Comprehensive proteomic analysis of JC polyomavirus-infected human astrocytes and their extracellular vesicles

Abstract

JC polyomavirus (JCPyV) is an opportunistic virus that remains in a latent state in the kidneys and lymphoid organs of more than half of the human adult population. In rare cases of severe immune suppression, the virus is able to establish a lytic infection of glial cells in the brain, resulting in a debilitating, demyelinating disease known as progressive multifocal leukoencephalopathy (PML). Because of the exceptional species and tissue specificity of the virus, appropriate models of JCPyV infection in the brain are lacking, thus hampering progress toward the development of novel antiviral strategies and biomarkers of disease activity. While PML has traditionally been characterized as a lytic infection of oligodendrocytes, more recent findings suggest an important role for astrocytes during the initial stages of disease. Here, using human-induced pluripotent stem cell (hiPSC)-derived astrocytes coupled with a multiparametric approach, we show that (i) JCPyV readily infects and replicates in astrocytes, (ii) JCPyV strongly dysregulates the cell biology, and (iii) these results adequately reflect ex vivo findings. We perform an in-depth characterization of the effect of JCPyV on the cell proteome over time, demonstrating a strong dysregulation of the cell cycle and activation of the DNA damage response. Furthermore, we show that the proteomic signature observed for infected astrocytes is extended to extracellular vesicles, underlining their potential as a resource to gain valuable insights into JCPyV infection in the brain. IMPORTANCE Progressive multifocal leukoencephalopathy is a crimpling demyelinating disease of the central nervous system caused by JC polyomavirus (JCPyV). Much about JCPyV propagation in the brain remains obscure because of a lack of proper animal models to study the virus in the context of the disease, thus hampering efforts toward the development of new antiviral strategies. Here, having established a robust and representative model of JCPyV infection in human-induced pluripotent stem cell-derived astrocytes, we are able to fully characterize the effect of JCPyV on the biology of the cells and show that the proteomic signature observed for JCPyV-infected astrocytes is extended to extracellular vesicles (EVs). These data suggest that astrocyte-derived EVs found in body fluids might serve as a rich source of information relevant to JCPyV infection in the brain, opening avenues toward better understanding the pathogenesis of the virus and, ultimately, the identification of new antiviral targets.

Smart Nanoscale Extracellular Vesicles in the Brain: Unveiling their Biology, Diagnostic Potential, and Therapeutic Applications

“…250,251 The oligodendrocytes and astrocytes, the prime targets, lack the sialic acid binding-lactoseries tetrasaccharide C (LSTc) receptors, 252,253 which are required for JCPyV endocytosis, 254−256 (hiPSCs)-derived astrocytes that were infected with JCPyV and observed altered levels of cell cycle and DNA damage proteins. 257 Moreover, EVs containing the virus are highly transmissible and resistant to neutralizing antibodies, suggesting viral entry occurs in a receptor-independent fashion 258,259 and the host-derived EVs shield the viral cargo from immune or enzyme attacks. As EVs are the only carriers identified for JCPyV and the virus-containing EVs are detected in the CSF 258 and the plasma of patients, 260 they can serve as diagnostic tools to predict the infection.…”

“…The virus infects immunocompromised individuals with AIDS, rheumatoid arthritis (RA), and MS. EVs derived from the epithelial cells of the choroid plexus are the prominent carriers of the virus. , The oligodendrocytes and astrocytes, the prime targets, lack the sialic acid binding-lactoseries tetrasaccharide C (LSTc) receptors, , which are required for JCPyV endocytosis, − thus making EVs the only source of JCPyV infection. Indeed, Oberholster et al characterized the EVs isolated from the human-induced pluripotent stem cell (hiPSCs)-derived astrocytes that were infected with JCPyV and observed altered levels of cell cycle and DNA damage proteins . Moreover, EVs containing the virus are highly transmissible and resistant to neutralizing antibodies, suggesting viral entry occurs in a receptor-independent fashion , and the host-derived EVs shield the viral cargo from immune or enzyme attacks.…”

Human stem cell derived neurons and astrocytes to detect novel auto-reactive IgG signature in immune-mediated neurological diseases

“…All cells were cultivated at 37°C in 5% CO2. Importantly, astrocyte and neuron differentiation profiles were assessed by routine RT-qPCR 22,26 and immunofluorescence 22,26,27 , confirming the expression of astrocytic or neuronal markers, respectively (See Supplementary Table 1 for primer list, Supplementary Table 2 for the list of reagents used for immunofluorescence stainings, Supplementary Figure 1 for representative results from differentiation profile assessments)…”

“…Expression of astrocytic and neuronal markers were analyzed by qPCR as done previously. 22,26,27 Briefly, human iPSC-derived astrocytes and neurons from two healthy donors (HD#002 and HD#003) were lysed in RLT Plus Buffer (Qiagen). RNA was extracted using the RNeasy Plus Mini Kit (Qiagen) and stored at −20 °C until RNA extraction.…”