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.

项目摘要/摘要 Kaposi的肉瘤（KS）是与人类免疫缺陷感染有关的最常见的恶性肿瘤 病毒（HIV）。作为通常在皮肤或粘膜下生长的内皮起源癌，KS AIDS患者主要表现为口腔病变。 Kaposi的肉瘤相关疱疹病毒（KSHV），成员 疱疹病毒家族的伽马广播病毒亚科是所有人的病因学剂 KS，一级积液淋巴瘤和多中心Castleman病的形式。 当前，没有专门针对KSHV裂解复制的药物。另外，原子结构 KSHV病毒基因组包装/射血机制和融合融合的糖蛋白所需的理性糖蛋白 抗病毒药物和疫苗的设计是不可用的。以前的四年资金对这一多本金 研究人员（MPI）R01项目导致了自然界KSHV CAPSID的原子结构的发布。 结构引导的诱变研究已经确定了衣壳蛋白之间的氨基酸相互作用 对于衣壳组件至关重要，并告知了抑制病毒成熟的小胡椒模仿的设计。 共同的MPI组还发布了KSHV DNA包装门户复合体的第一个原子模型和 CAPSID相关的团队复合物中的综合体。该更新应用程序的初步数据已经建立 获得基因组包装门户末端酶复合物和细胞进入的原位结构的可行性 融合前和融合后构象中的糖蛋白B（GB）。这些门户蛋白和信封 糖蛋白结构和结构引导的诱变结果导致了三个假设：（1）门户网站 - 相关的蛋白质和末端酶相互作用对于KSHV基因组封装至关重要。 （2）相互作用 在包膜中，包膜糖蛋白之间的构象变化是KSHV与宿主细胞融合在一起的需要的 细胞进入； （3）在原子结构中揭示的这种相互作用可以帮助设计抑制剂和阴道 针对KSHV裂解感染。 本应用程序中描述的研究将通过利用技术来检验上述假设 在两个MPI的实验室中已经证明了高分辨率冷冻和KSHV诱变的突破 当前的资金周期。在AIM 1中，我们将确定KSHV门户相关蛋白的原位结构 终端酶通过冷冻酶〜3Å。从这些结构中，我们将得出原子模型并确定氨基 酸在相互作用的蛋白质相互作用的6Å内保留（即，保留对DNA包装和射精至关重要）。在AIM 2中，我们 将确定主要包膜糖蛋白的结构至〜3Å。具体而言，我们将表征 GB的融合和后融合状态以及GH/GL，GM/GN和K8.1a与其结合伴侣的相互作用。 在AIM 3中，我们将通过结构引导的诱变来完善我们的结构解释，并确定目标 抑制基因组封装和膜融合的位点。该研究计划的结果将 为未来的药物开发和针对KSHV感染和扩散的新型疫苗开发。小说 建立的方法通常适用于其他病毒和复合物。