Latent and lytic viral gene expression in KSHV- and rKSHV.219-infected CD34⁺HPCs. (A) CD34⁺ HPCs infected with KSHV or rKSHV.219 and cells were permeabilized and incubated with primary rat monoclonal antibody (mAb) directed against KSHV ORF 73 (LANA-1) followed by incubation with either an FITC-labeled goat anti-rat IgG secondary antibody (KSHV-infected cells) or AMCA-labeled rabbit anti-rat IgG secondary antibody (rKSHV.219-infected cells). Samples were analyzed by flow cytometry for the presence of LANA-1, and cells were gated according to patterns demonstrated by staining with secondary antibody alone. (B-D) CD34⁺ HPCs infected with KSHV and rKSHV.219 were incubated with primary rabbit polyclonal antibodies directed against (B) ORF K2 (vIL-6), primary mouse mAbs directed against (C) ORF 59 (PF-8) and (D) ORF K8.1A of KSHV, followed by incubation with Cy5-conjugated goat anti-rabbit or goat anti-mouse IgG secondary antibody. Samples were analyzed by flow cytometry for the presence of vIL-6, ORF 59, and ORF K8.1A, and gated as described in panel A. (E) CD34⁺ HPCs infected with KSHV were fixed onto siliconized glass slides, permeabilized, and incubated with primary rat mAb directed against KSHV LANA-1, primary rabbit polyclonal antibodies directed against KSHV vIL-6, and primary mouse mAbs directed against KSHV ORF59 and ORF K8.1A. Cells were washed with PBS, followed by incubation with TRITC-conjugated goat anti-rat immunoglobulin (IgG), FITC-conjugated goat anti-rabbit IgG, or FITC-conjugated goat anti-mouse IgG. Cells were washed and counterstained with TO-PRO-3 iodide nuclear stain. KSHV LANA-1 staining is represented by red. KSHV vIL-6, ORF 59, and ORF K8.1A staining are represented by green. TO-PRO-3 nuclear staining is represented by blue. KSHV-infected CD34⁺ HPCs were analyzed at 3 and 15 days after infection (dpi) and are from the cultures presented in panels A-D. TPA-treated BCBL-1 cells and mock-infected CD34⁺ HPCs were similarly analyzed. Cells were visualized through a 100 ×1.40 NA oil-immersion Plan-Apochromat objective lens (Nikon, Tokyo, Japan); total magnification was × 1000.