Identification and rapid quantification of early- and late-lytic human herpesvirus 8 infection in single cells by flow cytometric analysis: Characterization of antiherpesvirus agents

Human herpesvirus 8 (HHV-8) infection is associated with Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease. In t his study, me used monoclonal antibodies (MAbs) directed against HHV-8 lyti c cycle-associated proteins encoded by open reading frame (ORF) 59 (nuclear PF-8 protein) and ORF K8.1 (viral envelope glycoprotein K8.1 [gpK8.1]) to investigate HHV-8 lytic infection in single cells. Lytically infected cells were labeled with MAbs, stained with fluorescently conjugated secondary Ab s, and analyzed by Bow cytometry. A 3-day stimulation of HHV-8-positive PEL cell lines (BCBL-1 and BC-3) with 12-O-tetradecanoylphorbol-13-acetate (30 nM) or n-butyric acid (0.3 mM) maximized the expression of lytic-phase vir al proteins and minimized cell toxicity. The absolute number of expressing cells was inducer and cell line dependent. Expression of PF-8 occurred earl ier and more frequently tin up to 20% of cells) than did expression of gpK8 .1. A subset of PF-8 positive cells (25%) co-expressed,gpK8.1., representin g the majority of gpK8.1 expressing cells. Acyclovir, foscarnet, cidofovir, and PMEA reduced the number of cells expressing gpK8.1, but not the number expressing the nonstructural early lytic gene product PF-8. By contrast, a lpha interferon (IFN-alpha) and IFN-beta reduced expression of both PF-8 an d gpK8.1, implying an overall inhibitory effect on viral gene transcription or translation. In summary, we have characterized and quantified HHV-8 lyt ic infection in single cells by dual measurement of early- and late-lytic-c ycle HHV-8 protein expression. This technique should prove useful for scree ning of possible antiherpesvirus agents and for detailed phenotypic charact erization of HHV-8-infected cells in vitro and in patients with HHV-8-assoc iated diseases.