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1.
Int J Mol Sci ; 24(10)2023 May 11.
Article in English | MEDLINE | ID: mdl-37239927

ABSTRACT

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.


Subject(s)
JC Virus , Leukoencephalopathy, Progressive Multifocal , Polyomavirus Infections , Humans , Leukoencephalopathy, Progressive Multifocal/drug therapy , JC Virus/physiology , Signal Transduction
2.
mBio ; 14(2): e0358322, 2023 04 25.
Article in English | MEDLINE | ID: mdl-36786589

ABSTRACT

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.


Subject(s)
JC Virus , Leukoencephalopathy, Progressive Multifocal , Polyomavirus Infections , Polyomavirus , Humans , Oxindoles/pharmacology , Leukoencephalopathy, Progressive Multifocal/drug therapy , Leukoencephalopathy, Progressive Multifocal/genetics , JC Virus/genetics , MAP Kinase Signaling System , Antiviral Agents
3.
J Extracell Biol ; 1(5)2022 May.
Article in English | MEDLINE | ID: mdl-36688929

ABSTRACT

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.

4.
Int J Mol Sci ; 22(18)2021 Sep 10.
Article in English | MEDLINE | ID: mdl-34575975

ABSTRACT

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.


Subject(s)
Crotonates/pharmacology , DNA Viruses/drug effects , Hydroxybutyrates/pharmacology , Leukoencephalopathy, Progressive Multifocal/drug therapy , Multiple Sclerosis, Relapsing-Remitting/drug therapy , Neuroglia/drug effects , Nitriles/pharmacology , Toluidines/pharmacology , Astrocytes/drug effects , Astrocytes/virology , Cell Line , Choroid Plexus/drug effects , Choroid Plexus/virology , DNA Viruses/pathogenicity , Demyelinating Diseases/drug therapy , Demyelinating Diseases/pathology , Demyelinating Diseases/virology , Epithelial Cells/drug effects , Epithelial Cells/virology , Extracellular Vesicles/drug effects , Extracellular Vesicles/virology , Humans , Immunologic Factors/adverse effects , Immunologic Factors/therapeutic use , JC Virus/drug effects , JC Virus/pathogenicity , Leukoencephalopathy, Progressive Multifocal/chemically induced , Leukoencephalopathy, Progressive Multifocal/pathology , Leukoencephalopathy, Progressive Multifocal/virology , Multiple Sclerosis, Relapsing-Remitting/genetics , Multiple Sclerosis, Relapsing-Remitting/pathology , Multiple Sclerosis, Relapsing-Remitting/virology , Neuroglia/virology , Virus Diseases/drug therapy , Virus Diseases/genetics , Virus Diseases/virology
5.
Virology ; 548: 17-24, 2020 09.
Article in English | MEDLINE | ID: mdl-32838939

ABSTRACT

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.


Subject(s)
JC Virus/physiology , Neuroglia/metabolism , Polyomavirus Infections/metabolism , Cell Line , Host-Pathogen Interactions , Humans , JC Virus/genetics , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Membrane Proteins , Neuroglia/virology , Oligodendroglia/metabolism , Oligodendroglia/virology , Polyomavirus Infections/genetics , Polyomavirus Infections/virology
6.
PLoS Pathog ; 16(3): e1008371, 2020 03.
Article in English | MEDLINE | ID: mdl-32130281

ABSTRACT

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.


Subject(s)
Choroid Plexus/metabolism , Epithelial Cells/metabolism , Extracellular Vesicles/metabolism , JC Virus/metabolism , Leukoencephalopathy, Progressive Multifocal/metabolism , Neuroglia/metabolism , Receptors, Virus/metabolism , Cell Line, Transformed , Choroid Plexus/pathology , Choroid Plexus/virology , Epithelial Cells/pathology , Epithelial Cells/virology , Extracellular Vesicles/pathology , Extracellular Vesicles/virology , Humans , Leukoencephalopathy, Progressive Multifocal/pathology , Neuroglia/pathology , Neuroglia/virology
8.
Cell Rep ; 27(7): 1960-1966.e6, 2019 05 14.
Article in English | MEDLINE | ID: mdl-31091436

ABSTRACT

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.


