Figure 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.