EVJC+ were first treated with DNAse 1 (New England Biolabs) for 30 minutes at 37C, followed by a 10 minute inactivation at 75C according to the manufacturers protocol

EVJC+ were first treated with DNAse 1 (New England Biolabs) for 30 minutes at 37C, followed by a 10 minute inactivation at 75C according to the manufacturers protocol. seen in the fluorescent micrographs and in the histograms obtained by circulation cytometric analysis. When cells were shifted to 37C EV are internalized and the addition of trypan blue has no effect on the intracellular transmission.(TIF) ppat.1008371.s002.tif (6.4M) GUID:?307FA7EE-E634-4219-B3CD-1D052CB28CBE S2 Fig: Infectivity of SEC fractions. (A) Extracellular vesicles from JCPyV infected CPEL cells were purified by ultracentrifugation and size exclusion chromatography (SEC). SEC portion 5C13 were used to challenge SVG-A cells. Infectivity was scored by indirect immunofluorescence analysis of VP1 positive cells (green). The cells were counterstained with DAPI. Fractions 7 and 8 contained the majority of infectious extracellular vesicles. (B) Extracellular vesicles from uninfected CPEL cells were purified by ultracentrifugation and then spiked with purified JCPyV virion particles. This combination was then further purified by SEC and the producing fractions tested for infectivity. Fractions 8 and 9 contained the majority of infectious extracellular vesicles but infectious material also was apparent in fractions 10C13.(TIF) ppat.1008371.s003.tif (7.6M) GUID:?66CFBCFC-C212-47E4-897E-1929A830BF21 S3 Fig: MTS assay of Pitstop2, and EIPA treated SVG-A cells. An MTS assay was used to assess the metabolic activity of cells being treated with compounds that antagonize TMB specific cellular access pathways. None of the compounds used negatively affected metabolic activity of the cells at the concentrations used in the uptake assays.(TIF) ppat.1008371.s004.tif (4.1M) GUID:?2B3F4C26-D78F-4637-9D70-DFE2699FA631 Data Availability StatementAll relevant data are within the manuscript and its Supporting Information files. Abstract The human polyomavirus, JCPyV, is the causative agent of progressive multifocal leukoencephalopathy (PML) in immunosuppressed and immunomodulated patients. Initial contamination with JCPyV is usually common and the computer virus establishes a long-term prolonged contamination in the urogenital system of 50C70% of the human population worldwide. A major space in the field is usually that we do not know how the computer virus traffics from your periphery to the brain to cause disease. Our recent discovery that human choroid plexus epithelial cells are fully susceptible to computer virus infection together with reports of JCPyV contamination of choroid plexus in vivo has led us to hypothesize that this choroid plexus plays a fundamental role in this process. The choroid plexus is known to relay information between the blood and the Mouse monoclonal to ESR1 brain by the release of extracellular vesicles. This is particularly important because human macroglia (oligodendrocytes and astrocytes), the major TMB targets of computer virus contamination in the central nervous system (CNS), do not express the known attachment receptors for the computer virus and do not bind computer virus in human tissue sections. In this statement we show that JCPyV infected choroid plexus epithelial cells produce extracellular vesicles that contain JCPyV and readily transmit the infection to TMB human glial cells. Transmission of the computer virus by extracellular vesicles is usually independent of the known computer virus attachment receptors and is not neutralized by antisera directed at the computer virus. We also show that extracellular vesicles made up of computer virus are taken into target glial cells by both clathrin dependent endocytosis and macropinocytosis. Our data support the hypothesis that this choroid plexus plays a fundamental role in the dissemination of computer virus to brain parenchyma. Author summary JC polyomavirus (JCPyV) is usually a common human pathogen that causes a central nervous system demyelinating disease known as progressive multifocal leukoencephalopathy (PML). To cause PML, JCPyV has to traffic from peripheral tissues to the central nervous system (CNS) and invade glial cells. In previous work we found that choroid plexus epithelial cells express receptors for JCPyV in vivo and are fully susceptible to computer virus contamination in vitro. In contrast, glial cells do not TMB express the receptors for JCPyV and computer virus does not bind to these cells in human tissue sections. Because choroid plexus epithelial cells are known to relay information between the blood and the brain using extracellular vesicles we hypothesized that this could be important for JCPyV neuroinvasion. We found that JCPyV infected choroid plexus epithelial cells produce extracellular vesicles made up of JCPyV virions and that these extracellular vesicles transmit the infection to human glial.