Subject(s)
JC Virus/genetics , Receptors, Serotonin, 5-HT2/genetics , Receptors, Serotonin, 5-HT2/metabolism , Receptors, Virus/genetics , Receptors, Virus/metabolism , Virus Internalization , HEK293 Cells , Host-Pathogen Interactions/genetics , Humans , JC Virus/pathogenicity , beta-Arrestins/genetics , beta-Arrestins/metabolism
9.
mBio ; 10(2)2019 04 09.
Article in English | MEDLINE | ID: mdl-30967463

ABSTRACT

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.


Subject(s)
Extracellular Vesicles/virology , JC Virus/physiology , Virus Internalization , Cell Line , Humans
10.
J Virol ; 92(8)2018 04 15.
Article in English | MEDLINE | ID: mdl-29437972

ABSTRACT

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.


Subject(s)
Choroid Plexus , Epithelial Cells , JC Virus/metabolism , Leukoencephalopathy, Progressive Multifocal , Meninges , Ritanserin/pharmacology , Cell Line , Choroid Plexus/metabolism , Choroid Plexus/pathology , Choroid Plexus/virology , Epithelial Cells/metabolism , Epithelial Cells/pathology , Epithelial Cells/virology , Humans , Leukoencephalopathy, Progressive Multifocal/drug therapy , Leukoencephalopathy, Progressive Multifocal/metabolism , Leukoencephalopathy, Progressive Multifocal/pathology , Leukoencephalopathy, Progressive Multifocal/virology , Meninges/metabolism , Meninges/pathology , Meninges/virology
11.
mBio ; 7(4)2016 07 05.
Article in English | MEDLINE | ID: mdl-27381292

ABSTRACT

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.


Subject(s)
BK Virus/immunology , BK Virus/physiology , Epithelial Cells/immunology , Host-Pathogen Interactions , Interferon Type I/metabolism , JC Virus/immunology , JC Virus/physiology , Cell Nucleus/chemistry , Cell Survival , Cells, Cultured , Epithelial Cells/virology , Gene Expression Profiling , Humans , Interferon-Stimulated Gene Factor 3, gamma Subunit/metabolism , Protein Transport , STAT1 Transcription Factor/metabolism , Suppressor of Cytokine Signaling 1 Protein/metabolism , Suppressor of Cytokine Signaling 3 Protein/metabolism , Virus Latency
12.
Am J Pathol ; 185(8): 2246-58, 2015 Aug.
Article in English | MEDLINE | ID: mdl-26056932

ABSTRACT

The human polyomavirus, JCPyV, is the causative agent of progressive multifocal leukoencephalopathy, a rare demyelinating disease that occurs in the setting of prolonged immunosuppression. After initial asymptomatic infection, the virus establishes lifelong persistence in the kidney and possibly other extraneural sites. In rare instances, the virus traffics to the central nervous system, where oligodendrocytes, astrocytes, and glial precursors are susceptible to lytic infection, resulting in progressive multifocal leukoencephalopathy. The mechanisms by which the virus traffics to the central nervous system from peripheral sites remain unknown. Lactoseries tetrasaccharide c (LSTc), a pentasaccharide containing a terminal α2,6-linked sialic acid, is the major attachment receptor for polyomavirus. In addition to LSTc, type 2 serotonin receptors are required for facilitating virus entry into susceptible cells. We studied the distribution of virus receptors in kidney and brain using lectins, antibodies, and labeled virus. The distribution of LSTc, serotonin receptors, and virus binding sites overlapped in kidney and in the choroid plexus. In brain parenchyma, serotonin receptors were expressed on oligodendrocytes and astrocytes, but these cells were negative for LSTc and did not bind virus. LSTc was instead found on microglia and vascular endothelium, to which virus bound abundantly. Receptor distribution was not changed in the brains of patients with progressive multifocal leukoencephalopathy. Virus infection of oligodendrocytes and astrocytes during disease progression is LSTc independent.


