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. 2009 Apr 22;83(13):6641–6651. doi: 10.1128/JVI.00049-09

FIG. 3.

FIG. 3.

RPA, Rad51, Rad52, and MRN complexes are recruited to EBV replication compartments. (A) Subnuclear localization of RPA, phosphorylated RPA, Rad51, and Rad52 in latent Tet-BZLF1/B95-8 cells treated with 0.5% Triton X-100-mCSK buffer, fixed with methanol, and stained with the indicated combinations of antibodies as described in Materials and Methods. (a) DAPI (blue), anti-BMRF1 (green), and anti-RPA32 Ser-4/Ser-8 (red); (b) DAPI (blue), anti-BMRF1 (green), and anti-RPA32 Ser-33 (red); (c) anti-BALF2 (green) and anti-RPA32 (red); (d) anti-BALF2 (green) and anti-Rad51 (red); (e) anti-BMRF1 (green) and anti-Rad52 (red) antibodies. Panels labeled DAPI (blue) are DAPI-stained nuclei. (Right) Merged images. (B) Phosphorylated RPA32, Rad51, Rad52, and Mre11 are colocalized to EBV replication compartments. Tet-BZLF1/B95-8 cells were cultured in the presence of 2 μg of doxycycline/ml and harvested at 24 h p.i. Cells were treated with 0.5% Triton X-100-mCSK buffer, fixed with methanol, and stained with the indicated combinations of anti-BMRF1 (green), anti-BALF2 (green), anti-Rad52 (red), anti-RPA32 Ser-33 (red), anti-RPA32 Ser-4/Ser-8 (red), anti-RPA32 (red), anti-Mre11 (red), and anti-Rad51 (red) antibodies as described in Materials and Methods. Panels labeled DAPI (blue) are DAPI-stained nuclei. (Right) Merged images. (C) Phosphorylated RPA32 and NBS1 are colocalized to discrete nuclear foci in EBV lytic replication compartments. Tet-BZLF1/B95-8 cells were cultured in the presence of 2 μg of doxycycline/ml and harvested at 24 h p.i. Cells were treated with 0.5% Triton X-100-mCSK buffer, fixed with methanol, and stained with the indicated combinations of anti-NBS1 antibody (green), Alexa Fluor 647-labeled BMRF1 antibody (blue), and phospho-specific anti-RPA32 Ser-4/Ser-8 or anti-RPA32 Ser-33 antibody (red). (Right) Merged images. (D) Recruitment of phosphorylated RPA32 to the EBV replication compartment in Tet-BZLF1/Akata cell lines. Tet-BZLF1/Akata cells were cultured in the presence of 2 μg of doxycycline/ml and harvested at 24 h p.i. Cells were treated as described above (B) and stained with the indicated combinations of anti-BMRF1 (green), anti-RPA32 Ser-33 (red), and anti-RPA32 Ser-4/Ser-8 (red) antibodies as described in Materials and Methods. (Right) Merged images.