An Elusive Target: Inhibitors of JC Polyomavirus Infection and Their Development as Therapeutics for the Treatment of Progressive Multifocal Leukoencephalopathy.

Progressive multifocal leukoencephalopathy (PML) is a rare demyelinating disease caused by infection with JC Polyomavirus (JCPyV). Despite the identification of the disease and isolation of the causative pathogen over fifty years ago, no antiviral treatments or prophylactic vaccines exist. Disease onset is usually associated with immunosuppression, and current treatment guidelines are limited to restoring immune function. This review summarizes the drugs and small molecules that have been shown to inhibit JCPyV infection and spread. Paying attention to historical developments in the field, we discuss key steps of the virus lifecycle and antivirals known to inhibit each event. We review current obstacles in PML drug discovery, including the difficulties associated with compound penetrance into the central nervous system. We also summarize recent findings in our laboratory regarding the potent anti-JCPyV activity of a novel compound that antagonizes the virus-induced signaling events necessary to establish a productive infection. Understanding the current panel of antiviral compounds will help center the field for future drug discovery efforts.

The Oxindole GW-5074 Inhibits JC Polyomavirus Infection and Spread by Antagonizing the MAPK-ERK Signaling Pathway.

JC polyomavirus (JCPyV) is a ubiquitous, double-stranded DNA virus that causes the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML) in immunocompromised patients. Current treatments for PML are limited to immune reconstitution, and no effective antivirals exist. In this report, we show that the oxindole GW-5074 (3-(3,5-dibromo-4-hydroxybenzylidene)-5-iodoindolin-2-one) reduces JCPyV infection in primary and immortalized cells. This compound potently inhibits virus spread, which suggests that it could control infection in PML patients. We demonstrate that GW-5074 inhibits endogenous ERK phosphorylation, and that JCPyV infection in GW-5074-treated cells cannot be rescued with ERK agonists, which indicates that the antiviral mechanism may involve its antagonistic effects on MAPK-ERK signaling. Importantly, GW-5074 exceeds thresholds of common pharmacological parameters that identify promising compounds for further development. This MAPK-ERK antagonist warrants further investigation as a potential treatment for PML. IMPORTANCE Human polyomaviruses, such as JCPyV and BKPyV, cause significant morbidity and mortality in immunocompromised or immunomodulated patients. There are no treatments for polyomavirus-induced diseases other than restoration of immune function. We discovered that the oxindole GW-5074 potently inhibits infection by both JCPyV and BKPyV. Further optimization of this compound could result in the development of antiviral therapies for polyomavirus-induced diseases.

Complexities of JC Polyomavirus Receptor-Dependent and -Independent Mechanisms of Infection.

JC polyomavirus (JCPyV) is a small non-enveloped virus that establishes lifelong, persistent infection in most of the adult population. Immune-competent patients are generally asymptomatic, but immune-compromised and immune-suppressed patients are at risk for the neurodegenerative disease progressive multifocal leukoencephalopathy (PML). Studies with purified JCPyV found it undergoes receptor-dependent infectious entry requiring both lactoseries tetrasaccharide C (LSTc) attachment and 5-hydroxytryptamine type 2 entry receptors. Subsequent work discovered the major targets of JCPyV infection in the central nervous system (oligodendrocytes and astrocytes) do not express the required attachment receptor at detectable levels, virus could not bind these cells in tissue sections, and viral quasi-species harboring recurrent mutations in the binding pocket for attachment. While several research groups found evidence JCPyV can use novel receptors for infection, it was also discovered that extracellular vesicles (EVs) can mediate receptor independent JCPyV infection. Recent work also found JCPyV associated EVs include both exosomes and secretory autophagosomes. EVs effectively present a means of immune evasion and increased tissue tropism that complicates viral studies and anti-viral therapeutics. This review focuses on JCPyV infection mechanisms and EV associated and outlines key areas of study necessary to understand the interplay between virus and extracellular vesicles.

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.

Teriflunomide Inhibits JCPyV Infection and Spread in Glial Cells and Choroid Plexus Epithelial Cells.

