DOUVILLE LAB
Sheena: Re-activation of Human Endogenous Retrovirus-K (ERVK) during neuroinflammation
Although majority of ERVK loci have been silenced through accumulation of mutations over evolutionary time, some of these proviruses do not sit as silent passengers within our genome. Re-activation of ERVK has been observed in many inflammatory diseases, and most notably in Amyotrophic Lateral Sclerosis (ALS). Unfortunately, the mechanism(s) behind its re-activation is not clearly understood. Thus, the aim of my project is to determine the pro-inflammatory transcription factors and signalling molecules that may induce ERVK under conditions of inflammation. I have generated evidence that this provirus may be induced in neurons by the pro-inflammatory transcription factors Nuclear Factor-kappa B and Interferon Regulatory Factor-1 through exacerbated Tumor Necrosis Factor-alpha signalling. These results will be confirmed through fluorescent microscopy using our EVOS system and further validated in the brain tissue of ALS patients through our confocal imaging platform. The ERVK promoter will also be subjected to Chromatin Immunoprecipitation in order to elucidate the binding sites of these transcription factors.
Although majority of ERVK loci have been silenced through accumulation of mutations over evolutionary time, some of these proviruses do not sit as silent passengers within our genome. Re-activation of ERVK has been observed in many inflammatory diseases, and most notably in Amyotrophic Lateral Sclerosis (ALS). Unfortunately, the mechanism(s) behind its re-activation is not clearly understood. Thus, the aim of my project is to determine the pro-inflammatory transcription factors and signalling molecules that may induce ERVK under conditions of inflammation. I have generated evidence that this provirus may be induced in neurons by the pro-inflammatory transcription factors Nuclear Factor-kappa B and Interferon Regulatory Factor-1 through exacerbated Tumor Necrosis Factor-alpha signalling. These results will be confirmed through fluorescent microscopy using our EVOS system and further validated in the brain tissue of ALS patients through our confocal imaging platform. The ERVK promoter will also be subjected to Chromatin Immunoprecipitation in order to elucidate the binding sites of these transcription factors.
Current Research Projects
Samah: Innate anti-retroviral sensors in astrocytes and neurons
The goal of my project is to determine cellular viral sensors which recognize ERVK derived nucleic acids, and thus elicit an innate immune response against ERVK in neurons and astrocytes. To achieve this aim, I am in the process of making knock-out neuronal and astrocytic cell lines, lacking specific Pattern Recognition Receptors (PRRs) such as RIG-I. These knock-out cells will be treated with ERVK RNA isolated from virions and well as with artificially synthesized ERVK RNA. Cell-type specific innate immune response will then be characterized by measuring up-regulation of IFN stimulated genes (ISGs) through Q-PCR, Western Blot, and confocal microscopy.
The goal of my project is to determine cellular viral sensors which recognize ERVK derived nucleic acids, and thus elicit an innate immune response against ERVK in neurons and astrocytes. To achieve this aim, I am in the process of making knock-out neuronal and astrocytic cell lines, lacking specific Pattern Recognition Receptors (PRRs) such as RIG-I. These knock-out cells will be treated with ERVK RNA isolated from virions and well as with artificially synthesized ERVK RNA. Cell-type specific innate immune response will then be characterized by measuring up-regulation of IFN stimulated genes (ISGs) through Q-PCR, Western Blot, and confocal microscopy.
Matt: The structure, function, and evolution of ERVK family proteases
Viral protease is absolutely critical to the viral metabolism, and currently we do not know enough about ERVK protease activity to determine what role it might play in the human body. I am using bioinformatics to compare ERVK protease to other retroviral proteases in order to identify important structural elements to determine which ERVK loci have the potential to produce an active protease, which protease inhibitors are most likely to affect them, and what function the enzymes may have. I then plan to experimentally verify my predictions in human cell culture models by detecting the effects of protease inhibitors on viral activity.
Viral protease is absolutely critical to the viral metabolism, and currently we do not know enough about ERVK protease activity to determine what role it might play in the human body. I am using bioinformatics to compare ERVK protease to other retroviral proteases in order to identify important structural elements to determine which ERVK loci have the potential to produce an active protease, which protease inhibitors are most likely to affect them, and what function the enzymes may have. I then plan to experimentally verify my predictions in human cell culture models by detecting the effects of protease inhibitors on viral activity.
Sam and Michael: Characterizing a novel ERV protein with neurotoxic potential
Our lab has recently discovered a novel ERV protein which has neurotoxic potential. This protein may contribute to neuronal dysfunction in diseases such as ALS and Schizophrenia. We will use in vitro neuronal cell culture and established microbiology techniques - plasmid transfections, western blot, Q-PCR, and immunohistochemistry - to determine if this protein is neurotoxic. Using our confocal imaging platform, we will attempt to determine the mechanism(s) behind its toxicity. To validate our in vitro models, we will measure the expression of this novel protein in combination with neuro-degeneration markers in brain and spinal cord tissue from patients with ALS. Overall, this project will allow us to determine whether this previously uncharacterized endogenous retroviral protein is expressed in ALS, and provide evidence of a novel source of toxin-mediated neuro-degeneration in this disease. If successful, this will be the first study to concretely show that re-activation of ERVs has a direct neurotoxic effect in the central nervous system and will open future avenues into therapeutic antiviral and immunomodulatory regimens for ALS.
Our lab has recently discovered a novel ERV protein which has neurotoxic potential. This protein may contribute to neuronal dysfunction in diseases such as ALS and Schizophrenia. We will use in vitro neuronal cell culture and established microbiology techniques - plasmid transfections, western blot, Q-PCR, and immunohistochemistry - to determine if this protein is neurotoxic. Using our confocal imaging platform, we will attempt to determine the mechanism(s) behind its toxicity. To validate our in vitro models, we will measure the expression of this novel protein in combination with neuro-degeneration markers in brain and spinal cord tissue from patients with ALS. Overall, this project will allow us to determine whether this previously uncharacterized endogenous retroviral protein is expressed in ALS, and provide evidence of a novel source of toxin-mediated neuro-degeneration in this disease. If successful, this will be the first study to concretely show that re-activation of ERVs has a direct neurotoxic effect in the central nervous system and will open future avenues into therapeutic antiviral and immunomodulatory regimens for ALS.
Heather: Detection of the ERVK RNA by innate immune sensors
The key aim of my project is to determine whether the cytoplasmic viral sensors, particularly RIG-I, can detect ERVK RNA. RIG-I dependent recognition of viral RNA may be mediated by the secondary structure of the target RNA. Through bioinformatics analysis of ERVK sequences, I have determined that ERVK RNA folds into complex secondary structures - some of these folds are conserved between different ERVK strains. I am in the process of constructing synthetic versions of these RNA sequences. Synthetic ERVK RNA will be presented to a variety of cell lines used in our lab - neurons, astrocytes, and microglia - to see if they trigger an immune response.
The key aim of my project is to determine whether the cytoplasmic viral sensors, particularly RIG-I, can detect ERVK RNA. RIG-I dependent recognition of viral RNA may be mediated by the secondary structure of the target RNA. Through bioinformatics analysis of ERVK sequences, I have determined that ERVK RNA folds into complex secondary structures - some of these folds are conserved between different ERVK strains. I am in the process of constructing synthetic versions of these RNA sequences. Synthetic ERVK RNA will be presented to a variety of cell lines used in our lab - neurons, astrocytes, and microglia - to see if they trigger an immune response.
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