FIG 9.

Cytoplasmic LANA is sensitive to mild detergent extraction. Vero cells were infected with KSHV and induced to lytically replicate by superinfection with BacK50 as in Fig. 7 and were either left untreated (A to C) or briefly treated with 0.1% NP-40 (D to F), or the ProteoExtract fraction I extraction buffer (G to I) to solubilize cytoplasmic proteins. The cells were fixed and sequentially stained for LANA, ORF59, and cell nuclei, as in Fig. 4. In the untreated culture (A to C), the arrowheads indicate one of many cells with cytoplasmic LANA (A) and nuclear ORF59 fluorescence (B), merged in panel C. The arrow indicates one of many cells with punctate nuclear LANA (A) and no ORF59 fluorescence (B). After extraction with 0.1% NP-40 or fraction I buffer, the arrowhead indicates one of many cells, in which cytoplasmic LANA was removed (D and G), yet the nuclear ORF59 and LANA remain (E and H; merged in panels F and I). The arrows indicate cells with punctate nuclear LANA (D and G) or no nuclear ORF59 (E and H). Scale bar, 50 μm.