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 (KS), primary effusion lymphoma (PEL), and multicentric Castleman’s disease (5, 10, 26, 37). HHV-8 is present in both diseased tissue and peripheral blood mononuclear cells (PBMC) of patients with these conditions (4), a finding which strengthens the possibility that HHV-8 is involved in disease pathogenesis. Unfortunately, detailed characterization and quantification of single cells infected with HHV-8 has been limited and technically difficult. Recently, monoclonal antibodies (MAbs) have been developed against lytic-cycle viral proteins encoded by open reading frame (ORF) 59 and ORF K8.1 (8, 39). ORF 59 protein is a putative viral accessory protein (processivity factor 8 [PF-8]) located in the cell nucleus and expressed in the early lytic phase of HHV-8 replication (8, 21). PF-8 binds HHV-8 DNA polymerase, allowing it to synthesize greatly extended DNA products. By contrast, proteins encoded by ORF K8.1 (glycoprotein K8.1 [gpK8.1], also known as gp35–37) are immunogenic glycoproteins expressed in the late lytic phase and are associated with the viral envelope (9, 30). In this study, we used these new MAbs to assess early nonstructural and late structural viral protein expression during active HHV-8 replication in single cells by flow cytometry. This particular technique should prove useful for developing in vitro HHV-8 infection models and for performing detailed phenotypic characterization of HHV-8-infected cells in vivo, thus providing an important new tool in the study of HHV-8-associated diseases. Interestingly, past use of certain antiherpesvirus drugs (e.g., foscarnet and cidofovir) correlates with a reduced risk of developing KS (11, 15, 19, 24, 33). These and other potential antiherpesvirus agents may eventually prove to be useful in the prevention of HHV-8-associated disease in HHV-8-infected individuals (4). In concordance with these epidemiologic studies, several investigators have shown that certain antiherpesvirus drugs are capable of blocking HHV-8 replication in chronically infected PEL cell lines (14, 20, 22, 28). These previous in vitro studies, however, used laborious methods and relied on semiquantitative differences in band intensities to assess the efficacy of drugs. In this study, we have also applied our system to characterize the ability of potential antiherpesvirus agents to block the induction of HHV-8 replication. This method is rapid and quantitative and therefore can be easily used for future investigations of novel antiherpesvirus agents.