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

项目摘要/摘要 卡波西肉瘤(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)。作为一种起源于内皮的癌症，通常生长在皮肤或粘膜下，KS在 艾滋病患者多表现为口腔病变。卡波西肉瘤相关疱疹病毒 疱疹病毒科的伽马疱疹病毒亚家族中的一种，已被证明是所有 各种类型的KS、原发渗出性淋巴瘤和多中心性Castleman病。 目前，还没有针对KSHV裂解复制的药物。此外，原子结构 KSHV病毒基因组包装/喷射机制和融合调节糖蛋白的研究 目前还没有抗病毒药物和疫苗的设计。这一多重本金的前四年资金 调查者(MPI)R01项目导致在《自然》杂志上发表了KSHV衣壳的原子结构。 结构导向的突变研究已经确定了衣壳蛋白之间的氨基酸相互作用 对衣壳组装至关重要，并提供了抑制病毒成熟的小肽模拟物的设计。 MPIS的团队还联合发布了KSHV DNA包装门户复合体的第一个原子模型，并 细胞内衣壳伴生的被膜复合体。本次续签申请的初步数据已确定 原位获取基因组包装门户终末酶复合体结构及细胞进入的可行性 融合前和融合后构象中的糖蛋白B(GB)。这些门户蛋白和包膜 糖蛋白结构和结构导向的突变结果导致了三个假说：(1)门静脉- 相关蛋白和终止酶的相互作用是KSHV基因组囊化的关键；(2)相互作用 KSHV与宿主细胞的融合需要包膜糖蛋白之间的构象变化。 细胞进入；以及(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 抗KSHV裂解感染。 本申请中描述的研究将利用技术优势来检验上述假设 在高分辨率低温EM和KSHV突变方面的突破已经在#年的两个MPI实验室中展示。 当前的筹资周期。在目标1中，我们将确定KSHV门户相关蛋白的原位结构 以及通过CryoEM到~3ä的终止酶。从这些结构中，我们将推导出原子模型，并确定氨基- 相互作用蛋白质(即对DNA包装和排出至关重要的残基)内的酸性残留物。在目标2中，我们 将确定主要包膜糖蛋白的结构。具体地说，我们将描述Pre- GB的融合和融合后的状态以及Gh/gl、Gm/gn和K8.1a与其结合伙伴的相互作用。 在目标3中，我们将通过结构导向突变来完善我们的结构解释，并确定目标 抑制基因组包埋和膜融合的部位。这项研究计划的结果将 告知未来针对KSHV感染和传播的药物和新型疫苗的开发。这部小说 所建立的方法将普遍适用于其他病毒和复合体。

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.