RIPK3-dependent transcriptional program in flavivirus-infected neurons
Ontology highlight
ABSTRACT: Here, we characterize RIPK3-dependent transcriptional responses in cortical neurons following infection with neurotropic flaviviruses. Neurons were infected with either Zika virus (ZIKV) strain MR766 at an MOI of 0.1, West Nile virus (WNV) strain TX 2002-HC at an MOI of 0.001, or a saline mock solution. Neurons were derived from mice lacking RIPK3 expression (Ripk3-/-) or wildtype controls. These studies revealed a number of antiviral genes whose upregulation following viral infection is absent in neurons lacking RIPK3, a subset of which were validated using qRT-PCR.
Project description:This purpose of this experiment was to investigate the transcriptional differences between C57BL6, RIPK3 knock-out mice infected with influenza strain A/CA/04/2009 (H1N1) virus. Overview of Experiment: Groups of 6-8 week-old C57BL6 and RIPK3 knock-out mice were infected with influenza A/CA/04/2009 virus. Infections were done at 10^5 PFU or time-matched mock infected. Time points were 2 and 4 d.p.i. There were 2-3 animals/dose/time point. Lung samples were collected for virus load and transcriptional analysis. Weight loss and animal survival were also monitored.
Project description:This purpose of this experiment was to investigate the transcriptional differences between C57BL6, RIPK3 knock-out mice infected with influenza strain A/CA/04/2009 (H1N1) virus.
Project description:RNA-seq was utilized to characterize the transcriptome of human neural stem cells infected with Zika virus strains MR766 and Paraiba at MOI 1. Coding and noncoding genes were analyzed to determine transcriptional changes 3 days post-infection.
Project description:small RNA-seq was utilized to characterize the transcriptome of human neural stem cells infected with Zika virus strains MR766 and Paraiba at MOI 1. Coding and noncoding genes were analyzed to determine transcriptional changes 3 days post-infection.
Project description:Astrocyte activation is a common feature of neurodegenerative diseases. However, the ways in which dying neurons influence the activity of astrocytes is poorly understood. RIPK3 signaling has recently been described as a key regulator of neuroinflammation, but whether this kinase mediates astrocytic responsiveness to neuronal death has not yet been studied. Here, we used the MPTP model of Parkinson’s disease to show that activation of astrocytic RIPK3 drives dopaminergic cell death and axon damage. Transcriptomic profiling revealed that astrocytic RIPK3 promoted gene expression associated with neuroinflammation and movement disorders, and this coincided with significant engagement of DAMP signaling. Using human cell culture systems, we show that factors released from dying neurons signal through RAGE to induce RIPK3-dependent astrocyte activation. These findings highlight a mechanism of neuron-glia crosstalk in which neuronal death perpetuates further neurodegeneration by engaging inflammatory astrocyte activation via RIPK3.
Project description:Flaviviruses, including Dengue virus (DV) and Zika virus, extensively remodel the cellular endomembrane network to generate replication organelles that promote viral genome replication and virus production. However, it remains unclear how these membranes and associated cellular proteins act during the virus cycle. Here, we show that atlastins (ATLs), a subset of ER resident proteins involved in neurodegenerative diseases, have dichotomous effects on flaviviruses with ATL2 depletion leading to replication organelle defects and ATL3 depletion to changes in virus production pathways. We characterized non-conserved functional domains in ATL paralogues and show that the ATL interactome is profoundly reprogrammed upon DV infection. Screen analysis confirmed non-redundant ATL functions and identified a specific role for ATL3, and its interactor ARF4, in vesicle trafficking and virion maturation. Our data identify ATLs as central hubs targeted by flaviviruses to establish their replication organelle and to achieve efficient virion maturation and secretion.
Project description:Flaviviruses, including Dengue virus (DV) and Zika virus, extensively remodel the cellular endomembrane network to generate replication organelles that promote viral genome replication and virus production. However, it remains unclear how these membranes and associated cellular proteins act during the virus cycle. Here, we show that atlastins (ATLs), a subset of ER resident proteins involved in neurodegenerative diseases, have dichotomous effects on flaviviruses with ATL2 depletion leading to replication organelle defects and ATL3 depletion to changes in virus production pathways. We characterized non-conserved functional domains in ATL paralogues and show that the ATL interactome is profoundly reprogrammed upon DV infection. Screen analysis confirmed non-redundant ATL functions and identified a specific role for ATL3, and its interactor ARF4, in vesicle trafficking and virion maturation. Our data identify ATLs as central hubs targeted by flaviviruses to establish their replication organelle and to achieve efficient virion maturation and secretion.
Project description:Necroptosis is a lytic and inflammatory form of cell death that is highly constrained to mitigate detrimental collateral tissue damage and impaired immunity. These constraints make it difficult to define the relevance of necroptosis in diseases such as chronic and persistent viral infections and within individual organ systems. The role of necroptotic signalling is further complicated because proteins essential to this pathway, such as receptor interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL), have been implicated in roles outside of necroptotic signalling. We sought to address this issue by individually defining the role of RIPK3 and MLKL in chronic lymphocytic choriomeningitis virus (LCMV) infection. We investigated if necroptosis contributes to the death of LCMV-specific CD8+ T cells or virally infected target cells during infection. We provide evidence showing that necroptosis was redundant in the pathogenesis of acute forms of LCMV (Armstrong strain) and the early stages of chronic (Docile strain) LCMV infection in vivo. The number of immune cells, their specificity and reactivity towards viral antigens and viral loads are not altered in the absence of either MLKL or RIPK3 during acute and during the early stages of chronic LCMV infection. However, we identified that RIPK3 promotes immune dysfunction and prevents control of infection at later stages of chronic LCMV disease. This was not phenocopied by the loss of MLKL indicating that the phenotype was driven by a necroptosis-independent function of RIPK3. We provide evidence that RIPK3 signaling evoked a dysregulated type 1 interferone response which we linked to an impaired antiviral immune response and abrogated clearance of chronic LCMV infection.
Project description:RIPK4 but not the related kinases RIPK1, RIPK2, and RIPK3 caused similar transcriptional changes to Wnt3a. PA1 cells were transfected by 8ug RIPK1, RIPK2, RIPK3, or RIPK4 for 48h, RNA were extracted and sequenced.