Subject(s)
Brain/metabolism , Choroid Plexus/metabolism , JC Virus , Kidney/metabolism , Polysaccharides/metabolism , Receptors, Serotonin, 5-HT2/metabolism , Receptors, Virus/metabolism , Sialic Acids/metabolism , Adult , Aged , Aged, 80 and over , Astrocytes/metabolism , Female , Humans , Male , Middle Aged , Oligodendroglia/metabolism
13.
J Virol ; 89(12): 6364-75, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25855729

ABSTRACT

UNLABELLED: The human JC polyomavirus (JCPyV) establishes an asymptomatic, persistent infection in the kidneys of the majority of the population and is the causative agent of the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML) in immunosuppressed individuals. The Mad-1 strain of JCPyV, a brain isolate, was shown earlier to require α2,6-linked sialic acid on the lactoseries tetrasaccharide c (LSTc) glycan for attachment to host cells. In contrast, a JCPyV kidney isolate type 3 strain, WT3, has been reported to interact with sialic acid-containing gangliosides, but the role of these glycans in JCPyV infection has remained unclear. To help rationalize these findings and probe the effects of strain-specific differences on receptor binding, we performed a comprehensive analysis of the glycan receptor specificities of these two representative JCPyV strains using high-resolution X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, and correlated these data with the results of infectivity assays. We show here that capsid proteins of Mad-1 and WT3 JCPyV can both engage LSTc as well as multiple sialylated gangliosides. However, the binding affinities exhibit subtle differences, with the highest affinity observed for LSTc. Engagement of LSTc is a prerequisite for functional receptor engagement, while the more weakly binding gangliosides are not required for productive infection. Our findings highlight the complexity of virus-carbohydrate interactions and demonstrate that subtle differences in binding affinities, rather than the binding event alone, help determine tissue tropism and viral pathogenesis. IMPORTANCE: Viral infection is initiated by attachment to receptors on host cells, and this event plays an important role in viral disease. We investigated the receptor-binding properties of human JC polyomavirus (JCPyV), a virus that resides in the kidneys of the majority of the population and can cause the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML) in the brains of immunosuppressed individuals. JCPyV has been reported to interact with multiple carbohydrate receptors, and we sought to clarify how the interactions between JCPyV and cellular carbohydrate receptors influenced infection. Here we demonstrate that JCPyV can engage numerous sialylated carbohydrate receptors. However, the virus displays preferential binding to LSTc, and only LSTc mediates a productive infection. Our findings demonstrate that subtle differences in binding affinity, rather than receptor engagement alone, are a key determinant of viral infection.


Subject(s)
Capsid Proteins/metabolism , JC Virus/physiology , Polysaccharides/metabolism , Receptors, Virus/metabolism , Sialic Acids/metabolism , Virus Attachment , Animals , Capsid Proteins/chemistry , Crystallography, X-Ray , Humans , Magnetic Resonance Spectroscopy , Mice , Receptors, Virus/chemistry
14.
J Virol ; 89(7): 3910-21, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25609820

