title: Identification and rapid quantification of early- and late-lytic human herpesvirus 8 infection in single cells by flow cytometric analysis: characterization of antiherpesvirus agents.
abstract: Human herpesvirus 8 (HHV-8) infection is associated with Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease. In this study, we used monoclonal antibodies (MAbs) directed against HHV-8 lytic 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 Abs, and analyzed by flow 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 viral proteins and minimized cell toxicity. The absolute number of expressing cells was inducer and cell line dependent. Expression of PF-8 occurred earlier and more frequently (in up to 20% of cells) than did expression of gpK8.1. A subset of PF-8 positive cells (25%) co-expressed gpK8.1, representing 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, alpha interferon (IFN-alpha) and IFN-beta reduced expression of both PF-8 and gpK8.1, implying an overall inhibitory effect on viral gene transcription or translation. In summary, we have characterized and quantified HHV-8 lytic infection in single cells by dual measurement of early- and late-lytic-cycle HHV-8 protein expression. This technique should prove useful for screening of possible antiherpesvirus agents and for detailed phenotypic characterization of HHV-8-infected cells in vitro and in patients with HHV-8-associated diseases.
authors.affiliation: Dermatology Branch, National Cancer Institute,National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: J P
authors.initials: JP
authors.lastname: Zoeteweij
authors.forename: S T
authors.initials: ST
authors.lastname: Eyes
authors.forename: J M
authors.initials: JM
authors.lastname: Orenstein
authors.forename: T
authors.initials: T
authors.lastname: Kawamura
authors.forename: L
authors.initials: L
authors.lastname: Wu
authors.forename: B
authors.initials: B
authors.lastname: Chandran
authors.forename: B
authors.initials: B
authors.lastname: Forghani
authors.forename: A
authors.initials: A
authors.lastname: Blauvelt
mesh_terms: D000212:Acyclovir / Q000494:pharmacology; D000998:Antiviral Agents / Q000494:pharmacology*; D015153:Blotting, Western; D002460:Cell Line; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D017245:Foscarnet / Q000494:pharmacology; D006023:Glycoproteins; D019288:Herpesvirus 8, Human / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology / Q000648:ultrastructure; D006801:Humans; D016898:Interferon-alpha / Q000494:pharmacology; D016899:Interferon-beta / Q000494:pharmacology; D007371:Interferon-gamma / Q000494:pharmacology; D008854:Microscopy, Electron; D013997:Time Factors; D014764:Viral Proteins / Q000096:biosynthesis*
chemical_list: D000998:Antiviral Agents; D006023:Glycoproteins; D016898:Interferon-alpha; C113454:K8.1 protein, Human herpesvirus 8; C110348:ORF59 protein, Human herpesvirus 8; D014764:Viral Proteins; D017245:Foscarnet; D016899:Interferon-beta; D007371:Interferon-gamma; D000212:Acyclovir