Several classes of immunomodulators are used for treating relapsing-remitting multiple sclerosis (RRMS). Most of these disease-modifying therapies, except teriflunomide, carry the risk of progressive multifocal leukoencephalopathy (PML), a severely debilitating, often fatal virus-induced demyelinating disease. Because teriflunomide has been shown to have antiviral activity against DNA viruses, we investigated whether treatment of cells with teriflunomide inhibits infection and spread of JC polyomavirus (JCPyV), the causative agent of PML. Treatment of choroid plexus epithelial cells and astrocytes with teriflunomide reduced JCPyV infection and spread. We also used droplet digital PCR to quantify JCPyV DNA associated with extracellular vesicles isolated from RRMS patients. We detected JCPyV DNA in all patients with confirmed PML diagnosis (n = 2), and in six natalizumab-treated (n = 12), two teriflunomide-treated (n = 7), and two nonimmunomodulated (n = 2) patients. Of the 21 patients, 12 (57%) had detectable JCPyV in either plasma or serum. CSF was uniformly negative for JCPyV. Isolation of extracellular vesicles did not increase the level of detection of JCPyV DNA versus bulk unprocessed biofluid. Overall, our study demonstrated an effect of teriflunomide inhibiting JCPyV infection and spread in glial and choroid plexus epithelial cells. Larger studies using patient samples are needed to correlate these in vitro findings with patient data.

Control of Archetype BK Polyomavirus MicroRNA Expression.

BK polyomavirus (BKPyV) is a ubiquitous human pathogen, with over 80% of adults worldwide being persistently infected. BKPyV infection is usually asymptomatic in healthy people; however, it causes polyomavirus-associated nephropathy in renal transplant patients and hemorrhagic cystitis in bone marrow transplant patients. BKPyV has a circular, double-stranded DNA genome that is divided genetically into three parts: an early region, a late region, and a noncoding control region (NCCR). The NCCR contains the viral DNA replication origin and cis-acting elements regulating viral early and late gene expression. It was previously shown that a BKPyV microRNA (miRNA) expressed from the late strand regulates viral large-T-antigen expression and limits the replication capacity of archetype BKPyV. A major unanswered question in the field is how expression of the viral miRNA is regulated. Typically, miRNA is expressed from introns in cellular genes, but there is no intron readily apparent in BKPyV from which the miRNA could derive. Here, we provide evidence for primary RNA transcripts that circle the genome more than once and include the NCCR. We identified splice junctions resulting from splicing of primary transcripts circling the genome more than once, and Sanger sequencing of reverse transcription-PCR (RT-PCR) products indicates that there are viral transcripts that circle the genome up to four times. Our data suggest that the miRNA is expressed from an intron spliced out of these greater-than-genome-size primary transcripts.IMPORTANCE The BK polyomavirus (BKPyV) miRNA plays an important role in regulating viral large-T-antigen expression and limiting the replication of archetype BKPyV, suggesting that the miRNA regulates BKPyV persistence. However, how miRNA expression is regulated is poorly understood. Here, we present evidence that the miRNA is expressed from an intron that is generated by RNA polymerase II transcribing the circular viral genome more than once. We identified splice junctions that could be generated only from primary transcripts that contain tandemly repeated copies of the viral genome. The results indicate another way in which viruses optimize expression of their genes using limited coding capacity.

Phosphoinositide 3'-Kinase γ Facilitates Polyomavirus 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.

Fifty Years of JC Polyomavirus: A Brief Overview and Remaining Questions.

In the fifty years since the discovery of JC polyomavirus (JCPyV), the body of research representing our collective knowledge on this virus has grown substantially. As the causative agent of progressive multifocal leukoencephalopathy (PML), an often fatal central nervous system disease, JCPyV remains enigmatic in its ability to live a dual lifestyle. In most individuals, JCPyV reproduces benignly in renal tissues, but in a subset of immunocompromised individuals, JCPyV undergoes rearrangement and begins lytic infection of the central nervous system, subsequently becoming highly debilitating-and in many cases, deadly. Understanding the mechanisms allowing this process to occur is vital to the development of new and more effective diagnosis and treatment options for those at risk of developing PML. Here, we discuss the current state of affairs with regards to JCPyV and PML; first summarizing the history of PML as a disease and then discussing current treatment options and the viral biology of JCPyV as we understand it. We highlight the foundational research published in recent years on PML and JCPyV and attempt to outline which next steps are most necessary to reduce the disease burden of PML in populations at risk.

Natural Killer cell activation, reduced ACE2, TMPRSS2, cytokines G-CSF, M-CSF and SARS-CoV-2-S pseudovirus infectivity by MEK inhibitor treatment of human cells.

