Atomic structure of Kaposi's sarcoma-associated herpesvirus capsid

 描述(申请人提供：爱滋病患者的KS主要表现为口腔病变，是一种典型的皮下或粘膜下的内皮源性癌症。卡波西肉瘤相关疱疹病毒(KSHV)是疱疹病毒科伽马疱疹病毒亚家族的成员之一，已被证明是所有形式的KS、原发渗出性淋巴瘤和多中心Castleman病的病原体。KSHV致病和传播的中心是裂解复制，这一过程始于衣壳在宿主细胞内的组装，结束于感染性病毒粒子释放到细胞外空间 病毒传播。目前，还没有针对KSHV裂解复制的药物，也没有合理设计抗病毒药物和抗KSHV感染疫苗所需的原子衣壳结构。1999年，利用冷冻电子显微镜(CryoEM)，皮周的团队发表了从艾滋病患者来源的BCBL-1细胞中分离出的KSHV衣壳的第一个三维(3D)结构，随后是小鼠(与Pi Sun)和恒河猴伽马疱疹病毒的7个超分辨结构，揭示了伽马疱疹病毒衣壳蛋白之间的分子相互作用。最近，两个PI的合作努力提高了KSHV的冷冻EM和细菌人工染色体(BAC)的分辨率，并定位了KSHV最小衣壳蛋白(SCP，ORF65)的几个重要片段，这些片段与KSHV的主要衣壳蛋白(MCP)结合在一起。这些结构结果，加上已发表的残基扫描突变结果，导致了SCP-MCP相互作用对KSHV组装至关重要的假设，这种在原子结构中揭示的相互作用可以作为抑制KSHV裂解感染的靶点。本申请中描述的研究将利用两个PI实验室已经建立的高分辨率低温EM和KSHV BAC突变方面的技术突破来检验上述假设。在目标1中，我们将利用革命性的直接电子计数技术，通过低温电子显微镜来确定KSHV衣壳的结构。从这张低温EM图中，我们将得到KSHV衣壳的原子模型，并确定相互作用的衣壳蛋白6ä内的氨基酸残基--特别是SCP和MCP之间的氨基酸残基--即对衣壳组装至关重要的残基。在目标2a中，我们将完善我们对相互作用氨基酸的结构解释，将现有的和 来自定点突变的新数据，并评估它们对衣壳组装的影响。接下来，在SCP-SCP和SCP-MCP相互作用中确定的关键片段和特异键将被用于设计显性负突变(即细胞表达的)和新肽(即化学合成的)，以选择能够破坏KSHV裂解复制的有效抑制剂(目标2b)。这项研究计划的结果将为未来抗KSHV感染和传播的治疗方法的发展提供信息。所建立的新方法将普遍适用于其他病毒和复合体。

 DESCRIPTION (provided by applicant: As a cancer of endothelial origin that typically grows under the skin or mucous membranes, KS in AIDS patients mostly manifests as oral lesions. Kaposi's sarcoma-associated herpesvirus (KSHV), a member of the gammaherpesvirus subfamily of the Herpesviridae family, has been shown to be an etiologic agent of all forms of KS, primary effusion lymphoma and multicentric Castleman's disease. Central to pathogenesis and spread of KSHV is lytic replication, a process that begins with the assembly of capsids inside host cells and ends with the release of infectious virions into the extra-cellular space for viral propagation. Currently, no drugs specifically targeting lytic replication of KSHV are available and no atomic capsid structures are available needed for rational design of anti-viral drugs and vaccines against KSHV infection. By cryo electron microscopy (cryoEM) in 1999, PI Zhou's group published the first three-dimensional (3D) structure of KSHV capsid isolated from AIDS patient-derived BCBL-1 cells, followed by 7Å-resolution structures of the murine (with PI Sun) and rhesus monkey gammaherpesviruses, revealing molecular interactions among gammaherpesvirus capsid proteins. Recently, the two PIs' collaborative efforts with improved resolution (4.5Å) cryoEM and bacterial artificial chromosome (BAC) mutagenesis of KSHV have mapped several important segments of the smallest capsid protein (SCP, ORF65) in cementing the major capsid protein (MCP) of KSHV. These structure results, together with published results from residue-scanning mutagenesis, have led to the hypotheses that the SCP-MCP interactions are vital to KSHV assembly and such interactions revealed in an atomic structure can be targeted for inhibitors against KSHV lytic infections. The studies described in this application will test the above hypotheses by taking advantage of technology breakthroughs in high-resolution cryoEM and KSHV BAC mutagenesis already established in the two PIs' labs. In Aim 1, we will determine the structure of KSHV capsid to ~3Å by cryoEM with the revolutionary direct electron counting technology. From this cryoEM map, we will derive an atomic model of the KSHV capsid and identify amino-acid residues within 6 Å of interacting capsid proteins - particularly those between SCP and MCP - i.e., residues vital to capsid assembly. In Aim 2a, we will refine our structural interpretation of interacting amino acids by correlating existing and new data from site-specific mutagenesis and assess their impact on capsid assembly. Next, key segments and specific bonds identified among SCP-SCP and SCP-MCP interactions will be targeted to design both dominant negative mutants (i.e., cell-expressed) and novel peptides (i.e., chemically-synthesized) to select potent inhibitors that can disrupt KSHV lytic replication (Aim 2b). Results from this research program will inform future development of therapeutics against KSHV infection and spread. The novel approach established will be generally applicable to other viruses and complexes.