ABSTRACT

UNLABELLED: JC polyomavirus (JCPyV) infection of immunocompromised individuals results in the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). The viral capsid of JCPyV is composed primarily of the major capsid protein virus protein 1 (VP1), and pentameric arrangement of VP1 monomers results in the formation of a pore at the 5-fold axis of symmetry. While the presence of this pore is conserved among polyomaviruses, its functional role in infection or assembly is unknown. Here, we investigate the role of the 5-fold pore in assembly and infection of JCPyV by generating a panel of mutant viruses containing amino acid substitutions of the residues lining this pore. Multicycle growth assays demonstrated that the fitness of all mutants was reduced compared to that of the wild-type virus. Bacterial expression of VP1 pentamers containing substitutions to residues lining the 5-fold pore did not affect pentamer assembly or prevent association with the VP2 minor capsid protein. The X-ray crystal structures of selected pore mutants contained subtle changes to the 5-fold pore, and no other changes to VP1 were observed. Pore mutant pseudoviruses were not deficient in assembly, packaging of the minor capsid proteins, or binding to cells or in transport to the host cell endoplasmic reticulum. Instead, these mutant viruses were unable to expose VP2 upon arrival to the endoplasmic reticulum, a step that is critical for infection. This study demonstrated that the 5-fold pore is an important structural feature of JCPyV and that minor modifications to this structure have significant impacts on infectious entry. IMPORTANCE: JCPyV is an important human pathogen that causes a severe neurological disease in immunocompromised individuals. While the high-resolution X-ray structure of the major capsid protein of JCPyV has been solved, the importance of a major structural feature of the capsid, the 5-fold pore, remains poorly understood. This pore is conserved across polyomaviruses and suggests either that these viruses have limited structural plasticity in this region or that this pore is important in infection or assembly. Using a structure-guided mutational approach, we showed that modulation of this pore severely inhibits JCPyV infection. These mutants do not appear deficient in assembly or early steps in infectious entry and are instead reduced in their ability to expose a minor capsid protein in the host cell endoplasmic reticulum. Our work demonstrates that the 5-fold pore is an important structural feature for JCPyV.


Subject(s)
Capsid Proteins/metabolism , Capsid/physiology , JC Virus/physiology , Protein Multimerization , Virus Assembly , Virus Internalization , Amino Acid Substitution , Capsid/chemistry , Capsid Proteins/chemistry , Capsid Proteins/genetics , Crystallography, X-Ray , Humans , JC Virus/chemistry , JC Virus/genetics , JC Virus/growth & development , Macromolecular Substances/chemistry , Mutant Proteins/genetics , Mutant Proteins/metabolism , Protein Conformation
15.
J Pept Sci ; 21(3): 236-42, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25522925

ABSTRACT

The JC polyomavirus (JCPyV) infects approximately 50% of the human population. In healthy individuals, the infection remains dormant and asymptomatic, but in immuno-suppressed patients, it can cause progressive multifocal leukoencephalopathy (PML), a potentially fatal demyelinating disease. Currently, there are no drugs against JCPyV infection nor for the treatment of PML. Here, we report the development of small-molecule inhibitors of JCPyV that target the initial interaction between the virus and host cell and thereby block viral entry. Utilizing a combination of computational and NMR-based screening techniques, we target the LSTc tetrasaccharide binding site within the VP1 pentameric coat protein of JCPyV. Four of the compounds from the screen effectively block viral infection in our in vitro assays using SVG-A cells. For the most potent compound, we used saturation transfer difference NMR to determine the mode of binding to purified pentamers of JCPyV VP1. Collectively, these results demonstrate the viability of this class of compounds for eventual development of JCPyV-antiviral therapeutics.


Subject(s)
Antiviral Agents/chemistry , Capsid Proteins/antagonists & inhibitors , JC Virus/drug effects , Small Molecule Libraries/pharmacology , Virus Internalization/drug effects , Animals , Antiviral Agents/chemical synthesis , Binding Sites , Biological Assay , COS Cells , Capsid Proteins/chemistry , Capsid Proteins/genetics , Cell Line, Transformed , Chlorocebus aethiops , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , HEK293 Cells , Humans , JC Virus/growth & development , JC Virus/metabolism , Molecular Docking Simulation , Neuroglia/drug effects , Neuroglia/virology , Protein Binding/drug effects , Protein Multimerization , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Small Molecule Libraries/chemical synthesis , Small Molecule Libraries/chemistry
16.
Virus Res ; 189: 280-5, 2014 Aug 30.
Article in English | MEDLINE | ID: mdl-24960120