COVID-19 affects vulnerable populations including elderly individuals and patients with cancer. Natural Killer (NK) cells and innate-immune TRAIL suppress transformed and virally-infected cells. ACE2, and TMPRSS2 protease promote SARS-CoV-2 infectivity, while inflammatory cytokines IL-6, or G-CSF worsen COVID-19 severity. We show MEK inhibitors (MEKi) VS-6766, trametinib and selumetinib reduce ACE2 expression in human cells. In some human cells, remdesivir increases ACE2-promoter luciferase-reporter expression, ACE2 mRNA and protein, and ACE2 expression is attenuated by MEKi. In serum-deprived and stimulated cells treated with remdesivir and MEKi we observed correlations between pRB, pERK, and ACE2 expression further supporting role of proliferative state and MAPK pathway in ACE2 regulation. We show elevated cytokines in COVID-19-(+) patient plasma (N=9) versus control (N=11). TMPRSS2, inflammatory cytokines G-CSF, M-CSF, IL-1α, IL-6 and MCP-1 are suppressed by MEKi alone or with remdesivir. We observed MEKi stimulation of NK-cell killing of target-cells, without suppressing TRAIL-mediated cytotoxicity. Pseudotyped SARS-CoV-2 virus with a lentiviral core and SARS-CoV-2 D614 or G614 SPIKE (S) protein on its envelope infected human bronchial epithelial cells, small airway epithelial cells, or lung cancer cells and MEKi suppressed infectivity of the pseudovirus. We show a drug class-effect with MEKi to stimulate NK cells, inhibit inflammatory cytokines and block host-factors for SARS-CoV-2 infection leading also to suppression of SARS-CoV-2-S pseudovirus infection of human cells. MEKi may attenuate SARS-CoV-2 infection to allow immune responses and antiviral agents to control disease progression.

Adipocyte Plasma Membrane Protein (APMAP) promotes JC Virus (JCPyV) infection in human glial cells.

The demyelinating disease progressive multifocal leukoencephalopathy (PML) is caused by the human polyomavirus, JCPyV, under conditions of prolonged immunosuppression. Initial infection is asymptomatic, and the virus establishes lifelong persistence in the host. Following the loss of immune surveillance, the virus can traffic to the central nervous system and infect oligodendrocytes to cause demyelination and PML. The mechanisms involved in glial cell infection are not completely understood. In a screen for N-glycosylated proteins that influence JCPyV pathology, we identified Adipocyte Plasma Membrane Associated Protein (APMAP) as a host cell modulator of JCPyV infection. The removal of APMAP by small interfering siRNA as well as by CRISPR-Cas9 gene editing resulted in a significant decrease in JCPyV infection. Exogenous expression of APMAP in APMAP knockout cell lines rescued susceptibility to infection. These data suggest that virus infection of glial cells is dependent on APMAP.

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.

Genetic and Functional Dissection of the Role of Individual 5-HT2 Receptors as Entry Receptors for JC Polyomavirus.

JC polyomavirus (JCPyV) is a ubiquitous human pathogen that causes progressive multifocal leukoencephalopathy (PML). The entry receptors for JCPyV belong to the 5-hydroxytryptamine 2 receptor (5-HT2R) family, but how individual members of the family function to facilitate infection is not known. We used proximity ligation assay (PLA) to determine that JCPyV interacts with each of the 5-HT2 receptors (5-HT2Rs) in a narrow window of time during entry. We used CRISPR-Cas9 to randomly introduce stop codons in the gene for each receptor and discovered that the second intracellular loop of each was necessary for infection. This loop contains a motif possibly involved in receptor internalization by ß-arrestin. Mutation of this motif and small interfering RNA (siRNA) knockdown of ß-arrestin recapitulated the results of our CRISPR-Cas9 screen, showing that this motif is critical. Our results have implications for the role these receptors play in virus infection and for their normal functioning as receptors for serotonin.

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.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.

Susceptibility of Primary Human Choroid Plexus Epithelial Cells and Meningeal Cells to Infection by JC Virus.

