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Journal of Virology, July 1999, p. 5894-5902, Vol. 73, No. 7
Dermatology Branch,
Received 17 February 1999/Accepted 29 March 1999
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-
0022-538X/99/$04.00+0
Identification and Rapid Quantification of Early- and Late-Lytic
Human Herpesvirus 8 Infection in Single Cells by Flow Cytometric
Analysis: Characterization of Antiherpesvirus Agents
) and IFN-
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.
*
Corresponding author. Mailing address: Dermatology
Branch, NCI, Building 10/Room 12N238, 10 Center Dr. MSC 1908, Bethesda, MD 20892-1908. Phone: (301) 402-4167. Fax: (301) 402-1439. E-mail: Andrew_Blauvelt{at}nih.gov
Journal of Virology, July 1999, p. 5894-5902, Vol. 73, No. 7
0022-538X/99/$04.00+0
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