ABSTRACT

JCPyV and BKPyV are common human polyomaviruses that cause lifelong asymptomatic persistent infections in their hosts. In immunosuppressed individuals, increased replication of JCPyV and BKPyV cause significant disease. JCPyV causes a fatal and rapidly progressing demyelinating disease known as progressive multifocal leukoencephalopathy. BKPyV causes hemorrhagic cystitis and polyomavirus associated nephropathy in bone marrow transplant recipients and in renal transplant recipients respectively. There are no specific anti-viral therapies to treat polyomavirus induced diseases. Based on detailed studies of the structures of these viruses bound to their receptors we screened several compounds that possessed similar chemical space as sialic acid for their ability to bind the virus. Positive hits in the assay were restricted to gallic acid based compounds that mimic the viruses known cellular glycan receptors. Pre-treatment of virions with these inhibitors reduced virus infection in cell culture and as such may form the basis for the development of virion specific antagonists to treat these infections.


Subject(s)
BK Virus/drug effects , Gallic Acid/pharmacology , JC Virus/drug effects , Virus Attachment/drug effects , Antiviral Agents/isolation & purification , Antiviral Agents/pharmacology , Cell Line , Drug Evaluation, Preclinical , Gallic Acid/isolation & purification , Humans
17.
Virology ; 456-457: 87-95, 2014 May.
Article in English | MEDLINE | ID: mdl-24889228

ABSTRACT

Autophagy is important for a variety for virus life cycles. We sought to determine the role of autophagy in human BK polyomavirus (BKPyV) infection. The addition excess amino acids during viral infection reduced BKPyV infection. Perturbing autophagy levels using inhibitors, 3-MA, bafilomycin A1, and spautin-1, also reduced infection, while rapamycin treatment of host cells increased infection. siRNA knockdown of autophagy genes, ATG7 and Beclin-1, corresponded to a decrease in BKPyV infection. BKPyV infection not only correlated with autophagosome formation, but also virus particles localized to autophagy-specific compartments early in infection. These data support a novel role for autophagy in the promotion of BKPyV infection.


Subject(s)
Autophagy , BK Virus/physiology , Host-Pathogen Interactions , Virus Replication , HeLa Cells , Humans
18.
J Virol ; 88(2): 948-60, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24198413

ABSTRACT

Progressive multifocal leukoencephalopathy (PML) is a fatal disease with limited treatment options, both clinically and in the research pipeline. Potential therapies would target and neutralize its etiologic agent, JC polyomavirus (JCPyV). The innate immune response to JCPyV infection has not been studied, and little is known about the initial host response to polyomavirus infection. This study examined the ability of a human alpha defensin, HD5, to neutralize JCPyV infection in human fetal glial cells. We show that HD5, by binding to the virion, blocks infection. The JCPyV-HD5 complexes bind to and enter host cells but are reduced in their ability to reach the endoplasmic reticulum (ER), where virions are normally uncoated. Furthermore, HD5 binding to the virion stabilizes the capsid and prevents genome release. Our results show that HD5 neutralizes JCPyV infection at an early postentry step in the viral life cycle by stabilizing the viral capsid and disrupting JCPyV trafficking. This study provides a naturally occurring platform for developing antivirals to treat PML and also expands on the known capabilities of human defensins.


Subject(s)
Capsid/metabolism , Endoplasmic Reticulum/virology , JC Virus/physiology , Polyomavirus Infections/metabolism , Tumor Virus Infections/metabolism , alpha-Defensins/metabolism , Capsid/chemistry , Capsid Proteins/genetics , Capsid Proteins/metabolism , Endoplasmic Reticulum/metabolism , Humans , JC Virus/genetics , Polyomavirus Infections/genetics , Polyomavirus Infections/virology , Protein Binding , Protein Transport , Tumor Virus Infections/genetics , Tumor Virus Infections/virology , alpha-Defensins/genetics
19.
mBio ; 4(6): e00729-13, 2013 Nov 12.
Article in English | MEDLINE | ID: mdl-24222489

