Chemical Probes of Kaposi's Sarcoma Herpesvirus Latent Infection

描述（由申请人提供）：卡波西氏肉瘤相关疱疹病毒（KSHV）潜伏感染肿瘤细胞，在卡波西氏肉瘤和原发性积液性淋巴瘤中具有病因学作用。在潜伏性感染中，病毒基因组不整合到染色体中，而是以偶发体（质粒）的形式存在。病毒在快速分裂的细胞中存活依赖于一个精心安排的链

DESCRIPTION (provided by applicant): Kaposi's sarcoma-associated herpesvirus (KSHV) latently infects tumor cells and has an etiologic role in Kaposi's sarcoma and primary effusion lymphoma. In latent infection, the viral genome is not integrated into chromosomes and exists as an episome (plasmid). Virus survival in rapidly dividing cells depends on a carefully orchestrated chain of events. Episomes must replicate in concert with cellular genetic material, and then efficiently segregate to progeny nuclei. KSHV achieves this through its latency associated nuclear antigen (LANA), which tethers viral DNA to mitotic chromosomes to efficiently partition episomes. LANA's N-terminal region is essential for efficient KSHV DNA replication and tethering to mitotic chromosomes. We recently reported that N-terminal LANA binds histones H2A and H2B to attach to host chromosomes. Crystal structure of N-terminal LANA complexed with the nucleosome reveals that LANA peptide forms a hairpin that interacts exclusively with an acidic region of H2A/H2B on the nucleosome surface that is implicated in the formation of higher order chromatin structure. Currently, there are no small molecule inhibitors available that affect any aspect of LANA function. Since LANA is necessary for KSHV latent infection, chemical probes which block the essential LANA binding to the nucleosome would serve as extremely useful reagents to investigate LANA and KSHV biology. Such inhibitory small molecules would be of potential therapeutic benefit since tumor cell persistence is dependent on KSHV infection. Small molecules which bind to the acidic region on the nucleosomal surface to block LANA binding will also greatly facilitate investigation of this region, which is rapidly emerging as a critical functional region of the nucleosome. This work will develop a robust, reproducible, and miniaturized fluorescence polarization (FP) assay for a high throughput screen for small molecules that inhibit KSHV LANA binding to histones H2A/H2B. Pilot screens using the FP assay will be performed to validate the assay prior to high throughput screening. Secondary screens will be performed to confirm and prioritize compound "hits" that inhibit N-terminal LANA's interaction with histones H2A/H2B.