Skip to main content
. 2009 Feb 4;83(8):3904–3918. doi: 10.1128/JVI.02137-08

FIG. 4.

FIG. 4.

The single-stranded DNA binding protein ORF29 is targeted to IE62 nuclear domains that transform into viral replication compartments. (A) HELF cells were seeded on glass coverslips and infected with green-labeled inoculum cells for 4 h, 6 h, or 8 h and were double stained with anti-IE62 (green; mouse monoclonal antibody) and anti-ORF29 (red; polyclonal rabbit antibody) followed by secondary anti-mouse Alexa Fluor 488 or anti-rabbit Texas Red-conjugated secondary antibodies. Nuclei (upper panels a through d; blue) were counterstained with Hoechst 22358. Newly infected cells were identified by the absence of green cytoplasmic staining and were analyzed for IE62 and ORF29 expression by confocal microscopy. The upper panels (a through d) show an overview of the nuclei with merged blue, green, and red channels. Areas of the same nuclei (within the white squares in the upper panels) are shown at higher magnification in lower panels a' through d' (for better visualization of red and green signals, no blue channels are shown). White arrows point to red ORF29 punctae associated with green-stained IE62 nuclear domains. Scale bars are 10 μm. (B) HELF cells were seeded and infected and then additionally treated at 12 h after infection with a 30-min pulse of 0.1 mM BrdU (in medium) before being fixed at 12.5 h. Cells were then stained with anti-ORF29 (red) or -ORF62 (green; polyclonal antibody) and Texas Red (ORF29)- or FITC (IE62)-conjugated secondary antibodies, respectively, processed for DNA in situ hybridization (red; DIG-labeled VZV DNA probe), or stained for incorporated BrdU (red; biotinylated anti-BrdU monoclonal antibody and streptavidin-Texas Red conjugate for secondary detection). Scale bars are 10 μm. Each of these single stainings reveals similarly shaped globular nuclear compartments. (C) Cells from the same experiment as for panel B were double stained for either BrdU (a, red) with viral DNA (vDNA; b, green), BrdU (d, red) with ORF29 (e, green), or ORF29 (g, red) with IE62 (h, green). The merged channels are shown in panels c, f, and i. Control stainings for ORF29, viral DNA, and BrdU are shown in panels j, k, and l. Scale bars are 10 μm.