JC Virus infected choroid plexus epithelial cells produce extracellular vesicles that infect glial cells independently of the virus attachment receptor

The human polyomavirus, JCPyV, is the causative agent of progressive multifocal leukoencephalopathy (PML) in immunosuppressed and immunomodulated patients. Initial infection with JCPyV is common and the virus establishes a long-term persistent infection in the urogenital system of 50-70% of the human population worldwide. A major gap in the field is that we do not know how the virus traffics from the periphery to the brain to cause disease. Our recent discovery that human choroid plexus epithelial cells are fully susceptible to virus infection together with reports of JCPyV infection of choroid plexus in vivo has led us to hypothesize that the choroid plexus plays a fundamental role in this process. The choroid plexus is known to relay information between the blood and the brain by the release of extracellular vesicles. This is particularly important because human macroglia (oligodendrocytes and astrocytes), the major targets of virus infection in the central nervous system (CNS), do not express the known attachment receptors for the virus and do not bind virus in human tissue sections. In this report we show that JCPyV infected choroid plexus epithelial cells produce extracellular vesicles that contain JCPyV and readily transmit the infection to human glial cells. Transmission of the virus by extracellular vesicles is independent of the known virus attachment receptors and is not neutralized by antisera directed at the virus. We also show that extracellular vesicles containing virus are taken into target glial cells by both clathrin dependent endocytosis and macropinocytosis. Our data support the hypothesis that the choroid plexus plays a fundamental role in the dissemination of virus to brain parenchyma

JC Polyomavirus Uses Extracellular Vesicles To Infect Target Cells

The endemic human JC polyomavirus (JCPyV) causes progressive multifocal leukoencephalopathy in immune-suppressed patients. The mechanisms of virus infection in vivo are not understood because the major target cells for virus in the brain do not express virus receptors and do not bind virus. We found that JCPyV associates with extracellular vesicles (EVs) and can infect target cells independently of virus receptors. Virus particles were found packaged inside extracellular vesicles and attached to the outer side of vesicles. Anti-JCPyV antisera reduced infection by purified virus but had no effect on infection by EV-associated virus. Treatment of cells with the receptor-destroying enzyme neuraminidase inhibited infection with purified virus but did not inhibit infection by EV-associated virus. Mutant pseudoviruses defective in sialic acid receptor binding could not transduce cells as purified pseudovirions but could do so when associated with EVs. This alternative mechanism of infection likely plays a critical role in the dissemination and spread of JCPyV both to and within the central nervous system

Biogenesis of JC Polyomavirus Associated Extracellular Vesicles

JC polyomavirus (JCPyV) is a small, non-enveloped virus that persists in the kidney in about half the adult population. In severely immune-compromised individuals JCPyV causes the neurodegenerative disease progressive multifocal leukoencephalopathy (PML) in the brain. JCPyV has been shown to infect cells by both direct and indirect mechanisms, the latter involving extracellular vesicle (EV) mediated infection. While direct mechanisms of infection are well studied indirect EV mediated mechanisms are poorly understood. Using a combination of chemical and genetic approaches we show that several overlapping intracellular pathways are responsible for the biogenesis of virus containing EV. Here we show that targeting neutral sphingomyelinase 2 (nSMase2) with the drug cambinol decreased the spread of JCPyV over several viral life cycles. Genetic depletion of nSMase2 by either shRNA or CRISPR/Cas9 reduced EV-mediated infection. Individual knockdown of seven ESCRT-related proteins including HGS, ALIX, TSG101, VPS25, VPS20, CHMP4A, and VPS4A did not significantly reduce JCPyV associated EV (JCPyV(+) EV) infectivity, whereas knockdown of the tetraspanins CD9 and CD81 or trafficking and/or secretory autophagy-related proteins RAB8A, RAB27A, and GRASP65 all significantly reduced the spread of JCPyV and decreased EV-mediated infection. These findings point to a role for exosomes and secretory autophagosomes in the biogenesis of JCPyV associated EVs with specific roles for nSMase2, CD9, CD81, RAB8A, RAB27A, and GRASP65 proteins