Aspects of human gamma-herpesvirus lytic infection and possible therapeutic implications.

摘要

The gamma-herpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV, also referred to as human herpesvirs-8, HHV-8) and Epstein-Baff virus (EBV) are associated with several human malignancies and exist in both latent and lytic states. Latency is the predominant form of infection and is generally associated with tumors, whereas induction of lytic infection is associated with viral replication and cell lysis. Studies detailed here characterize a novel approach for specifically targeting KSHV- and EBV-infected tumor cells. Treatment of tumor cells with clinically suitable pharmacologic agents, in combination with nucleoside analogs, may be used to induce lytic infection and cause specific killing of virally infected cells. The goal of this thesis research was to investigate the aspects of gamma-herpesvirus lytic infection and to consider the feasibility of this novel therapeutic strategy.; In order to characterize lytic gene expression in KSHV-associated tumors, immunohistochemistry and in situ hybridization were applied to formalin-fixed tumor specimens. Lytic cycle expression was detected in a small percentage of cells in primary effusion lymphomas (PEL), in Castleman's disease, and in rare KS specimens, demonstrating that the lytic pathway is intact in these tumors.; The establishment and characterization of a new PEL-derived cell lines, JSC-1, provided a useful tool for studying KSHV lytic infection, investigating pharmacologic agents that induce lytic infection, and developing an in vitro infection assay using primary endothelial cell cultures.; In order to determine whether nucleoside analogs such as ganciclovir (GCV) are activated by lytic viral kinases, the KSHV-thymidine kinase (TK), KSHV-phosphotransferase (PT), and EBV-PT homologues were cloned and expressed in 293T cells. These homologues phosphorylated GCV and sensitized cells to GCV-mediated killing. Induction of viral kinase expression in tumor cells correlated with GCV phosphorylation and inhibition of viral replication.; The induction of KSHV and EBV lytic infection with pharmacologic agents was investigated. Histone deacetylase inhibitors induced lytic gene expression and resulted in viral-specific cell killing of PEL and 5-aza-2 ′-deoxycytidine pretreated Burkitt's lymphoma (BL) cell lines. While the presence of GCV inhibited viral replication, it did not enhance cell killing. The results of these experiments demonstrate the feasibility of this novel approach for killing KSHV- and EBV-positive tumors.