Atomic structure of Kaposi's sarcoma-associated herpesvirus capsid

卡波西肉瘤相关疱疹病毒衣壳的原子结构

• 批准号: 9185965
• 负责人: REN SUN
• 金额: $ 38.5万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-12-01 至 2020-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9185965
• 关键词: Acquired Immunodeficiency Syndrome; Affect; Amino Acids; Architecture; Bacterial Artificial Chromosomes; Binding; Capsid; Capsid Proteins; Cells; Chemicals; Complex; Cryoelectron Microscopy; DNA biosynthesis; Data; Development; Dimensions; Dimerization; Disease; Dominant-Negative Mutation; Electrons; Etiology; Extracellular Space; Family; Future; Genome; Growth; HIV; Herpesviridae; Herpesviridae Infections; Human Herpesvirus 8; Hydrogen Bonding; Hydrophobic Interactions; Individual; Infection; Interruption; Kaposi Sarcoma; Lead; Link; Lytic; Lytic Phase; Macaca mulatta; Malignant Neoplasms; Maps; Modeling; Mucous Membrane; Multicentric Angiofollicular Lymphoid Hyperplasia; Mus; Mutagenesis; Mutation; Nuclear; Pathogenesis; Patients; Peptides; Pharmaceutical Preparations; Process; Production; Proteins; Publishing; Research; Resolution; Sampling; Scanning; Side; Simplexvirus; Site; Site-Directed Mutagenesis; Skin; Sodium Chloride; Source; Structure; System; Technology; Testing; The Sun; Therapeutic Intervention; Vaccines; Vertebral column; Viral; Virion; Virus; chemical bond; density; design; dimer; fluorophore; gammaherpesvirus; improved; inhibitor/antagonist; lytic replication; member; monomer; mutant; novel; novel strategies; oral lesion; particle; peptide I; primary effusion lymphoma; programs; protein expression; protein protein interaction; public health relevance; reconstruction; therapeutic development; three dimensional structure; viral transmission

 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.

 描述（申请人提供：作为一种内皮来源的癌症，通常生长在皮肤或粘膜下，艾滋病患者的KS大多表现为口腔病变。卡波西肉瘤相关疱疹病毒（KSHV），属于疱疹病毒科伽马疱疹病毒亚科的成员家族，已被证明是所有形式的 KS、原发性渗出性淋巴瘤和多中心性中央病的病因。 KSHV 发病机制和传播的关键是裂解复制，这一过程始于宿主细胞内衣壳的组装，最终将感染性病毒粒子释放到细胞外空间，以维持病毒的传播。 目前，还没有专门针对 KSHV 裂解复制的药物，也没有可用的原子衣壳结构来合理设计抗 KSHV 感染的病毒药物和疫苗，PI Zhou 团队于 1999 年通过冷冻电子显微镜 (cryoEM) 发现。发表了第一个从艾滋病患者衍生的 BCBL-1 细胞中分离出的 KSHV 衣壳的三维 (3D) 结构，随后是 7Å 分辨率的结构最近，两位 PI 通过提高分辨率 (4.5Å) 冷冻电镜和 KSHV 细菌人工染色体 (BAC) 诱变技术合作，绘制了几个重要的图谱。最小衣壳蛋白（SCP、ORF65）的片段与 KSHV 的主要衣壳蛋白（MCP）结合在一起。已发表的残基扫描诱变结果得出了这样的假设：SCP-MCP 相互作用对于 KSHV 组装至关重要，并且原子结构中揭示的这种相互作用可以作为针对 KSHV 裂解性感染的抑制剂。本申请中描述的研究将进行测试。通过利用两个 PI 实验室已经建立的高分辨率冷冻电镜和 KSHV BAC 诱变技术的突破，我们将确定上述假设。在目标 1 中，我们将确定 KSHV 的结构。使用革命性的直接电子计数技术，通过冷冻电镜将衣壳缩小到约 3Å，从这张冷冻电镜图谱中，我们将得出 KSHV 衣壳的原子模型，并识别相互作用衣壳蛋白 6 Å 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和氨基酸来完善对相互作用氨基酸的结构解释。 接下来，将针对 SCP-SCP 和 SCP-MCP 相互作用中确定的关键片段和特定键来设计显性失活突变体（即细胞表达的）和新型突变体。肽（即化学合成的）来选择可以破坏 KSHV 裂解性复制的有效抑制剂（目标 2b），该研究计划的结果将为未来治疗方法的开发提供信息。 KSHV 感染和传播。所建立的新方法将普遍适用于其他病毒和复合物。