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. IMPORTANCE JC polyomavirus (JCPyV) is a ubiquitous human pathogen that causes progressive multifocal leukoencephalopathy (PML), a severe and often fatal neurodegenerative disease in immunocompromised or immunomodulated patients. The mechanisms responsible for initiating infection in susceptible cells are not completely known. The major attachment receptor for the virus, lactoseries tetrasaccharide c (LSTc), is paradoxically not expressed on oligodendrocytes or astrocytes in human brain, and virus does not bind to these cells. Because these are the major cell types targeted by the virus in the brain, we hypothesized that alternative mechanisms of infection must be responsible. Here we provide evidence that JCPyV is packaged in extracellular vesicles from infected cells. Infection of target cells by vesicle-associated virus is not dependent on LSTc and is not neutralized by antisera directed against the virus. This is the first demonstration of a polyomavirus using extracellular vesicles as a means of transmission.

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JC polyomavirus (JCPyV) is the causative agent of a fatal central nervous system demyelinating disease known as progressive multifocal leukoencephalopathy (PML). PML occurs in people with underlying immunodeficiency or in individuals being treated with potent immunomodulatory therapies. JCPyV is a DNA tumor virus with a double-stranded DNA genome and encodes a well-studied oncogene, large T antigen. Its host range is highly restricted to humans and only a few cell types support lytic infection in vivo or in vitro. Its oncogenic potential in humans has not been firmly established and the international committee on oncogenic viruses lists JCPyV as possibly carcinogenic. Significant progress has been made in understanding the biology of JCPyV and here we present an overview of the field and discuss some important questions that remain unanswered.

The JC and BK human polyomaviruses (JCPyV and BKPyV, respectively) establish lifelong persistent infections in the kidney. In immunosuppressed individuals, JCPyV causes progressive multifocal leukoencephalopathy (PML), a fatal neurodegenerative disease, and BKPyV causes polyomavirus-associated nephropathy (PVN). In this study, we compared JCPyV and BKPyV infections in primary human renal proximal tubule epithelial (HRPTE) cells. JCPyV established a persistent infection, but BKPyV killed the cells in 15 days. To identify the cellular factors responsible for controlling JCPyV infection and promoting viral persistence, we profiled the transcriptomes of JCPyV- and BKPyV-infected cells at several time points postinfection. We found that infection with both viruses induced interferon production but that interferon-stimulated genes (ISGs) were only activated in the JCPyV-infected cells. Phosphorylated STAT1 and IRF9, which are responsible for inducing ISGs, translocated to the nucleus of JCPyV-infected cells but did not in BKPyV-infected cells. In BKPyV-infected cells, two critical suppressors of cytokine signaling, SOCS3 and SOCS1, were induced. Infection with BKPyV but not JCPyV caused reorganization of PML bodies that are associated with inactivating antiviral responses. Blockade of the interferon receptor and neutralization of soluble interferon alpha (IFN-α) and IFN-β partially alleviated the block to JCPyV infection, leading to enhanced infectivity. Our results show that a type I IFN response contributes to the establishment of persistent infection by JCPyV in HRPTE cells.

Chitinase-3-like protein 1 (CHI3L1/YKL-40) has long been known as a biomarker for early detection of neuroinflammation and disease diagnosis of Alzheimer's disease (AD). In the brain, CHI3L1 is primarily provided by astrocytes and heralds the reactive, neurotoxic state triggered by inflammation and other stress signals. However, how CHI3L1 acts in neuroinflammation or how it contributes to AD and relevant neurodegenerative conditions remains unknown. In peripheral tissues, our group and others have uncovered that CHI3L1 is a master regulator for a wide range of injury and repair events, including the innate immunity pathway that resembles the neuroinflammation process governed by microglia and astrocytes. Based on assessment of current knowledge regarding CHI3L1 biology, we hypothesize that CHI3L1 functions as a signaling molecule mediating distinct neuroinflammatory responses in brain cells and misfunctions to precipitate neurodegeneration. We also recommend future research directions to validate such assertions for better understanding of disease mechanisms.

Highlights d JC virus interacts transiently with each of the 5-HT 2 receptors (5-HT 2 Rs) d CRISPR-Cas9 genetic knockout of 5-HT 2 receptors reduces infection d The second intracellular loop of each 5-HT 2 receptor is sufficient for infection d A proline in each 5-HT 2 receptor is critical to support infection

Polyomaviruses are small, non-enveloped DNA tumor viruses that cause serious disease in immunosuppressed people, including progressive multifocal leukoencephalopathy (PML) in patients infected with JC polyomavirus, but the molecular events mediating polyomavirus entry are poorly understood. Through genetic knockdown approaches, we identified phosphoinositide 3′-kinase γ (PI3Kγ) and its regulatory subunit PIK3R5 as cellular proteins that facilitate infection of human SVG-A glial cells by JCPyV. PI3Kα appears less important for polyomavirus infection than PI3Kγ. CRISPR/Cas9-mediated knockout of PIK3R5 or PI3Kγ inhibited infection by authentic JCPyV and by JC pseudovirus. PI3Kγ knockout also inhibited infection by BK and Merkel Cell pseudoviruses, other pathogenic human polyomaviruses, and SV40, an important model polyomavirus. Reintroduction of the wild-type PI3Kγ gene into the PI3Kγ knock-out SVG-A cells rescued the JCPyV infection defect. Disruption of the PI3Kγ pathway did not block binding of JCPyV to cells or virus internalization, implying that PI3Kγ facilitates some intracellular step(s) of infection. These results imply that agents that inhibit PI3Kγ signaling may have a role in managing polyomavirus infections.

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.