ABSTRACT

UNLABELLED: Polyomaviruses are ubiquitous pathogens that cause severe disease in immunocompromised individuals. JC polyomavirus (JCPyV) is the causative agent of the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML), whereas BK polyomavirus (BKPyV) causes polyomavirus-induced nephropathy and hemorrhagic cystitis. Vaccines or antiviral therapies targeting these viruses do not exist, and treatments focus on reducing the underlying causes of immunosuppression. We demonstrate that retro-2(cycl), an inhibitor of ricin and Shiga-like toxins (SLTs), inhibits infection by JCPyV, BKPyV, and simian virus 40. Retro-2(cycl) inhibits retrograde transport of polyomaviruses to the endoplasmic reticulum, a step necessary for productive infection. Retro-2(cycl) likely inhibits polyomaviruses in a way similar to its ricin and SLT inhibition, suggesting an overlap in the cellular host factors used by bacterial toxins and polyomaviruses. This work establishes retro-2(cycl) as a potential antiviral therapy that broadly inhibits polyomaviruses and possibly other pathogens that use retrograde trafficking. IMPORTANCE: The human polyomaviruses JC polyomavirus (JCPyV) and BK polyomavirus (BKPyV) cause rare but severe diseases in individuals with reduced immune function. During immunosuppression, JCPyV disseminates from the kidney to the central nervous system and destroys oligodendrocytes, resulting in the fatal disease progressive multifocal leukoencephalopathy. Kidney transplant recipients are at increased risk of BKPyV-induced nephropathy, which results in kidney necrosis and loss of the transplanted organ. There are currently no effective therapies for JCPyV and BKPyV. We show that a small molecule named retro-2(cycl) protects cells from infection with JCPyV and BKPyV by inhibiting intracellular viral transport. Retro-2(cycl) treatment reduces viral spreading in already established infections and may therefore be able to control infection in affected patients. Further optimization of retro-2(cycl) may result in the development of an effective antiviral therapy directed toward pathogens that use retrograde trafficking to infect their hosts.


Subject(s)
Antiviral Agents/metabolism , BK Virus/drug effects , Benzamides/metabolism , JC Virus/drug effects , Simian virus 40/drug effects , Thiophenes/metabolism , Virus Internalization/drug effects , Animals , BK Virus/physiology , Cell Line , Haplorhini , Humans , JC Virus/physiology , Protein Transport/drug effects , Ricin/antagonists & inhibitors , Shiga Toxins/antagonists & inhibitors , Simian virus 40/physiology
20.
Virus Res ; 178(2): 281-6, 2013 Dec 26.
Article in English | MEDLINE | ID: mdl-24100235

ABSTRACT

The normally asymptomatic human polyomavirus, JCPyV, is the causative agent of a rare but fatal demyelinating disease known as progressive multifocal leukoencephalopathy (PML). Individuals at risk for developing PML include those with AIDS, with other underlying immunosuppressive diseases, and in patients treated with immunomodulatory regimens. Drugs to prevent viral reactivation in the setting of immunosuppression or immunomodulation could be used to sustain lives. Development of such drugs has been impeded by the difficulty of growing and studying the virus. We sought to develop a more efficient method for screening drugs that inhibit viral infection. Pseudovirus models have been developed which may be of use in pharmaceutical research. The use of pseudoviruses as models for viral infection is dependent on them using similar pathways for infection as virus. We screened known inhibitors of viral entry for their ability to block pseudovirus infection. Here we show that the pseudovirus based on the human polyomavirus JCPyV recapitulates virus binding, entry and trafficking. This system can be used for high-throughput screening of antiviral drugs.


Subject(s)
Antiviral Agents/isolation & purification , Antiviral Agents/pharmacology , Drug Evaluation, Preclinical/methods , JC Virus/physiology , Virology/methods , Virus Internalization/drug effects , Cell Line , Humans , JC Virus/genetics
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