CAPSID STRUCTURE AND ASSEMBLY IN KSHV

卡波西肉瘤(KS)--混合性多中心血管生成瘤 细胞性，是获得性缺陷患者的主要肿瘤 综合症(艾滋病)。目前的分子和血清流行病学数据表明 KS相关疱疹病毒(KSHV)，又称人类疱疹病毒8型(HHV8)，是 KS的感染原因。在大多数感染病例中，健康的细胞 免疫反应可能会摧毁绝大多数活跃的细胞 复制中的病毒。相反，免疫抑制会使这一平衡发生倾斜。 从这种以潜伏为主的状态转变为病毒重新激活的状态， 复制和广泛传播。这种致病进程取决于， 在一定程度上，KSHV的高水平生产。最引人注目的前病毒粒子 病毒复制开始后出现的结构是内部 衣壳--填充在核中的二十面体对称粒子，当 完全成熟，拥有线性病毒基因组。形成鲜明对比的是 成功地调查了衣壳的结构和组装 甲型和乙型疱疹病毒(如单纯疱疹病毒1型和 巨细胞病毒)，试图在 伽马疱疹病毒(如爱泼斯坦-巴尔病毒)进展甚微。这个 我们在提案中描述的研究的长期目标是 与其他成员相比，了解KSHV的结构和组件 并将这些发现放在KS的背景下 发病机制。最近，我们设计了一些方法来提纯结构完整的 KSHV衣壳。在本申请中，我们建议： 1.提纯和鉴定KSHV裂解过程中形成的不同衣壳物种 使用生化和物理分离技术进行生长。 2.用生化方法表征这些衣壳的结构成分， 电子显微镜和质谱学方法。 3.利用计算机辅助确定衣壳的三维结构 电子冷冻显微镜图像分析。 4.研究KSHV衣壳组装体。 了解衣壳结构和组装的细节，这是复制KSHV的早期和关键步骤，可能同时确定新的潜在治疗干预靶点。

Kaposi's sarcoma (KS), a multicentric angiogenic tumor of mixed cellularity, is the leading neoplasm of patients with acquired deficiency syndrome (AIDS). Current molecular- and seroepidemiologic data demonstrate that KS-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV8), is the infectious cause of KS. In most cases of infection, a healthy cellular immune response likely destroys the vast majority of cells that harbor actively replicating virus. In contrast, immunosuppression can tilt this equilibrium away from this predominantly latent state to one of viral reactivation, replication and widespread dissemination. This pathogenic progression depends, in part, on high levels of KSHV production. The most remarkable pre-virion structures to appear after the initiation of viral replication are the inner capsid--the icosahedrally symmetric particles that fill the nucleus and, when fully mature, harbor the linear viral genome. In stark contrast to the successful investigations into the structure and assembly of the capsids of alpha- and betaherpesviruses, (such as herpes simplex virus 1 and cytomegalovirus, respectively), attempts to explore these aspects in the gammaherpesviruses (such as Epstein-Barr virus) have made little progress. The long-term objectives of the research we describe in the proposal are to understand KSHV structure and assembly in comparison to members of the other herpesvirus subfamilies and to place these findings into the context of KS pathogenesis. Recently, we have devised methods to purify structurally intact KSHV capsids. In this application, we propose to: 1.Purify and identify the distinct capsid species formed during lytic KSHV growth using biochemical and physical separation techniques. 2.Characterize the structural components of these capsids with biochemical, electron microscopic and mass spectrometric approaches. 3.Determine the three-dimensional structure of the capsids using computer-aided analyses of electron cryomicroscopic images. 4.Investigate KSHV capsid assembly. Understanding the details of capsid structure and assembly, an early and essential step in KSHV replication, may simultaneously identify new potential targets for therapeutic intervention.