Fig 2: Protein phosphatase 2A (PP2A) dephosphorylation of pATR changes ATR to the higher form (ATR-H). (A) UV irradiation of A549 cells at 40 J/m2 increases the formation of ATR-H in the cytoplasm. Also, the ATR-H is not phosphorylated while the ATR-L band is phosphorylated in both the nucleus and the cytoplasm (cytoplasm to nucleus loading ratio = 3:1). The samples were analyzed on a 3–8% gradient gel. (B) A549 cells were pretreated with DMSO (Ctrl) or transfected with PP2A siRNAs, then UV-irradiated at 40 J/m2 (–/+UV) followed by a 2 h recovery. The cytoplasmic fraction was analyzed by electrophoresis through a 3–8% gradient gel and WB to show the form of ATR in the cytoplasm. PP2A depletion reduces the levels of cytoplasmic ATR and the amount of ATR-H formed following UV treatment. (C) WB showing PP2A siRNA knockdown efficiency in A549 cells.

Fig 3: Depletion of HERC2 inhibit ATR-mediated phosphorylation of RPA2 induced by low-level replication stress. (a–d) HeLa-shHERC2 cells were induced or not with Dox, treated or not with the indicated genotoxic agents, and subjected to immunoblotting with the indicated antibodies. (e–h) HeLa-shHERC2 cells were transfected with siRNA specific to ATR (e,f) or RFWD3 (g,h) induced or not with Dox, treated or not with indicated concentration of HU (e,g) or APH (f,h), and subjected to immunoblotting with the indicated antibodies. The asterisks indicates non-specific bands.

Fig 4: RPA2 Recruitment of ATR Mediates Cytoplasmic Genome Replication(A) Analysis of F13 expression and virus growth in ATR- and RPA2-depleted HeLa cells 8 hr post-infection.(B) Immunofluorescence images reveal RPA is recruited to viral factories 8 hr post-infection (arrowheads). Quantitation of subcellular localization of RPA shows nuclear depletion of RPA2 during infection.(C) Immunoblot analysis of fractionated cells reveals that vaccinia increases the level of RPA2, ATR, and Ku70 in the cytoplasm 8 hr after infection.(D) Immunoblot analysis of whole-cell extracts reveals vaccinia induces phosphorylation of Ser33 of RPA2 from 4 hr post-infection. Immunofluorescence images showing pRPA2 (Ser33) co-localizes with viral factories 4 hr post-infection (green).(E) Immunofluorescence analysis of CldU incorporation after AraC washout reveals HAMNO treatment suppresses DNA replication and viral factory formation (arrowheads).(F) HAMNO inhibits viral genome replication, as well as late viral gene expression 12 hpi.All error bars represent SEM from three independent experiments, with ***p < 0.001 and ****p < 0.0001. Scale bars, 20 µm.

Fig 5: ATR Activation Is Independent of Viral Genome Uncoating(A) Immunoblot analysis reveals that cycloheximide (CHX) and the proteasome inhibitor MG-132, but not AraC, inhibit both RPA Ser33 phosphorylation and late viral protein expression (F13).(B) Quantitative immunofluorescence analysis reveals that MG-132 inhibits vaccinia-induced cytoplasmic pSQ/TQ immunoreactivity (green).(C) Quantitative immunofluorescence analysis reveals that loss of D5 does not inhibit vaccinia-induced cytoplasmic pSQ/TQ immunoreactivity or expression of I3.All error bars represent SEM from three independent experiments in which a minimum of 200 cells were counted, with *p < 0.05, **p < 0.005, and ***p < 0.001. Scale bars, 20 µm.