In situ atomic structures of the Kaposi's sarcoma-associated herpesvirus portal-terminase complex and glycoproteins

卡波西肉瘤相关疱疹病毒门静脉末端酶复合物和糖蛋白的原位原子结构

• 批准号: 10470289
• 负责人: TING-TING WU
• 金额: $ 41.7万
• 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 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10470289
• 关键词: AIDS related cancer; Acquired Immunodeficiency Syndrome; Affect; Amino Acids; Antiviral Agents; Artificial nanoparticles; Bacterial Artificial Chromosomes; Binding; Capsid; Capsid Proteins; Cells; Cercopithecine Herpesvirus 1; Complex; Cryoelectron Microscopy; Cytomegalovirus; DNA; DNA Packaging; Data; Development; Disease; Endothelium; Etiology; Family; Funding; Future; Genome; Glycoproteins; HIV; Herpesviridae; Herpesviridae Infections; Human; Human Herpesvirus 8; In Situ; Individual; Infection; Interruption; Kaposi Sarcoma; Lead; Link; Lymphoma; Lytic; Lytic Phase; Malignant Neoplasms; Mediating; Membrane Fusion; Modeling; Molecular Conformation; Mucous Membrane; Multicentric Angiofollicular Lymphoid Hyperplasia; Mutagenesis; Nature; Pathogenesis; Patients; Pharmaceutical Preparations; Principal Investigator; Production; Prophylactic treatment; Proteins; Proteomics; Publications; Publishing; Relaxation; Research; Resolution; Respiratory syncytial virus; Side; Site; Site-Directed Mutagenesis; Skin; Structure; System; Technology; Testing; The Sun; Therapeutic Intervention; Vaccine Design; Vaccines; Vertebral column; Viral; Viral Genome; Virion; Virus; Visualization; base; cancer type; chemical bond; chronic infection; crosslink; density; design; drug development; gammaherpesvirus; glycoprotein structure; high risk infant; inhibiting antibody; inhibitor; insight; interest; lytic replication; member; mutant; novel strategies; novel vaccines; oral lesion; particle; peptidomimetics; primary effusion lymphoma; programs; protein protein interaction; protein structure; rational design; receptor; reconstruction; terminase; vaccine candidate; viral transmission

Project Summary/Abstract Kaposi's sarcoma (KS) is the most common malignancy associated with infection by human immunodeficiency virus (HIV). 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. Currently, no drugs specifically targeting lytic replication of KSHV are available. Additionally, atomic structures of KSHV viral genome packaging/ejection machinery and fusion-mediating glycoproteins needed for rational design of antiviral drugs and vaccines are unavailable. The prior four-years' funding of this multiple principal investigator (MPI) R01 project has led to the publication of the atomic structure of the KSHV capsid in Nature. Structure-guided mutagenesis studies have identified amino acid interactions among capsid proteins that are essential to capsid assembly and informed the design of small peptide mimics that inhibited viral maturation. Jointly, the MPIs' groups also published the first atomic model of the KSHV DNA-packaging portal complex and capsid associated tegument complexes in Cell. Preliminary data for this renewal application have established the feasibility of obtaining in situ structures of genome-packaging portal-terminase complex and the cell-entry glycoprotein B (gB) in both pre-fusion and post-fusion conformations. These portal protein and envelope glycoprotein structures and structure-guided mutagenesis results have led to three hypotheses: (1) the portal- associated proteins and terminase interactions are vital to KSHV genome encapsidation; (2) the interactions and conformational changes among envelope glycoproteins are required for KSHV fusion with host cells during cell entry; and (3) such interactions revealed in atomic structures can help design inhibitors and vaccines against KSHV lytic infection. The studies described in this application will test the above hypotheses by taking advantage of technology breakthroughs in high-resolution cryoEM and KSHV mutagenesis already demonstrated in the two MPIs' labs in the current funding cycle. In Aim 1, we will determine the in situ structures of KSHV portal-associated proteins and the terminase to ~3Å by cryoEM. From these structures, we will derive atomic models and identify amino- acid residues within 6 Å of interacting proteins (i.e., residues vital to DNA packaging and ejection). In Aim 2, we will determine the structures of major envelope glycoproteins to ~3Å. Specifically, we will characterize the pre- fusion and post-fusion states of gB and the interactions of gH/gL, gM/gN, and K8.1A with their binding partners. In Aim 3, we will refine our structural interpretation through structure-guided mutagenesis and identify target sites for inhibition of genome encapsidation and membrane fusion. Results from this research program will inform future development of drugs and novel vaccines against KSHV infection and spread. The novel approach established will be generally applicable to other viruses and complexes.

项目概要/摘要 卡波西肉瘤 (KS) 是与人类免疫缺陷感染相关的最常见恶性肿瘤 病毒（艾滋病毒）。作为一种内皮来源的癌症，通常生长在皮肤或粘膜下，KS 艾滋病患者多表现为口腔病变。卡波西肉瘤相关疱疹病毒（KSHV），成员 疱疹病毒科伽马疱疹病毒亚科的一员，已被证明是所有疱疹病毒的病原体 KS、原发性渗出性淋巴瘤和多中心卡斯尔曼病。 目前，还没有专门针对 KSHV 裂解性复制的药物。此外，原子结构 KSHV 病毒基因组包装/喷射机制和融合介导的糖蛋白需要合理 抗病毒药物和疫苗的设计尚不可用。该多重本金的前四年资助情况 研究人员 (MPI) R01 项目已在《自然》杂志上发表了 KSHV 衣壳的原子结构。 结构引导诱变研究已经确定衣壳蛋白之间的氨基酸相互作用 对于衣壳组装至关重要，并为抑制病毒成熟的小肽模拟物的设计提供了信息。 MPI 小组还联合发布了 KSHV DNA 包装门户复合体的第一个原子模型和 细胞中衣壳相关的被膜复合物。本次续签申请的初步数据已确定 获得基因组包装门静脉末端酶复合物原位结构和细胞进入的可行性 融合前和融合后构象的糖蛋白 B (gB)。这些门蛋白和包膜 糖蛋白结构和结构引导诱变结果得出了三个假设：（1）门户- 相关蛋白和终止酶相互作用对于 KSHV 基因组衣壳化至关重要； (2) 相互作用 KSHV 与宿主细胞融合需要包膜糖蛋白之间的构象变化 细胞进入； （3）原子结构中揭示的这种相互作用可以帮助设计抑制剂和疫苗 对抗 KSHV 溶解性感染。 本申请中描述的研究将利用技术来检验上述假设 高分辨率冷冻电镜和 KSHV 诱变方面的突破已在两个 MPI 实验室中得到证实 当前的融资周期。在目标 1 中，我们将确定 KSHV 门脉相关蛋白的原位结构 通过冷冻电镜将终止酶终止至~3Å。从这些结构中，我们将推导出原子模型并识别氨基 相互作用蛋白质 6 Å 内的酸性残基（即对 DNA 包装和排出至关重要的残基）。在目标 2 中，我们 将确定主要包膜糖蛋白的结构~3Å。具体来说，我们将描述预 gB 的融合和融合后状态以及 gH/gL、gM/gN 和 K8.1A 与其结合伴侣的相互作用。 在目标 3 中，我们将通过结构引导诱变完善我们的结构解释并确定目标 抑制基因组衣壳化和膜融合的位点。该研究计划的结果将 为未来针对 KSHV 感染和传播的药物和新型疫苗的开发提供信息。小说 所建立的方法通常适用于其他病毒和复合体。