Targeting LANA Function to Develop KSHV Antivirals

DESCRIPTION (provided by applicant): Kaposi's sarcoma-associated herpesvirus (KSHV), also called human herpesvirus 8, is associated with Kaposi's sarcoma (KS), primary effusion lymphoma (PEL) and a plasmablastic variant of multicentric Castleman's disease (MCD). Common to these malignancies is that a majority of cells in these lesions are latently infected and express only a small subset of viral genes. One of these genes, the latency-associated nuclear antigen (LANA) has consistently shown to be expressed in all KSHV -associated malignancies. LANA is a multifunctional nuclear protein that interacts with a variety of host cellular proteins including- transcriptional modulators such as mSin3 and the tumor suppressors p53 and RB, thereby regulating viral and cellular gene expression. In addition, LANA is required for maintenance of the episomal viral DNA genome during latency in-dividing cells. Ballestas, et.al., demonstrated that LANA expressing cells efficiently maintain plasmids containing terminal repeat (TR) over many cell divisions. This suggested that the latent origin of replication is located within TR and that interaction of LANA and TR sequences are crucial for DNA replication. By performing detailed mapping and footprinting studies, we have identified two LANA-binding sites within TR that are bound by LANA in a cooperative fashion. Furthermore, in congruence with data from other laboratories, we have shown that LANA is required for supporting DNA replication of TR-containing plasmids. Based on the fact that LANA is required not only for long-term maintenance but also for DNA replication, we hypothesize that LANA is an attractive target for the development of antiviral compounds. Such potential drugs would specifically act on latently infected cells and conceivably, could also prevent the establishment of latency in de novo infected cells. To directly test our hypothesis, we propose to develop a fluorescence anisotropy-based high-throughput screening assay (HTS) to identify small molecule inhibitors of LANA function. We further propose to develop cell-based assays in order to investigate the ability of candidate compounds to interfere with DNA replication and long-term maintenance.