Roles of the UL148 glycoprotein in human cytomegalovirus infection

UL148糖蛋白在人巨细胞病毒感染中的作用

• 批准号: 10735696
• 负责人: Jeremy Phillip Kamil
• 金额: $ 41.68万
• 依托单位: LOUISIANA STATE UNIV HSC SHREVEPORT
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-12-15 至 2028-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10735696
• 关键词: 3-Dimensional; Address; Amino Acids; Antibodies; Antibody Response; Antibody titer measurement; Antigens; Binding; Binding Proteins; Biogenesis; Biological; Biological Process; Carbohydrates; Cells; Cellular Stress; Charge; Child; Complex; Congenital Abnormality; Consensus; Cysteine; Cytomegalovirus; Cytomegalovirus Infections; Cytomegalovirus Vaccines; DNA Viruses; Data; Development; Endothelial Cells; Environment; Enzymes; Epithelial Cells; Epithelium; Exhibits; Fabaceae; Fibroblasts; Generations; Genes; Genetic; Glycobiology; Glycocalyx; Glycoproteins; Goals; Herpesviridae; Homologous Gene; Human; Immune; Immune Evasion; Immune response; Immunity; Immunize; Immunoglobulins; Immunologic Deficiency Syndromes; Infection; Ions; Knowledge; Lectin; Life; Link; Lymphocyte; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Measures; Mediating; Membrane; Membrane Proteins; Messenger RNA; Metals; Modification; Molecular; Mus; Mutagenesis; Mutation; Neuraminidase; Neurodevelopmental Disability; Neuropilin-2; Neutralization Tests; Neutralizing antibody assay; Oligosaccharides; Opportunistic Infections; Orthologous Gene; Pathway interactions; Patients; Pattern; Persons; Platelet-Derived Growth Factor alpha Receptor; Play; Polysaccharides; Production; Protein Glycosylation; Proteins; Proteomics; Quality Control; Reagent; Recombinants; Research; Resistance; Role; Sialic Acids; Sialyltransferases; Structural Models; Structure; Surface; Testing; Tissues; Tropism; Vaccines; Viral; Viral Envelope Proteins; Viral Proteins; Virion; Virus; acute infection; adaptive immunity; analog; biological adaptation to stress; cell type; chronic infection; congenital infection; env Gene Products; genetic approach; glycosylation; immunogenicity; mouse model; mutant; neutralizing antibody; nonhuman primate; permanent hearing loss; pharmacologic; polyclonal antibody; prevent; programs; protein structure; receptor; response; sialic acid binding Ig-like lectin; sialylation; sugar; treatment strategy; vaccination strategy; virus host interaction

This project focuses on the role of glycobiology in human cytomegalovirus (HCMV) immune evasion strategies. Our long-term goal is to understand how the virus exploits host protein glycosylation pathways to shield its virions and decorate the surfaces of infected cells with oligosaccharide patterns that prevent antibody binding and/or suppress immune responses. The Specific Aims are (1) to evaluate how the HCMV ER resident protein UL148 impacts the glycocalyx, or “sugar cloak,” of virions and infected cells, (2) to carry out a structure function analysis of UL148 to determine the mechanisms by which UL148 activates sculpts the host cell to enhance glycosylation of virion envelope proteins, and (3) to evaluate how UL148 contributes to evasion of antibody responses. Throughout the three Specific Aims, wild-type (WT) and UL148-null mutant viruses are evaluated in comparison to each other. In Specific Aim 1, mass spectrometry based ‘glycomics’ is applied to profile N-linked oligosaccharide structures conjugated to virion envelope proteins and surface proteins of infected cells in the presence versus absence of UL148. A second aspect of Aim 1 makes use of mass spectrometry-based proteomics together with genetic and pharmacological perturbations targeting the unfolded protein response (UPR) and cellular genes whose expression is positively regulated by UL148 to determine how the viral protein harnesses the host cell’s stress response circuitry toward the generation of virions that resist neutralization by antibodies. In Specific Aim 2, computational predictions of UL148’s three-dimensional protein structure are used to guide mutagenesis studies aimed at understanding the molecular basis for its biological functions, focusing in large part on the roles of (i) a consensus metal binding motif and (ii) two non- conserved cysteine residues. Aim 2 also leverages structural models non-human primate cytomegaloviruses homologs to elucidate the determinants of HCMV UL148’s unique biological functions. These studies will make use of recombinant HCMVs to test the impact of selected mutations and chimeric UL148 proteins in the context of infected cells. In Specific Aim 3, we will (i) characterize UL148’s impact on neutralization resistance of virions while targeting glycan modification enzymes hypothesized to be important for biogenesis of neutralization resistant virions, (ii) map specific antigenic domains on virion glycoproteins whose reactivity to neutralizing antibodies is impacted by UL148, (iii) compare the humoral immunogenicity of UL148 competent versus UL148-null HCMV virions in a mouse model, testing for neutralizing antibody titers, and overall polyclonal antibody titers against whole virions, viral envelope proteins and internal virion antigens. Collectively, these studies will address a key knowledge gap concerning how enveloped large DNA viruses exploit glycobiology to evade adaptive immunity and establish persistent infection.

