GENETIC AND BIOCHEMICAL STUDIES OF THE KSHV LANA GENE

卡波西肉瘤（KS）相关疱疹病毒（KSHV或HHV8）是最近发现的一种与恶性肿瘤相关的病毒，特别是与艾滋病患者有关。KSHV见于KS、原发性积液淋巴细胞（PELs）和多中心Castleman病（一种淋巴细胞增生性疾病）。与一种密切相关的疱疹病毒Epstein-Barr病毒类似，KSHV在恶性肿瘤和PEL细胞系中的感染主要是潜伏的，病毒DNA以染色体外圆形片段的形式持续存在。潜伏期相关核抗原（LANA或LNA）是一种含有1162个氨基酸的KSHV蛋白，在潜伏感染细胞的细胞核中以点状分布表达。KSHV感染患者的血清通常与LANA反应，流行病学研究经常使用这种反应性来确定KSHV血清阳性。LANA的功能尚不清楚。我们现在表明LANA与细胞染色体上的KSHV片段共定位，LANA允许细胞中KSHV片段的维持。这些结果与LANA在有丝分裂期间通过栓系插曲体向细胞染色体的分离介导插曲体向子细胞分离的模型一致。该项目将通过确定LANA与染色体上的插曲体共定位是否对插曲体的维持至关重要来测试该模型。首先，将对LANA进行广泛的突变分析，以确定介导与染色体和KSHV发作共定位的LANA结构域。不能与染色体或片段共定位的LANA突变体将被检测是否丧失维持细胞中KSHV片段的能力。接下来，将定义LANA介导的片段维持所必需的KSHV DNA元件。然后，实验将研究LANA和基本KSHV DNA元件之间相互作用的性质。

Kaposi's sarcoma (KS) associated herpesvirus (KSHV or HHV8) is a recently discovered virus which is associated with malignancies in patients, especially those with AIDS. KSHV is found in KS, primary effusion lymphocytes (PELs) and multicentric Castleman's disease, a lymphoproliferative disorder. Similar to a closely related herpesvirus, Epstein-Barr virus, KSHV infection in malignancy and in PEL cell lines is primarily latent, with viral DNA persisting as an extra-chromosomal, circular episome. The latency associated nuclear antigen (LANA or LNA) is an 1162 amino acid KSHV protein expressed in a punctate distribution in the nuclei of latently infected cells. The serum of KSHV infected patients usually reacts with LANA and epidemiologic studies have often used this reactivity to determine KSHV seropositivity. The function of LANA is unknown. We now show that LANA colocalizes with KSHV episomes on cell chromosomes and LANA permits maintenance of KSHV episomes in cells. These results are consistent with a model in which LANA mediates segregation of episomes to daughter cells by tether episomes to cell chromosomes during mitosis. This project will test this model by determine if LANA colocalization with episomes on chromosomes is essential for episome maintenance. First, extensive mutational analysis of LANA will be performed to determine the LANA domains that mediate colocalization with chromosomes and with KSHV episomes. LANA mutants which cannot colocalize with chromosomes or episomes will be tested for loss of the ability to permit maintenance of KSHV episomes in cells. Next, the KSHV DNA element essential for LANA mediated episome maintenance will be defined. Experiments will then investigate the nature of the interaction between LANA and the essential KSHV DNA element.