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, ?4?1-integrin, may also contribute to early signaling events. We hypothesize that multivalent binding of MPyV to gangliosides and ?4?1-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.