该项目的重点是糖生物学在人类巨细胞病毒（HCMV）免疫逃避策略中的作用。 我们的长期目标是了解病毒如何利用宿主蛋白质糖基化途径来保护它的免疫系统。 病毒粒子和装饰感染细胞表面的寡糖模式，阻止抗体结合 和/或抑制免疫应答。本研究的具体目的是：（1）探讨HCMV ER驻留蛋白在细胞内的表达， UL 148影响病毒粒子和受感染细胞的糖萼或“糖衣”，（2）实现结构 对UL 148进行功能分析以确定UL 148激活的机制， 增强病毒体包膜蛋白的糖基化，以及（3）评估UL 148如何有助于病毒体包膜蛋白的糖基化。 抗体反应。在这三个特定目的中，野生型（WT）和UL 148无效突变病毒是 相互比较来评价。在具体目标1中，基于质谱的“糖组学”应用于 与病毒体包膜蛋白和表面蛋白偶联的N-连接寡糖结构 感染的细胞在存在与不存在UL 148的情况下。目标1的第二个方面利用质量 基于光谱的蛋白质组学以及针对未折叠的 蛋白质应答（UPR）和其表达受UL 148正调控的细胞基因，以确定 病毒蛋白如何利用宿主细胞的应激反应电路产生病毒体， 抵抗抗体中和。在具体目标2中，UL 148的三维计算预测 蛋白质结构用于指导诱变研究，旨在了解其分子基础， 生物学功能，在很大程度上集中在（i）一个共识金属结合基序和（ii）两个非 保守的半胱氨酸残基。AIM 2还利用了非人灵长类动物巨细胞病毒的结构模型 同源物，以阐明HCMV UL 148的独特生物学功能的决定因素。这些研究将使 使用重组HCMV测试所选突变和嵌合UL 148蛋白在背景下的影响 感染的细胞。在具体目标3中，我们将（i）表征UL 148对以下物质的耐中和性的影响： 病毒粒子，而靶向聚糖修饰酶假设是重要的生物合成 中和抗性病毒粒子，（ii）定位病毒粒子糖蛋白上的特异性抗原结构域，所述病毒粒子糖蛋白对 中和抗体受UL 148的影响，（iii）比较UL 148活性的体液免疫原性 与小鼠模型中的UL 148-null HCMV病毒体相比，测试中和抗体滴度，以及总体 针对完整病毒体、病毒包膜蛋白和内部病毒体抗原的多克隆抗体滴度。 总的来说，这些研究将解决一个关键的知识差距，关于如何包膜大DNA病毒 利用糖生物学来逃避适应性免疫并建立持续感染。

项目成果

Expression Levels of Glycoprotein O (gO) Vary between Strains of Human Cytomegalovirus, Influencing the Assembly of gH/gL Complexes and Virion Infectivity.

糖蛋白 O (gO) 的表达水平在人巨细胞病毒株之间存在差异，影响 gH/gL 复合物的组装和病毒粒子的感染性。

• DOI: 10.1128/jvi.00606-18
• 发表时间: 2018
• 期刊: Journal of virology
• 影响因子: 5.4
• 作者: Zhang,Le;Zhou,Momei;Stanton,Richard;Kamil,Jeremy;Ryckman,BrentJ
• 通讯作者: Ryckman,BrentJ

The Human Cytomegalovirus Nonstructural Glycoprotein UL148 Reorganizes the Endoplasmic Reticulum.

人类巨细胞病毒非结构糖蛋白 UL148 重组内质网。

• DOI: 10.1128/mbio.02110-19
• 发表时间: 2019
• 期刊: mBio
• 影响因子: 6.4
• 作者: Zhang,Hongbo;Read,Clarissa;Nguyen,ChristopherC;Siddiquey,MohammedNA;Shang,Chaowei;Hall,CameronM;vonEinem,Jens;Kamil,JeremyP
• 通讯作者: Kamil,JeremyP

The Human Cytomegalovirus Protein UL116 Interacts with the Viral Endoplasmic-Reticulum-Resident Glycoprotein UL148 and Promotes the Incorporation of gH/gL Complexes into Virions.

人类巨细胞病毒蛋白 UL116 与病毒内质网糖蛋白 UL148 相互作用，促进 gH/gL 复合物掺入病毒粒子。

• DOI: 10.1128/jvi.02207-20
• 发表时间: 2021
• 期刊: Journal of virology
• 影响因子: 5.4
• 作者: Siddiquey,MohammedNA;Schultz,EricP;Yu,Qin;Amendola,Diego;Vezzani,Giacomo;Yu,Dong;Maione,Domenico;Lanchy,Jean-Marc;Ryckman,BrentJ;Merola,Marcello;Kamil,JeremyP
• 通讯作者: Kamil,JeremyP