Mouse polyomavirus (MPyV) activation of intracellular signaling pathways upon binding to cell surface gangliosides and the alpha-4-integrin receptor

小鼠多瘤病毒 (MPyV) 与细胞表面神经节苷脂和 α-4-整合素受体结合后激活细胞内信号通路

• 批准号: 9180680
• 负责人: Samantha Dawn O'Hara
• 金额: $ 1.37万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-12-01 至 2016-12-16
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9180680
• 关键词: Antibodies; Binding; Binding Sites; Biological Assay; Capsid; Cell Surface Receptors; Cell membrane; Cell surface; Cells; Complex; Dimerization; Dose; Embryo; Endocytosis; Environment; Event; Extracellular Matrix Proteins; FOS gene; Fibroblasts; Fibronectins; Flow Cytometry; Fluorescence Microscopy; Focal Adhesions; Gangliosides; Genes; Genetic Transcription; Glycosphingolipids; Growth Factor Receptors; Infection; Integrin Binding; Integrin alpha4beta1; Integrins; JUN gene; Knock-out; Knockout Mice; Lead; Life Cycle Stages; Ligands; Lipids; Measures; Mediating; Membrane Microdomains; Mus; Mutation; Pathway interactions; Phosphoproteins; Plasmids; Polyomavirus; Polyomavirus Infections; Polysaccharides; Process; Protein Tyrosine Kinase; Receptor Activation; Receptor Cell; Recombinants; Reporter; Resistance; Role; Sialic Acids; Signal Pathway; Signal Transduction; Simian virus 40; Supplementation; Time; Transcriptional Activation; Tropism; Variant; Virion; Virus; Virus Replication; Work; c-myc Genes; dimer; ganglioside receptor; gene induction; in vivo; mouse polyomavirus; mutant; public health relevance; receptor; response; transcriptome sequencing; viron

 DESCRIPTION (provided by applicant): Murine polyomavirus (MPyV) binding to the cell surface activates intracellular signaling pathways that lead to rapid transcription of the primary response genes (PRG) c-myc, c-fos, and c-jun. Similar transcriptional responses have been demonstrated for both SV40 and JCV infections; however, the host-cell receptors that are required for PyV early signaling events are unknown. Gangliosides are receptors for many PyVs, and have been implicated in modulating signals elicited through cell surface growth factor receptors (GFRs). The MPyV co-receptor, α4β1-integrin, may also contribute to early signaling events. We hypothesize that multivalent binding of MPyV to gangliosides and α4β1-integrin clusters associated GFRs, activating intracellular signaling pathways leading to PRG induction and endocytosis of MPyV. We will use various VP1-ligands (recombinant VP1 capsomeres, MPyV pseudoviruses (PsVs) and MPyV virons) to activate signaling pathways upon addition to MEFs. PsVs containing mutations in sialic acid or integrin binding sites will be used to identify required receptor interactions for MPyV-activation of signal transduction. Additionally, mouse embryonic fibroblasts (MEFs) knocked-out for major ganglioside synthesis pathways (KO-MEFs) will be used to study the role of gangliosides in these signaling events. KO-MEFs are resistant to MPyV infection; however, MPyV binds the KO-MEF cell surface to a similar level as WT MEFs. To determine specific GFRs activated upon MPyV binding we will use phospho-protein arrays in combination with PsV mutants deficient in specific receptor interactions. Low valency (VP1 capsomeres) and high valency (PsV/virions) capsid ligands will be used to measure the effect of MPyV multivalent binding on GFR activation. RNA-seq will be used to measure the host cell transcriptional response after PsV addition to WT and KO-MEFs. Supplementation of different gangliosides to KO-MEFs will assess the requirement for specific MPyV-ganglioside interactions in signal transduction and altered host transcription. Activation of GFRs upon MPyV binding is likely an important step for proper MPyV endocytosis and targeting of virus to infectious pathways. We will assay endocytosis of mutant PsVs by flow cytometry in both WT and KO-MEFs. Proper targeting of pseudovirions to infectious pathways will be measured by expression of PsV-encapsidated reporter plasmids. This work will illuminate the importance of PyV-receptor glycan interactions for activation of PyV signal transduction, virus endocytosis, and altered host transcription.

 描述（由申请人提供）：与细胞表面结合的鼠多瘤病毒（MPyV）激活细胞内信号传导途径，导致主要应答基因（PRG）c-myc、c-fos和c-jun的快速转录。已证明SV 40和JCV感染具有相似的转录应答;然而，PyV早期信号传导事件所需的宿主细胞受体尚不清楚。神经节苷脂是许多PyV的受体，并且已经涉及调节通过细胞表面生长因子受体（GFR）引起的信号。MPyV共受体α4β1-整联蛋白也可能有助于早期信号传导事件。我们假设MPyV与神经节苷脂和α4β1-整联蛋白簇的多价结合与GFR相关，激活导致PRG诱导和MPyV内吞的细胞内信号传导途径。我们将使用各种VP 1配体（重组VP 1衣壳、MPyV假病毒（PsVs）和MPyV病毒粒子）在加入MEF后激活信号传导途径。在唾液酸或整联蛋白结合位点中含有突变的PsV将用于鉴定MPyV激活信号转导所需的受体相互作用。此外，敲除主要神经节苷脂合成途径（KO-MEFs）的小鼠胚胎成纤维细胞（MEFs）将用于研究神经节苷脂在这些信号传导事件中的作用。KO-MEF对MPyV感染具有抗性;然而，MPyV结合KO-MEF细胞表面的水平与WT MEF相似。为了确定MPyV结合后激活的特异性GFR，我们将使用磷酸蛋白阵列与特异性受体相互作用缺陷的PsV突变体组合。低价（VP 1衣壳）和高价（PsV/病毒体）衣壳配体将用于测量MPyV多价结合对GFR活化的影响。RNA-seq将用于测量PsV添加至WT和KO-MEF后的宿主细胞转录应答。向KO-MEF补充不同的神经节苷脂将评估在信号转导和改变的宿主转录中对特异性MPyV-神经节苷脂相互作用的需求。MPyV结合后GFR的活化可能是适当的MPyV内吞作用和病毒靶向感染途径的重要步骤。我们将在WT和KO-MEF中通过流式细胞术测定突变体PsV的内吞作用。将通过PsV标记的报告质粒的表达来测量假病毒体对感染途径的适当靶向。这项工作将阐明PyV-受体聚糖相互作用对激活PyV信号转导，病毒内吞作用和改变宿主转录的重要性。