vIRF-3 is constitutively expressed in primary effusion lymphoma cells. Whole-cell lysates from the human KSHV⁻ B-cell line BJAB (lane 1), KSHV⁺ BCBL-1 cells (lane 2), and BCBL-1 cells activated by TPA for 4 days (lane 3) were separated on 8% (A) or 12% (B) discontinuous SDS–polyacrylamide gels. Separated proteins were transferred onto nitrocellulose membranes and incubated with either monoclonal antibody 3G7 generated against a N-terminal fragment of vIRF-3 (A), or antibody BS555 directed against the virion glycoprotein K8.1.⁴⁵  Whereas the various forms of gpK8.1 were abundantly expressed in BCBL-1 cells induced with TPA (panel B lane 3) and not detectable without induction (panel B lane 2), the relatively moderate expression level of vIRF-3 remains essentially unaltered by TPA treatment (panel A lanes 2-3). The immunofluorescence in panel C shows that vIRF-3 is present in the nuclei of latently infected BCBL-1 cells. BCBL-1 cells (lower photomicrograph) or KSHV⁻ BJAB cells were fixed onto glass slides and incubated with monoclonal antibody 3G7 directed against recombinant vIRF-3, followed by detection with fluorescein-labeled antibodies against rat IgG. A fine-grained nuclear fluorescence was clearly detectable in virtually all BCBL-1 cells, but not in the KSHV⁻ human B-cell line BJAB.