Visualizing PABPC Localization in Viral Replication Compartments

NIH 3T3 cells were plated on coverslips (7.5 x 10⁴ cells/well of a 12-well), infected the following day for 25–27 h, and then fixed in 4% formaldehyde for 10 min. iSLK-Bac16 cells were plated on coverslips (5 x 10⁴ cells/well of a 12-well), reactivated 24h later with doxycycline and sodium butyrate for 48 hours and then fixed as above. Cells were permeabilized with ice-cold methanol at -20°C for at least 20 min and incubated with anti-Pol II antibody (Biolegend, 8WG16 at 1:200), anti-PABPC antibody (Abcam ab21060 at 1:200 for mouse cells and Santa Cruz sc32318 at 1:25 for human cells) or anti-ORF59 antibody (Advanced Biotechnologies 13-211-100 at 1:200) in 5% BSA overnight at 4°C. Secondary antibodies were added (1:1000) for 1 h at 37°C. Coverslips were mounted in DAPI- containing Vectashield (VectorLabs). We identified RCs by Pol II recruitment or ORF59 staining corresponding with DAPI-poor regions [34 (link),36 (link),61 (link)]. Images were collected on a Zeiss LSM 710 AxioObserver with a 40x oil objective. Cells with RCs were first identified, then PABPC co-localization was determined by counting cells with at least 2x nuclear pixel intensity relative to non-RC containing cells in the same image (ImageJ).