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

 描述(由申请人提供)：小鼠多瘤病毒(MPyV)结合到细胞表面激活细胞内信号通路，导致主要反应基因(PRG)c-myc、c-fos和c-jun的快速转录。在SV40和JCV感染中发现了相似的转录反应；然而，PYV早期信号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，并参与调节通过细胞表面生长因子受体(GFRs)产生的信号。MPyV联合受体α4β1-整合素也可能参与早期信号事件。我们推测，MPyV与神经节苷脂和α4β1-整合素簇的多价结合与GFRs相关，激活细胞内信号通路，导致MPyV的PRG诱导和内吞。我们将使用不同的VP1配体(重组VP1胶囊、MPyV伪病毒(PSV)和MPyV病毒)来激活加入MEF的信号通路。含有唾液酸或整合素结合位点突变的PSV将被用来确定MPyV激活信号转导所需的受体相互作用。此外，主要神经节苷脂合成途径(KO-MEF)敲除的小鼠胚胎成纤维细胞(MEF)将被用来研究神经节苷脂在这些信号事件中的作用。KO-MEF对MPyV感染具有抵抗力；然而，MPyV结合KO-MEF细胞表面的水平与WT MEF相似。为了确定MPyV结合后激活的特定GFRs，我们将使用磷酸蛋白阵列与缺乏特定受体相互作用的PSV突变体相结合。低价(VP1衣壳)和高价(PSV/病毒)衣壳配体将被用来测量MPyV多价结合对GFR激活的影响。RNA-seq将被用来测量PSV加入WT和KO-MEF后宿主细胞的转录反应。在KO-MEF中添加不同的神经节苷脂将评估在信号转导和宿主转录改变中对MPyV-神经节苷脂相互作用的要求。MPyV结合后GFRs的激活可能是正确的MPyV内吞作用和病毒靶向感染途径的重要步骤。我们将用流式细胞术检测突变的PSV在WT和KO-MEF中的内吞作用。假病毒粒子对感染途径的适当靶向将通过PSV包裹的报告质粒的表达来测量。这项工作将阐明PYV-受体多糖相互作用对于激活PYV信号转导、病毒内吞作用和改变宿主转录的重要性。

 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.