig 4: Pol ι interacts with RAD23A and RAD23B. (a) Schematic representing the protein microarray used to detect Pol ι protein interactions in vitro. The graph below represents the 2211 immobilized proteins for which statistically significant differences in signal intensity were detected between Pol ι-incubated and control plates. (b) Immunoprecipitation of WT FLAG-tagged RAD23A or RAD23B from cells co-expressing WT HA-Pol ι. Eluent and lysate (input) were immunoblotted as indicated. * = mono-ubiquitinated Pol ι, < = poly-ubiquitinated Pol ι. The bar graph represents the relative proportion of mono and poly-ubiquitinated Pol ι in the eluent, normalized to the proportion of Pol ι ubiquitination detected in the lysate. (c) Immunoprecipitation of endogenous RAD23A from 293 T cells co-expressing WT HA-Pol ι. Eluent and lysate (input) were immunoblotted as indicated. (d) Recombinant His-Pol ι (1.5 μg) was incubated with equimolar quantities of GST (0.5 μg) or GST-tagged RAD23A (2.5 μg) and GST-tagged RAD23B (2.7 μg). GST was captured on glutathione sepharose beads, washed, and eluted. Proteins were separated by gel electrophoresis and proteins stained with Coomassie blue (shown in greyscale). I = 10% input, E = 50% eluent.

Fig 5: HCV core and E1 mutations modulate the HA-E2 protein interactome. Heat map showing alterations of the E2 interactome between the mutant viruses. All primary and accessory HA-E2 binders and RAD23A and B were plotted. For statistical testing, we used multi-parametric two-way analysis of variance (ANOVA) (FDR ≤0.05; S0 = 0; second ANOVA P-value ≤0.05). Significant interactomic changes are highlighted with asterisks. Boxplots on the right show IBAQ values of those proteins whose HA-E2 interaction significantly changed between the viral mutants. Values were normalized to wt E2 (bait) (n = 3–4, mean ± SD).