FIG. 3.

In situ localization of KSHV GCR gene expression to lytically infected cells in KS and BCBL-1 cell culture. In situ hybridization of ³⁵S-labeled riboprobes specific for KSHV RNAs to thin sections of a dermal KS lesion reveals that few cells are transcribing ORF 74 (visualized as silver grains that have developed in a photographic emulsion coating the specimen) (A, arrow; 7-day exposure). The frequency of ORF 74-positive cells is similar to that of cells transcribing the lytic gene, nut-1 (B; 4-h exposure) and represents only a fraction of the total population of infected cells that are revealed by hybridization with probe to detect the viral K12 (T0.7) gene, which transcribed in latent as well as lytically infected cells (C; 3-day exposure). Double-label in situ hybridization with ³⁵S-labeled ORF 74 riboprobe (yellow-green silver grains as viewed with epipolarized illumination) and digoxigenin-labeled nut-1 riboprobe (dark purple nuclei) shows colocalization to the same subpopulation of spindle tumor cells (D; 7-day exposure). Hybridization of the ORF 74 probe to thin sections of paraffin-embedded BCBL-1 cells before TPA treatment (E, arrows; 3-day exposure) and 2 days after treatment with TPA (F; 3-day exposure) reveals that uninduced cultures contain a minor subset (1 to 3%) of cells expressing ORF 74 and that GCR transcription is turned on in cells induced to undergo lytic replication. (A to C, E, F) Counterstained with hematoxylin and eosin; (D) lightly counterstained with hematoxylin.