Atomic structure of Kaposi's sarcoma-associated herpesvirus capsid

 描述（由申请人提供）：作为一种内皮源性癌症，通常生长在皮肤或粘膜下，艾滋病患者的KS主要表现为口腔病变。卡波济肉瘤相关疱疹病毒（Kaposi's sarcoma-associated herpesvirus，KSHV）是疱疹病毒科γ疱疹病毒亚科的一个成员，是各种KS、原发性渗出性淋巴瘤和多中心Castleman病的病原体。KSHV的致病和传播的中心是裂解复制，这一过程始于宿主细胞内衣壳的组装，并以感染性病毒体释放到细胞外空间中，从而使病毒在宿主细胞内传播结束。 病毒繁殖目前，没有特异性靶向KSHV裂解复制的药物可用，也没有合理设计抗KSHV感染的抗病毒药物和疫苗所需的原子衣壳结构可用。1999年，通过冷冻电镜（cryoEM），PI Zhou的小组发表了从AIDS患者来源的BCBL-1细胞中分离的KSHV衣壳的第一个三维（3D）结构，随后是小鼠（与PI Sun）和恒河猴γ疱疹病毒的7 μ m分辨率结构，揭示了γ疱疹病毒衣壳蛋白之间的分子相互作用。最近，这两个PI的合作努力，提高分辨率（4.5kb）cryoEM和细菌人工染色体（BAC）诱变KSHV的定位几个重要的片段的最小衣壳蛋白（SCP，ORF 65）在巩固KSHV的主要衣壳蛋白（MCP）。这些结构的结果，连同公布的结果从残基扫描诱变，导致的假设，SCP-MCP的相互作用是至关重要的KSHV组装和这种相互作用揭示的原子结构可以针对抑制剂对KSHV裂解感染。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cryoEM和KSHV BAC诱变技术突破来测试上述假设。目的一：利用冷冻电镜技术，采用革命性的直接电子计数技术，对~ 3 HNMR进行KSHV衣壳结构的测定。从这个cryoEM图中，我们将推导出KSHV衣壳的原子模型，并鉴定相互作用衣壳蛋白的6个碱基内的氨基酸残基-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，我们将通过关联现有的和 新的数据，从位点特异性诱变，并评估其对衣壳组装的影响。接下来，在SCP-SCP和SCP-MCP相互作用中鉴定的关键片段和特异性键将被靶向以设计两种显性负突变体（即，细胞表达的）和新的肽（即，化学合成），以选择能够破坏KSHV裂解复制的有效抑制剂（目的2b）。这项研究计划的结果将为未来开发抗KSHV感染和传播的治疗方法提供信息。所建立的新方法将普遍适用于其他病毒和复合物。

 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.