JC polyomavirus (JCPyV) establishes a lifelong persistence in roughly half the human population worldwide. The cells and tissues that harbor persistent virus in vivo are not known, but renal tubules and other urogenital epithelial cells are likely candidates as virus is shed in the urine of healthy individuals. In an immunosuppressed host, JCPyV can become reactivated and cause progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease of the central nervous system. Recent observations indicate that JCPyV may productively interact with cells in the choroid plexus and leptomeninges. To further study JCPyV infection in these cells, primary human choroid plexus epithelial cells and meningeal cells were challenged with virus, and their susceptibility to infection was compared to the human glial cell line, SVG-A. We found that JCPyV productively infects both choroid plexus epithelial cells and meningeal cells in vitro Competition with the soluble receptor fragment LSTc reduced virus infection in these cells. Treatment of cells with neuraminidase also inhibited both viral infection and binding. Treatment with the serotonin receptor antagonist, ritanserin, reduced infection in SVG-A and meningeal cells. We also compared the ability of wild-type and sialic acid-binding mutant pseudoviruses to transduce these cells. Wild-type pseudovirus readily transduced all three cell types, but pseudoviruses harboring mutations in the sialic acid-binding pocket of the virus failed to transduce the cells. These data establish a novel role for choroid plexus and meninges in harboring virus that likely contributes not only to meningoencephalopathies but also to PML.IMPORTANCE JCPyV infects greater than half the human population worldwide and causes central nervous system disease in patients with weakened immune systems. Several recent reports have found JCPyV in the choroid plexus and leptomeninges of patients with encephalitis. Due to their role in forming the blood-cerebrospinal fluid barrier, the choroid plexus and leptomeninges are also poised to play roles in virus invasion of brain parenchyma, where infection of macroglial cells leads to the development of progressive multifocal leukoencephalopathy, a severely debilitating and often fatal infection. In this paper we show for the first time that primary choroid plexus epithelial cells and meningeal cells are infected by JCPyV, lending support to the association of JCPyV with meningoencephalopathies. These data also suggest that JCPyV could use these cells as reservoirs for the subsequent invasion of brain parenchyma.

Progressive Multifocal Leukoencephalopathy: Endemic Viruses and Lethal Brain Disease.

In 1971, the first human polyomavirus was isolated from the brain of a patient who died from a rapidly progressing demyelinating disease known as progressive multifocal leukoencephalopathy. The virus was named JC virus after the initials of the patient. In that same year a second human polyomavirus was discovered in the urine of a kidney transplant patient and named BK virus. In the intervening years it became clear that both viruses were widespread in the human population but only rarely caused disease. The past decade has witnessed the discovery of eleven new human polyomaviruses, two of which cause unusual and rare cancers. We present an overview of the history of these viruses and the evolution of JC polyomavirus-induced progressive multifocal leukoencephalopathy over three different epochs. We review what is currently known about JC polyomavirus, what is suspected, and what remains to be done to understand the biology of how this mostly harmless endemic virus gives rise to lethal disease.

The biology of JC polyomavirus.

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.

Decreased function of survival motor neuron protein impairs endocytic pathways.

Spinal muscular atrophy (SMA) is caused by depletion of the ubiquitously expressed survival motor neuron (SMN) protein, with 1 in 40 Caucasians being heterozygous for a disease allele. SMN is critical for the assembly of numerous ribonucleoprotein complexes, yet it is still unclear how reduced SMN levels affect motor neuron function. Here, we examined the impact of SMN depletion in Caenorhabditis elegans and found that decreased function of the SMN ortholog SMN-1 perturbed endocytic pathways at motor neuron synapses and in other tissues. Diminished SMN-1 levels caused defects in C. elegans neuromuscular function, and smn-1 genetic interactions were consistent with an endocytic defect. Changes were observed in synaptic endocytic proteins when SMN-1 levels decreased. At the ultrastructural level, defects were observed in endosomal compartments, including significantly fewer docked synaptic vesicles. Finally, endocytosis-dependent infection by JC polyomavirus (JCPyV) was reduced in human cells with decreased SMN levels. Collectively, these results demonstrate for the first time, to our knowledge, that SMN depletion causes defects in endosomal trafficking that impair synaptic function, even in the absence of motor neuron cell death.

JC Polyomavirus Infection of Primary Human Renal Epithelial Cells Is Controlled by a Type I IFN-Induced Response.

UNLABELLED: 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-ß 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. IMPORTANCE: The human polyomaviruses JCPyV and BKPyV both establish lifelong persistent infection in the kidneys. In immunosuppressed patients, BKPyV causes significant pathology in the kidney, but JCPyV is only rarely associated with disease in this organ. The reasons behind this striking difference in kidney pathology are unknown. In this study, we show that infection of primary human renal tubule epithelial cells with JCPyV and BKPyV results in divergent innate immune responses that control JCPyV but fail to control BKPyV. This is the first study that directly compares JCPyV and BKPyV infection in vitro in the same cell type they naturally infect, and the significant differences that have been uncovered could in part explain the distinct disease outcomes.